U.S. patent application number 17/090665 was filed with the patent office on 2021-04-15 for methods and systems for microfluidic screening.
The applicant listed for this patent is 1859, Inc.. Invention is credited to Devon CAYER, Pavel CHUBUKOV, Andrew MACCONNELL, Ramesh RAMJI, Sean STROMBERG.
Application Number | 20210106998 17/090665 |
Document ID | / |
Family ID | 1000005197324 |
Filed Date | 2021-04-15 |
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United States Patent
Application |
20210106998 |
Kind Code |
A1 |
CAYER; Devon ; et
al. |
April 15, 2021 |
METHODS AND SYSTEMS FOR MICROFLUIDIC SCREENING
Abstract
Provided are methods and systems useful for screening large
libraries of effector molecules. Such methods and systems are
particularly useful in microfluidic systems and devices. The
methods and systems provided herein utilize encoded effectors to
screen large libraries of effectors.
Inventors: |
CAYER; Devon; (Del Mar,
CA) ; MACCONNELL; Andrew; (Del Mar, CA) ;
CHUBUKOV; Pavel; (Del Mar, CA) ; RAMJI; Ramesh;
(Del Mar, CA) ; STROMBERG; Sean; (Del Mar,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
1859, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
1000005197324 |
Appl. No.: |
17/090665 |
Filed: |
November 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17067534 |
Oct 9, 2020 |
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17090665 |
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62954348 |
Dec 27, 2019 |
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62913624 |
Oct 10, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/0652 20130101;
B01L 3/502761 20130101; C12Q 1/686 20130101; C12N 15/1093 20130101;
B01L 2400/0403 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12Q 1/686 20060101 C12Q001/686; C12N 15/10 20060101
C12N015/10 |
Claims
1. A method for screening an encoded effector, the method
comprising: (a) providing at least one cell and a scaffold in an
encapsulation, wherein the scaffold comprises the encoded effector
bound to the scaffold by a cleavable linker and an encoding
corresponding to the encoded effector; (b) cleaving the cleavable
linker to release the encoded effector from the scaffold; (c)
detecting a signal from the encapsulation, wherein the signal
results from an interaction between the encoded effector and the at
least one cell; and (d) sorting the encapsulation, barcoding the
encoding, or both, based on the signal.
2. The method of claim 1, wherein the at least one cell comprises a
single cell.
3. The method of claim 1, wherein the sorting comprises using a
waveform pulse generator to move the encapsulation to a collection
tube by i) an electrical field gradient; ii) by sound; iii) by a
diaphragm; iv) by modifying geometry of the microfluidic channel;
or v) by changing the pressure of the microfluidic channel.
4. The method of claim 1, wherein the encoding comprises a nucleic
acid and the method further comprises identifying the encoded
effector by sequencing the nucleic acid.
5. The method of claim 1, wherein the barcoding comprises adding a
barcoding reagent into the encapsulation.
6. The method of claim 1, wherein the signal comprises
electromagnetic radiation, thermal radiation, a visual change in
the at least one cell, or combinations thereof.
7. The method of claim 1, wherein the cleavable linker is a
photocleavable linker.
8. The method of claim 7, wherein the cleaving releases a
pre-determined amount of the encoded effector into the
encapsulation.
9. The method of claim 1, wherein the interaction between the
encoded effector and the at least one cell comprises inhibition of
one or more cellular components of the at least one cell.
10. The method of claim 1, further comprising providing an
activating reagent to activate the photocleavable linker, so as to
enable the photocleavable linker to be cleaved.
11. The method of claim 10, wherein the activating reagent is added
into the encapsulation through pico-injection or droplet
merging.
12. The method claim 1, further comprising lysing the at least one
cell.
13. The method of claim 1, wherein the encoded effector is selected
from the group consisting of: a peptide, a compound, a protein, an
enzyme, a macrocycle compound, and a nucleic acid.
14. The method of claim 13, wherein the compound is a drug-like
small molecule.
15-20. (canceled)
21. The method of claim 7, wherein the cleaving comprises cleaving
the photocleavable linker using electromagnetic radiation.
22. The method of claim 7, wherein the cleaving comprises exposing
the encapsulation to a light from a light source.
23. The method of claim 22, wherein a light intensity of the light
is from about 0.01 J/cm.sup.2 to about 200 J/cm.sup.2.
24. The method of claim 12, wherein the lysing comprises adding a
lysis buffer to the encapsulation.
25. The method of claim 1, wherein the scaffold is selected from
the group consisting of: a bead, a fiber, a nanofibrous scaffold, a
molecular cage, a dendrimer, and a multi-valent molecular
assembly.
26. The method of claim 1, wherein the encapsulation is a droplet.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 17/067,534 filed Oct. 9, 2020 which claims priority to U.S.
Provisional Application No. 62/913,624, filed Oct. 10, 2019, and
U.S. Provisional Application No. 62/954,348, filed Dec. 27, 2019,
both of which are incorporated by reference herein in its
entirety.
BACKGROUND
[0002] Drug development often requires a significant amount of
testing and analysis to determine how specific chemical substances
impact cellular and other biological components. As such, devices
and specific methodologies that focus on correlating a relationship
between specific chemical substances and biological components is
an integral component for a drug developer, such as pharmaceutical
companies.
SUMMARY
[0003] Provided herein are systems and methods for performing
high-throughput assays using microfluidic systems and encoded
effectors. The systems and methods described herein can be used to
perform nearly any assay in a high-throughput manner and provide
detailed information about the effect of various effector molecules
on biological systems. The systems and methods provided herein
utilize encoded effectors, which allow a user to readily ascertain
which of the effectors has an effect on a biological sample.
[0004] Other systems have various drawbacks, including an inability
to customize the addition of reagents at concentrations of interest
at different unit operations during a screen. The systems and
methods described herein address these drawbacks. For example,
methods and systems described herein allow for the introduction of
reagents at specified concentrations at different steps in a
screening procedure. In some instances, adding reagents at defined
concentrations allows uniform doses of effectors to be administered
across a library being screened. This may allow for decreased false
positives in a screen because low potency but highly-loaded
effectors may be dosed against samples at a uniform concentration
across a library screen. In some instances, the customizable
additions of reagents allow for facile deconvolution of screening
hits without a step of physical sorting of effectors that elicit a
positive or negative response in the screen.
[0005] In another aspect are methods of monitoring biological
samples in a microfluidic based screen without utilizing light
(e.g. fluorescence) emitted from a sample. These methods may allow
for more detailed information about a sample being analyzed than is
available by other methods. Further provided herein are methods and
systems for incorporating genetic or cellular information from a
sample into the encoded effectors. This incorporation step can
allow for an improved analysis of the response of a cell or other
biological sample contacted with an effector than is available by
other methods. In another aspect, information encoded in a sample,
such as a DNA barcode, is incorporated from the sample into the
encoding to allow determination of synergistic benefits of multiple
effectors. This can be used for conducting a small-molecule
fragment-based screen to generate compound leads.
[0006] The methods provided herein provide advantages over existing
DNA encoded libraries being used for drug screening. In some
embodiments, the methods enclosed herein are functional
"activity-based" assays, not just "affinity-based" assays: they
allows the screening of functional assays. In some embodiments, the
methods herein are not limited to testing if a candidate drug binds
to a disease target. In contrast, the methods herein may be capable
of testing whether the candidate drug functions against that
disease target. Such functions may comprise inhibition, disruption
of protein-protein interactions, or activating an enzyme or
allosteric pocket.
[0007] In some instances, the methods provided herein can screen in
complex environments such as cell lysates, cells, or other
multi-component mixtures in a single assay. In some embodiments,
the functional activity test is orthogonal to all other components
in a mixture and is specifically testing for functional activity of
a target of interest. The screening modalities provided herein are
diverse. Such modalities can screen for potency, selectivity,
toxicity, liabilities, or other key metrics critical for drug
discovery campaigns. The methods provided herein may allow for
speed and diversity at 1000 times lower operational cost than other
methods. In some instances, the speed, low reagent needs, and
exceptional validation rates allow fast, iterative screening of
potentially an unlimited set of chemically diverse compounds. The
flexibility and speed allow for testing or screening of compounds
in many different assays or formats for a single target, allowing
multiple sampling of conditions, easy "restarts", fast "hit to
lead" starts, and "immediate" validation of library designs.
[0008] In some instances, the methods provided herein do not
require high sequencing depth, thus reducing costs for analysis.
Additionally, the methods disclosed herein may allow for the
quantification of yields of each chemistry step, allowing
normalized dose-response curves and possibly quantitative
analysis.
[0009] In some instances, the methods provided herein enable the
use of DNA damaging chemistries that require organic solvents, or
conditions that would otherwise be DNA damaging in the synthesis of
encoded beads. For example, some chemistries needed to construct
small-molecules may degrade or cause DNA to become non-amplifiable
and thus the DNA barcode information can no longer be read. In some
instances, this challenge is overcome by providing DNA encodings
bound to scaffolds at high levels. In some embodiments, the
scaffolds comprise 10 million or more encodings bound to a
scaffold. Additionally, in some embodiments, as few as 10 encodings
are required to be present in order to detect a positive hit.
[0010] Provided herein are methods for cell phenotypic screening.
Cells directly within droplets can be tested and probed for a
variety of different phenotypes. For example, an entire library can
be screened for toxicity against a particular cell type, or an
entire library can be screened for its ability to affect a
particular disease target in its native cell context, or an entire
library can be screened for its ability to affect a panel of
targets (transcriptome, protein panel, etc.). This is allowed
because a small molecule can be liberated off of the bead where it
can then penetrate intracellularly a cell (or affect an
extracellular target) and affect a particular disease target
[0011] Further provided herein are methods for normalizing the
results of screens of encoded effectors. Other methods of
ascertaining the results from a screen suffer from high rates of
false negative results, where an effector displays potency against
a target sample, but due to damage to the encoding during the
screen or low abundance of the encoding during the synthesis of the
encoded effector, the "hit" is missed in the subsequent analysis.
Provided herein are methods for normalizing the amount of encodings
present after a screen has been performed in order to minimize
false negative results due to low abundance of encodings of potent
effectors.
[0012] Also provided herein are devices for performing the methods
provided herein. In some instances, the method provided herein are
performed on microfluidic devices or in microfluidic channels.
[0013] Further provided herein are devices useful for the
performance of high-throughput screen using encoded libraries.
These devices can allow for fixing a target sample, in some
instances a single cell, in a fixed location in space with a single
encoded effector. Such devices can allow for screening single
compounds against cells to determine desired effects without the
need to create in situ encapsulations separating each individual
sample/effector combination.
[0014] Disclosed herein, in some embodiments is a method for
screening an encoded effector, the method comprising: a) providing
at least one cell and a scaffold in an encapsulation, wherein the
scaffold comprises an encoded effector bound to the scaffold by a
photocleavable linker and a nucleic acid encoding the effector; b)
cleaving the photocleavable linker to release the encoded effector
from the scaffold; and c) detecting a signal from the droplet,
wherein the signal results from an interaction between the encoded
effector and the at least one cell. In some embodiments, cleaving
the photocleavable linker releases a pre-determined amount of the
encoded effector into the droplet. In some embodiments, the
photocleavable linker is cleaved using electromagnetic radiation.
In some embodiments, cleaving the photocleavable linker comprises
exposing the encapsulation to a light from a light source. In some
embodiments, the light intensity of the light is from about 0.01
J/cm.sup.2 to about 200 J/cm.sup.2. In some embodiments, the method
further comprising the step of lysing the one or more cells. In
some embodiments, the method further comprising providing an
activating reagent to activate the photocleavable linker, so as to
enable the photocleavable linker to be cleaved from the encoded
effector.
[0015] Disclosed herein, in some embodiments, is a system for
screening an encoded effector, the system comprising: a) one or
more cells; b) a scaffold, wherein an encoded effector is bound to
the scaffold by a cleavable linker, wherein a nucleic acid encoding
the effector is bound to the scaffold; and c) a microfluidic device
configured to: i) receive the one or more cells and scaffold; ii)
encapsulate the one or more cells and scaffold within an
encapsulation; iii) cleave the cleavable linker from the encoded
effector to release a predetermined amount of the encoded effector
within the encapsulation; iv) incubate the encoded effector with
the one or more cells for a period of time; v) detect a signal from
the encapsulation, wherein the signal results from an interaction
between the encoded effector and one or more cells; and vi) sort
the encapsulation based on the detection of the signal. In some
embodiments, the cleavable linker is a photocleavable linker. In
some embodiments, the microfluidic device further comprises a first
collection tube and second collection tube for sorting the
encapsulation, wherein the encapsulation is placed in 1) the first
collection tube if the signal is at or above a predetermined
threshold or 2) the second collection tube if the signal is below a
predetermined threshold. In some embodiments, the system further
comprising a waveform pulse generator to move the encapsulation to
the first or second collection tube by an electrical field
gradient, by sound, by a diaphragm, by modifying geometry of the
microfluidic channel, or by changing the pressure of a microfluidic
channel of the microfluidic device. In some embodiments, the signal
is detected based on detecting morphological changes in the one or
more cells measured by recording a series of images of the droplet
or detecting fluorescence emitted by a molecular beacon or probe.
In some embodiments, the period of time is controlled by residence
time as the encapsulation travels through a microfluidic channel of
the microfluidic device.
[0016] Disclosed herein, is a method for amplifying a primer to
maximize cellular nucleic acid capture comprising: a) providing an
encapsulation comprising a nucleic acid encoded scaffold with one
or more cells, an amplification mix, and a nicking enzyme, wherein
a nucleic acid encoding is bound to the nucleic acid encoded
scaffold; b) lysing the one or more cells to release one or more
cellular nucleic acids; c) nicking the nucleic acid encoding with
the nicking enzyme, thereby creating an encoded nucleic acid
primer; d) amplifying the encoded nucleic acid primer via the
nicking site and amplification mix; and e) labeling a released
cellular nucleic acid with the encoded nucleic acid primer. In some
embodiments, the specific site comprises a specific nucleotide
sequence. In some embodiments, amplifying the encoded nucleic acid
primer comprises 1) creating a copy of the nucleic acid encoding
that extends from the nicking site, and 2) nicking the nucleic acid
encoding copy to create another encoded nucleic acid primer. In
some embodiments, amplifying the encoded nucleic acid primer
comprises simultaneously 1) creating a copy of the nucleic acid
encoding that extends from the nicking site, and 2) displacing the
nucleic acid encoding copy to create another encoded nucleic acid
primer. In some embodiments, the amplification mix comprises an
amplification enzyme, such that the amplification enzyme enables
for a copy of the nucleic acid encoding to be simultaneously
created and displaced. In some embodiments, the amplification
enzyme comprises a polymerase. In some embodiments, each nucleic
acid encoding comprises a capture site that prescribes a target
cellular coding or a target cellular nucleic acid to label a
released cellular nucleic acid.
INCORPORATION BY REFERENCE
[0017] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The novel features of the disclosure are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present disclosure will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the disclosure
are utilized, and the accompanying drawings of which:
[0019] FIG. 1 provides a depiction of a nucleic acid encoded
effector bound to a bead along with the nucleic acid encoding.
[0020] FIG. 2A shows an exemplary workflow of a screen using a
nucleic acid encoded effector bound to a bead.
[0021] FIG. 2B shows an exemplary workflow of a screen using a
microfluidic device.
[0022] FIG. 2C shows an exemplary workflow for an encapsulation
assay screen.
[0023] FIG. 2D shows an exemplary workflow for an encapsulation
assay screen using pico-injection.
[0024] FIG. 3 illustrates an exemplary method for amplifying a
primer to maximize cellular nucleic acid capture.
[0025] FIG. 4 shows an exemplary microfluidic device for performing
a screen according to the methods provided herein.
[0026] FIG. 5 shows an exemplary microfluidic device for performing
a screen according to the methods provided herein.
[0027] FIG. 6 shows an exemplary microfluidic device for performing
a screen according to the methods provided herein.
[0028] FIG. 7 shows an exemplary microfluidic device for performing
a screen according to the methods provided herein.
[0029] FIG. 8 shows an overview of specifically designed IC chips
and their related development workflow.
[0030] FIG. 9A provides a depiction of an exemplary microfluidic
device provided with the methods and systems described herein.
[0031] FIG. 9B provides a depiction of a specific section of an
exemplary microfluidic device provided herein.
[0032] FIG. 9C shows a picture of an exemplary microfluidic device
provided herein
[0033] FIG. 10 provides another exemplary depiction of the
microfluidic device provided in FIG. 9A.
[0034] FIG. 11 provides a depiction of another exemplary
microfluidic device used with the methods and systems described
herein.
[0035] FIG. 12A provides a depiction of a library of beads attached
with an encoded-effector modified with fluorophore.
[0036] FIG. 12B provides a depiction of an encoded-effector
modified with a fluorophore dye being liberated from a bead upon
being exposed to UV light.
[0037] FIG. 12C provides a depiction of the released encoded
effector-fluorophore from FIG. 12B.
[0038] FIG. 12D provides a depiction of the cleavage region of a
microfluidic device described herein.
[0039] FIG. 12E shows a depiction of a correlation between UV light
and a calibrant fluid.
[0040] FIG. 12F shows a depiction of an exemplary device for
confocal laser and PMT emission capture.
[0041] FIG. 13A shows measured intensity peaks of a fluorophore dye
using 100 mV UV light.
[0042] FIG. 13B shows a droplet map corresponding to intensity
peaks of a fluorophore dye using 100 mV UV light.
[0043] FIG. 14A shows measured intensity peaks of a fluorophore dye
using 600 mV UV light.
[0044] FIG. 14B shows a droplet map corresponding to intensity
peaks of a fluorophore dye using 600 mV UV light.
[0045] FIG. 15A provides a known correlation between UV power and
PMT count of a fluorophore dye.
[0046] FIG. 15B provides a histogram of distributed intensity
values of encoded effector-fluorophore compared with UV power
exposure.
[0047] FIG. 16 provides exemplary data of UV confinement in a
microfluidic device described herein.
[0048] FIG. 17A-B shows exemplary molecules being activated for
photocleavage.
[0049] FIG. 18 shows exemplary data for uniform incubation in the
microfluidic device shown in FIG. 9A
[0050] FIG. 19 shows exemplary data for uniform incubation in the
microfluidic device shown in FIG. 11.
[0051] FIG. 20 shows the microfluidic device of FIG. 9A with
various detector points along an assay flow path.
[0052] FIG. 21 shows an exemplary detection of a specific location
along an assay flow path in a microfluidic device described
herein.
[0053] FIG. 22A-B shows an exemplary fluorescence detection device
used with a microfluidic device described herein, and description
of related components.
[0054] FIG. 23A provides the detection of raw intensity levels at
an incubation time of 0 s for a fluorophore dye.
[0055] FIG. 23B provides the detection of real-time smoothing of
the intensity levels from FIG. 23A.
[0056] FIG. 24A provides the detection of raw intensity levels at
an incubation time of 1333 s for a fluorophore dye.
[0057] FIG. 24B provides the detection of real-time smoothing of
the intensity levels from FIG. 24A.
[0058] FIG. 25 shows increasing measured intensity peaks for a
fluorophore dye across an incubation period of an assay.
[0059] FIG. 26A shows an exemplary bead attached with a TR1-TAMRA
fluorophore.
[0060] FIG. 26B shows an exemplary intensity peak detected for the
TR1-TAMRA after it has been released from the bead.
[0061] FIG. 27A shows an exemplary bead attached with a TR3
inhibitor.
[0062] FIG. 27B shows an exemplary intensity inhibited
corresponding to activity by the TR3 inhibitor after it has been
released from the bead.
[0063] FIG. 27C shows an exemplary variation of Cathepsin D
activity based on increasing concentration of a TR3 inhibitor.
[0064] FIG. 28A provides an exemplary depiction of a sorting
schematic for beads that exhibit an intensity below an inhibition
threshold.
[0065] FIG. 28B shows an exemplary intensity peak detected for the
TR1-TAMRA that is above a threshold for positive sorting.
[0066] FIG. 28C shows an exemplary intensity peak inhibited for the
TR3 inhibitor that is below a threshold for positive sorting.
[0067] FIG. 28D shows an exemplary a device being used for sorting
encapsulations.
DETAILED DESCRIPTION
Screening Methods and Systems
[0068] Provided herein are methods and systems for screening
various effectors against samples in a high-throughput,
low-material manner. The systems and methods, in some embodiments,
utilize encoded effectors to probe various responses from samples.
In some embodiments, encoded effectors are molecules whose
structures can be measured by measuring a property of the
corresponding encoding. Generally, samples are incubated with
effectors in encapsulations. In response to an interaction with the
effector, some type of signal can then be detected. Based on this
signal, the effector can be determined to have efficacy against the
sample in inducing a particular response. The systems and methods
described herein, in some embodiments, utilize small
encapsulations, such as droplets. In some instances, each
individual encapsulation carries out an assay of the effector and
the sample in a small volume. A large library of such effectors can
be screened against the sample at the same time and in the same
experiment, thus providing high-throughput methods for conducting
screens. Effectors that produce a desired signal from a sample can
then be sorted, and the encoding of the effector can be measured to
deconvolute which effectors were efficacious in the assay.
[0069] Encoded Effectors
[0070] The systems and methods provided herein utilize encoded
effectors. An encoded effector, in some embodiments, is an effector
that has been linked with an encoding such that ascertaining a
property of the encoding allows a researcher to readily determine
the structure of the effector. An effector can be any type of
molecule or substance whose effect on a sample is being
investigated. In some embodiments, the effector is a compound, a
protein, a peptide, an enzyme, a nucleic acid, or any other
substance. In some instances, the encoding allows a user to
determine the structure of the effector by determining a property
of the encoding. Thus, each encoding moiety has a measurable
property that, when measured, can be used to determine the
structure of the effector which is encoded. Many different encoding
modalities can be used, including without limitation nucleic acids
and peptides. When the encoding modalities are nucleic acids, the
sequence of the nucleic acid may provide information about the
structure of its corresponding effector. In some instances, the
encoded effectors are described by what kind of molecules is used
in the encoding. For example, "nucleic acid encoded effectors"
comprise an effector encoded by a nucleic acid.
[0071] In some instances, the effectors and their corresponding
encodings are bound to a scaffold. This can allow the
effector/encoding pair to remain linked in space. In some
instances, when encoded effectors are placed into solutions or
other environments, the link between the pairing is not lost. Many
materials can be used as scaffolds, as any material capable of
binding both the effector and the encoding may accomplish the
desired goal of keeping the pair linked in space.
[0072] Various methods for preparing encoded effectors linked to
scaffolds can be used. In some embodiments, the methods use
orthogonal, compatible methodologies to create an effector and its
encoding in a parallel synthesis scheme. This is sometimes referred
to as "split and pool synthesis." For illustrative purposes only,
an exemplary, non-limiting, workflow for the preparation of a
scaffold containing an effector and encoding is described as
follows: A first effector subunit is attached at an attachment
point of a scaffold. The scaffold is then washed to remove
unreacted and excess reagents from the scaffold. A first encoding
subunit is then attached at another attachment point on the
scaffold, and a wash step performed. Following this, a second
effector subunit is then attached to the first effector subunit,
followed by another wash step. Then, a second encoding subunit is
attached to the first encoding subunit, followed by a wash step.
This process is repeated as many times as desired to prepare the
desired effectors and corresponding encodings. This process can be
repeated on a massively parallel scale in small volumes to prepare
vast libraries of compounds at low cost and with low amounts of
reagents. In some instances, pre-synthesized compounds are loaded
onto scaffolds which contain encodings. The encodings may be
pre-synthesized and loaded onto the scaffolds or are synthesized
directly onto the scaffolds using methods analogous to the split
and pool synthesis described above. In some instances, each
scaffold comprises numerous copies of a unique effector and its
corresponding encoding.
[0073] An example of a nucleic acid encoded effector linked with a
bead is shown in FIG. 1. A bead linked encoded effector 100
comprises a bead 101. Attached at one position is a nucleic acid
encoding 102, which is covalently attached to the scaffold in this
example. The nucleic acid encoding comprises encoding subunits A,
B, and C. The encoding subunits correspond with effector subunits
A, B, and C, which make up effector 103. The effector 103 is linked
to the bead 101 through a linker 104. The linker 104 may be a
cleavable linker, such a linker cleavable by electromagnetic
radiation (photocleavable) or selectively cleavable by a cleaving
reagent (chemically cleavable). Cleavable linkers can be used to
liberate effectors from a bead or other scaffold to allow the
effector to interact with a sample.
[0074] In some embodiments, the scaffolds further comprise
impurities in the effector and/or its encoding. In some instances,
impurities of the effector and its corresponding encoding occur due
to damage during a screen, during manufacturing of the bead,
effector, or encoding combination, or during storage. In some
embodiments, impurities of the effector and its corresponding
encoding are present due to defects in the methodologies used to
synthesize the encoded effectors. In some embodiments, scaffolds as
described herein can comprise a single encoder, an encoding and its
impurities, or combinations thereof. In some embodiments, at least
5%, at least 10%, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%, or at least 99% of the effectors attached to a
scaffold comprise an identical structure. In some embodiments, at
least 5%, at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, or at least 99% of the encodings attached to a
scaffold comprise an identical structure.
Screening System Components and Methods
[0075] Provided herein are methods and systems for screening
encoded effectors on samples using encapsulations. In some
embodiments, methods and systems for screening encoded effectors on
samples are capable of being performed in a high-throughput manner.
In some embodiments, the methods and systems provided herein allow
for screening large libraries of encoded effectors using small
volumes, minimal amounts of reagents, and small amounts of the
effectors being screened. In some embodiments, the methods and
systems provided herein allow for uniform dosing of effectors in a
library against samples. In some embodiments, the methods and
systems described herein allow for measurement of cellular features
in a high throughput manner. In some embodiments, the methods and
systems provided herein measure genomic, metabolomic, and/or
proteomic data from cells screened against the encoded effectors.
In some embodiments, the methods and systems provided herein allow
for synergistic effects of using multiple effectors against a
particular sample to be determined. In some embodiments, the
methods and systems provided herein allow for a library of mutant
proteins to be screened for a desired activity or improvement in
activity.
[0076] A non-limiting example workflow of a screen utilizing a
single encoded effector bound to a scaffold is shown in FIG. 2A.
The nucleic acid encoded effector bound to a scaffold is
encapsulated with a target of interest, in this case a cell. In
step 1, the effector, in this case a drug, is then cleaved from the
bead within the encapsulation. In step 2, the effector is allowed
to interact with the cell. If the drug has a desired effect on the
cell, a reporter signal indicates that the drug is a positive hit.
If there is no reporter signal detected, then the result for that
drug is negative. In step 3, positive and negative results are
sorted based on the detection of the signal. At the end of a
screen, in step 4, the positive hits, which have been pooled
together, are then sequenced (in the case of nucleic acid
encodings) to reveal which effectors had the desired effect. In
step 5, this information can then be used to guide synthesis of
further libraries or identify lead molecules for further
development.
[0077] FIG. 2B shows an additional exemplary, non-limiting workflow
of an effector screen on a microfluidic device. In the exemplary
workflow shown, a nucleic acid encoded effector bound to a bead is
placed in an inlet and merged with an additional aqueous stream,
which, in some embodiments, contains a sample to be tested. The
merged fluids are driven through an "extrusion region" or "droplet
formation region," wherein beads and sample are encapsulated within
a carrier fluid immiscible with the aqueous fluids. An effector is
then cleaved from bead at the effector cleavage region, which in
some embodiments utilizes a light source to cleave a photocleavable
linker. The encapsulations containing cleaved effectors are then
allowed to continue flowing along the flow path of the device
through the incubation region, which in some embodiments contains
widened or enlarged chambers to control flow rate or residence time
on the device. As the encapsulations travel through the incubation
region, a detectable signal is generated if the released effectors
have a desired activity. This signal is then detected in a
detection region of the device. In some embodiments, this
detectable signal is a fluorescent signal, though any detectable
signal can be employed. This signal is then measured or detected at
a detection region, which is in some embodiments equipped with a
light source (e.g. a laser or LED) and a detector (e.g. a
photomultiplier tube (PMT), a charged coupled device (CCD), or a
photodiode) coupled to a sorting device (e.g. a dielectrophoresis
electrode or any other sorting mechanism). In some embodiments, the
detection region comprises an interrogation region, which is
coupled to a sensor or an array of sensors. Based on the signal,
the encapsulations are sorted into a waste outlet or a hit outlet.
Following completion of the screen, the encodings of the hits are
amplified (e.g. by PCR or emulsion PCR) and the encodings sequenced
(e.g. by next generation sequencing). The sequenced encodings can
then be decoded to reveal the effectors which had the desired
activity. In some embodiments, each bead further comprises barcode
unique to the bead itself (independent of the effector). Thus, in
some embodiments, it is possible to ascertain if multiple beads
bearing identical effectors were selected as hits within multiple
encapsulations.
[0078] An exemplary, non-limiting droplet assay screen workflow is
shown in FIG. 2C. A bead buffer comprising a probe substrate and
nucleic acid encoded effectors bound to beads are merged with an
assay buffer comprising a disease target (e.g. a protein such as an
enzyme). An encapsulation comprising probe substrate, a bead
bearing a nucleic acid encoded effector, and the disease target is
then formed in an immiscible carrier fluid. The effector is then
released from the bead and allowed to interact with the disease
target. The sample is then incubated within a delay line (or any
such suitable channel or reservoir configured to incubate the
encapsulation for a desired time). In this embodiment, the probe
substrate is cleaved by the disease target. Upon cleavage, a change
in fluorescence properties of the substrate is observed, for
example due to FRET interactions of the probe substrate. If the
disease target is inhibited by the effector, the probe substrate
will not be cleaved. After a desired incubation time, the
fluorescence of the encapsulation is measured (e.g. by a PMT, CCD,
or photodiode) after excitation (e.g. by a laser or LED) and the
encapsulation is sorted (e.g. by electrophoresis or
dielectrophoresis) based on the result. FIG. 2D shows a similar
workflow but contains an additional step of adding a substrate
detection reagent (e.g. by pico-injection or droplet merging) in
order to allow detection of substrate that has or has not reacted
with the disease target. In some embodiments, an electrode is
employed at the pico-injection site in order to destabilize the
interface of the encapsulation to facilitate incorporation of the
pico-injected fluid into the encapsulation.
[0079] Provided herein are methods and systems for screening
encoded effectors on samples using encapsulations, wherein the
sample and an encoded effector are encapsulated. In some
embodiments, the encoded effector and the sample are encapsulated
by mixing a first solution comprising the encoded effector with a
second solution comprising the sample. In some embodiments, the
first and second solutions are mixed together with an oil. In some
embodiments, mixing the first and second solutions with an oil
forms an emulsion, wherein the first and second solutions combine
to form droplets. In some embodiments, encapsulations are formed in
a microfluidic device. In some embodiments, the encapsulation step
comprises merging the first and second solution at a T-junction of
microfluidic channels. In some embodiments, creating an
encapsulation comprises converging aqueous streams in a
microfluidic device. Creating an encapsulation can occur by
numerous methods, any of which may be compatible with the methods
described herein. In some embodiments, encapsulations are formed on
microfluidic devices. In some embodiments, encapsulations flow
through a microfluidic device.
[0080] In some embodiments, provided herein are methods and systems
for screening a library of encoded effectors. In some embodiments,
for any method or system described herein, the library of encoded
effectors comprises at least about 1, 1,000, 10,000, 100,000,
250,000, 1,000,000, or 10,000,000 unique encoded effectors. In some
embodiments, a plurality of scaffolds (as described herein) are
encapsulated in a plurality of encapsulations (as described herein)
with a sample in a microfluidic channel. In some embodiments, the
plurality of scaffolds (e.g., beads) are bound to a library of
unique encoded effectors. In some embodiments, each scaffold is
bound to one or more unique encoded effectors. In some embodiments,
the library of unique encoded effectors comprise at least about
250,000 unique encoded effectors. In some embodiments, the library
of unique encoded effectors comprise about 1 unique encoded
effector to about 10,000,000 unique encoded effectors. In some
embodiments, the library of unique encoded effectors comprise about
1 unique encoded effector to about 1,000 unique encoded effectors,
about 1 unique encoded effector to about 10,000 unique encoded
effectors, about 1 unique encoded effector to about 100,000 unique
encoded effectors, about 1 unique encoded effector to about 250,000
unique encoded effectors, about 1 unique encoded effector to about
1,000,000 unique encoded effectors, about 1 unique encoded effector
to about 10,000,000 unique encoded effectors, about 1 unique
encoded effector to about 200 unique encoded effectors, about 1,000
unique encoded effectors to about 10,000 unique encoded effectors,
about 1,000 unique encoded effectors to about 100,000 unique
encoded effectors, about 1,000 unique encoded effectors to about
250,000 unique encoded effectors, about 1,000 unique encoded
effectors to about 1,000,000 unique encoded effectors, about 1,000
unique encoded effectors to about 10,000,000 unique encoded
effectors, about 1,000 unique encoded effectors to about 200 unique
encoded effectors, about 10,000 unique encoded effectors to about
100,000 unique encoded effectors, about 10,000 unique encoded
effectors to about 250,000 unique encoded effectors, about 10,000
unique encoded effectors to about 1,000,000 unique encoded
effectors, about 10,000 unique encoded effectors to about
10,000,000 unique encoded effectors, about 10,000 unique encoded
effectors to about 200 unique encoded effectors, about 100,000
unique encoded effectors to about 250,000 unique encoded effectors,
about 100,000 unique encoded effectors to about 1,000,000 unique
encoded effectors, about 100,000 unique encoded effectors to about
10,000,000 unique encoded effectors, about 100,000 unique encoded
effectors to about 200 unique encoded effectors, about 250,000
unique encoded effectors to about 1,000,000 unique encoded
effectors, about 250,000 unique encoded effectors to about
10,000,000 unique encoded effectors, about 250,000 unique encoded
effectors to about 200 unique encoded effectors, about 1,000,000
unique encoded effectors to about 10,000,000 unique encoded
effectors, about 1,000,000 unique encoded effectors to about 200
unique encoded effectors, or about 10,000,000 unique encoded
effectors to about 200 unique encoded effectors, including
increments therein. In some embodiments, the library of unique
encoded effectors comprise about 1 unique encoded effector, about
1,000 unique encoded effectors, about 10,000 unique encoded
effectors, about 100,000 unique encoded effectors, about 250,000
unique encoded effectors, about 1,000,000 unique encoded effectors,
about 10,000,000 unique encoded effectors, or about 200 unique
encoded effectors. In some embodiments, the library of unique
encoded effectors comprise at least about 1 unique encoded
effector, about 1,000 unique encoded effectors, about 10,000 unique
encoded effectors, about 100,000 unique encoded effectors, about
250,000 unique encoded effectors, about 1,000,000 unique encoded
effectors, or about 10,000,000 unique encoded effectors. In some
embodiments, the library of unique encoded effectors comprise at
most about 1,000 unique encoded effectors, about 10,000 unique
encoded effectors, about 100,000 unique encoded effectors, about
250,000 unique encoded effectors, about 1,000,000 unique encoded
effectors, about 10,000,000 unique encoded effectors, or about 200
unique encoded effectors.
[0081] In some embodiments, each unique encoded effector is encoded
with a corresponding encoding. In some embodiments, at least one
encoding comprises a nucleic acid encoding. In some embodiments, at
least one encoded effector is bound to a respective scaffold
through a cleavable linker. In some embodiments, the cleavable
linker comprises a photocleavable linker, or a chemically cleavable
linker (e.g., linker cleaved through contact with a reagent). In
some embodiments, one or more photocleavable linkers between an
encoded effector and corresponding bead is cleaved. In some
embodiments, cleaving a photocleavable linker releases the
corresponding encoded effector from the bead. In some embodiments,
a released encoded effector interacts with the corresponding sample
within the respective encapsulation. In some embodiments, the
interaction between the encoded effector and the sample creates a
signal. In some embodiments, the signal is configured to be
detected. In some embodiments, the plurality of encapsulations are
sorted based on a corresponding signal being detected from each
encapsulation. In some embodiments, the plurality of encapsulations
are sorted based on a corresponding signal not being detected from
each encapsulation. In some embodiments, the encoding(s) associated
with the encapsulations having a detected signal(s) are barcoded,
as an alternative sorting the encapsulation. In some embodiments,
the encoding(s) associated with the encapsulations not having a
detected signal(s) are barcoded, as an alternative sorting the
encapsulation. In some embodiments, encapsulations are formed on
microfluidic devices. In some embodiments, encapsulations flow
through a microfluidic device.
[0082] Provided herein are methods and systems for screening
encoded effectors on samples using encapsulations, wherein a signal
is detected from the encapsulation. In some embodiments, the signal
results from an interaction between an effector and the sample. In
some embodiments, the signal is detected with a detector. In some
embodiments, detecting the signal comprises providing the
encapsulation through a microfluidic channel. In some embodiments,
detecting the signal comprises providing the encapsulation through
a microfluidic channel equipped with a detector. In some
embodiments, the detector is configured to detect the signal.
[0083] Signals of the methods and systems provided herein can be
any signal capable of detection in an encapsulation. In some
embodiments, the signal is electromagnetic radiation, thermal
radiation, a visual change in the sample, or combinations thereof.
In some embodiments, the electromagnetic radiation is fluorescence
or luminescence. In some embodiments, the electromagnetic radiation
is in the visible spectrum. In some embodiments, the signal is
absorbance of electromagnetic radiation.
[0084] Provided herein are methods and systems for screening
encoded effectors on samples using encapsulations, wherein the
encapsulation is sorted. In some embodiments, the encapsulation is
sorted based on the detection of a signal. In some embodiments, the
encapsulation is optionally sorted based on the detection of a
signal.
Alternative Signal Detection
[0085] Provided herein are methods and systems for screening
encoded effectors, wherein various alternative signal detection
methods and systems may be used to identify activity by an effector
or within an encapsulation. In some embodiments, the signal is a
thermal radiation. In some embodiments, the thermal radiation is
detected using an infrared camera. In some embodiments, the thermal
radiation is a change in thermal radiation emitted by a sample. In
some embodiments, the change in thermal radiation is due to
metabolic activity in a sample. In some embodiments, the change in
thermal radiation comprises a change in metabolic activity in the
sample. In some embodiments, the change in thermal radiation
comprises a change in metabolic activity in the sample due to an
effect of the effector on the sample. In some embodiments the
effect on the sample is a change in metabolic activity. In some
embodiments, detecting the signal comprises detecting a change in
metabolic activity in the sample by detecting a change in thermal
radiation. In some embodiments, the sample is a cell and the signal
is thermal radiation.
[0086] In some embodiments, the sample displays a change in
emission of thermal radiation compared to a sample not encapsulated
with the effector. In some embodiments, the change in thermal
radiation is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
or 100% emission of thermal radiation. In some embodiments, the
change in thermal radiation is at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 100% emission of thermal radiation relative
to sample not treated with the effector. In some embodiments, the
change in thermal radiation is at least 2-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold emission of
thermal radiation relative to a sample not treated with the
effector.
[0087] In some embodiments, the signal is luminescence. In some
embodiments, detecting the signal comprises monitoring
encapsulations for a period of time. In some embodiments, detecting
the signal comprises monitoring luminescence from the sample over a
period of time. In some embodiments, the luminescence is integrated
over a period of time. In some embodiments, the luminescence is
integrated over a period of at least 1 minute, at least 5 minutes,
at least 30 minutes, at least 4 hours, or at least 12 hours. In
some embodiments, the luminescence is integrated over a period of
at most 1 minutes, at most 5 minutes, at most 30 minutes, at most 4
hours, or at most 12 hours. In some embodiments, the luminescence
is integrated over a distance traveled by an encapsulation. In some
embodiments, the luminescence is integrated over a distance
travelled by an encapsulation through a microfluidic channel. In
some embodiments, the luminescence is integrated over a distance of
at least 1 .mu.m, at least 10 .mu.m, at least 50 .mu.m, at least
100 .mu.m, at least 250 .mu.m, at least 500 .mu.m, at least 1 mm,
at least 10 mm, or at least 100 mm travelled by an encapsulation
through a microfluidic channel. In some embodiments, the
luminescence is integrated over a distance of at most 1 .mu.m, at
most 10 .mu.m, at most 50 .mu.m, at most 100 .mu.m, at most 250
.mu.m, at most 500 .mu.m, at most 1 mm, at most 10 mm, or at most
100 mm travelled by an encapsulation through a microfluidic
channel.
[0088] The signal from the sample may be a morphological or visual
change in the sample which can be measured by imaging the
encapsulation. In some embodiments, detecting the signal comprises
recording images of the sample in the encapsulation. In some
embodiments, detecting the signal comprises recording a series of
images of the sample in the encapsulation. In some embodiments,
detecting a signal comprises recording a series of images of
samples in encapsulations and superimposing the series of images of
the sample. In some embodiments, detecting a signal comprises
detecting morphological or visual changes in the sample measured by
recording a series of images of the encapsulation.
[0089] In some embodiments, morphology changes in a sample, such as
one or more cells, can be detected by an imaging sensor, capturing
trans illuminated light with a high-speed shutter, where composite
video frames offers multiple full-cell images that can aid in shape
determination. In some embodiments, morphology changes in a sample,
such as one or more cells, can be detected by an imaging sensor,
capturing trans illuminated light from a high-frequency pulsed
light source, increasing temporal resolution and sharpening the
perimeter of the cell. In one manifestation, morphology changes can
be detected by fluorescence emission from a cell traversing a
laser-light sheet excitation region. In some embodiments, the
emission is captured by Avalanche Photodiode (APD) or charged
coupled detector (CCD), in a one-dimensional array of pixels,
binned by time, then restitched into a composite
fluorescence-microscopy image.
[0090] In some embodiments, detecting the signal comprises
recording images of the sample, wherein the sample is a cell. In
some embodiments, recording images of the cell provides information
about cell morphology, mitotic stage, levels of expressed proteins,
levels of cellular components, cell health, or combinations
thereof. In some embodiments, the encapsulation comprises a
detection agent. In some embodiments, the detection agent is an
intercalation dye. In some embodiments, the intercalation dye is
ethidium bromide, propidium iodide, crystal violet, a
dUTP-conjugated probe, DAPI (4', 6-diamidino-2-phenylindole), 7-AAD
(7-aminoactinomycin D), Hoechst 33258, Hoechst 33342, Hoechst
34580, combinations thereof, or derivatives thereof. In some
embodiments, the detection agent highlights different regions of
the cell. In some embodiments, the detection agent highlights a
particular organelle. In some embodiments, the organelle is a
mitochondrion, Golgi apparatus, endoplasmic reticulum, nucleus,
ribosomes, cellular membrane, nucleolus, liposome, lipid vesicle,
lysosome, or vacuole. In some embodiments, the organelle is a
mitochondrion. In some embodiments, the organelle is the
nucleus.
[0091] In some embodiments, detecting the signal comprises
detecting the presence of a target nucleic acid. In some
embodiments, the encapsulation further comprises a molecular
beacon. In some embodiments, the molecular beacon is complementary
to a portion of the target nucleic acid sequence of the sample. In
some embodiments, the methods further comprise adding a molecular
beacon to the encapsulation. In some embodiments, the target
nucleic acid is detected by a molecular beacon. In some
embodiments, the encapsulation further comprises a probe and a
polymerase. In some embodiments, the encapsulation further
comprises a TaqMan probe and a Taq polymerase. In some embodiments,
the methods further comprise adding a TaqMan probe and a Taq
polymerase to the encapsulation. In some embodiments, the TaqMan
probe is complementary to a portion of the target nucleic acid
sequence. In some embodiments, the TaqMan probe and Taq polymerase
are added to the encapsulation at the same time. In some
embodiments, the TaqMan probe and Taq polymerase are added
sequentially. In some embodiments, the signal is fluorescence
emitted by a molecular beacon. In some embodiments, the signal is
fluorescence emitted by TaqMan probe. In some embodiments, the
signal is fluorescence emitted by a molecular beacon or TaqMan
probe.
[0092] Various molecular beacons can be used with the methods and
systems described herein. In general, a molecular beacon comprises
a nucleic acid binding region that binds to a complementary nucleic
acid of interest. The molecular beacon can typically have a
secondary structure wherein a fluorophore and a quencher are in
proximity when the nucleic acid binding region is not bound to the
complementary nucleic acid of interest. Upon binding of the nucleic
acid binding region to the complementary nucleic acid of interest,
the fluorophore and quencher may be separated in space such that a
fluorescent signal can be detected. Thus, the amount of
fluorescence detected can be used to quantify the amount of nucleic
acid of interest present in a sample. In some embodiments, an
inhibitor is used wherein activity between an effector and a sample
inhibits or limits the intensity of a fluorescence signal.
[0093] In some embodiments, two or more signal detection methods
are used in combination for detecting a signal. In some
embodiments, detecting a signal comprises detecting morphological
changes in the sample as well as detecting fluorescence emitted by
a molecular beacon or probe. For example, in some embodiments,
fluorescence emission from a molecular beacon in the encapsulation
(e.g., droplet) can be measured by PMT or Avalanche Photodiode
(APD). In some embodiments, simultaneous image capture by
transillumination can identify other features in the encapsulation
(e.g., droplet), such as encoded effectors and cells. In some
embodiments, these streams of information together determine
outcome at the sorting junction.
[0094] In some embodiments, detecting the presence of the target
nucleic acid comprises amplifying the target nucleic acid. In some
embodiments, the target nucleic acid is amplified by an isothermal
amplification method. In some embodiments, the isothermal
amplification method is loop-mediated isothermal amplification
(LAMP), strand displacement amplification (SDA), helicase-dependent
amplification (HAD), recombinase polymerase amplification (RPA),
rolling circle replication (RCA) or nicking enzyme amplification
reaction (NEAR). In some embodiments, the encapsulation further
comprises reagents for isothermal amplification of the target
nucleic acid. In some embodiments, the methods comprise adding
reagents for isothermal amplification to the encapsulation. In some
embodiments, the reagents for isothermal amplification are specific
to the target nucleic acid sequence.
[0095] In some embodiments, the target nucleic acid is DNA. In some
embodiments, the target nucleic acids are cellular DNA. In some
embodiments, the target nucleic acids are genomic DNA. In some
embodiments, the target nucleic acid is RNA. In some embodiments,
the RNA is mRNA, ribosomal RNA, tRNA, non-protein-coding RNA
(npcRNA), non-messenger RNA, functional RNA (fRNA), long non-coding
RNA (lncRNA), pre-mRNAs, or primary miRNAs (pri-miRNAs). In some
embodiments, the target nucleic acids are mRNA.
Scaffold and Beads
[0096] An exemplary embodiment of screening encoded effectors on
samples using encapsulations comprises use of a scaffold. In some
embodiments, the effector is bound to a scaffold. In some
embodiments, the scaffold acts as a solid support and keeps the
encoded effector molecules linked in space to their encodings. In
some embodiments, the scaffold is a structure with a plurality of
attachment points that allow linkage of one or more molecules. In
some embodiments, the encoded effector is bound to a scaffold. In
some embodiments, the scaffold is a solid support. In some
embodiments, the scaffold is a bead, a fiber, nanofibrous scaffold,
a molecular cage, a dendrimer, or a multi-valent molecular
assembly.
[0097] In some embodiments, the scaffold is a bead. In some
embodiments, the bead is a polymer bead, a glass bead, a metal
bead, or a magnetic bead. In some embodiments, the bead is a
polymer bead. In some embodiments, the bead is a glass bead. In
some embodiments, the bead is a metal bead. In some embodiments,
the bead is a magnetic bead.
[0098] The beads utilized in the methods provided herein may be
made of any material. In some embodiments, the bead is a polymer
bead. In some embodiments, the bead comprises a polystyrene core.
In some embodiments, the beads are derivatized with polyethylene
glycol. In some embodiments, the beads are grafted with
polyethylene glycol. In some embodiments, the polyethylene glycol
contains reactive groups for the attachment of other
functionalities, such as effectors or encodings. In some
embodiments, the reactive group is an amino or carboxylate group.
In some embodiments, the reactive group is at the terminal end of
the polyethylene glycol chain. In some embodiments, the bead is a
TentaGel.RTM. bead.
[0099] The polyethylene glycol (PEG) attached to the beads may be
any size. In some embodiments, the PEG is up to 20 kDa. In some
embodiments, the PEG is up to 5 kDa. In some embodiments, the PEG
is about 3 kDa. In some embodiments, the PEG is about 2 to 3
kDa.
[0100] In some embodiments, the PEG group is attached to the bead
by an alkyl linkage. In some embodiments, the PEG group is attached
to a polystyrene bead by an alkyl linkage. In some embodiments, the
bead is a TentaGel.RTM. M resin.
[0101] In some embodiments, the bead comprises a PEG attached to a
bead through an alkyl linkage and the bead comprises two
bifunctional species. In some embodiments, the beads comprise
surface modification on the outer surface of the beads that are
orthogonally protected to reactive sites in the internal section of
the beads. In some embodiments the beads comprise both cleavable
and non-cleavable ligands. In some embodiments, the bead is a
TentaGel.RTM. B resin.
[0102] Beads for use in the systems and methods as described herein
can be any size. In some embodiments, the beads are at most 10 nm,
at most 100 nm, at most 1 .mu.m, at most 10 .mu.m, or at most 100
.mu.m in diameter. In some embodiments, the beads are at least 10
nm, at least 100 nm, at least 1 .mu.m, at least 10 .mu.m, or at
least 100 .mu.m in diameter. In some embodiments, the beads are
about 10 .mu.m to about 100 .mu.m in diameter.
[0103] In some embodiments, the effector is covalently bound to the
scaffold. In some embodiments, the effector is non-covalently bound
to the scaffold. In some embodiments, the effector is bound to the
scaffold through ionic interactions. In some embodiments, the
effector is bound to the scaffold through hydrophobic
interactions.
Cleavable Linker and Effector Release
[0104] Cleavable linkers can be used to attach effectors to
scaffolds. In some embodiments, the effector is bound to a scaffold
by a cleavable linker. In some embodiments, the cleavable linker is
cleavable by electromagnetic radiation, an enzyme, a chemical
reagent, heat, pH adjustment, sound, or electrochemical reactivity.
In some embodiments, the cleavable linker is cleavable by
electromagnetic radiation. In some embodiments, the cleavable
linker is cleavable by electromagnetic radiation such as UV light.
In some embodiments, the cleavable linker is a photocleavable
linker. In some embodiments the photocleavable linker is cleavable
by electromagnetic radiation. In some embodiments the
photocleavable linker is cleavable through exposure to light. In
some embodiments, the light comprises UV light. In some
embodiments, the cleavable linker is cleavable by a cleaving
reagent. In some embodiments, the cleavable linker must first be
activated in order to be able to be cleaved. In some embodiments,
the cleavable linker is activated through interaction with a
reagent.
[0105] In some embodiments, the cleavable linker is a disulfide
bond. In some embodiments, the cleavable linker is a disulfide bond
and the cleavable reagent is a reducing agent. In some embodiments,
the reducing agent is a disulfide reducing agent. In some
embodiments, the disulfide reducing agent is a phosphine. In some
embodiments, the reducing agent is 2-mercapto ethanol,
2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP),
dithiothreitol, a combination thereof, or a derivative thereof.
[0106] In some embodiments, the cleavable linker and cleaving
reagent are biorthogonal reagents. Bioorthogonal reagents are
combinations of reagents that selectively react with each other,
but do not have significant reactivity with other biological
components. Such reagents allow for minimal cross-reactivity with
other components of the reaction mixture, which allows for less off
target events.
[0107] In some embodiments, the cleavable linker is a substituted
trans-cyclooctene. In some embodiments, the cleavable linker is a
substituted trans-cyclooctene and the cleaving reagent is a
tetrazine. In some embodiments, the cleavable linker as the
structure
##STR00001##
wherein X is --C(.dbd.O)NR--, --C(.dbd.O)O--, --C(.dbd.O)-- or a
bond, and R is H or alkyl. In some embodiments, the cleaving
reagent is a tetrazine. In some embodiments, the cleaving reagent
is dimethyl tetrazine (DMT). Further examples of tetrazine
cleavable linkers and methods of use are described in
Tetrazine-triggered release of carboxylic-acid-containing molecules
for activation of an anti-inflammatory drug, ChemBioChem 2019, 20,
1541-1546, which is hereby incorporated by reference.
[0108] In some embodiments, the cleavable linker comprises an azido
group attached to the same carbon as an ether linkage. In some
embodiments, the cleavable linker has the structure
##STR00002##
In some embodiments, the cleaving reagent is a reagent that reduces
an azido group. In some embodiments, the cleaving reagent is a
phosphine. In some embodiments, the cleaving reagent is hydrogen
and a palladium catalyst.
[0109] In some embodiments, the cleavable linker is cleaved by a
transition metal catalyst. In some embodiments, the cleavage
reagent is a transition metal catalyst. In some embodiments, the
transition metal catalyst is a ruthenium metal complex. In some
embodiments, the cleavable linker is an O-allylic alkene. In some
embodiments, the cleavable linker has the structure
##STR00003##
A non-limiting example of such a catalyst is described in
Bioorthogonal catalysis: a general method to evaluate
metal-catalyzed reaction in real time in living systems using a
cellular luciferase reporter system, Bioconjugate Chem. 2016, 27,
376-382, which is hereby incorporated by reference. In some
embodiments, the transition metal complex is a palladium complex.
In some embodiments, the cleavable linker has the structure
##STR00004##
Such cleavable linkers are described in 3'-O-modified nucleotides
as reversible terminators for pyrosequencing, PNAS Oct. 16, 2007,
104 (42) 16462-16467, which is hereby incorporated by
reference.
[0110] In some embodiments, the number of effectors cleaved from
the scaffold is controlled. In some embodiments, the number of
effectors cleaved from a scaffold is controlled by controlling the
amount of stimulus used to cleave the cleavable linker. In this
context, a "stimulus" is any method or chemical used to
specifically cleave a cleavable linker. In some embodiments, the
stimulus is a chemical reaction with a cleaving reagent. In some
embodiments, the stimulus is electromagnetic radiation. In some
embodiments, the stimulus is a change in pH. In some embodiments,
the change in pH is acidification. In some embodiments, the change
in pH is basification.
[0111] In some embodiments, methods described herein comprise
cleaving the cleavable linker with a cleaving reagent. In some
embodiments, the methods comprise adding the cleaving reagent to an
encapsulation comprising an effector bound to a scaffold through a
cleavable linker. In some embodiments, the methods comprise adding
the cleaving reagent to an encapsulation comprising an encoding
bound to a scaffold through a cleavable linker.
[0112] In some embodiments, the number of effectors cleaved from
the scaffold is controlled by controlling the concentration of the
cleaving reagent. In some embodiments, the concentration of the
cleavage reagent is controlled in an encapsulation containing an
encoded effector bound to a scaffold. In some embodiments, the
concentration of chemical reagent used to cleave the cleavable
linker is at least 100 pM, at least 500 pM, at least 1 nM, at last
10 nM, at least 100 nM, at least 1 .mu.M, at least 10 .mu.M, at
least 100 .mu.M, at least 1 mM, at least 10 mM, at least 100 mM, or
at least 500 mM. In some embodiments, the concentration of cleaving
reagent used to cleave the cleavable linker is at most 100 pM, at
most 500 pM, at most 1 nM, at most 10 nM, at most 100 nM, at most 1
.mu.M, at most 10 .mu.M, at most 100 .mu.M, at most 1 mM, at most
10 mM, at most 100 mM, or at most 500 mM.
[0113] In some embodiments, the cleaving reagent is added to a
plurality of encapsulations. In some embodiments, the concentration
of cleaving reagent added to the plurality of encapsulations is
substantially uniform among individual encapsulations of the
plurality. In some embodiments, the concentration of cleaving
reagent used to cleave the cleavable linker in a plurality of
encapsulations is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
or 90% identical in each individual encapsulation. In some
embodiments, concentration of cleaving reagent used to cleave the
cleavable linker in a plurality of encapsulations differs by no
more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,
9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold among each
individual encapsulation of the plurality.
[0114] In some embodiments, the cleaving reagent is added to the
encapsulation by pico-injection. In some embodiments, the
encapsulation is passed through a microfluidic channel comprising a
pico-injection site. In some embodiments, pico-injections are timed
such that the rate of pico-injection matches the rate at which
encapsulation cross the pico-injection site. In some embodiments,
at least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing
a pico-injection site receive a pico-injection. In some
embodiments, the pico-injections are at least 2-fold, at least
5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at
least 50-fold, at least 100-fold, at least 500-fold, or at least
1000-fold smaller in volume than the passing droplets. In some
embodiments, the cleaving reagent is added to the encapsulation by
droplet merging.
[0115] In some embodiments, the cleaving reagent is added from a
stock solution to the encapsulation. In some embodiments, the stock
solution is at least 2.times., 5.times., 10.times., 20.times.,
30.times., 50.times., 100.times., 500.times., or 1000.times. more
concentrated than the desired final concentration in the
encapsulation.
[0116] In some embodiments, methods and systems described herein
comprise cleaving a photocleavable linker between an encoded
effector and a scaffold. In some embodiments, the methods and
systems described herein comprise exposing an encapsulation to
electromagnetic radiation comprising an effector bound to a
scaffold through a photocleavable linker. In some embodiments, the
methods and systems described herein comprise exposing an
encapsulation to light (for e.g., UV light) comprising an effector
bound to a scaffold through a photocleavable linker. In some
embodiments, the encapsulation is exposed to the light using a
microfluidic device.
[0117] In some embodiments, the photocleavable linker is cleaved by
exposure to light (e.g., UV light). In some embodiments, the
concentration of the number of effector molecules released from a
scaffold is controlled by controlling the intensity and/or duration
of exposure to UV light. In some embodiments, the light intensity
of a light (e.g., UV light) that an encapsulation (e.g., droplet)
described herein is exposed to is at least about 0.1 J/cm.sup.2 to
about 200 J/cm.sup.2. In some embodiments, the light intensity of a
light (e.g., UV light) that an encapsulation (e.g., droplet)
described herein is exposed to is about 0.1 J/cm.sup.2 to about 200
J/cm.sup.2. In some embodiments, the light intensity of a light
(e.g., UV light) that an encapsulation (e.g., droplet) described
herein is exposed to is about 0.1 J/cm.sup.2 to about 5 J/cm.sup.2,
about 0.1 J/cm.sup.2 to about 25 J/cm.sup.2, about 0.1 J/cm.sup.2
to about 100 J/cm.sup.2, about 0.1 J/cm.sup.2 to about 150
J/cm.sup.2, about 0.1 J/cm.sup.2 to about 200 J/cm.sup.2, about 5
J/cm.sup.2 to about 25 J/cm.sup.2, about 5 J/cm.sup.2 to about 100
J/cm.sup.2, about 5 J/cm.sup.2 to about 150 J/cm.sup.2, about 5
J/cm.sup.2 to about 200 J/cm.sup.2, about 25 J/cm.sup.2 to about
100 J/cm.sup.2, about 25 J/cm.sup.2 to about 150 J/cm.sup.2, about
25 J/cm.sup.2 to about 200 J/cm.sup.2, about 100 J/cm.sup.2 to
about 150 J/cm.sup.2, about 100 J/cm.sup.2 to about 200 J/cm.sup.2,
or about 150 J/cm.sup.2 to about 200 J/cm.sup.2, including
increments therein. In some embodiments, the light intensity of a
light (e.g., UV light) that an encapsulation (e.g., droplet)
described herein is exposed to is about 0.1 J/cm.sup.2, about 5
J/cm.sup.2, about 25 J/cm.sup.2, about 100 J/cm.sup.2, about 150
J/cm.sup.2, or about 200 J/cm.sup.2. In some embodiments, the light
intensity of a light (e.g., UV light) that an encapsulation (e.g.,
droplet) described herein is exposed to is at least about 0.1
J/cm.sup.2, about 5 J/cm.sup.2, about 25 J/cm.sup.2, about 100
J/cm.sup.2, or about 150 J/cm.sup.2. In some embodiments, the light
intensity of a light (e.g., UV light) that an encapsulation (e.g.,
droplet) described herein is exposed to is at most about 5
J/cm.sup.2, about 25 J/cm.sup.2, about 100 J/cm.sup.2, about 150
J/cm.sup.2, or about 200 J/cm.sup.2.
[0118] In some embodiments, the light (e.g., UV light) that an
encapsulation (e.g., droplet) described herein is exposed to is at
least about 5 mV. In some embodiments, the light (e.g., UV light)
that an encapsulation (e.g., droplet) described herein is exposed
to is from about 5 mV to about 10,000 mV. In some embodiments, the
light (e.g., UV light) that an encapsulation (e.g., droplet)
described herein is exposed to is about 100 mV, 200 mV, 400 mV, 600
mV, 800 mV, 1000 mv, 1250 mV, 1500 mV, 2000 mV, 4000 mV, 5000 mV.
In some embodiments, the light that an encapsulation (e.g.,
droplet) is exposed to is a calibrated amount of light.
[0119] In some embodiments, the cleavable linker is cleaved by
electromagnetic radiation. In some embodiments, the concentration
of the number of effector molecules released from a scaffold is
controlled by controlling the intensity or duration of
electromagnetic radiation.
[0120] Any suitable photoreactive or photocleavable linker can be
used as a cleavable linker cleaved by electromagnetic radiation
(e.g., exposure to UV light). A non-limiting list of linkers
cleavable by electromagnetic radiation includes (i)
o-nitrobenzyloxy linkers, (ii) o-nitrobenzylamino linkers, (iii)
.alpha.-substituted o-nitrobenzyl linkers, (iv) o-nitroveratryl
linkers, (v) phenacyl linkers, (vi) p-alkoxyphenacyl linkers, (vii)
benzoin linkers, (viii) pivaloyl linkers, and (ix) other
photolabile linkers. Further examples of photocleavable linkers are
described in Photolabile linkers for solid-phase synthesis, ACS
Comb Sci. 2018 Jul. 9; 20(7):377-99, which is hereby incorporated
by reference. In some embodiments, the cleavable linker is an
o-nitrobenzyloxy linker, an o-nitrobenzylamino linker, an
.alpha.-substituted o-nitrobenzyl linker, an o-nitroveratryl
linker, a phenacyl linker, p-alkoxyphenacyl linker, a benzoin
linker, or a pivaloyl linker.
[0121] In some embodiments, the photocleavable linker requires to
be first activated through exposure to a reagent before being able
to be cleaved through exposure to electromagnetic radiation (e.g.,
UV light). In some embodiments, the desired number of effectors
released can be further controlled by selectively exposing reagents
to encapsulations (e.g., droplets). In some embodiments, providing
photocleavable linkers that need to be activated before being
cleaved through exposure to UV light enables for improved
bead-handling, synthesis, storage, and preparation due to minimized
or eliminated encoded effector release through incident UV
exposure. FIG. 17A provides an exemplary molecule configured to be
transformed upon interaction with a reagent, such that it becomes
activated for UV photocleavage (reference: J. AM. CHEM. SOC. 2003,
125, 8118-8119; 10.1021/ja035616d). As depicted, the azide group
functionally reduces the sensitivity of the photocleavable-linker
moiety, such that linker is more stable, thus advantageous for
handling and storing under ambient lighting. As depicted in FIG.
17A, the azide can be converted upon reagent treatment (HOF--CH3CN)
to generate the photo-sensitive Nitro-benzyl motif (molecule
depicted in the middle), wherein the product photocleavable-linker
can be calibrated to release a known quantity of effector upon
UV-exposure. FIG. 17B provides another exemplary molecule
configured to be transformed upon interaction with a reagent, such
that it becomes activated for UV photocleavage (reference: J. Comb.
Chem. 2000, 2, 3, 266-275). As depicted, the thio-phenol ester
provides a stable covalent linker to compound (R). Specific
oxidation of the thio-phenol (shown in middle molecule) can
generate an "activated" linker-moiety. Kinetic control of the
oxidation step may allow for quantitative "activation" to prescribe
compound release. In some embodiments, base treatment causes linker
scission through elimination, thereby generating a free acid
compound, or with subsequent decarboxylation generates just a
compound.
[0122] In some embodiments, the cleavable linker is cleaved by an
enzyme. In some embodiments, the cleavable linker is cleaved by a
protease, a nuclease, or a hydrolase. In some embodiments, the
cleavable linker is a peptide. In some embodiments, the cleavable
linker is a cleavable nucleic acid sequence. In some embodiments,
the cleavable linker is a carbohydrate. In some embodiments, the
number of effector molecules cleaved from the scaffold is
controlled by controlling the concentration of the enzyme. In some
embodiments, the rate at which effector molecules are cleaved from
the scaffold is controlled by controlling the concentration of the
enzyme.
[0123] In some embodiments, the methods comprise cleaving the
cleavable linker. In some embodiments, the methods comprise
cleaving the cleavable linker with a cleaving reagent. In some
embodiments, the cleaving reagent is added to the encapsulation by
pico-injection. In some embodiments, the cleaving reagent is added
to the encapsulation by pico-injection at a concentration
configured to release a predetermined amount of effector. In some
embodiments, the cleaving reagent is added to the encapsulation by
pico-injection at a concentration configured to release a desired
amount of effector.
[0124] In some embodiments, methods described herein comprise first
activating the cleavable linker to enable the cleavable linker to
be cleaved. In some embodiments, upon activating the cleavable
linker, the cleavable linker can be cleaved using methods described
herein, such as through photocleavage, interaction with an enzyme,
using a cleaving reagent, and so on. In some embodiments, the
cleavable linker is activated through interaction with an
activating reagent. In some embodiments, the methods comprise
adding the activating reagent to an encapsulation comprising an
effector bound to a scaffold. In some embodiments, the methods
comprise adding the activating reagent to an encapsulation
comprising an encoding bound to a scaffold. In some embodiments,
the activating reagent comprises any reagent described herein as a
cleaving reagent. In some embodiments, the activating reagent
comprises a disulfide reducing reagent. In some embodiments, the
activating reagent comprises tetrazine.
[0125] In some embodiments, the activating reagent is added to the
encapsulation by pico-injection. In some embodiments, the
encapsulation is passed through a microfluidic channel comprising a
pico-injection site. In some embodiments, pico-injections are timed
such that the rate of pico-injection matches the rate at which
encapsulation cross the pico-injection site. In some embodiments,
at least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing
a pico-injection site receive a pico-injection. In some
embodiments, the pico-injections are at least 2-fold, at least
5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at
least 50-fold, at least 100-fold, at least 500-fold, or at least
1000-fold smaller in volume than the passing droplets. In some
embodiments, the activating reagent is added to the encapsulation
by droplet merging.
[0126] In some embodiments, the concentration of the activating
reagent used to activate the cleavable linker is at most 100
picomolar (pM), at most 500 pM, at most 1 nanomolar (nM), at most
10 nM, at most 100 nM, at most 1 micromolar (.quadrature.M), at
most 10 .quadrature.M, at most 100 .quadrature.M, at most 1
millimolar (mM), at most 10 mM, at most 100 mM, or at most 500
mM.
[0127] In some embodiments, the activating reagent is added from a
stock solution to the encapsulation. In some embodiments, the stock
solution is at least 2.times., 5.times., 10.times., 20.times.,
30.times., 50.times., 100.times., 500.times., or 1000.times. more
concentrated than the desired final concentration in the
encapsulation.
[0128] In some embodiments, effectors are released from scaffolds.
In some embodiments, releasing effectors from scaffolds allows the
effectors to move freely in solution. This free movement may allow
the effector to interact with the sample or target being
interrogated. In some embodiments, these effectors are released in
a controlled fashion. This controlled fashion may allow for a
predetermined and/or known dose of effectors to be released form
the scaffold. Such a procedure may allow for improved
quantification and analysis of hits from a screen, as dose response
can be measured. Additionally, releasing a known amount of
effectors across a library of effectors being screened may remove
bias from the sample set. Bias can occur in library screens using
encoded scaffolds when individual scaffolds possess attachments of
effectors that vary in amount among the scaffolds of the library.
For example, one scaffold may contain 10 copies of an effector
molecule, and another scaffold may contain 1000 copies of an
effector molecule. Consequently, different concentrations of
effector being screened against a sample or target may be released,
making a determination of the efficacy of individual effectors
difficult to ascertain. By releasing a uniform amount of effectors
from each scaffold in a screen, a uniform dose across the screen is
employed, removing bias from lower potency, higher concentration
effectors.
[0129] In some embodiments, the effectors are released to a desired
concentration. In some embodiments, the effectors are released to a
desired concentration within an encapsulation. In some embodiments,
the desired concentration is at least 100 pM, at least 500 pM, at
least 1 nM, at least 10 nM, at least 100 nM, at least 1 .mu.M, at
least 10 .mu.M, at least 100 .mu.M, at least 1 mM, at least 10 mM,
at least 50 mM, at least 100 mM, or at least 250 mM. In some
embodiments, the desired concentration is at most 100 pM, at most
500 pM, at most 1 nM, at most 10 nM, at most 100 nM, at most 1
.mu.M, at most 10 .mu.M, at most 100 .mu.M, at most 1 mM, at most
10 mM, at most 50 mM, at most 100 mM, or at most 250 mM.
[0130] In some embodiments, the effectors are released to a
predetermined concentration. In some embodiments, the effectors are
released to a predetermined concentration within an encapsulation.
In some embodiments, the predetermined concentration is at least
100 pM, at least 500 pM, at least 1 nM, at least 10 nM, at least
100 nM, at least 1 .mu.M, at least 10 .mu.M, at least 100 .mu.M, at
least 1 mM, at least 10 mM, at least 50 mM, at least 100 mM, or at
least 250 mM. In some embodiments, the predetermined concentration
is at most 100 pM, at most 500 pM, at most 1 nM, at most 10 nM, at
most 100 nM, at most 1 .mu.M, at most 10 .mu.M, at most 100 .mu.M,
at most 1 mM, at most 10 mM, at most 50 mM, at most 100 mM, or at
most 250 mM.
[0131] In some embodiments, effector molecules are released from
scaffolds in a plurality of encapsulations. In some embodiments,
the concentration of effector molecules released from scaffolds in
a plurality of encapsulations is uniform among the encapsulations.
In some embodiments, the concentration of effector molecules
released from scaffolds in a plurality of encapsulations is
substantially uniform among the encapsulations. In some
embodiments, the concentration of effector molecules released from
scaffolds in a plurality of encapsulations is at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, or 90% identical in each individual
encapsulation. In some embodiments, the concentration of effector
molecules released from scaffolds in a plurality of encapsulations
differs by no more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,
7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or
100-fold among each individual encapsulation of the plurality.
[0132] In some embodiments, the methods described herein comprise
incubating the encapsulation for a period of time. In some
embodiments, the methods comprise incubating the encapsulation for
a period of time to allow the effector and sample to interact. In
some embodiments, the encapsulations are incubated for a period of
time to allow the effector and the sample to react. In some
embodiments, the period of time is at least 1 millisecond, 1
second, 1 minute, at least 10 minutes, at least 1 hour, at least 4
hours, or at least 24 hours. In some embodiments, the period of
time is at most 1 minutes, at most 10 minutes, at most 1 hour, at
most for hours, or at most 24 hours. In some embodiments, the
incubation time is measured after releasing effectors from a
scaffold.
[0133] In some embodiments, the period of time is controlled by a
residence time as the encapsulation travels through a microfluidic
channel. In some embodiments, the residence time is controlled by a
flow valve, a geometry of the microfluidic channel, the length of
the microfluidic channel, by removing the encapsulations from the
microfluidic channel, or combinations thereof.
[0134] The effectors of the methods and systems provided herein can
be any type of molecule. In some embodiments, an effector is a
biochemical, chemical, or biological moiety. In some embodiments,
an effector is a cell, a protein, peptide, small molecule, small
molecule fragment, or a nucleic acid. An effector is any molecule
that is capable interacting with a target. The term "effector" is
used broadly to encompass any moiety whose effect on a sample is
being interrogated.
[0135] In some embodiments, the effectors have a handle that allows
for attachment to a scaffold. A handle is a reactive functional
group that can be used to tether the effector to an attachment site
on a scaffold. This handle may be any functional group capable of
forming a bond. Handles may include, without limitation, sulfhydryl
groups, CLICK chemistry reagents, amino groups, carboxylate groups,
or numerous other groups.
[0136] In some embodiments, effectors are comprised of individual
subunits. These individual subunits may be joined using various
chemical reactions to form the full effector. In some embodiments,
iterative chemical processes are used to generate the effectors,
similar to methodologies used in solid-phase peptide synthesis.
Similar methods can be used to create non-peptide effectors,
wherein a first reaction is performed to link two subunits, the two
linked subunits are subjected to a second reaction to activate the
linked subunits, and a third subunit is then attached, and so on.
Any type of such an iterative chemical synthesis scheme may be
employed to create the effectors used in the methods and systems
provided herein.
[0137] In some embodiments, the effectors elicit a response from
the target being interrogated. The response elicited can take any
form and depends on the sample being interrogated. As a
non-limiting example, when the sample comprises a cell, the
response may be a change in expression pattern, apoptosis,
expression of a particular molecule, or a morphological change in
the cell. As another non-limiting example, when the sample
comprises a protein, the effector may inhibit protein activity,
enhance protein activity, alter protein folding, or measure protein
activity.
[0138] In some embodiments, the effector is a protein. In some
embodiments, the protein may be a naturally occurring or mutant
protein. In some embodiments, the protein is a fragment of a
naturally occurring protein. In some embodiments, the protein is an
antibody. In some embodiments, the protein is an antibody-fragment.
In some embodiments, the protein is an enzyme. In some embodiments,
the protein is a recombinant protein. In some embodiments, the
protein is a signaling protein, an enzyme, a binding protein, an
antibody or antibody fragment, a structural protein, a storage
protein, or a transport protein, or any mutant thereof
[0139] In some embodiments, the effector is a peptide. In some
embodiments, the effector is a non-natural peptide. In some
embodiments, the effector is a polymer. In some embodiments, the
peptide is 5 amino acids to 50 amino acids in length. In some
embodiments, the peptide is 5 amino acids to 10 amino acids, 5
amino acids to 15 amino acids, 5 amino acids to 20 amino acids, 5
amino acids to 30 amino acids, 5 amino acids to 50 amino acids, 10
amino acids to 15 amino acids, 10 amino acids to 20 amino acids, 10
amino acids to 30 amino acids, 10 amino acids to 50 amino acids, 15
amino acids to 20 amino acids, 15 amino acids to 30 amino acids, 15
amino acids to 50 amino acids, 20 amino acids to 30 amino acids, 20
amino acids to 50 amino acids, or 30 amino acids to 50 amino acids
in length. In some embodiments, the peptide is 5 amino acids, 10
amino acids, 15 amino acids, 20 amino acids, 30 amino acids, or 50
amino acids in length. In some embodiments, the peptide comprises
at least 5 amino acids, 10 amino acids, 15 amino acids, 20 amino
acids, or 30 amino acids. In some embodiments, the peptide
comprises at most 10 amino acids, 15 amino acids, 20 amino acids,
30 amino acids, or 50 amino acids. In some embodiments, the peptide
comprises unnatural amino acids. In some embodiments, the peptide
comprises a non-peptide region. In some embodiments, the peptide is
a cyclic peptide. In some embodiments, the peptide has a secondary
structure that mimics a protein.
[0140] In some embodiments, the effector is a compound. In some
embodiments, the compound is an organic molecule. In some
embodiments, the compound is an inorganic molecule. In some
embodiments, the compounds used as effectors contain organic and
inorganic atoms. In some embodiments, the compound is a drug-like
small molecule. In some embodiments, the compound is an organic
compound. In some embodiments, the compound comprises one or more
inorganic atoms, such as one or more metal atoms. In some
embodiments, the effector is a small molecule. In some embodiments,
the effector is a macro molecule.
[0141] In some embodiments, the compound is a completed chemical
that is synthesized by connecting a plurality of chemical monomers
to each other. In some embodiments, the effector is a
pre-synthesized compound loaded onto a bead after synthesis.
[0142] In some embodiments, the compound is a small molecule
fragment. Small molecule fragments are small organic molecules
which are small in size and low in molecular weight. In some
embodiments, the small molecule fragments are less than 500 Dalton
(Da), less than 400 Da, less than 300 Da, less than 200 Da, or less
than 100 Da in molecular weight (MW).
[0143] In some embodiments, the effector is an effector nucleic
acid. In some embodiments, the effector nucleic acid is 5
nucleotides to 50 nucleotides in length. In some embodiments, the
effector nucleic acid is 5 nucleotides to 10 nucleotides, 5
nucleotides to 15 nucleotides, 5 nucleotides to 20 nucleotides, 5
nucleotides to 30 nucleotides, 5 nucleotides to 50 nucleotides, 10
nucleotides to 15 nucleotides, 10 nucleotides to 20 nucleotides, 10
nucleotides to 30 nucleotides, 10 nucleotides to 50 nucleotides, 15
nucleotides to 20 nucleotides, 15 nucleotides to 30 nucleotides, 15
nucleotides to 50 nucleotides, 20 nucleotides to 30 nucleotides, 20
nucleotides to 50 nucleotides, or 30 nucleotides to 50 nucleotides
in length. In some embodiments, the effector nucleic acid comprises
5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 30
nucleotides, or 50 nucleotides. In some embodiments, the effector
nucleic acid comprises at least 5 nucleotides, 10 nucleotides, 15
nucleotides, 20 nucleotides, or 30 nucleotides. In some
embodiments, the effector nucleic acid is at most 10 nucleotides,
15 nucleotides, 20 nucleotides, 30 nucleotides, or 50 nucleotides
in length. In some embodiments, the effector nucleic acid comprises
unnatural nucleotides. In some embodiments, the nucleic acid is an
aptamer. In some embodiments, the effector nucleic acid comprises
DNA, RNA, or combinations thereof.
Enzyme Evolution Screen
[0144] The methods and systems herein are further useful for
screening effector proteins for the possession of various
activities. In these embodiments, the effector is a protein. A
variety of mutant variants of a protein can be screened by linking
plasmids or other nucleic acids coding for the expression of a
protein to scaffolds. The "coding" referred to in this aspect
refers to the genetic code, and "encoding" refers to an alternative
strategy for elucidating the structure of the protein. In some
instances, each nucleic acid has a barcode that is unique to the
specific mutant protein which can be sequenced to reveal the
mutations therein without conducting a full sequence read on the
whole plasmid or other nucleic acid which codes for the protein.
The barcode thus acts as its own encoding to delineate the
structure and sequence of the protein without relying on the full
coding sequence. In this aspect, a library of mutant proteins can
be screened against samples with the components provided herein in
encapsulation-based assays.
[0145] In a non-limiting example, a scaffold containing the nucleic
acid encoding the protein of interest is encapsulated. The protein
can then be expressed in the encapsulation using an expression
system, such as any in vitro transcription/translation system. In
some embodiments, one or more detection reagents can be added to
the encapsulation for which the protein may exhibit a certain
desired activity. In some instances, these detection reagents may
be present during the expression of the protein or may be added
later. These detection reagents may be used to assess any desired
activity, including protein binding, enzymatic activity, or the
detection reagents may be capable of probing protein structure. In
some embodiments, each detection reagent comprises one or more
chemical compounds or molecules, which the expressed protein (e.g.,
an enzyme of interest) can bind together. In some embodiments, at
least two detection reagents are provided, each comprising a
molecular probe, such that the expressed protein (e.g., an enzyme)
can bind the molecular probes from the respective detection
reagents together. In some embodiments, at least two detection
reagents are provided, each comprising one or more chemical
compounds, such that the expressed protein (e.g., an enzyme) can
bind one or more of the chemical compounds from the respective
detection reagents together. In some embodiments, the binding of
the molecular probes or chemical compounds by the protein leads to
the production of a signal. In some embodiments, the binding of the
molecular probes or chemical compounds by the protein is a certain
desired activity that leads to the production of a signal.
[0146] If the protein in an encapsulation has the certain desired
activity, the activity can lead to the production of a signal. The
signal can be any of the signals described herein. In some
embodiments, the signal is a fluorescent signal created due to the
ligation of two molecules of interest. In some embodiments, the
molecules of interest have FRET pairings affixed to them, or
fluorophore/quencher pairings affixed to them, or any other type of
moieties that lead to a change in signal due to brining the two
moieties into proximity to each other. In some embodiments, the two
moieties are brought into proximity to each other due to the
formation of a bond between the molecules of interest. The signal
produced can then be detected, indicating that the protein being
screened has the desired activity. The encapsulation can then be
sorted based on the detectable signal, such as the signals
presence, absence, or level. In some embodiments, the encoded
effector is a protein and the encoding comprises a barcoded nucleic
acid which further codes for the expression of the protein.
[0147] Provided herein are methods for screening nucleic acid
encoded proteins against a sample. In some embodiments, the methods
comprise providing an encapsulation comprising a nucleic acid
encoding attached to a scaffold, the nucleic acid encoding
comprising an encoding barcode and a coding section for the
expression of an encoded effector protein. In some embodiments, the
encapsulation further comprises an expression system for the
production of the encoded protein. In some embodiments, the encoded
protein is expressed within the encapsulation. In some embodiments,
detection reagents are introduced to the encapsulation. In some
embodiments the detection reagents are present in the encapsulation
during protein expression. In some embodiments, the detection
reagents produce a signal upon interaction with the encoded protein
if the encoded protein has a certain activity. In some embodiments,
the signal produced due to this interaction is measured. In some
embodiments, the encapsulation is sorted based on the measurement
of the signal. In some embodiments, the nucleic acid encoding is
sequenced. In some embodiments, this nucleic acid encoding is
sequenced by next-generation sequencing.
[0148] The nucleic acid encoding which comprises a coding section
for the expression of the encoded protein may be of any form that
allows for the expression to occur. In some embodiments, the
nucleic acid encoding comprising a coding section for the
expression of an encoded effector protein is a linear nucleic acid.
In some embodiments, the nucleic acid encoding comprising a coding
section for the expression of an encoded effector protein is a
plasmid. In some embodiments, the nucleic acid encoding comprising
a coding section for the expression of an encoded effector protein
is single stranded. In some embodiments, the nucleic acid encoding
comprising a coding section for the expression of an encoded
effector protein is double stranded.
[0149] In some embodiments, the nucleic acid encoding comprising a
coding section for the expression of an encoded effector protein
comprises a barcode. In some embodiments, the barcode acts as the
encoding for the encoded effector protein. In some embodiments, the
barcode is upstream of the coding section for the expression of the
encoded effector protein. In some embodiments, the barcode is
downstream of the coding section for the expression of the encoded
effector protein. In some embodiments, the nucleic acid encoding
comprising a coding section for the expression of an encoded
effector protein further comprises a sequencing primer. In some
embodiments, the sequencing primer is upstream of the barcode. In
some embodiments, the sequencing primer is downstream of the
barcode.
[0150] In some embodiments, the effector is a nucleic acid encoded
protein. In some embodiments, the corresponding nucleic acid
encoding comprises a coding section for the expression of the
encoded protein. In some embodiments, the nucleic acid encoded
protein is an enzyme or mutant thereof. In some embodiments, the
enzyme or mutant thereof is being screened for an enzymatic
activity.
[0151] In some embodiments, the enzymatic activity is oxidation,
reduction, ligation, polymerization, bond cleavage, bond formation,
or isomerization. In some embodiments, the enzymatic activity is
covalent bond formation. In some embodiments, the enzyme is an
amino acid dehydrogenase, a natural amine dehydrogenase, an opine
dehydrogenase, or an imine reductase. In some embodiments, the
enzymatic activity is an enantiospecific activity. In some
embodiments, the enzymatic activity is a stereospecific
activity.
[0152] A variety of protein characteristics can be probed or
screened for using the methods and systems provided herein. In some
embodiments, the certain characteristic being screened for
comprises an enzymatic activity, a binding ability, a catalytic
activity, a physical property, an inhibitory activity, or a
structure. In some embodiments, the certain characteristic being
screened for comprises a binding ability. In some embodiments, the
certain characteristic being screened for comprises a catalytic
activity. In some embodiments, the certain characteristic being
screened for comprises a physical property. In some embodiments,
the certain characteristic being screened for comprises an
inhibitory activity. In some embodiments, the certain
characteristic being screened for comprises a secondary, tertiary,
or quaternary structure.
[0153] In some embodiments, the enzymatic activity is the ability
to form a bond between molecular probes from a first detection
reagent and a second detection reagent. In some embodiments, the
enzymatic activity comprises forming a bond between molecular
probes from a first detection reagent and a second detection
reagent. In some embodiments, the enzymatic activity is the ability
to form a bond between one or more chemical compounds from a first
detection reagent and a second detection reagent. In some
embodiments, the enzymatic activity comprises forming a bond
between one or more chemical compounds from a first detection
reagent and a second detection reagent. In some embodiments, the
bond is a covalent bond. In some embodiments, the bond is an
irreversible covalent bond. In some embodiments, the first
detection reagent and the second detection reagent exhibit a
fluorescent signal when the molecules from the first and second
detection reagents are bound together. In some embodiments, the
first detection reagent and the second detection reagent exhibit a
changed fluorescent signal when molecular probes from the first and
second detection reagents are bound together compared to when the
molecular probes from the first detection reagent and second
detection reagent are not bound together. In some embodiments, the
first detection reagent and the second detection reagent exhibit a
fluorescent signal when the one or more chemical compounds from the
first and second detection reagents are bound together. In some
embodiments, the first detection reagent and the second detection
reagent exhibit a changed fluorescent signal when one or more
chemical compounds from the first and second detection reagents are
bound together compared to when the one or more chemical compounds
from the first detection reagent and second detection reagent are
not bound together. In some embodiments, the fluorescent signal is
due to fluorescence resonance energy transfer (FRET),
bioluminescent resonance energy transfer (BRET), lanthanide chelate
excite time resolved fluorescence resonance energy transfer (LANCE
TR-FRET), or an amplified luminescent proximity homogeneous assay.
In some embodiments, the first and second reagents are chemical
compounds.
[0154] In some embodiments, the molecular probes from the first and
second detection reagents comprise a FRET pair or a
fluorophore/quencher pair. In some embodiments, the molecular
probes from the first and second detection reagents comprise
fluorophores or quenchers independently selected from
4-(4-dimethylaminophenyl azo), 5-((3-aminoethyl)amino)-1-napthalene
sulfonic acid, 5-((2-aminoethyl)amino)-1-napthalene sulfonic acid
(EDANS), 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL),
and fluorescein-isothiocyanate (FITC), or derivatives thereof. In
some embodiments, the FRET pair or fluorophore/quencher pair
comprise different fluorophores. In some embodiments, the FRET
pairing is duplicate copies of the same fluorophore.
[0155] In some embodiments, the one or more chemical compounds from
the first and second detection reagents comprise a FRET pair or a
fluorophore/quencher pair. In some embodiments, the one or more
chemical compounds from the first and second detection reagents
comprise fluorophores or quenchers independently selected from
4-(4-dimethylaminophenyl azo), 5-((3-aminoethyl)amino)-1-napthalene
sulfonic acid, 5-((2-aminoethyl)amino)-1-napthalene sulfonic acid
(EDANS), 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL),
and fluorescein-isothiocyanate (FITC), or derivatives thereof. In
some embodiments, the FRET pair or fluorophore/quencher pair
comprise different fluorophores. In some embodiments, the FRET
pairing is duplicate copies of the same fluorophore.
[0156] In some embodiments, the ability to form a bond is an imine
reduction. In some embodiments, the imine reduction is
enantiospecific. In some embodiments, the imine reduction is
stereospecific. In some embodiments, the imine reduction favors an
S-enantiomer at a substituted carbon adjacent to the reduced imine
bond. In some embodiments, the imine reduction favors an
R-enantiomer at a substituted carbon adjacent to the reduced imine
bond. In some embodiments, the imine reduction is an intramolecular
reaction. In some embodiments, the imine reduction is
diastereospecific.
[0157] In some embodiments, a library of nucleic acid encoded
proteins are screened against the sample. In some embodiments, the
methods comprise performing any of the described screens against a
library of nucleic acid encoded proteins, wherein the library of
nucleic acid encoded proteins comprises a plurality of different
mutant versions of the nucleic acid encoded protein. In some
embodiments, each mutant version of the nucleic acid encoded
protein is encoded by a unique barcode.
[0158] The methods and systems provided herein sometimes comprise
the addition of detection reagents to the encapsulation. In some
embodiments, the detection reagents are added by pico-injection. In
some embodiments, the detection reagents are added by droplet
merging. In some embodiments, the detection reagents are added
before the signal is detected. In some embodiments, the detection
reagents are added after the signal is detected. In some
embodiments, the detection reagents facilitate the detection of the
signal.
[0159] In some embodiments, the encapsulation further comprises a
reporter enzyme. In some embodiments, the reporter enzyme reacts
with another reagent to produce a functional readout. In some
embodiments, a bond between the first and second molecular probes
creates a new molecule that inhibits the reporter enzyme.
[0160] Additional reagents may also be used to add barcodes to
nucleic acids of the sample or the encoding. In some embodiments,
the additional reagents add a nucleic acid barcode to one or more
contents of the encapsulation. In some embodiments, the nucleic
acid barcode is added to the encoding. In some embodiments, the
nucleic acid barcode is added to nucleic acids from the sample.
Encodings for Effectors
[0161] The effectors provided herein can be linked with encodings.
In some embodiments, the effectors are linked with an encoding. In
some instances, the encoding allows a user to determine the
structure of the effector by determining a property of the
encoding. Thus, each encoding moiety has a measurable property
that, when measured, can be used to determine the structure of the
effector which is encoded.
[0162] In some embodiments, the encoding is a nucleic acid. In some
embodiments, the sequence of the nucleic acid provides information
about the structure of the effector. In some embodiments, the
encoding comprises a nucleic acid barcode. In some embodiments, the
barcode is unique to a specific effector. In some embodiments, the
encoding comprises a sequencing primer. In some embodiments,
sequencing the nucleic acid encoding allows the user to ascertain
the structure of the corresponding effector.
[0163] In some embodiments, the encoding is DNA. In some
embodiments, the encoding is double stranded DNA. In some
embodiments, the encoding is single stranded DNA. In some
embodiments, the encoding is RNA. In some embodiments, the encoding
is single stranded RNA. In some embodiments, the encoding is double
stranded RNA.
[0164] In some embodiments, the encoding nucleic acid comprises at
least 20 nucleotides, at least 40 nucleotides, at least 60
nucleotides, at least 80 nucleotides, at least 100 nucleotides, at
least 200 nucleotides, or at least 500 nucleotides. In some
embodiments, the encoding nucleic acid comprises 20 nucleotides to
100 nucleotides in length. In some embodiments, the encoding
nucleic acid is 20 nucleotides to 40 nucleotides, 20 nucleotides to
60 nucleotides, 20 nucleotides to 80 nucleotides, 20 nucleotides to
100 nucleotides, 40 nucleotides to 60 nucleotides, 40 nucleotides
to 80 nucleotides, 40 nucleotides to 100 nucleotides, 60
nucleotides to 80 nucleotides, 60 nucleotides to 100 nucleotides,
or 80 nucleotides to 100 nucleotides in length. In some
embodiments, the encoding nucleic acid comprises about 20
nucleotides, about 40 nucleotides, about 60 nucleotides, about 80
nucleotides, or about 100 nucleotides. In some embodiments, the
encoding nucleic acid comprises at least 20 nucleotides, 40
nucleotides, 60 nucleotides, or 80 nucleotides. In some
embodiments, the encoding nucleic acid is at most 40 nucleotides,
60 nucleotides, 80 nucleotides, or 100 nucleotides in length.
[0165] In some embodiments, the encoding is made up of individual
subunits that encode a corresponding effector subunit.
Consequently, an entire encoding can specify which individual
subunits have been linked or combined to form the effector. In some
embodiments, each subunit may comprise up to 5, 10, 15, 20, 25, 30,
40, 50, or more individual nucleotides. The full encoding sequence
can comprise any number of these individual subunits. In some
embodiments, the full encoding sequence comprises 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more encoding subunits. These encoding subunits can
be ligated together using many known methods, including enzymatic
ligation, template-free synthesis, templated polymerase extension,
chemical ligation, recombination, or solid phase nucleic acid
synthesis techniques.
[0166] In some embodiments, the encoding is a molecular weight
barcode. In some embodiments, the molecular weight barcode is at
least 1,000, at least 5,000, at least 10,000, or at least 15,000
Daltons in molecular weight. In some embodiments, the molecular
weight barcode is a peptide. In some embodiments, the molecular
weight barcode peptide comprises 5 amino acids to 10 amino acids, 5
amino acids to 15 amino acids, 5 amino acids to 20 amino acids, 5
amino acids to 30 amino acids, 5 amino acids to 50 amino acids, 10
amino acids to 15 amino acids, 10 amino acids to 20 amino acids, 10
amino acids to 30 amino acids, 10 amino acids to 50 amino acids, 15
amino acids to 20 amino acids, 15 amino acids to 30 amino acids, 15
amino acids to 50 amino acids, 20 amino acids to 30 amino acids, 20
amino acids to 50 amino acids, or 30 amino acids to 50 amino acids.
In some embodiments, the molecular weight barcode peptide comprises
5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 30
amino acids, or 50 amino acids. In some embodiments, the molecular
weight barcode peptide comprises at least 5 amino acids, 10 amino
acids, 15 amino acids, 20 amino acids, or 30 amino acids. In some
embodiments, the peptide comprises at most 10 amino acids, 15 amino
acids, 20 amino acids, 30 amino acids, or 50 amino acids. In some
embodiments, the molecular weight barcode peptides comprise
unnatural amino acids.
[0167] In some embodiments, the encoding is loaded onto a scaffold.
In some embodiments, the scaffold comprises a high loading of the
encoding. In some embodiments, the scaffold comprises about
1,000,000 copies to about 50,000,000 copies of the encoding. In
some embodiments, the scaffold comprises about 1,000,000 copies to
about 2,000,000 copies, about 1,000,000 copies to about 5,000,000
copies, about 1,000,000 copies to about 10,000,000 copies, about
1,000,000 copies to about 15,000,000 copies, about 1,000,000 copies
to about 20,000,000 copies, about 1,000,000 copies to about
50,000,000 copies, about 2,000,000 copies to about 5,000,000
copies, about 2,000,000 copies to about 10,000,000 copies, about
2,000,000 copies to about 15,000,000 copies, about 2,000,000 copies
to about 20,000,000 copies, about 2,000,000 copies to about
50,000,000 copies, about 5,000,000 copies to about 10,000,000
copies, about 5,000,000 copies to about 15,000,000 copies, about
5,000,000 copies to about 20,000,000 copies, about 5,000,000 copies
to about 50,000,000 copies, about 10,000,000 copies to about
15,000,000 copies, about 10,000,000 copies to about 20,000,000
copies, about 10,000,000 copies to about 50,000,000 copies, about
15,000,000 copies to about 20,000,000 copies, about 15,000,000
copies to about 50,000,000 copies, or about 20,000,000 copies to
about 50,000,000 copies of the encoding. In some embodiments, the
scaffold comprises about 1,000,000 copies, about 2,000,000 copies,
about 5,000,000 copies, about 10,000,000 copies, about 15,000,000
copies, about 20,000,000 copies, or about 50,000,000 copies of the
encoding. In some embodiments, the scaffold comprises at least
about 1,000,000 copies, about 2,000,000 copies, about 5,000,000
copies, about 10,000,000 copies, about 15,000,000 copies, or about
20,000,000 copies. In some embodiments, the scaffold comprises at
most about 2,000,000 copies, about 5,000,000 copies, about
10,000,000 copies, about 15,000,000 copies, about 20,000,000
copies, or about 50,000,000 copies of the encoding.
[0168] In some embodiments, the encoding is nucleic acid comprising
a barcode sequence. In some embodiments, the encoding comprises a
DNA barcode. In some embodiments, there is at least 1 DNA barcode
per bead, at least 10 copies of the DNA barcode per bead, at least
100 copies, at least 1,000 copies, at least 100,000 copies, at
least 1 million copies, or at least 10 million copies of the DNA
barcode per bead. In some embodiments, the scaffold comprises at
least 10 million copies of the DNA barcode per bead.
[0169] In some embodiments, DNA barcodes are used to identify a
scaffold. In some instances, the scaffold is a bead. In some
instances, only 1 DNA barcode out of 10 million DNA barcodes is
required to identify the bead. In some instances, only 5 DNA
barcodes, 10 DNA barcodes, 20 DNA barcodes, 50 DNA barcodes, 100
DNA barcodes, 1000 DNA barcodes, 10,000 DNA barcodes, 100,000 DNA
barcodes, or 1 million DNA barcodes out of 10 million barcodes is
required to identify the bead.
Sample
[0170] Samples of any type can be utilized with the methods and
systems provided herein. In some embodiments, the sample is a
biological sample. In some embodiments, the sample comprises one or
more cells, one or more proteins, one or more enzymes, one or more
nucleic acids, one or more cellular lysates, or one or more tissue
extracts.
[0171] In some embodiments, the sample is a cell. In some
embodiments, the cell is a eukaryotic cell. In some embodiments,
the cell is a prokaryotic cell. In some embodiments, the cell is a
mammalian cell. In some embodiments, the cell is a bacterial cell.
In some embodiments, the cell is a human cell. In some embodiments,
the cell is a cancer cell. In some embodiments, the cell is
SH-SYSY, Human neuroblastoma; Hep G2, Human Caucasian hepatocyte
carcinoma; 293 (also known as HEK 293), Human Embryo Kidney; RAW
264.7, Mouse monocyte macrophage; HeLa, Human cervix epitheloid
carcinoma; MRC-5 (PD 19), Human fetal lung; A2780, Human ovarian
carcinoma; CACO-2, Human Caucasian colon adenocarcinoma; THP 1,
Human monocytic leukemia; A549, Human Caucasian lung carcinoma;
MRC-5 (PD 30), Human fetal lung; MCF7, Human Caucasian breast
adenocarcinoma; SNL 76/7, Mouse SIM strain embryonic fibroblast;
C2Cl2, Mouse C3H muscle myoblast; Jurkat E6.1, Human leukemic T
cell lymphoblast; U937, Human Caucasian histiocytic lymphoma; L929,
Mouse C3H/An connective tissue; 3T3 L1, Mouse Embryo; HL60, Human
Caucasian promyelocytic leukaemia; PC-12, Rat adrenal
phaeochromocytoma; HT29, Human Caucasian colon adenocarcinoma;
OE33, Human Caucasian oesophageal carcinoma; OE19, Human Caucasian
oesophageal carcinoma; NIH 3T3, Mouse Swiss NIH embryo; MDA-MB-231,
Human Caucasian breast adenocarcinoma; K562, Human Caucasian
chronic myelogenous leukemia; U-87 MG, Human glioblastoma
astrocytoma; MRC-5 (PD 25), Human fetal lung; A2780cis, Human
ovarian carcinoma; B9, Mouse B cell hybridoma; CHO-K1, Hamster
Chinese ovary; MDCK, Canine Cocker Spaniel kidney; 1321N1, Human
brain astrocytoma; A431, Human squamous carcinoma; ATDC5, Mouse 129
teratocarcinoma AT805 derived; RCC4 PLUS VECTOR ALONE, Renal cell
carcinoma cell line RCC4 stably transfected with an empty
expression vector, pcDNA3, conferring neomycin resistance; HUVEC
(5200-05n), Human Pre-screened Umbilical Vein Endothelial Cells
(HUVEC); neonatal; Vero, Monkey African Green kidney; RCC4 PLUS
VHL, Renal cell carcinoma cell line RCC4 stably transfected with
pcDNA3-VHL; Fao, Rat hepatoma; J774A.1, Mouse BALB/c monocyte
macrophage; MC3T3-E1, Mouse C57BL/6 calvaria; J774.2, Mouse BALB/c
monocyte macrophage; PNT1A, Human post pubertal prostate normal,
immortalised with SV40; U-2 OS, Human Osteosarcoma; HCT 116, Human
colon carcinoma; MA104, Monkey African Green kidney; BEAS-2B, Human
bronchial epithelium, normal; NB2-11, Rat lymphoma; BHK 21 (clone
13), Hamster Syrian kidney; NS0, Mouse myeloma; Neuro 2a, Mouse
Albino neuroblastoma; SP2/0-Ag14, Mouse.times.Mouse myeloma,
non-producing; T47D, Human breast tumor; 1301, Human T-cell
leukemia; MDCK-II, Canine Cocker Spaniel Kidney; PNT2, Human
prostate normal, immortalized with SV40; PC-3, Human Caucasian
prostate adenocarcinoma; TF1, Human erythroleukaemia; COS-7, Monkey
African green kidney, SV40 transformed; MDCK, Canine Cocker Spaniel
kidney; HUVEC (200-05n), Human Umbilical Vein Endothelial Cells
(HUVEC); neonatal; NCI-H322, Human Caucasian bronchioalveolar
carcinoma; SK.N.SH, Human Caucasian neuroblastoma; LNCaP.FGC, Human
Caucasian prostate carcinoma; OE21, Human Caucasian oesophageal
squamous cell carcinoma; PSN1, Human pancreatic adenocarcinoma;
ISHIKAWA, Human Asian endometrial adenocarcinoma; MFE-280, Human
Caucasian endometrial adenocarcinoma; MG-63, Human osteosarcoma; RK
13, Rabbit kidney, BVDV negative; EoL-1 cell, Human eosinophilic
leukemia; VCaP, Human Prostate Cancer Metastasis; tsA201, Human
embryonal kidney, SV40 transformed; CHO, Hamster Chinese ovary; HT
1080, Human fibrosarcoma; PANC-1, Human Caucasian pancreas; Saos-2,
Human primary osteogenic sarcoma; Fibroblast Growth Medium
(116K-500), Fibroblast Growth Medium Kit; ND7/23, Mouse
neuroblastoma.times.Rat neuron hybrid; SK-OV-3, Human Caucasian
ovary adenocarcinoma; COV434, Human ovarian granulosa tumor; Hep
3B, Human hepatocyte carcinoma; Vero (WHO), Monkey African Green
kidney; Nthy-ori 3-1, Human thyroid follicular epithelial; U373 MG
(Uppsala), Human glioblastoma astrocytoma; A375, Human malignant
melanoma; AGS, Human Caucasian gastric adenocarcinoma; CAKI 2,
Human Caucasian kidney carcinoma; COLO 205, Human Caucasian colon
adenocarcinoma; COR-L23, Human Caucasian lung large cell carcinoma;
IMR 32, Human Caucasian neuroblastoma; QT 35, Quail Japanese
fibrosarcoma; WI 38, Human Caucasian fetal lung; HMVII, Human
vaginal malignant melanoma; HT55, Human colon carcinoma; TK6, Human
lymphoblast, thymidine kinase heterozygote; SP2/0-AG14 (AC-FREE),
Mouse.times.mouse hybridoma non-secreting, serum-free, animal
component (AC) free; AR42J, or Rat exocrine pancreatic tumor, or
any combination thereof.
[0172] In some embodiments, the sample is a protein. In some
embodiments, the sample is a recombinant protein. In some
embodiments, the sample is a mutant protein. In some embodiments,
the sample is an enzyme. In some embodiments, the sample is a
mutant enzyme. In some embodiments, the enzyme is a protease, a
hydrolase, a kinase, a recombinase, a reductase, a dehydrogenase,
an isomerase, a synthetase, an oxidoreductase, a transferase, a
lyase, a ligase, or any mutant thereof.
[0173] In some embodiments, the sample is a single cell. In some
embodiments, sample is 2 or more cells. In some embodiments, the
sample is at least 2, at least 3, at least 4, at least 5, at least
10, at least 100, at least 1000, or at least 10000 cells.
[0174] In some embodiments, the cells comprise transfected nucleic
acids. In some embodiments, the cells comprise stably integrated
nucleic acids.
Ion Channel Screen
[0175] In some embodiments, the cells comprise ion channels. In
some embodiments, the ion channels are endogenous to the cells. In
some embodiments, the ion channels are non-endogenous to the cells.
In some embodiments, the ion channels are mutant ion channels. In
some embodiments, the ion channels comprise a mutation. In some
embodiments, the mutation sensitizes the ion channel to optical
stimulation. In some embodiments, the optical stimulation is
stimulation with electromagnetic radiation. In some embodiments,
the optical stimulation is stimulation with visible light.
[0176] In some embodiments, the methods comprise stimulating ion
channels. Stimulating ion channels may comprise activating or
deactivating an ion channel. In some embodiments, the ion channels
are stimulated by electrostimulation, optical stimulation, or
chemical stimulation. In some embodiments, the stimulation is
electrostimulation. In some embodiments, electrostimulation
comprises delivering an electric field to an ion channel. In some
embodiments, the electrostimulation is performed by an electrode.
In some embodiments, the electrostimulation is performed by an
electrode on a microfluidic device. In some embodiments, the
electrode is within a flow path of a microfluidic device. In some
embodiments, the electrode is within a flow path of an
encapsulation. In some embodiments, the electrode is outside of a
flow path of a microfluidic device. In some embodiments, the
electrode is outside a flow path of an encapsulation.
[0177] In some embodiments, provided herein, is a method for
screening ion channel modulators. In some embodiments, the ion
channel modulator is an inhibitor. In some embodiments, the ion
channel modulator is an agonist. In some embodiments, the method
comprises providing an encapsulation. In some embodiments, the
encapsulation comprises a cell expressing an ion channel protein.
In some embodiments, the encapsulation comprises a set of voltage
sensor probes. In some embodiments, the encapsulation comprises an
encoded effector and its corresponding encoding. In some
embodiments, the encapsulation comprises a cell expressing an ion
channel protein, a set of voltage sensor probes, and an encoded
effector and its corresponding encoding. In some embodiments, the
method comprises stimulating an ion channel of the cell. In some
embodiments, the method comprises detecting a signal from at least
one member of the set of voltage sensor probes. In some
embodiments, the method comprises sorting the encapsulation. In
some embodiments, the method comprises sorting the encapsulation
based on the presence, absence, level, or change of the signal. In
some embodiments, the method comprises measuring a property of the
encoding to ascertain the identity of the effector. In some
embodiments, the encoding is a nucleic acid and the property
measured to ascertain the identity of the effector is the nucleic
acid sequence of the encoding.
[0178] The ion channel protein may be any such protein. In some
embodiments, the ion channel protein comprises a sodium, calcium,
chloride, proton, or potassium ion channel protein. In some
embodiments, the ion channel protein comprises a sodium ion channel
protein. In some embodiments, the ion channel protein comprises a
potassium ion channel protein. In some embodiments, the ion channel
protein comprises a calcium ion channel protein. In some
embodiments, the ion channel protein comprises a chloride ion
channel protein. In some embodiments, the ion channel protein
comprises a proton ion channel protein.
[0179] In some embodiments, the ion channel protein comprises a
voltage gated ion channel protein. Any voltage gated ion channel
protein may be used. In some embodiments, the voltage gated ion
channel protein comprises a sodium, calcium, chloride, proton, or
potassium voltage gated ion channel protein. In some embodiments,
the voltage gated ion channel protein comprises a voltage gated
calcium ion channel protein. In some embodiments, the voltage gated
ion channel protein comprises a voltage gated sodium ion channel
protein. In some embodiments, the voltage gated ion channel protein
comprises a voltage gated potassium ion channel protein. In some
embodiments, the voltage gated ion channel protein comprises a
voltage gated chloride ion channel protein. In some embodiments,
the voltage gated ion channel protein comprises a voltage gated
proton ion channel protein.
[0180] In some embodiments, the ion channel protein is endogenous
to the cell. In some embodiments, the ion channel protein is an
exogenous ion channel protein. In some embodiments, the ion channel
protein is incorporated into the cell through a vector. In some
embodiments, the ion channel protein stably expressed in the cell
through the addition of a vector. In some embodiments, a gene
encoding the ion channel protein is transiently transfected into
the cell. In some embodiments, a gene encoding the ion channel is
stably incorporated into the cell. In some embodiments, the ion
channel protein is overexpressed.
[0181] In some embodiments, the voltage gated ion channel protein
comprises a voltage-gated calcium channel protein (VGCC). Any VGCC
or any mutant, fragment, or conjugate thereof may be used. In some
embodiments, the VGCC comprises an L-type calcium channel (e.g.
Ca.sub.v1.1, Ca.sub.v1.2, Ca.sub.v1.3, or Ca.sub.v1.4), a P-type
calcium channel (e.g. Ca.sub.v2.1), an N-type calcium channel (e.g.
Ca.sub.v2.2), an R-type calcium channel (e.g. Ca.sub.v2.3), or a
T-type calcium channel (e.g. Ca.sub.v3.1, Ca.sub.v3.2, or
Ca.sub.v3.3), or any mutant, fragment, or conjugate thereof. In
some embodiments, the VGCC comprises an L-type calcium channel. In
some embodiments, the VGCC comprises a P-type calcium channel. In
some embodiments, the VGCC comprises an N-type calcium channel. In
some embodiments, the VGCC comprises an R-type calcium channel. In
some embodiments, the VGCC comprises a T-type calcium channel.
[0182] In some embodiments, the ion channel protein comprises a
voltage gated sodium channel protein (Nay) or any mutant, fragment,
or conjugate thereof. Any voltage gated sodium channel protein may
be used. In some embodiments, the voltage gated sodium channel
protein comprises Na.sub.v1.1, Na.sub.v1.2, Na.sub.v1.3,
Na.sub.v1.4, Na.sub.v1.5, Na.sub.v1.6, Na.sub.v1.7, Na.sub.v1.8,
Na.sub.v1.9, Na.sub.v2.1, Na.sub.v2.2, Na.sub.v2.3, or Na.sub.v3.1,
or any mutant, fragment, or conjugate thereof.
[0183] In some embodiments, the ion channel protein comprises a
voltage gated potassium channel protein (VGKC) or any mutant,
fragment, or conjugate thereof. Any VGKC protein may be used. The
VGKC protein may have any alpha subunit. In some embodiments, the
VGKC comprises a delayed rectifier potassium channel (e.g.
K.sub.v1.1, K.sub.v1.2, K.sub.v1.3, K.sub.v1.5, K.sub.v1.6,
K.sub.v1.7, K.sub.v1.8, K.sub.v2.1, K.sub.v2.1, K.sub.v3.1,
K.sub.v3.2, K.sub.v7.1, K.sub.v7.2, K.sub.v7.3, K.sub.v7.4,
K.sub.v7.5, or K.sub.v10.1). In some embodiments, the VGKC
comprises an A-type potassium channel (E.g. K.sub.v1.4, K.sub.v3.3,
K.sub.v3.4, K.sub.v4.1, K.sub.v4.1, K.sub.v4.2, or K.sub.v4.3). In
some embodiments, the VGKC comprises an outward-rectifying
potassium channel (e.g. K.sub.v10.2). In some embodiments, the VGKC
comprises an inwardly-rectifying potassium channel (e.g. an
ether-a-go-go potassium channel, such as K.sub.v11.1, K.sub.v11.2,
or K.sub.v11.3). In some embodiments, the VGKC comprises a slowly
activating potassium channel (e.g. K.sub.v12.1, K.sub.v12.2, or
K.sub.v12.3). In some embodiments, the VGKC comprises a
modifier/silencer potassium channel (e.g. K.sub.v5.1, K.sub.v6.1,
K.sub.v6.2, K.sub.v6.3, K.sub.v6.4, K.sub.v8.1, K.sub.v8.2,
K.sub.v9.1, K.sub.v9.2, or K.sub.v9.3). Any mutant, fragment, or
conjugate of any of the preceding potassium channels may be
used.
[0184] In some embodiments, the ion channel protein comprises a
voltage gated chloride channel protein. Any voltage gated chloride
channel protein may be used. In some embodiments, the voltage gated
chloride channel protein is from the CLCN family (e.g. CLCN1,
CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, CLCNKB). In some
embodiments, the voltage gated chloride channel protein is from the
epithelial chloride channel family (e.g. CLCA1, CLCA2, CLCA3, or
CLCA4). In some embodiments, the voltage gated chloride channel
protein is from the chloride intracellular channel (CLIC) family
(e.g. CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, or CLIC6).
[0185] In some embodiments, the ion channel protein comprises a
voltage gated proton channel. Any voltage gated proton channel
protein may be used. In some embodiments, the voltage gated proton
channel comprise voltage-gated hydrogen channel 1 protein.
[0186] In some embodiments, the ion channel protein comprises a
channelrhodopsin or any mutant, fragment, or conjugate thereof. In
some embodiments, wherein the channelrhodopsin is ChrimsonR or any
mutant, fragment, or conjugate thereof. In some embodiments, the
channelrhodopsin is a ChrimsonR mutant comprising a K176R mutation,
S267M mutation, Y268F mutation, Y261F mutation, or any combination
thereof.
[0187] The set of voltage sensor probes may comprise any suitable
probe. In some embodiments, the set of voltage sensor probes
comprise a FRET pair. In some embodiments, the set of voltage
sensor probes comprises a voltage-sensitive oxonol, a fluorescent
coumarin, or both. In some embodiments, the set of voltage sensor
probes comprises a voltage-sensitive oxonol. In some embodiments,
the set of voltage sensor probes comprises a fluorescent coumarin.
In some embodiments, the set of voltage sensor probes comprises a
DiSBAC compound, a coumarin phospholipid, or any combination or
derivative thereof. In some embodiments, the set of voltage sensor
probes comprises a DiSBAC compound. In some embodiments, the set of
voltage sensor probes comprises a coumarin phospholipid, the set of
voltage sensors comprises a DiSBAC.sub.2, DiSBAC.sub.4,
DiSBAC.sub.6, CC1-DMPE, CC2-DMPE, or any combination or derivative
thereof. In some embodiments, the set of voltage sensors comprises
a DiSBAC.sub.2(3), DiSBAC.sub.2(5), DiSBAC.sub.4(3),
DiSBAC.sub.4(5), DiSBAC.sub.6(3), DiSBAC.sub.6(5), CC1-DMPE,
CC2-DMPE, or any combination or derivative thereof. In some
embodiments, the set of voltage sensors comprises DiSBAC.sub.6 and
CC2-DMPE.
[0188] The encapsulation may further comprise a voltage assay
background suppression compound. In some embodiments, the voltage
assay background suppression compound comprises VABSC-1.
[0189] In some embodiments, the stimulation is optical stimulation.
In some embodiments, the optical stimulation is electromagnetic
radiation. In some embodiments, the optical stimulation is visible
light. In some embodiments, the optical stimulation is UV, VIS, or
near-infrared radiation. In some embodiments, the optical
stimulation is UV radiation. In some embodiments, the optical
stimulation is visible light. In some embodiments, the optical
stimulation is near-infrared radiation.
[0190] In some embodiments, the wavelength of light for optical
stimulation is about 660 nm. In some embodiments, the wavelength of
light for optical stimulation is about 100 nm to about 1,000 nm. In
some embodiments, the wavelength of light for optical stimulation
is about 100 nm to about 200 nm, about 100 nm to about 400 nm,
about 100 nm to about 450 nm, about 100 nm to about 500 nm, about
100 nm to about 550 nm, about 100 nm to about 600 nm, about 100 nm
to about 650 nm, about 100 nm to about 700 nm, about 100 nm to
about 750 nm, about 100 nm to about 800 nm, about 100 nm to about
1,000 nm, about 200 nm to about 400 nm, about 200 nm to about 450
nm, about 200 nm to about 500 nm, about 200 nm to about 550 nm,
about 200 nm to about 600 nm, about 200 nm to about 650 nm, about
200 nm to about 700 nm, about 200 nm to about 750 nm, about 200 nm
to about 800 nm, about 200 nm to about 1,000 nm, about 400 nm to
about 450 nm, about 400 nm to about 500 nm, about 400 nm to about
550 nm, about 400 nm to about 600 nm, about 400 nm to about 650 nm,
about 400 nm to about 700 nm, about 400 nm to about 750 nm, about
400 nm to about 800 nm, about 400 nm to about 1,000 nm, about 450
nm to about 500 nm, about 450 nm to about 550 nm, about 450 nm to
about 600 nm, about 450 nm to about 650 nm, about 450 nm to about
700 nm, about 450 nm to about 750 nm, about 450 nm to about 800 nm,
about 450 nm to about 1,000 nm, about 500 nm to about 550 nm, about
500 nm to about 600 nm, about 500 nm to about 650 nm, about 500 nm
to about 700 nm, about 500 nm to about 750 nm, about 500 nm to
about 800 nm, about 500 nm to about 1,000 nm, about 550 nm to about
600 nm, about 550 nm to about 650 nm, about 550 nm to about 700 nm,
about 550 nm to about 750 nm, about 550 nm to about 800 nm, about
550 nm to about 1,000 nm, about 600 nm to about 650 nm, about 600
nm to about 700 nm, about 600 nm to about 750 nm, about 600 nm to
about 800 nm, about 600 nm to about 1,000 nm, about 650 nm to about
700 nm, about 650 nm to about 750 nm, about 650 nm to about 800 nm,
about 650 nm to about 1,000 nm, about 700 nm to about 750 nm, about
700 nm to about 800 nm, about 700 nm to about 1,000 nm, about 750
nm to about 800 nm, about 750 nm to about 1,000 nm, or about 800 nm
to about 1,000 nm. In some embodiments, the wavelength of light for
optical stimulation is about 100 nm, about 200 nm, about 400 nm,
about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650
nm, about 700 nm, about 750 nm, about 800 nm, or about 1,000 nm. In
some embodiments, the wavelength of light for optical stimulation
is at least about 100 nm, about 200 nm, about 400 nm, about 450 nm,
about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700
nm, about 750 nm, or about 800 nm. In some embodiments, the
wavelength of light for optical stimulation is at most about 200
nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about
600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, or
about 1,000 nm.
[0191] In some embodiments, the intensity of light for optical
stimulation is about 500 mJ/s/cm.sup.2. In some embodiments,
intensity of light for optical stimulation is about 50 to about
1,000 mJ/S/cm.sup.2. In some embodiments, intensity of light for
optical stimulation is about 50 to about 100, about 50 to about
250, about 50 to about 500, about 50 to about 750, about 50 to
about 1,000, about 100 to about 250, about 100 to about 500, about
100 to about 750, about 100 to about 1,000, about 250 to about 500,
about 250 to about 750, about 250 to about 1,000, about 500 to
about 750, about 500 to about 1,000, or about 750 to about 1,000
mJ/S/cm.sup.2. In some embodiments, intensity of light for optical
stimulation is about 50, about 100, about 250, about 500, about
750, or about 1,000 mJ/S/cm.sup.2. In some embodiments, intensity
of light for optical stimulation is at least about 50, about 100,
about 250, about 500, or about 750. In some embodiments, intensity
of light for optical stimulation is at most about 100, about 250,
about 500, about 750, or about 1,000 mJ/S/cm.sup.2.
[0192] In some embodiments, the frequency of optical stimulation is
about 10 Hz. In some embodiments, the frequency of optical
stimulation is about 1 Hz to about 100 Hz. In some embodiments, the
frequency of optical stimulation is about 1 Hz to about 2 Hz, about
1 Hz to about 5 Hz, about 1 Hz to about 10 Hz, about 1 Hz to about
20 Hz, about 1 Hz to about 50 Hz, about 1 Hz to about 100 Hz, about
2 Hz to about 5 Hz, about 2 Hz to about 10 Hz, about 2 Hz to about
20 Hz, about 2 Hz to about 50 Hz, about 2 Hz to about 100 Hz, about
5 Hz to about 10 Hz, about 5 Hz to about 20 Hz, about 5 Hz to about
50 Hz, about 5 Hz to about 100 Hz, about 10 Hz to about 20 Hz,
about 10 Hz to about 50 Hz, about 10 Hz to about 100 Hz, about 20
Hz to about 50 Hz, about 20 Hz to about 100 Hz, or about 50 Hz to
about 100 Hz. In some embodiments, the frequency of optical
stimulation is about 1 Hz, about 2 Hz, about 5 Hz, about 10 Hz,
about 20 Hz, about 50 Hz, or about 100 Hz. In some embodiments, the
frequency of optical stimulation is at least about 1 Hz, about 2
Hz, about 5 Hz, about 10 Hz, about 20 Hz, or about 50 Hz. In some
embodiments, the frequency of optical stimulation is at most about
2 Hz, about 5 Hz, about 10 Hz, about 20 Hz, about 50 Hz, about 100
Hz, about 150 Hz, or about 200 Hz.
[0193] In some embodiments, stimulation is chemical stimulation. In
some embodiments, the chemical stimulation comprises contacting the
ion channel with a toxin. In some embodiments, the toxin is an ion
channel toxin. In some embodiments, the toxin is added to an
encapsulation by pico-injection. In some embodiments, the toxin is
added to an encapsulation by conditional pico-injection. In some
embodiments, chemical stimulation comprises contacting the ion
channel with an ion channel toxin. In some embodiments, the ion
channel toxin comprises veratridine, OD-1, or another ion channel
toxin, or any combination thereof. In some embodiments, the ion
channel toxin comprises veratridine. In some embodiments, the ion
channel toxin comprises OD-1.
[0194] In some embodiments, the ion channel toxin as added to the
encapsulation by pico-injection, droplet fusion, or through a
pre-arranged architecture of a microfluidic device which contains
the encapsulation. In some embodiments, the ion channel toxin as
added to the encapsulation by pico-injection. In some embodiments,
the ion channel toxin as added to the encapsulation by droplet
fusion. In some embodiments, the ion channel toxin as added to the
encapsulation through a pre-arranged architecture of a microfluidic
device which contains the encapsulation.
[0195] The ion channel may be stimulated by electrical stimulation.
In some embodiments, stimulating the ion channel is performed by at
least one electrode. In some embodiments, the at least one
electrode is in the flow path of the encapsulation. In some
embodiments, the at least one electrode is outside the flow path of
the encapsulation. In some embodiments, electrostimulation is
performed by non-contact electrodes to generate electric fields,
dielectrophoretic forces, or embedded metal-contact electrodes. In
some embodiments, electrostimulation is performed by non-contact
electrodes to generate electric fields. In some embodiments,
electrostimulation is performed dielectrophoretic forces. In some
embodiments, electrostimulation is performed by embedded
metal-contact electrodes.
[0196] In some embodiments, electrostimulation is dictated by
geometry of a microfluidic device containing the encapsulation. In
some embodiments, the frequency of electrostimulation is about 10
Hz. In some embodiments, the frequency of electrostimulation is
about 1 Hz to about 100 Hz. In some embodiments, the frequency of
electrostimulation is about 1 Hz to about 2 Hz, about 1 Hz to about
5 Hz, about 1 Hz to about 10 Hz, about 1 Hz to about 20 Hz, about 1
Hz to about 50 Hz, about 1 Hz to about 100 Hz, about 2 Hz to about
5 Hz, about 2 Hz to about 10 Hz, about 2 Hz to about 20 Hz, about 2
Hz to about 50 Hz, about 2 Hz to about 100 Hz, about 5 Hz to about
10 Hz, about 5 Hz to about 20 Hz, about 5 Hz to about 50 Hz, about
5 Hz to about 100 Hz, about 10 Hz to about 20 Hz, about 10 Hz to
about 50 Hz, about 10 Hz to about 100 Hz, about 20 Hz to about 50
Hz, about 20 Hz to about 100 Hz, or about 50 Hz to about 100 Hz. In
some embodiments, the frequency of electrostimulation is about 1
Hz, about 2 Hz, about 5 Hz, about 10 Hz, about 20 Hz, about 50 Hz,
or about 100 Hz. In some embodiments, the frequency of
electrostimulation is at least about 1 Hz, about 2 Hz, about 5 Hz,
about 10 Hz, about 20 Hz, or about 50 Hz. In some embodiments, the
frequency of electrostimulation is at most about 2 Hz, about 5 Hz,
about 10 Hz, about 20 Hz, about 50 Hz, about 100 Hz, about 150 Hz,
or about 200 Hz.
[0197] The stimulation of the ion channels can be performed
numerous times, or only a single time. In some embodiments, the ion
channel of the cell is stimulated about 1 time to about 20 times.
In some embodiments, the ion channel of the cell is stimulated
about 1 time to about 2 times, about 1 time to about 3 times, about
1 time to about 5 times, about 1 time to about 7 times, about 1
time to about 10 times, about 1 time to about 20 times, about 2
times to about 3 times, about 2 times to about 5 times, about 2
times to about 7 times, about 2 times to about 10 times, about 2
times to about 20 times, about 3 times to about 5 times, about 3
times to about 7 times, about 3 times to about 10 times, about 3
times to about 20 times, about 5 times to about 7 times, about 5
times to about 10 times, about 5 times to about 20 times, about 7
times to about 10 times, about 7 times to about 20 times, or about
10 times to about 20 times. In some embodiments, the ion channel of
the cell is stimulated about 1 time, about 2 times, about 3 times,
about 5 times, about 7 times, about 10 times, or about 20 times. In
some embodiments, the ion channel of the cell is stimulated at
least about 1 time, about 2 times, about 3 times, about 5 times,
about 7 times, or about 10 times. In some embodiments, the ion
channel of the cell is stimulated at most about 2 times, about 3
times, about 5 times, about 7 times, about 10 times, or about 20
times. In some embodiments, the ion channel is stimulated a single
time. In embodiments where stimulation occurs by the addition of an
ion channel toxin or other ion channel inhibitor, the ion channel
toxin need only be added at a single step.
[0198] In some embodiments, provided herein, are methods for
stimulating an ion channel. In some embodiments, the methods
comprise providing a cell in an encapsulation. In some embodiments,
the methods comprise stimulating an ion channel of the cell by
electrostimulation, optical stimulation, or chemical stimulation.
In some embodiments, the methods comprise detecting a signal from
the cell by capturing images of the cell in the encapsulation.
[0199] In some embodiments, the method comprises detecting a signal
from at least one member of the set of voltage sensor probes. In
some embodiments, the signal is electromagnetic radiation. In some
embodiments, the electromagnetic radiation is luminescence or
fluorescence. In some embodiments, the electromagnetic radiation is
fluorescence. In some embodiments, the electromagnetic radiation is
emitted due to a FRET interaction. In some embodiments, the signal
is an increase, decrease, or change in electromagnetic radiation as
compared to an identical encapsulation without the encoded
effector. In some embodiments, the signal is an increase, decrease,
or change in electromagnetic radiation as compared to the
encapsulation before the stimulation of the ion channel.
[0200] In some embodiments, the method comprises the step of
sorting the encapsulation based on the presence, absence, level, or
change of the signal. In some embodiments, the method further
comprises measuring a property of the encoding to ascertain the
identity of the effector.
[0201] In some embodiments, the sample is a protein. In some
embodiments, the sample is a recombinant protein. In some
embodiments, the sample is a mutant protein. In some embodiments,
the sample is an enzyme. In some embodiments, the sample is a
mutant enzyme. In some embodiments, the enzyme is a protease, a
hydrolase, a kinase, a recombinase, a reductase, a dehydrogenase,
an isomerase, a synthetase, an oxidoreductase, a transferase, a
lyase, a ligase, or any mutant thereof.
[0202] The sample may further comprise a nucleic acid which codes
for the expression of a target protein and the target protein
itself. These sample nucleic acids may be barcoded. The presence of
a barcode on the nucleic acids may allow for the transfer of the
barcode to nucleic acid encodings of effectors that are
co-encapsulated with the target protein and the nucleic acid which
codes for the expression of the target protein. This in turn allows
for a determination of which combinations of effectors were
encapsulated together and produced a synergistic effect against the
target protein. Such methods can be used to conduct fragment-based
screens to identify lead molecules of interest in further drug
discovery.
Fragment Based Screen and Enzyme Evolution Method
[0203] In some embodiments, the sample is a target protein and a
nucleic acid coding the expression of a target protein. In some
embodiments, the nucleic acid coding the expression of the target
protein further comprises a barcode region. In some embodiments,
the nucleic acid coding the expression of a target protein is bound
to a scaffold. In some embodiments, the barcode from the nucleic
acid that codes for the target protein can be transferred to
nucleic acid encodings of effectors. In some embodiments, the
sample target protein and nucleic acid coding the expression of the
target protein are co-encapsulated with an in vitro
transcription/translation system. In some embodiments, the in vitro
transcription/translation system is used to amplify the target
protein.
[0204] In some embodiments, two or more nucleic acid encoded
effectors with their corresponding nucleic acid encodings are
introduced into the encapsulation comprising the target protein and
nucleic acid encoding the expression of the target protein. In some
embodiments, the barcode is transferred to the nucleic acids
encoding the effectors. In some embodiments, the encapsulation is
incubated for a period of time to allow the two or more effectors
to interact with the target protein. In some embodiments, a signal
is produced by the interaction of the two or more effectors and the
target protein. In some embodiments, the encapsulation is sorted
based on the measurement of the signal. In some embodiments, the
nucleic acid encodings which now comprise the barcode from nucleic
acid coding for the target protein are sequenced. In some
embodiments, the sequencing allows for identifying combinations of
effectors that conferred efficacy against the target protein.
[0205] In some embodiments, the target protein coded by the nucleic
acid is a signaling protein, an enzyme, a binding protein, an
antibody or antibody fragment, a structure protein, a storage
protein, or a transport protein. In some embodiments, the target
protein is an enzyme. In some embodiments, the target protein is
trypsin, macrophage metalloelastase 12 (MMP-12), extracellular
signal-related kinase 1 (ERK1), or extracellular signal-regulated
kinase 2 (EKR2).
[0206] In embodiments wherein the sample is a target protein and a
nucleic acid coding the expression of the target protein, the
nucleic acid may comprise a sequence complementary to the nucleic
acid encoding an effector. This complementarity can be utilized for
amplification of the barcode onto the nucleic acid encoding the
effector. In embodiments wherein the sample is a target protein and
a nucleic acid coding the expression of the target protein, the
nucleic acid may contain a promoter sequence. In some embodiments,
the promoter sequence allows for amplification of the nucleic acid
sequence and/or the nucleic acid sequence encoding the effector
after the barcode has been transferred.
[0207] In vitro transcription/translation systems are systems which
can express proteins from nucleic acids which code for the protein
without requiring any living tissue or cells. In some embodiments,
the in vitro transcription/translation system is used to express
the target protein within an encapsulation. In some embodiments,
the in vitro transcription/translation system is used to express
the target protein within an encapsulation to a target
concentration. In some embodiments, the in vitro
transcription/translation system is used to amplify the target
protein within an encapsulation. In some embodiments, the in vitro
transcription/translation system is used to amplify the target
protein within an encapsulation to a desired concentration.
Encapsulation
[0208] An encapsulation can refer to the formation of a compartment
within a larger system. In preferred embodiments, the encapsulation
is a droplet within a microfluidic channel. In some embodiments,
the encapsulation is a droplet, an emulsion, a macrowell, a
microwell, bubble, or a microfluidic confinement. Once an
encapsulation is formed, any component inside the encapsulation can
remain in the encapsulation until the encapsulation is destroyed or
broken down. In some embodiments, the encapsulations used herein
remain stable for at least 4 hours, at least 12 hours, at least 1
day, at least 2 days, at least 3 days, or at least 1 week. In some
embodiments, the encapsulations are stable for the duration of the
screen to be performed so that no intermingling of reagents between
encapsulations occurs.
[0209] In some embodiments, the encapsulation is a droplet. In some
embodiments, the droplet is at most 1 picoliter, at most 10
picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10
nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. In some embodiments, the droplet is at least 1 picoliter,
at least 10 picoliters, at least 100 picoliters, at least 1
nanoliter, at least 10 nanoliters, at least 100 nanoliters, or at
least 1 microliter in volume. In some embodiments, the droplet is
between about 200 picoliters and about 10 nanoliters.
[0210] In some embodiments, the droplet is an aqueous droplet in a
larger body of oil. In some embodiments, the droplets are placed in
an oil emulsion. In some embodiments, the oil comprises a silicone
oil, a fluorosilicone oil, a hydrocarbon oil, a mineral oil, a
paraffin oil, a halogenated oil, a fluorocarbon oil, or any
combination thereof. In some embodiments, the oil comprises a
silicone oil. In some embodiments, the oil comprises a
fluorosilicone oil. In some embodiments, the oil comprises a
hydrocarbon oil. In some embodiments, the oil comprises a mineral
oil. In some embodiments, the oil comprises a paraffin oil. In some
embodiments, the oil comprises a halogenated oil. In some
embodiments, the oil comprises a fluorocarbon oil.
[0211] In embodiments wherein there are a plurality of
encapsulations, each individual encapsulation may be any size. In
some embodiments, each encapsulation is approximately the same
size. In some embodiments, each encapsulation is within 5%, 10%,
15%, 20%, or 25% of the average size encapsulation within the
plurality. In some embodiments, at least 80%, 85%, 90%, or 95% of
the encapsulations are within about 5%, 10%, 15%, 20%, or 25% of
the average size encapsulation within the plurality.
[0212] The encapsulations may be formed by any method. In some
embodiments, an encapsulation is formed by flowing an aqueous
stream into an immiscible carrier fluid. In some embodiment, the
aqueous stream flows into an immiscible carrier fluid at a junction
of microfluidic channels. In some embodiments, the junction is a
T-junction. In some embodiments, the junction is a meeting of two
perpendicular microfluidic channels. The junction may be a meeting
of any number of microfluidic channels. The junction may be at any
angle. The aqueous stream may be formed by an upstream junction of
two or more aqueous streams. In some embodiments, sample solutions
and effector solutions are joined upstream of the aqueous stream
junction with the immiscible carrier fluid.
[0213] The size of the droplets may be controlled by modulating a
variety of parameters. These parameters include the geometry of the
junction of two microfluidic channels, the flow rate of the two
streams, the type of oil used, the presence of surfactants, the
pressure applied to the flow streams, or any combination
thereof.
[0214] In some embodiments, a single encoded effector is present in
an encapsulation. In some embodiments, a single scaffold comprising
an encoded effector and its encoding are present in an
encapsulation. In some embodiments, a plurality of scaffolds, each
scaffold comprising a different encoded effector and its respective
encoding, are present in an encapsulation.
[0215] In some embodiments, encapsulations comprise biological
samples. In some embodiments, encapsulations comprise single cells.
In some embodiments, encapsulations comprise one or more cells. In
some embodiments, the encapsulations comprise nucleic acids. In
some embodiments, the encapsulations comprise proteins. In some
embodiments, the encapsulations comprise.
Sorting
[0216] The methods and systems provided herein may comprise sorting
steps. The sorting step can be accomplished in a variety of ways.
One way of sorting the "hit" effectors from the non-hit effectors
is to physically separate the hits from non-hits in space. This can
be accomplished in a variety of manners. In some embodiments,
sorting the encapsulations comprises providing the encapsulation
through a microfluidic channel. In some embodiments, the
microfluidic channel is equipped with a detector. In some
embodiments, the "hit" effectors are placed into one collection
vessel if the "hit" criteria is met, and the "non-hit" effectors
are placed into another collection vessel. As described herein, in
some embodiments, such "hit" effectors are sorted based on the
presence or absence of a signal resulting from an interaction with
the effector (or another component) and the sample, a reagent, or
combinations thereof. In some embodiments, the sorting is based on
the level of a signal detected. In some embodiments, the sorting is
based on the presence of a signal detected. In some embodiments,
the sorting is based on the absence of a signal.
[0217] In some embodiments, sorting droplets is accomplished by
activity-based screening. Activity based sorting is accomplished by
the ability to sort based on detecting a response emitted by the
droplet as it passes by a detecting region on the microfluidic
chip. As an example, certain small-molecules inhibit particular
enzymes which can be screened by an activity-based assay that
detects for that inhibition. Thus, sorting is based on the
"activity" of the enzyme and thus screening for small-molecules
that functionally inhibit the enzyme rather than simply bind to the
enzyme. It is a more relevant screen and is much more similar to
conventional HTS screening which screens for activity.
[0218] In some embodiments, sorting the encapsulations comprises
placing the encapsulations (e.g., droplets) into a first collection
tube if the signal is at or above a predetermine threshold. In some
embodiments, sorting the encapsulation comprises placing the
droplet into a second collection tube if the signal is below a
predetermined threshold. In some embodiments, sorting the
encapsulation comprises placing the droplet into a first collection
tube if the signal is at or above a predetermine threshold or
placing the droplet into a second collection tube if the signal is
below a predetermined threshold. In some embodiments, sorting the
encapsulation comprises placing encapsulations in two or more
collection tubes, or bins. In some embodiments, "hit" effectors or
positive "hits` are stored in two or more collection tubes or bins.
In some embodiments, the "hit" effectors, or positive "hits" are
sorted based on the signal or activity measured.
[0219] FIGS. 26A to 28C depict sorting droplets based on two types
of detection signals. FIGS. 26A-B depict the use of a bead attached
with fluorophore TR1-TAMRA, which upon release from the bead,
provides a detectable intensity level (FIG. 26B). By contrast,
FIGS. 27A-B depict the use of a bead attached with an inhibitor
TR3, which upon release inhibits or minimizes the intensity of
fluorescence detected (FIG. 27B). FIG. 27C depicts a decrease in
Cathepsin D activity with increasing concentration of the TR3
inhibitor. FIG. 28A provides an exemplary depiction of droplets
being sorted based on a certain inhibition threshold being met,
wherein for those droplets exhibiting a fluorescence intensity
level below a certain threshold will be a "positive" hit, and those
droplets exhibiting fluorescence intensity levels above the
threshold, will be a "negative" hit. FIG. 28C provides an exemplary
threshold level for such inhibitory activity. In some embodiments,
the threshold for sorting will be based on a minimum fluorescence
intensity level being measured (e.g., as occurring through use of
TAMRA fluorophore). FIG. 28B provides an exemplary threshold level
for such fluorescence detection activity. FIG. 28D provides an
exemplary illustration of a device as used in a method or system
described herein.
[0220] In some embodiments, sorting the encapsulation comprises
using a waveform pulse generator to move the encapsulation to a
collection tube by an electrical field gradient, by sound, by a
diaphragm, by modifying geometry of microfluidic channel, or by
changing the pressure of the microfluidic channel. In some
embodiments, the waveform pulse generator moves the encapsulation
by an electrical field gradient. In some embodiments, the waveform
pulse generator moves the encapsulation by sound. In some
embodiments, the waveform pulse generator moves the encapsulation
by a diaphragm. In some embodiments, the waveform pulse generator
modifies the geometry of the microfluidic channel. In some
embodiments, the waveform pulse generator changes the pressure of
the microfluidic channel.
[0221] Various methods for determining which effectors had the
desired effect may be used. In some instances, physical sorting of
"hit" effectors is used to determine which effectors had the
desired effect. In some instances, selective addition of a
detectable label to encapsulations comprising a "hit" effector is
used. In some instances, a detectable label is used to determine
which effectors had the desired effect by linking detectable label
with the encoding. For example, the addition of a nucleic acid
barcode to nucleic acid encodings of effectors can accomplish
tagging the "hit" effectors in a way that can be ascertained by
sequencing. If only "hit" effectors encodings are tagged with the
nucleic acid barcode, then these samples can be picked out during a
subsequent sequencing step, as effectors which lacked the desired
activity will lack the barcode. The barcode may additionally
comprise a unique primer sequence to allow for amplification of
only the "hit" effector encodings. In this way, all encapsulations
can be pooled together, regardless of activity or efficacy, and the
resulting hits can still be ascertained.
Barcode Non-Sorting Method
[0222] In some embodiments provided herein, the methods do not
comprise a physical sorting step. In these embodiments,
deconvolution of which effectors had the desired effect on a sample
is accomplished in a different manner. In some embodiments, the
method further comprises the step of adding additional reagents to
the encapsulation which add a barcode to the encoding. In some
embodiments, the method further comprises the step of adding
additional reagents to the encapsulation which add a barcode to a
nucleic acid encoding. In some embodiments, the additional reagents
add a barcode to the encoding by annealing the barcode to the
encoding, ligating the barcode to the encoding, or amplifying the
barcode onto the encoding. In some embodiments, the additional
reagents comprise a tagging nucleic acid comprising a sequence
complementary to a sequence on the nucleic acid encoding which acts
as a primer for the nucleic acid encoding and the barcode. In some
embodiments, the additional reagents comprise enzymes to add the
barcode to the nucleic acid encoding.
[0223] Provided herein, in some embodiments, are methods for
screening an encoded effector without a physical sorting step. In
some embodiments, the method comprises providing a sample, a
nucleic acid encoded effector, and a nucleic acid encoding in an
encapsulation. In some embodiments, a signal is detected in the
encapsulation. In some embodiments, the signal results from an
interaction between the effector and the sample. In some
embodiments, a first capping mix is added to the droplet based on
the detection, absence, or level of the signal. In some
embodiments, the first capping mix adds a first nucleic acid cap to
the nucleic acid encoding. In some embodiments, a second capping
mix is added to the encapsulation. In some embodiments, the second
capping mix is only added if the first capping mix is not added to
the encapsulation. In some embodiments, the first nucleic acid cap
and the second nucleic acid cap have different sequences. In some
embodiments, only the first nucleic acid cap or only the second
nucleic acid cap is added to the nucleic acid encoding.
[0224] The first and second nucleic acid caps can have different
significance and indicate different things when added to nucleic
acid encodings. In some embodiments, the first nucleic acid cap
indicates that the effector had a desired activity. In some
embodiments, the desired activity resulted in the signal being
above a pre-determined threshold. In some embodiments, the desired
activity resulted in the signal being below a pre-determined
threshold. In some embodiments, the desired activity resulted in
the presence of the signal. In some embodiments, the desired
activity resulted in the absence of the signal.
[0225] In some embodiments, the second nucleic acid cap indicates
that the effector lacked a desired activity. In some embodiments,
the lack of desired activity resulted in the signal being below a
pre-determined threshold. In some embodiments, the lack of desired
activity resulted in the signal being above a pre-determined
threshold. In some embodiments, the lack of desired activity
resulted in the absence of the signal. In some embodiments, the
lack of desired activity resulted in the presence of the
signal.
[0226] The nucleic acid caps can be added to nucleic acid encodings
by a variety of methods. In some embodiments, the nucleic acid cap
is added to the nucleic acid encoding by ligation, hybridization,
extension of the nucleic acid encoding, or combinations thereof. In
some embodiments, the nucleic acid cap is added to the nucleic acid
encoding by ligation. In some embodiments, the nucleic acid cap is
added to the nucleic acid encoding by hybridization. In some
embodiments, the nucleic acid cap is added to the nucleic acid
encoding by extension of the nucleic acid encoding. In some
embodiments, the nucleic acid cap is added to the nucleic acid
encoding by chemically crosslinking the nucleic acids. In some
embodiments, the nucleic acid cap is added to the nucleic acid
encoding by chemical crosslinking with psoralen. In some
embodiments, a complementary sequence the nucleic acid cap is
located on the terminal end of the nucleic acid encoding to allow
for the addition of the nucleic acid cap. In some embodiments, the
nucleic acid caps comprise a barcode sequence.
[0227] In some embodiments, the capping mix comprises additional
reagents for adding the nucleic acid cap to the encoding. In some
embodiments, the additional reagents comprise an enzyme. In some
embodiments, the enzyme is a polymerase, a ligase, a restriction
enzyme, or a recombinase. In some embodiments, the enzyme is a
polymerase.
Bead Capture of Nucleic Acids
[0228] In addition to measuring activity from detectable signals,
additional information can be gathered from a screen by
incorporating nucleic acids from the sample onto encodings. In some
embodiments, the method comprises transferring one or more nucleic
acids from the sample to the encoding. The transfer of nucleic
acids from the sample to the encoding allows substantial
information about the sample, and information about the effect the
effector has on the sample to be ascertained, particularly when the
sample is a cell. The transfer of the nucleic acids from the sample
can allow for quantification of expressed protein by quantifying
the amount of target mRNA, as well as provide global proteomic and
genomic data about the cell. This data can be collected and
compared to cells that did not receive a dose of the indicated
effector
[0229] In one aspect, provided herein, is a method for detecting
sample nucleic acids in a nucleic acid encoded effector screen. In
some embodiments, the method comprises providing one or more cells,
a nucleic acid encoded effector, and a nucleic acid encoding in an
encapsulation. In some embodiments, the encapsulation is incubated
for a period of time to allow for the effector and the cell to
interact. In some embodiments, as described herein, the interaction
between the effector and the cell produces a signal. In some
embodiments, the period of time is sufficient to allow for changes
in transcription and/or translation to occur in the cell in
response to the effector. In some embodiments, the method comprises
transferring cellular nucleic acids to the nucleic acid encoding.
In some embodiments, the cellular nucleic acids are quantified by
sequencing the nucleic acid encodings after the cellular nucleic
acids have been transferred. In this way, an expression fingerprint
of the cell can be generated in response to treatment with the
effector. As described herein, in some embodiments, the method
further comprises detecting a signal produced through interaction
between the effector and one or more cells, and sorting the
encapsulation based on the detection of the signal.
[0230] In order to release the cellular nucleic acids, the cell may
be lysed. In some embodiments, the method further comprises the
step of lysing the cell. In some embodiments, lysing the cell
comprises adding lysis buffer to the encapsulation. In some
embodiments, the lysis buffer is added by pico-injection. In some
embodiments, the lysis buffer comprises a salt. In some
embodiments, the lysis buffer comprises a detergent. In some
embodiments, the detergent is SDS, Triton, or Tween. In some
embodiments, the lysis buffer comprises a chemical which causes
cell lysis.
[0231] Any type of cellular nucleic acid can be transferred to the
nucleic acid encoding. In some embodiments, the method comprises
transferring one or more cellular nucleic acids from the sample to
the nucleic acid encoding. In some embodiments, the nucleic acids
are mRNA. In some embodiments, the nucleic acids are mRNA that
express a protein of interest. In some embodiments, the nucleic
acids are genomic DNA. In some embodiments, the nucleic acids are
added as antibody-DNA constructs. In some embodiments, the nucleic
acids added are proximity ligation products. In some embodiments,
the nucleic acids added are proximity extension products. In some
embodiments, a plurality of different cellular nucleic acids are
attached to nucleic acid encodings.
[0232] In some embodiments, the nucleic acids transferred to the
encoding comprise a complementary sequence to a sequence on the
encoding. This may allow for the ligation of the sample nucleic
acid with the encoding nucleic acid via various methods. These
methods include, without limitation, annealing, ligating,
chemically cross-linking, or amplifying the cellular contents on to
the nucleic acid encoding the effector. In some embodiments, the
nucleic acid encodings comprise a sequence complementary to the
nucleic acid of interest to be transferred to the encoding. This
complementary sequence allows for the nucleic acids to hybridize
with the encoding, which in turn allows for extension of the
encoding with the cellular nucleic acid and vice versa.
[0233] In some embodiments, additional reagents are added to the
encapsulation to facilitate the transfer of the nucleic acids to
the encoding. In some embodiments, the additional reagents comprise
an enzyme that facilitates the transfer of the nucleic acids. In
some embodiments, the reagents for transferring the nucleic acids
to the encoding are added during encapsulation step. In some
embodiments, the reagents for transferring the nucleic acids to the
encoding are added during an incubation step. In some embodiments,
the reagents for transferring the nucleic acids to the encoding are
added after an incubation step.
[0234] In some embodiments, the additional reagents to facilitate
the transfer of the nucleic acids comprise an enzyme. In some
embodiments, the enzyme is a polymerase, a ligase, a restriction
enzyme, or a recombinase. In some embodiments, the enzyme is a
polymerase. In some embodiments, the additional reagents comprise a
chemical cross-linking reagent. In some embodiments, the chemical
cross-linking reagent is psoralen.
Adding Reagents to an Encapsulation
[0235] Methods and systems described herein may include adding one
or more reagents to an encapsulation. In some embodiments,
additional reagents can be added during a screen to encapsulations
by pico-injection. In some embodiments, additional reagents are
added by pico-injection. In some embodiments, each encapsulation
passing by a pico-injection site receive a pico-injection. In some
embodiments, at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% of encapsulations
passing a pico-injection site receive pico-injections. In some
embodiments, at least 80% of encapsulations passing a
pico-injection site receive pico-injections. In some embodiments,
at least 85% of encapsulations passing a pico-injection site
receive pico-injections. In some embodiments, at least 90% of
encapsulations passing a pico-injection site receive
pico-injections. In some embodiments, at least 95% of
encapsulations passing a pico-injection site receive
pico-injections. In some embodiments, at least 97% of
encapsulations passing a pico-injection site receive
pico-injections. In some embodiments, at least 98% of
encapsulations passing a pico-injection site receive
pico-injections. In some embodiments, at least 99% of
encapsulations passing a pico-injection site receive
pico-injections.
[0236] In some embodiments, pico-injections are performed at the
same frequency at which encapsulations pass by a pico-injection
site. In some embodiments, pico-injections are performed at
substantially the same frequency at which encapsulations pass by a
pico-injection site. In some embodiments, the frequency at which
encapsulations pass by a pico-injection site is determined by
monitoring the encapsulations. In some embodiments, the frequency
at which encapsulations pass by a pico-injection site is determined
by monitoring the encapsulations in flow. In some embodiments, the
encapsulations are monitored by taking images in real time. In some
embodiments, the encapsulations are monitored with a detector.
[0237] In some embodiments, the pico-injections are conditional.
Conditional pico-injections may only occur after a certain
condition is met. In some embodiments, a conditional pico-injection
only occurs when a signal is detected. In some embodiments, a
reagent is injected by pico-injection if a signal is detected. In
some embodiments, a reagent is added to an encapsulation by
pico-injections if a signal is detected. In some embodiments, the
signal must be above a pre-determined threshold.
[0238] In some embodiments, a method for screening an encoded
effector comprises providing an encapsulation comprising a sample
and one or more scaffolds, wherein the scaffold comprises: an
encoded effector bound to the scaffold by a cleavable linker and a
nucleic acid encoding the effector; adding one or more reagents to
the encapsulation through pico-injection or by droplet merging;
cleaving the cleavable linker to release a pre-determined amount of
the effector; detecting one or more signals from the encapsulation,
wherein the signal results from an interaction between the encoded
effector and the sample; and sorting the encapsulation based on the
detection of the signal.
[0239] In some embodiments, one or more reagents added to an
encapsulation comprises one or more fluorophores, one or more
antibodies, one or more chemical compounds, or any combination
thereof.
Post-Sorting of Encapsulations
[0240] After a sorting step or barcoding step based on the
detection of the signal of interest, the results are deconvoluted
in order to determine which effectors displayed the activity of
interest against the target sample. In some embodiments, the
methods described herein comprise the step of ascertaining which
encodings are present in the samples sorted based on the detection
of the signal. In some embodiments wherein the encoding is a
nucleic acid, the methods described herein further comprise the
step of sequencing the encodings. In some embodiments, the
encodings are sequenced by next generation sequencing. In some
embodiments, the sequences are compared to a reference to ascertain
which effectors displayed the activity of interest in the
screen.
[0241] In some embodiments, sequencing the nucleic acid encoding
comprises sequencing the encoding while the encoding is still
attached to the scaffold. In some embodiments, sequencing the
nucleic acid encoding comprises cleaving the nucleic acid encoding
from the scaffold. In some embodiments, sequencing the nucleic acid
encoding comprises cleaving the nucleic acid encoding from the
scaffold prior to sequencing. In some embodiments, cleaving the
nucleic acid encoding from the scaffold comprises cleaving a
cleavable linker with a cleaving reagent. In some embodiments,
cleaving the nucleic acid encoding from the scaffold comprises
cleaving a cleavable linker with electromagnetic radiation. In some
embodiments, any of the cleavable linkers and cleaving reagents
described herein work for this purpose. In some embodiments, a
nicking enzyme or a restriction enzyme can be used to cleave. In
some embodiments enzymatic, chemical reagent, photocleavage can be
used to cleave the encodings.
[0242] In some embodiments, the nucleic acid encoding comprises a
sequencing primer. The sequencing primer allows for facile
amplification of the nucleic acid encoding. In some embodiments
comprising a library of encoded effectors, the sequencing primer is
the same for each encoding. In some embodiments comprising a
library of encoded effectors, the sequencing primer differs among
the encodings. In some embodiments, the sequencing primer is
upstream of the encoding. In some embodiments, the sequencing
primer is downstream of the encoding.
[0243] In some embodiments, the methods provided herein are
performed using microfluidic devices. Microfluidic devices may
perform the encapsulation steps. Additionally, microfluidic devices
may be equipped with pico-injectors and other components which
allow for the methods provided herein to be performed. In some
embodiments, pico-injectors are in place along microfluidic
channels defining a flow path through the microfluidic device. In
some embodiments, the pico-injectors are positioned such that
reagents are added at desired times while performing the methods
provided herein.
[0244] In some embodiments, the methods and systems provided herein
utilize libraries of encoded effectors. Libraries of encoded
effectors comprise a plurality of different effectors, each
uniquely encoded by a known encoding modality, such as those
described above. Libraries may contain any number of encoded
effectors. In some embodiments, the libraries comprise at least
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14,
10.sup.15, or 10.sup.16 unique effectors. In some embodiments, the
libraries comprise at least about 10.sup.3, 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.1.degree.,
10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, 10.sup.15, or 10.sup.16
unique effectors.
[0245] In some embodiments, libraries of encoded effectors are
linked to scaffolds. These scaffolds may be referred to as
"scaffold encoded libraries." Scaffold encoded libraries comprise a
plurality of encoded effector molecules linked to the scaffold. The
scaffold acts as a solid support and keeps the encoded effector
molecules linked in space to their encodings. In some embodiments,
the libraries comprise at least 10.sup.3, 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11,
10.sup.12, 10.sup.13, 10.sup.14, 10.sup.15, or 10.sup.16 scaffolds.
In some embodiments, the libraries comprise at least about
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14,
10.sup.15, or 10.sup.16 scaffolds.
[0246] Any of the methods or systems described herein for a single
encoded effector may be utilized by a library of encoded effectors.
In some embodiments, provided herein, is a method of screening a
library of encoded effectors, the method comprising using any of
the methods previously described herein with a library of encoded
effectors.
[0247] In some embodiments, libraries of encoded effectors comprise
a plurality of different encoded effectors. In some embodiments,
libraries comprise multiple copies of substantially identical
effectors or scaffold encoded effectors.
Microfluidic Devices
[0248] The methods and systems provided herein may be performed on
a microfluidic device. Device architecture and methods may be
accomplished in a variety of ways. An analyzer or sorter device
according to the disclosure comprises at least one analysis unit
having an inlet region in communication with a main channel at a
droplet extrusion region (e.g., for introducing droplets of a
sample into the main channel), a detection region within or
coincident with all or a portion of the main channel or droplet
extrusion region, and a detector associated with the detection
region. In certain embodiments the device may have two or more
droplet extrusion regions. For example, embodiments are provided in
which the analysis unit has a first inlet region in communication
with the main channel at a first droplet extrusion region, a second
inlet region in communication with the main channel at a second
droplet extrusion region (for example, downstream from the first
droplet extrusion region), and so forth.
[0249] In some embodiments, a microfluidic device described herein
is configured for a droplet generation frequency of about 5 Hz to
about 200 Hz. In some embodiments, a microfluidic device described
herein is configured for a throughput of about 5 Hz to about 15 Hz,
about 5 Hz to about 25 Hz, about 5 Hz to about 50 Hz, about 5 Hz to
about 80 Hz, about 5 Hz to about 100 Hz, about 5 Hz to about 150
Hz, about 5 Hz to about 200 Hz, about 15 Hz to about 25 Hz, about
15 Hz to about 50 Hz, about 15 Hz to about 80 Hz, about 15 Hz to
about 100 Hz, about 15 Hz to about 150 Hz, about 15 Hz to about 200
Hz, about 25 Hz to about 50 Hz, about 25 Hz to about 80 Hz, about
25 Hz to about 100 Hz, about 25 Hz to about 150 Hz, about 25 Hz to
about 200 Hz, about 50 Hz to about 80 Hz, about 50 Hz to about 100
Hz, about 50 Hz to about 150 Hz, about 50 Hz to about 200 Hz, about
80 Hz to about 100 Hz, about 80 Hz to about 150 Hz, about 80 Hz to
about 200 Hz, about 100 Hz to about 150 Hz, about 100 Hz to about
200 Hz, or about 150 Hz to about 200 Hz, including increments
therein. In some embodiments, a microfluidic device described
herein is configured for a droplet generation frequency of about 5
Hz, about 15 Hz, about 25 Hz, about 50 Hz, about 80 Hz, about 100
Hz, about 150 Hz, or about 200 Hz. In some embodiments, a
microfluidic device described herein is configured for a droplet
generation frequency of at least about 5 Hz, about 15 Hz, about 25
Hz, about 50 Hz, about 80 Hz, about 100 Hz, or about 150 Hz. In
some embodiments, a microfluidic device described herein is
configured for a droplet generation frequency of at most about 15
Hz, about 25 Hz, about 50 Hz, about 80 Hz, about 100 Hz, about 150
Hz, or about 200 Hz.
[0250] Sorter embodiments of the device also have a discrimination
region or branch point in communication with the main channel and
with branch channels, and a flow control responsive to the
detector. There may be a plurality of detection regions and
detectors, working independently or together, e.g., to analyze one
or more properties of a sample or encapsulation. The branch
channels may each lead to an outlet region and to a well or
reservoir. There may also be a plurality of inlet regions, each of
which introduces droplets of a different sample (e.g., of cells, of
virions or of molecules such as molecules of an enzyme or a
substrate) into the main channel. Each of the one or more inlet
regions may also communicate with a well or reservoir.
[0251] As each droplet passes into the detection region, it is
examined for a predetermined characteristic or activity (i.e.,
using the detector) and a corresponding signal is produced, for
example indicating that "yes" the characteristic or activity is
present, or "no" it is not. The signal may correspond to a
characteristic qualitatively or quantitatively. That is, the amount
of the signal can be measured and can correspond to the degree to
which a characteristic or activity is present. For example, the
strength of the signal may indicate the size of a molecule, or the
potency or amount of an enzyme expressed by a cell, or a positive
or negative reaction such as binding or hybridization of one
molecule to another, a chemical reaction of a substrate catalyzed
by an enzyme, or the activation or inhibition of an enzyme, or any
other type of response. In response to the signal, data can be
collected and/or a flow control can be activated to divert a
droplet into one branch channel or another. Thus, samples within a
droplet at a discrimination region can be sorted into an
appropriate branch channel according to a signal produced by the
corresponding examination at a detection region. In some
embodiments, optical detection of molecular, cellular, viral, or
other sample characteristics is used, for example directly or by
use of a reporter associated with a characteristic chosen for
sorting. However, other detection techniques may also be
employed.
[0252] A variety of channels for sample flow and mixing can be
microfabricated on a single chip and can be positioned at any
location on the chip as the detection and discrimination or sorting
points, e.g., for kinetic studies. A plurality of analysis units of
the disclosure may be combined in one device. Microfabrication
applied according to the disclosure eliminates the dead time
occurring in conventional gel electrophoresis or flow cytometric
kinetic studies, and achieves a better time-resolution.
Furthermore, linear arrays of channels on a single chip, i.e., a
multiplex system, can simultaneously detect and sort a sample by
using an array of photo multiplier tubes (PMT) for parallel
analysis of different channels. This arrangement can be used to
improve throughput or for successive sample enrichment, and can be
adapted to provide a very high throughput to the microfluidic
devices that exceeds the capacity permitted by conventional flow
sorters. Circulation systems can be used in cooperation with these
and other features of the disclosure. Microfluidic pumps and valves
are one way of controlling fluid and sample flow. See, for example,
U.S. patent application Ser. No. 60/186,856.
[0253] Microfabrication permits other technologies to be integrated
or combined with flow cytometry on a single chip, such as PCR,
moving cells using optical tweezer/cell trapping, transformation of
cells by electroporation, .mu.TAS, and DNA hybridization. Detectors
and/or light filters that are used to detect viral (or cell)
characteristics of the reporters can also be fabricated directly on
the chip.
[0254] A device of the disclosure can be microfabricated with a
sample solution reservoir or well at the inlet region, which is
typically in fluid communication with an inlet channel. A reservoir
may facilitate introduction of molecules or cells into the device
and into the sample inlet channel of each analysis unit. An inlet
region may have an opening such as in the floor of the
microfabricated chip, to permit entry of the sample into the
device. The inlet region may also contain a connector adapted to
receive a suitable piece of tubing, such as liquid chromatography
or HPLC tubing, through which a sample may be supplied. Such an
arrangement facilitates introducing the sample solution under
positive pressure in order to achieve a desired pressure at the
droplet extrusion region.
[0255] A device of the disclosure may have an additional inlet
region, in direct communication with the main channel at a location
upstream of the droplet extrusion region, through which a
pressurized stream or "flow" of a fluid is introduced into the main
channel. In some embodiments, this fluid is one which is not
miscible with the solvent or fluid of the sample. For example, in
some embodiments, the fluid is a non-polar solvent, such as decane
(e.g., tetradecane or hexadecane), and the sample (e.g., of cells,
virions or molecules) is dissolved or suspended in an aqueous
solution so that aqueous droplets of the sample are introduced into
the pressurized stream of non-polar solvent at the droplet
extrusion region.
[0256] Substrate and flow channels may be accomplished in a variety
of ways. A typical analysis unit of the disclosure comprises a main
inlet that is part of and feeds or communicates directly with a
main channel, along with one or more sample inlets in communication
with the main channel at a droplet extrusion region situated
downstream from the main inlet (each different sample inlet may
communicate with the main channel at a different droplet extrusion
region). The droplet extrusion region generally comprises a
junction between the sample inlet and the main channel such that a
pressurized solution of a sample (i.e., a fluid containing a sample
such as cells, virions or molecules) is introduced to the main
channel in droplets. In some embodiment, the sample inlet
intersects the main channel such that the pressurized sample
solution is introduced into the main channel at an angle
perpendicular to a stream of fluid passing through the main
channel. For example, in some embodiments, the sample inlet and
main channel intercept at a T-shaped junction; i.e., such that the
sample inlet is perpendicular (90 degrees) to the main channel.
However, the sample inlet may intercept the main channel at any
angle, and need not introduce the sample fluid to the main channel
at an angle that is perpendicular to that flow. In some embodiments
the angle between intersecting channels is in the range of from
about 60 to about 120 degrees. Particular exemplary angles are 45,
60, 90, and 120 degrees. In some embodiments, the angle between the
intersecting channels is in the range of about 5 to about 60
degrees. In some embodiments, the angle between the intersecting
channels is in the range of about 5 to about 60 degrees. In some
embodiments, the angle between the intersecting channels is in the
range of about 5 to about 10, about 5 to about 15, about 5 to about
20, about 5 to about 25, about 5 to about 30, about 5 to about 40,
about 5 to about 50, about 5 to about 60, about 10 to about 15,
about 10 to about 20, about 10 to about 25, about 10 to about 30,
about 10 to about 40, about 10 to about 50, about 10 to about 60,
about 15 to about 20, about 15 to about 25, about 15 to about 30,
about 15 to about 40, about 15 to about 50, about 15 to about 60,
about 20 to about 25, about 20 to about 30, about 20 to about 40,
about 20 to about 50, about 20 to about 60, about 25 to about 30,
about 25 to about 40, about 25 to about 50, about 25 to about 60,
about 30 to about 40, about 30 to about 50, about 30 to about 60,
about 40 to about 50, about 40 to about 60, or about 50 to about 60
degrees. In some embodiments, the angle between the intersecting
channels is in the range of about 5, about 10, about 15, about 20,
about 25, about 30, about 40, about 50, or about 60 degrees. In
some embodiments, the angle between the intersecting channels is in
the range of at least about 5, about 10, about 15, about 20, about
25, about 30, about 40, or about 50. In some embodiments, the angle
between the intersecting channels is in the range of at most about
10, about 15, about 20, about 25, about 30, about 40, about 50, or
about 60 degrees. In some embodiments, the angle between the
intersecting channels is in the range of about 120 to about 175
degrees. In some embodiments, the angle between the intersecting
channels is in the range of about 120 to about 130, about 120 to
about 140, about 120 to about 150, about 120 to about 160, about
120 to about 170, about 120 to about 175, about 130 to about 140,
about 130 to about 150, about 130 to about 160, about 130 to about
170, about 130 to about 175, about 140 to about 150, about 140 to
about 160, about 140 to about 170, about 140 to about 175, about
150 to about 160, about 150 to about 170, about 150 to about 175,
about 160 to about 170, about 160 to about 175, or about 170 to
about 175 degrees. In some embodiments, the angle between the
intersecting channels is in the range of about 120, about 130,
about 140, about 150, about 160, about 170, or about 175 degrees.
In some embodiments, the angle between the intersecting channels is
in the range of at least about 120, about 130, about 140, about
150, about 160, or about 170 degrees. In some embodiments, the
angle between the intersecting channels is in the range of at most
about 130, about 140, about 150, about 160, about 170, or about 175
degrees.
[0257] The droplet extrusion or droplet formation region may also
comprise two microfluidic channels carrying immiscible carrier
fluid that are introduced on opposite sides of a main microfluidic
channel. In some embodiments, the two microfluidic channels are
substantially collinear. In some embodiments, such a junction
resembles and X-shape. In some embodiments, the main microfluidic
channel contains the sample or assay fluid.
[0258] The main channel in turn communicates with two or more
branch channels at another junction or "branch point", forming, for
example, a T-shape or a Y-shape. Other shapes and channel
geometries may be used as desired. In sorting embodiments, the
region at or surrounding the junction can also be referred to as a
discrimination region or a sorting region. Precise boundaries for
the discrimination region are not required, but are preferred.
[0259] A detection region may be within, communicating or
coincident with a portion of the main channel at or downstream of
the droplet extrusion region and, in sorting embodiments, at or
upstream of the discrimination region or branch point. Precise
boundaries for the detection region are not required, but are
preferred. The discrimination region may be located immediately
downstream of the detection region or it may be separated by a
suitable distance consistent with the size of the molecules, the
channel dimensions and the detection system. It will be appreciated
that the channels may have any suitable shape or cross-section (for
example, tubular or grooved), and can be arranged in any suitable
manner so long as flow can be directed from inlet to outlet and
from one channel into another.
[0260] The channels of the disclosure may be microfabricated, for
example by etching a silicon chip using conventional
photolithography techniques, or using a micromachining technology
called "soft lithography". These and other microfabrication methods
may be used to provide inexpensive miniaturized devices, and in the
case of soft lithography, can provide robust devices having
beneficial properties such as improved flexibility, stability, and
mechanical strength. When optical detection is employed, the
devices provided herein may also provide minimal light scatter from
molecule or cell (including virion) suspension and chamber
material. In some embodiments, devices provided herein are
relatively inexpensive and easy to set up. They can also be
disposable, which greatly relieves many of the concerns of gel
electrophoresis (for molecules), and of sterilization and permanent
adsorption of particles into the flow chambers and channels of
conventional FACS machines (for cells, virions and other particle
suspensions).
[0261] A microfabricated device of the disclosure may be fabricated
from a silicon microchip or silicon elastomer. In some embodiments,
the dimensions of the chip are those of typical microchips, ranging
between about 0.5 cm to about 5 cm per side and about 1 micron to
about 1 cm in thickness. The device may contain at least one
analysis unit having a main channel with a droplet extrusion region
and a coincident detection region. The device may also contain at
least one inlet region (which may contain an inlet channel) and one
or more outlet regions (which may have fluid communication with a
branch channel in each region). In a sorting embodiment, at least
one detection region cooperates with at least one discrimination
region to divert flow via a detector-originated signal. It shall be
appreciated that the "regions" and "channels" are in fluid
communication with each other and therefore may overlap; i.e.,
there may be no clear boundary where a region or channel begins or
ends. A microfabricated device can be transparent and can be
covered with a material having transparent properties, such as a
glass coverslip, to permit detection of a reporter, for example, by
an optical device such as an optical microscope.
[0262] The dimensions of the detection region are influenced by the
nature of the sample under study and, in particular, by the size of
the molecules or cells (including virions) under study. For
example, viruses can have a diameter from about 20 nm to about 500
nm, although some extremely large viruses may reach lengths of
about 2000 nm (i.e., as large or larger than some bacterial cells).
By contrast, biological cells are typically many times larger. For
example, mammalian cells can have a diameter of about 1 to 50
microns, more typically 10 to 30 microns, although some mammalian
cells (e.g., fat cells) can be larger than 120 microns. Plant cells
are generally 10 to 100 microns.
[0263] Detection regions used for detecting molecules and cells
(including virions) have a cross-sectional area large enough to
allow a desired molecule to pass through without being
substantially slowed down relative to the flow carrying it. To
avoid "bottlenecks" and/or turbulence, and promote single-file
flow, the channel dimensions, particularly in the detection region,
should generally be at least about twice, or at least about five
times as large per side or in diameter as the diameter of the
largest molecule, cell or droplet that will be passing through
it.
[0264] For particles (e.g., cells, including virions) or molecules
that are in encapsulations (i.e., deposited by the droplet
extrusion region) within the flow of the main channel, the channels
of the device may be rounded, with a diameter between about 2 and
100 microns. In some embodiments, the round channels of the device
are about 60 microns in diameter or about 30 microns at the
crossflow area or droplet extrusion region. This geometry
facilitates an orderly flow of droplets in the channels. Similarly,
the volume of the detection region in an analysis device may be in
the range of between about 10 femtoliters (fl) and 5000 fl, about
40 or 50 fl to about 1000 or 2000 fl, or on the order of about 200
fl. In some embodiments, the channels of the device, and
particularly the channels of the inlet connecting to a droplet
extrusion region, are between about 2 and 50 microns, or about 30
microns.
[0265] In one embodiment, droplets at these dimensions tend to
conform to the size and shape of the channels, while maintaining
their respective volumes. Thus, as droplets move from a wider
channel to a narrower channel they become longer and thinner, and
vice versa. In some embodiments, droplets are at least about four
times as long as they are wide. This droplet configuration, which
can be envisioned as a lozenge shape, flows smoothly and well
through the channels. Longer droplets, produced in narrower
channels, provides a higher shear, meaning that droplets can more
easily be sheared or broken off from a flow, i.e. using less force.
Droplets may also tend to adhere to channel surfaces, which can
slow or block the flow, or produce turbulence. Droplet adherence is
overcome when the droplet is massive enough in relation to the
channel size to break free. Thus, droplets of varying size, if
present, may combine to form uniform droplets having a so-called
critical mass or volume that results in smooth or laminar droplet
flow. Droplets that are longer than they are wide, for example
about four times longer than they are wide, generally have the
ability to overcome channel adherence and move freely through the
microfluidic device. Thus, in an exemplary embodiment with 60
micron channels, a typical free-flowing droplet is about 60 microns
wide and 240 microns long. Droplet dimensions and flow
characteristics can be influenced as desired, in part by changing
the channel dimensions, e.g. the channel width.
[0266] In some embodiments, the devices provided herein generate
round, monodisperse droplets. In some embodiments, the droplets
have a diameter that is smaller than the diameter of the
microchannel; i.e., less than 60 .mu.m. Monodisperse droplets may
be particularly preferable, e.g., in high throughput devices and
other embodiments where it is desirable to generate droplets at
high frequency.
[0267] To prevent sample (e.g., cells, virions and other particles
or molecules) or other material from adhering to the sides of the
channels, the channels (and coverslip, if used) may have a coating
which minimizes adhesion. Such a coating may be intrinsic to the
material from which the device is manufactured, or it may be
applied after the structural aspects of the channels have been
microfabricated. "TEFLON" is an example of a coating that has
suitable surface properties. Alternatively, the channels may be
coated with a surfactant.
[0268] Non-limiting examples of surfactants that may be used
include, but are not limited to, surfactants such as sorbitan-based
carboxylic acid esters (e.g., the "Span" surfactants, Fluka
Chemika), including sorbitan monolaurate (Span20), sorbitan
monopalmitate (Spa n 40), sorbitan monostearate (Span60) and
sorbitan monooleate (Span80). Other non-limiting examples of
non-ionic surfactants which may be used include polyoxyethylenated
alkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols),
polyoxyethylenated straight chain alcohols, polyoxyethylenated
polyoxypropylene glycols, polyoxyethylenated mercaptans, long chain
carboxylic acid esters (for example, glyceryl and polyglycerl
esters of natural fatty acids, propylene glycol, sorbitol,
polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters,
etc.) and alkanolamines (e.g., diethanolamine-fatty acid
condensates and isopropanolamine-fatty acid condensates). In
addition, ionic surfactants such as sodium dodecyl sulfate (SDS)
may also be used.
[0269] A silicon substrate containing the microfabricated flow
channels and other components may be covered and sealed, including
with a transparent cover, e.g., thin glass or quartz, although
other clear or opaque cover materials may be used. When external
radiation sources or detectors are employed, the detection region
may be covered with a clear cover material to allow optical access
to the cells. For example, anodic bonding to a "PYREX" cover slip
can be accomplished by washing both components in an aqueous
H.sub.2SO.sub.4/H.sub.2O.sub.2 bath, rinsing in water, and then,
for example, heating to about 350.degree. C. while applying a
voltage of 450V.
[0270] Switching and flow control can be accomplished in a variety
of ways. Some embodiments of the disclosure use pressure drive flow
control, e.g., utilizing valves and pumps, to manipulate the flow
of cells virions, particles, molecules, enzymes or reagents in one
or more directions and/or into one or more channels of a
microfluidic device. However, other methods may also be used, alone
or in combination with pumps and valves, such as electro-osmotic
flow control, electrophoresis and dielectrophoresis. In certain
embodiments of the disclosure, the flow moves in one "forward"
direction, e.g. from the main inlet region through the main and
branch channels to an outlet region. In other embodiments the
direction of flow is reversible. Application of these techniques
according to the disclosure provides more rapid and accurate
devices and methods for analysis or sorting, for example, because
the sorting occurs at or in a discrimination region that can be
placed at or immediately after a detection region. This provides a
shorter distance for molecules or cells to travel, they can move
more rapidly and with less turbulence, and can more readily be
moved, examined, and sorted in single file, i.e., one at a time. In
a reversible embodiment, potential sorting errors can be avoided,
for example by reversing and slowing the flow to re-read or resort
a molecule, cell or virion (or pluralities thereof) before
irretrievably committing the cell or cells to a particular branch
channel.
[0271] Without being bound by any theory, electro-osmosis is
believed to produce motion in a stream containing ions, e.g. a
liquid such as a buffer, by application of a voltage differential
or charge gradient between two or more electrodes. Neutral
(uncharged) molecules or cells (including virions) can be carried
by the stream. Electro-osmosis is particularly suitable for rapidly
changing the course, direction or speed of flow. Electrophoresis is
believed to produce movement of charged objects in a fluid toward
one or more electrodes of opposite charge, and away from one on or
more electrodes of like charge. In embodiments of the disclosure
where an aqueous phase is combined with an oil phase, aqueous
droplet encapsulations are encapsulated or separated from each
other by oil. In some embodiments, the oil phase is not an
electrical conductor and may insulate the encapsulations from the
electro-osmotic field. In these embodiment, electro-osmosis may be
used to drive the flow of encapsulations if the oil is modified to
carry or react to an electrical field, or if the oil is substituted
for another phase that is immiscible in water but which does not
insulate the water phase from electrical fields.
[0272] Dielectrophoresis produces dielectric objects, which have no
net charge, but have regions that are positively or negatively
charged in relation to each other. Alternating, non-homogeneous
electric fields in the presence of encapsulations, including
droplets, and/or particles, such as cells or virions, cause the
encapsulations and/or particles to become electrically polarized
and thus to experience dielectrophoretic forces. Depending on the
dielectric polarizability of the particles and the suspending
medium, dielectric particles will move either toward the regions of
high field strength or low field strength. For example, the
polarizability of living cells and virions depends on their
composition, morphology, and phenotype and is highly dependent on
the frequency of the applied electrical field. Thus, cells and
virions of different types and in different physiological states
generally possess distinctly different dielectric properties, which
may provide a basis for cell separation, e.g., by differential
dielectrophoretic forces. Likewise, the polarizability of
encapsulations, including droplets, also depends upon their size,
shape and composition. For example, droplets that contain salts can
be polarized. Individual manipulation of single encapsulations
requires field differences (inhomogeneities) with dimensions close
to the encapsulations.
[0273] Manipulation is also dependent on permittivity (a dielectric
property) of the encapsulations and/or particles with the
suspending medium. Thus, polymer particles, living cells and
virions show negative dielectrophoresis at high-field frequencies
in water. For example, dielectrophoretic forces experienced by a
latex sphere in a 0.5 MV/m field (10V for a 20 micron electrode
gap) in water are predicted to be about 0.2 piconewtons (pN) for a
3.4 micron latex sphere to 15 pN for a 15 micron latex sphere.
These values are mostly greater than the hydrodynamic forces
experienced by the sphere in a stream (about 0.3 pN for a 3.4
micron sphere and 1.5 pN for a 15 micron sphere). Therefore,
manipulation of individual cells or particles can be accomplished
in a streaming fluid, such as in a cell sorter device, using
dielectrophoresis. Using conventional semiconductor technologies,
electrodes can be microfabricated onto a substrate to control the
force fields in a microfabricated sorting device of the disclosure.
Dielectrophoresis is particularly suitable for moving objects that
are electrical conductors. AC current may be used to prevent
permanent alignment of ions. Megahertz frequencies are suitable to
provide a net alignment, attractive force, and motion over
relatively long distances.
[0274] Radiation pressure can also be used in the disclosure to
deflect and move objects, e.g. encapsulations, droplets, and
particles (molecules, cells, virions, etc.) contained therein, with
focused beams of light such as lasers. Flow can also be obtained
and controlled by providing a pressure differential or gradient
between one or more channels of a device or in a method of the
disclosure.
[0275] In some embodiments, molecules, cells or virions (or
droplets containing molecules, cells or virions) can be moved by
direct mechanical switching, e.g., with on-off valves or by
squeezing the channels. Pressure control may also be used, for
example, by raising or lowering an output well to change the
pressure inside the channels on the chip. Different switching and
flow control mechanisms can be combined on one chip or in one
device and can work independently or together as desired.
[0276] Detection and discrimination for sorting can be accomplished
in a variety of ways. The detector can be any device or method for
interrogating a molecule, a cell or a virion as it passes through
the detection region. Typically, molecules, cells or virions (or
droplets containing such particles) are to be analyzed or sorted
according to a predetermined characteristic that is directly or
indirectly detectable, and the detector is selected or adapted to
detect that characteristic. One detector is an optical detector,
such as a microscope, which may be coupled with a computer and/or
other image processing or enhancement devices to process images or
information produced by the microscope using known techniques. For
example, molecules can be analyzed and/or sorted by size or
molecular weight. Enzymes can be analyzed and/or sorted by the
extent to which they catalyze chemical reaction of a substrate
(conversely, substrate can be analyzed and/or sorted by the level
of chemical reactivity catalyzed by an enzyme). Cells and virions
can be sorted according to whether they contain or produce a
particular protein, by using an optical detector to examine each
cell or virion for an optical indication of the presence or amount
of that protein. The protein may itself be detectable, for example
by a characteristic fluorescence, or it may be labeled or
associated with a reporter that produces a detectable signal when
the desired protein is present, or is present in at least a
threshold amount. There is no limit to the kind or number of
characteristics that can be identified or measured using the
techniques of the disclosure, which include without limitation
surface characteristics of the cell or virion and intracellular
characteristics, provided only that the characteristic or
characteristics of interest for sorting can be sufficiently
identified and detected or measured to distinguish cells having the
desired characteristic(s) from those which do not. For example, any
label or reporter as described herein can be used as the basis for
analyzing and/or sorting molecules or cells (including virions),
i.e. detecting molecules or cells to be collected.
[0277] In some embodiments, the samples (or encapsulations
containing them) are analyzed and/or separated based on the
intensity of a signal from an optically-detectable reporter bound
to or associated with them as they pass through a detection window
or "detection region" in the device. In some embodiments, the
samples are analyzed and/or separated based on the intensity of a
signal from a detectable reporter. Molecules or cells or virions
having an amount or level of the reporter at a selected threshold
or within a selected range are diverted into a predetermined outlet
or branch channel of the device. The reporter signal may be
collected by a microscope and measured by a photo multiplier tube
(PMT). A computer digitizes the PMT signal and controls the flow
via valve action or electro-osmotic potentials. Alternatively, the
signal can be recorded or quantified as a measure of the reporter
and/or its corresponding characteristic or marker, e.g., for the
purpose of evaluation and without necessarily proceeding to sort
the molecules or cells.
[0278] In one embodiment, the chip is mounted on an inverted
optical microscope. Fluorescence produced by a reporter is excited
using a laser beam focused on molecules (e.g., DNA, protein, enzyme
or substrate) or cells passing through a detection region.
Fluorescent reporters include, e.g., rhodamine, fluorescein, Texas
red, Cy 3, Cy 5, phycobiliprotein, green fluorescent protein (GFP),
YOYO-1 and PicoGreen, to name a few. In molecular fingerprinting
applications, the reporter labels are optionally fluorescently
labeled single nucleotides, such as fluorescein-dNTP,
rhodamine-dNTP, Cy3-dNTP, etc.; where dNTP represents dATP, dTTP,
dUTP or dCTP. The reporter can also be chemically-modified single
nucleotides, such as biotin-dNTP. In other embodiments, the
reporter can be fluorescently or chemically labeled amino acids or
antibodies (which bind to a particular antigen, or fragment
thereof, when expressed or displayed by a cell or virus).
[0279] Thus, in one aspect of the disclosure, the device can
analyze and/or sort cells or virions based on the level of
expression of selected cell markers, such as cell surface markers,
which have a detectable reporter bound thereto, in a manner similar
to that currently employed using fluorescence-activated cell
sorting (FACS) machines. Proteins or other characteristics within a
cell, and which do not necessarily appear on the cell surface, can
also be identified and used as a basis for sorting. In another
aspect of the disclosure, the device can determine the size or
molecular weight of molecules such as polynucleotides or
polypeptides (including enzymes and other proteins) or fragments
thereof passing through the detection region. Alternatively, the
device can determine the presence or degree of some other
characteristic indicated by a reporter. If desired, the cells,
virions or molecules can be sorted based on this analysis. The
sorted cells, virions or molecules can be collected from the outlet
channels and used as needed.
[0280] To detect a reporter or determine whether a molecule, cell
or virion has a desired characteristic, the detection region may
include an apparatus for stimulating a reporter for that
characteristic to emit measurable light energy, e.g., a light
source such as a laser, laser diode, high-intensity lamp, (e.g.,
mercury lamp), and the like. In embodiments where a lamp is used,
the channels may be shielded from light in all regions except the
detection region. In embodiments where a laser is used, the laser
can be set to scan across a set of detection regions from different
analysis units. In addition, laser diodes may be microfabricated
into the same chip that contains the analysis units. Alternatively,
laser diodes may be incorporated into a second chip (i.e., a laser
diode chip) that is placed adjacent to the microfabricated analysis
or sorter chip such that the laser light from the diodes shines on
the detection region(s).
[0281] In some embodiments, an integrated semiconductor laser
and/or an integrated photodiode detector are included on the
silicon wafer in the vicinity of the detection region. This design
provides the advantages of compactness and a shorter optical path
for exciting and/or emitted radiation, thus minimizing
distortion.
[0282] Sorting schemes can be accomplished in a variety of ways.
According to the disclosure, molecules (such as DNA, protein,
enzyme or substrate) or particles (i.e., cells, including virions)
are sorted dynamically in a flow stream of microscopic dimensions
based on the detection or measurement of a characteristic, marker
or reporter that is associated with the molecules or particles.
More specifically, encapsulations of a solution (for example an
aqueous solution or buffer), containing a sample of molecules,
cells or virions, are introduced through a droplet extrusion region
into a stream of fluid (for example, a non-polar fluid such as
decane or other oil) in the main channel. The individual droplet
encapsulations are then analyzed and/or sorted in the flow stream,
thereby sorting the molecules, cells or virions contained within
the droplets.
[0283] The flow stream in the main channel is typically, but not
necessarily continuous and may be stopped and started, reversed or
changed in speed. Prior to sorting, a liquid that does not contain
samples molecules, cells or virions can be introduced into a sample
inlet region (such as an inlet well or channel) and directed
through the droplet extrusion region, e.g., by capillary action, to
hydrate and prepare the device for use. Likewise, buffer or oil can
also be introduced into a main inlet region that communicates
directly with the main channel to purge the device (e.g., or "dead"
air) and prepare it for use. If desired, the pressure can be
adjusted or equalized, for example, by adding buffer or oil to an
outlet region.
[0284] The pressure at the droplet extrusion region can also be
regulated by adjusting the pressure on the main and sample inlets,
for example, with pressurized syringes feeding into those inlets.
By controlling the pressure difference between the oil and water
sources at the droplet extrusion region, the size and periodicity
of the droplets generated may be regulated. Alternatively, a valve
may be placed at or coincident to either the droplet extrusion
region or the sample inlet connected thereto to control the flow of
solution into the droplet extrusion region, thereby controlling the
size and periodicity of the droplets. Periodicity and droplet
volume may also depend on channel diameter, the viscosity of the
fluids, and shear pressure.
[0285] The droplet forming liquid is typically an aqueous buffer
solution, such as ultrapure water (e.g., 18 mega-ohm resistivity,
obtained, for example by column chromatography), 10 mM Tris HCl and
1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate
buffer. Any liquid or buffer that is physiologically compatible
with the population of molecules, cells or virions to be analyzed
and/or sorted can be used. The fluid passing through the main
channel and in which the droplets are formed is preferably one that
is not miscible with the droplet forming fluid. In some
embodiments, the fluid passing through the main channel is a
non-polar solvent, for example decane (e.g., tetradecane or
hexadecane) or another oil.
[0286] The fluids used in the disclosure may contain additives,
such as agents which reduce surface tensions (surfactants).
Exemplary surfactants include Tween, Span, fluorinated oils, and
other agents that are soluble in oil relative to water. Surfactants
may aid in controlling or optimizing droplet size, flow and
uniformity, for example by reducing the shear force needed to
extrude or inject droplets into an intersecting channel. This may
affect droplet volume and periodicity, or the rate or frequency at
which droplets break off into an intersecting channel.
[0287] Channels of the disclosure may be formed from silicon
elastomer (e.g. RTV), urethane compositions, of from
silicon-urethane composites such as those available from Polymer
Technology Group (Berkeley, Calif.), e.g. PurSil.TM. and
CarboSil.TM.. The channels may also be coated with additives or
agents, such as surfactants, TEFLON, or fluorinated oils such as
octadecafluoroctane (98%, Aldrich) or fluorononane. TEFLON is
particularly suitable for silicon elastomer (RTV) channels, which
are hydrophobic and advantageously do not absorb water, but they
may tend to swell when exposed to an oil phase. Swelling may alter
channel dimensions and shape, and may even close off channels, or
may affect the integrity of the chip, for example by stressing the
seal between the elastomer and a coverslip. Urethane substrates do
not tend to swell in oil but are hydrophilic, they may undesirably
absorb water, and tend to use higher operating pressures.
Hydrophobic coatings may be used to reduce or eliminate water
absorption. Absorption or swelling issues may also be addressed by
altering or optimizing pressure or droplet frequency (e.g.
increasing periodicity to reduce absorption). RTV-urethane hybrids
may be used to combine the hydrophobic properties of silicon with
the hydrophilic properties of urethane.
[0288] Embodiments of the disclosure are also provided in which
there are two or more droplet formation regions introducing
droplets of samples into the main channel. For example, a first
droplet extrusion region may introduce droplets of a first sample
into a flow of fluid (e.g., oil) in the main channel and a second
droplet extrusion region may introduce droplets of a second sample
into the flow of fluid in main channel, and so forth. Optionally,
the second droplet extrusion region is downstream from the first
droplet extrusion region (e.g., about 30 .mu.m). In one embodiment,
the fluids introduced into the two or more different droplet
extrusion regions comprise the same fluid or the same type of fluid
(e.g., different aqueous solutions). For example, in one embodiment
droplets of an aqueous solution containing an enzyme are introduced
into the main channel at the first droplet extrusion region and
droplets of aqueous solution containing a substrate for the enzyme
are introduced into the main channel at the second droplet
extrusion region. The introduction of droplets through the
different extrusion regions may be controlled, e.g., so that the
droplets combine (allowing, for example, the enzyme to catalyze a
chemical reaction of the substrate). Alternatively, the droplets
introduced at the different droplet extrusion regions may be
droplets of different fluids which may be compatible or
incompatible. For example, the different droplets may be different
aqueous solutions, or droplets introduced at a first droplet
extrusion region may be droplets of one fluid (e.g., an aqueous
solution) whereas droplets introduced at a second droplet extrusion
region may be another fluid (e.g., alcohol or oil).
[0289] The concentration (i.e., number) of scaffolds, molecules,
cells or virions in a droplet can influence sorting efficiently and
therefore may be optimized. In particular, the sample concentration
should be dilute enough that most of the droplets contain no more
than a singles scaffold, molecule, cell or virion, with only a
small statistical chance that a droplet will contain two or more
molecules, cells or virions. In some embodiments, the sample
concentration should be such that a single cell is encapsulated
with a single scaffold. This is to ensure that for the large
majority of measurements, the level of reporter measured in each
droplet as it passes through the detection region corresponds to a
single molecule, cell or virion and not to two or more molecules,
cells or virions. Additionally, ensuring that a single cell or
virion is encapsulated with only a single encoded effector scaffold
ensures that positive "hits" are correctly correlated with the
correct effectors.
[0290] The parameters which govern this relationship are the volume
of the droplets and the concentration of molecules, cells or
virions in the sample solution. The probability that a droplet will
contain two or more scaffolds, molecules, cells, or virions
(P.ltoreq.2) can be expressed as
P.ltoreq.2=1-{1+[virion].times.V}.times.e-[virion].times.V
[0291] where "[virion]" is the concentration of molecules, cells or
virions in units of number of molecules, cells or virions per cubic
micron (.mu.m3), and V is the volume of the droplet in units of
.mu.m3.
[0292] It will be appreciated that P.ltoreq.2 can be minimized by
decreasing the concentration of scaffolds, molecules, cells or
virions in the sample solution. However, decreasing the
concentration of molecules, cells or virions in the sample solution
also results in an increased volume of solution processed through
the device and can result in longer run times. Accordingly, it is
desirable to minimize to presence of multiple molecules, cells or
virions in the droplets (thereby increasing the accuracy of the
sorting) and to reduce the volume of sample, thereby permitting a
sorted sample in a reasonable time in a reasonable volume
containing an acceptable concentration of molecules, cells or
virions.
[0293] The maximum tolerable P.ltoreq.2 depends on the desired
"purity" of the sorted sample. The "purity" in this case refers to
the fraction of sorted molecules, cells or virions that possess a
desired characteristic (e.g., display a particular antigen, are in
a specified size range or are a particular type of molecule, cell
or virion). The purity of the sorted sample is inversely
proportional to P.ltoreq.2. For example, in applications where high
purity is not needed or desired a relatively high P.ltoreq.2 (e.g.,
P.ltoreq.2=0.2) may be acceptable. For most applications,
maintaining P.ltoreq.2 at or below about 0.1, or at or below about
0.01, provides satisfactory results.
[0294] A sample solution containing a mixture or population of
molecule, cells or virions in a suitable carrier fluid (such as a
liquid or buffer described above) is supplied to the sample inlet
region, and droplets of the sample solution are introduced, at the
droplet extrusion region, into the flow passing through the main
channel. The force and direction of flow can be controlled by any
desired method for controlling flow, for example, by a pressure
differential, by valve action or by electro-osmotic flow (e.g.,
produced by electrodes at inlet and outlet channels). This permits
the movement of the cells into one or more desired branch channels
or outlet regions.
[0295] A "forward" sorting algorithm, according to the disclosure,
includes embodiments where droplets from a droplet extrusion region
flow through the device to a predetermined branch or outlet channel
(which can be called a "waste channel"), until the level of
measurable reporter of a molecule, cell or virion within a droplet
is above a pre-set threshold. At that time, the flow is diverted to
deliver the droplet (and the scaffold, molecule, cell, and/or
virion contained therein) to another channel. For example, in an
electro-osmotic embodiment, where switching is virtually
instantaneous and throughput is limited by the highest voltage, the
voltages are temporarily changed to divert the chosen droplet to
another predetermined outlet channel (which can be called a
"collection channel"). Sorting, including synchronizing detection
of a reporter and diversion of the flow, can be controlled by
various methods including computer or microprocessor control.
Different algorithms for sorting in the microfluidic device can be
implemented by different computer programs, such as programs used
in conventional FACS devices. For example, a programmable card can
be used to control switching, such as a Lab PC 1200 Card, available
from National Instruments, Austin, Tex. Algorithms as sorting
procedures can be programmed using C++, LAB VIEW, or any suitable
software.
[0296] A "reversible" sorting algorithm can be used in place of a
"forward" mode, for example in embodiments where switching speed
may be limited. For example, a pressure-switched scheme can be used
instead of electro-osmotic flow and does not require high voltages
and may be more robust for longer runs. However, mechanical
constraints may cause the fluid switching speed to become
rate-limiting. In a pressure-switched scheme the flow is stopped
when a molecule or cell or virion of interest is detected within a
droplet. By the time the flow stops, the droplet containing the
molecule, cell or virion may be past the junction or branch point
and be part of the way down the waste channel. In this situation, a
reversible embodiment can be used. The system can be run backwards
at a slower (switchable) speed (e.g., from waste to inlet), and the
droplet is then switched to a different branch or collection
channel. At that point, a potentially mis-sorted droplet (and the
molecule, cell or virion therein) is "saved", and the device can
again be run at high speed in the forward direction. This
"reversible" sorting method is not possible with standard FACS
machines. FACS machines mostly sort aerosol droplets which cannot
be reversed back to the chamber, in order to be redirected. The
aerosol droplet sorters are virtually irreversible. Reversible
sorting is particularly useful for identifying molecules, cells or
virions that are rare (e.g., in molecular evolution and cancer
cytological identification) or few in number, which may be
misdirected due to a margin of error inherent to any fluidic
device. The reversible nature of the device of the disclosure
permits a reduction in this possible error.
[0297] In addition, a "reversible" sorting method permits multiple
time course measurements of a molecule, cell or virion contained
within a single droplet. This allows for observations or
measurements of the same molecule, cell or virion at different
times, because the flow reverses the cell back into the detection
window again before redirecting the cell into a different channel.
Thus, measurements can be compared or confirmed, and changes in
properties over time can be examined, for example in kinetic
studies.
[0298] When trying to separate scaffolds, molecules, cells or
virions in a sample at a very low ratio to the total number of
scaffolds, molecules, cells or virions, a sorting algorithm can be
implemented that is not limited by the intrinsic switching speed of
the device. Consequently, the droplets flow at the highest possible
static (non-switching) speed from the inlet channel to the waste
channel. Unwanted droplets (i.e., containing unwanted molecules,
cells or virions) can be directed into the waste channel at the
highest speed possible, and when a droplet containing a desired
molecule, cell or virion is detected, the flow can be slowed down
and then reversed, to direct the droplet back into the detection
region, from where it can be redirected (i.e. to accomplish
efficient switching). Hence the droplets (and the molecules, cells
or virions contained therein) can flow at the highest possible
static speed.
[0299] Provided herein are methods for controlling for variables
such as temperature, pH and concentration. This may be accomplished
by converging two aqueous streams to form droplets, where, for
example, the first aqueous stream would contain 2.times. the
concentration of component "A" desired in the droplet and the
second aqueous stream would contain 2.times. the concentration of
component "B" desired in the droplet, thus when the streams merge
they would form a 1.times. solution of both "A" and "B". Different
ratios of aqueous streams converging with different concentrations
of reagents may also be sued to reach desired final concentrations
of samples, scaffolds, and/or reagents. The concentrations in
droplets are controlled by knowing what the concentrations are of
components in each aqueous stream. This concept can be applied to
pH, salt, concentration, etc. For temperature control a transparent
stage may be used to heat the chip to a desired temperature.
[0300] Both the fluid comprising the droplets and the fluid
carrying the droplets (i.e., the aqueous and non-polar fluids) may
have a relatively low Reynolds Number, for example 10-2. The
Reynolds Number represents an inverse relationship between the
density and velocity of a fluid and its viscosity in a channel of
given length. More viscous, less dense, slower moving fluids over a
shorter distance will have a lower Reynolds Number, and are easier
to divert, stop, start, or reverse without turbulence. Because of
the small sizes and slow velocities, microfabricated fluid systems
are often in a low Reynolds number regime (Re<<1). In this
regime, inertial effects, which cause turbulence and secondary
flows, are negligible; viscous effects dominate the dynamics. These
conditions are advantageous for sorting, and are provided by
microfabricated devices of the disclosure. Accordingly, the
microfabricated devices of the disclosure are optionally operated
at a low or very low Reynold's number.
[0301] In one aspect provided herein is a microfluidic device
designed for droplet based encoded library screening. In some
embodiments, the device comprises a first microfluidic channel
comprising an aqueous fluid. In some embodiments, the device
comprises a second microfluidic channel comprising a fluid
immiscible with the aqueous stream. In some embodiments, the device
comprises a junction at which the first microfluidic channel is in
fluid communication with the second microfluidic channel. In some
embodiments, the junction of the first and second microfluidic
channels defines a device plane. In some embodiments, the junction
is configured to form droplets of the aqueous fluid within the
fluid from the second microfluidic channel. In some embodiments,
the second microfluidic channel is configured to continue past the
junction thereby defining an assay flow path. In some embodiments,
the fluid from the second microfluidic channel with the droplets
therein moves past the junction in a third microfluidic channel
that defines an assay flow path. The assay flow path may also be
called an incubation region. In some embodiments, the device
comprises a cleavage region for cleaving effectors from scaffolds
disposed within the assay flow path. In some embodiments, the
device comprises a detection region. In some embodiments, the
device comprises a sorting region. In some embodiments, the device
comprises a stimulation region.
[0302] In some embodiments, the device comprises a third
microfluidic channel. The third microfluidic channel may be in
fluidic communication with the first microfluidic channel upstream
of the junction of the first and second microfluidic channels. This
third microfluidic channel may be used to mix an additional aqueous
fluid with the first aqueous fluid prior to droplet formation, thus
allowing the mixing of different sets of reagents shortly before
the droplets are formed.
[0303] The junction of the first and second microfluidic channels
is configured to create aqueous droplets encapsulated in the
immiscible fluid of the second microfluidic channel. This junction
may be of any configuration. In some embodiments, the junction is a
T-junction. In some embodiments, the junction is at an oblique
angle. In some embodiments, the junction further comprises a
supplementary microfluidic channel. In some embodiments, the
supplementary microfluidic channel comprises a second fluid
immiscible with the aqueous stream. In some embodiments, the second
fluid immiscible with the aqueous stream is the same as the fluid
immiscible with the aqueous stream from the second microfluidic
channel. In some embodiments, the second fluid immiscible with the
aqueous stream is different from the fluid immiscible with the
aqueous stream from the second microfluidic channel. In some
embodiments, the second microfluidic channel and the supplementary
microfluidic channel are positioned on opposite sides of the first
microfluidic channel. In some embodiments, the second microfluidic
channel and the supplementary microfluidic channel are configured
to add their respective fluids immiscible with the aqueous stream
simultaneously.
[0304] After the junction, the flow path of the second microfluidic
channel may continue along the same trajectory for a least a short
distance. After droplet formation, the channel downstream of the
junction forms an assay flow path. The assay flow path is the path
of the microfluidic channel where the screening assay is performed
in the droplet. As the droplet continues along this assay flow
path, additional unit operations can be performed on the droplet in
sequences that allow an assay with a detectable readout to occur
within the droplet. In some embodiments, the assay flow path
comprises a cleavage region. In some embodiments, the assay flow
path comprises a detection region. In some embodiments, the assay
flow path comprises a sorting region. In some embodiments, the
assay flow path comprises a stimulation region.
[0305] The assay flow path may be in any shape. In some
embodiments, the assay flow path acts as an incubation region,
allowing the assay to be performed over a desired length of time.
In some embodiments, the assay flow path comprises a serpentine
path region. The serpentine path region may contain a plurality of
curves or turns. Such a pathway allows for an extended flow path to
able to be embedded on a device of a small size. Additionally, the
curves of the flow path may be used to orient various detectors,
stimulators, sorters, or other components in a manner that
minimizes background signal, cross-talk, or bleed through of
various inputs into the droplets as they travel along the path. In
some embodiments, this is accomplished by orienting the various
inputs of unit operations along the curves or turns of the
serpentine path. This minimizes the amount of the input that can
travel along the flow path. For example, configuring a light source
to input the light at a location along a curve or turn of the flow
path minimizes the light that will travel along the path and reach
droplets not the target of the emission.
[0306] The serpentine path region can be any length of the
microfluidic device and can comprise any number of curves or turns.
In some embodiments, the serpentine flow path region comprises
about 10 curves to about 100 curves. In some embodiments, the
serpentine flow path region comprises about 10 curves to about 20
curves, about 10 curves to about 30 curves, about 10 curves to
about 40 curves, about 10 curves to about 50 curves, about 10
curves to about 100 curves, about 20 curves to about 30 curves,
about 20 curves to about 40 curves, about 20 curves to about 50
curves, about 20 curves to about 100 curves, about 30 curves to
about 40 curves, about 30 curves to about 50 curves, about 30
curves to about 100 curves, about 40 curves to about 50 curves,
about 40 curves to about 100 curves, or about 50 curves to about
100 curves. In some embodiments, the serpentine flow path region
comprises about 10 curves, about 20 curves, about 30 curves, about
40 curves, about 50 curves, or about 100 curves. In some
embodiments, the serpentine flow path region comprises at least
about 10 curves, about 20 curves, about 30 curves, about 40 curves,
or about 50 curves. In some embodiments, the serpentine flow path
region comprises at most about 20 curves, about 30 curves, about 40
curves, about 50 curves, or about 100 curves.
[0307] In some embodiments, the assay flow path comprises one or
more chambers disposed within the assay flow path. In some
embodiments, one or more of the chambers comprise an entrance and
exit microfluidic channel. In some embodiments, the entrance
microfluidic channel is at an upstream position and the exit
microfluidic channel is at a downstream position of the chamber. In
some embodiments, the entrance microfluidic channels and exit
microfluidic channels act as connecting channels between chambers.
In some embodiments, each droplet travelling through the assay flow
path travels through the one or more chambers. In some embodiments,
the chambers are configured to adjust the flow rate of the droplets
as they flow through the assay flow path. In some embodiments, the
chambers are configured to adjust the residence time of the
droplets as they flow through the assay flow path. In some
embodiments, the one or more chambers do not comprise an entrance
and exit microfluidic channel (e.g., there are no connecting
channels between the chambers). In some embodiments, the one or
more chambers are connected to each other. In some embodiments, the
one or more chambers are arranged to form serpentine assay flow
path.
[0308] Additional design considerations may be taken into mind when
selecting desired chamber and assay flow path geometry. For
example, characteristics of the immiscible carrier fluid can
influence suitability of a chamber or channel geometry for a
particular assay being performed on a device. For example,
immiscible carrier fluids with high viscosity contribute to greater
resistance to flow on the device, and thus are less compatible with
device flow path geometries which utilize substantial lengths of
narrow channels or chambers. However, widening of channels or
chambers on the device can increase dispersion of droplets
travelling through the chambers or channels, thereby yielding a
high variance of incubation times for individual droplets
travelling through the device. Thus, in some embodiments, it is
preferable that a device be operated with a low-viscosity
immiscible carrier fluid, such as
3-ethoxyperfluoro(2-methylhexane). In some embodiments, the device
is designed to optimize characteristics such as residence time,
modest flow pressures, and dispersion ratio with a particular
immiscible carrier fluid. In some embodiments, the device is
designed for optimal performance with low-density (e.g. less than
1.00 g/mL) immiscible carrier fluid with low viscosity. In some
embodiments, the device is designed for optimal performance with
3-ethoxyperfluoro(2-methylhexane) as the immiscible carrier
fluid.
[0309] In some embodiments, the chambers are configured to prevent
the trapping of droplets as the droplets travel through the flow
path. In embodiments wherein a carrier fluid denser than the
aqueous droplets is used (e.g. 3-ethoxyperfluoro(2-methylhexane)),
aqueous droplets may rise to the top of the widened, heightened
chambers and become trapped within the chamber as the droplets and
carrier fluid flow through the device. To counteract this, in some
embodiments, the chambers and entrance and or exit microfluidic
channels are configured to have only a small difference in channel
height between the chambers and the connecting channels. In some
embodiments, the height between the chambers and the exit channels
does not change until after the width of the channel has been
narrowed along the flow path. By adjusting the height only after
narrowing the width of the channel, droplets are more prone to
flowing along the desired path and not becoming trapped.
[0310] In some embodiments, the height of the chambers is only
slightly greater than the height of the connecting channels. In
some embodiments, the height of the chamber is about 1.1.times. to
about 3.times. greater than the height of the connecting channel.
In some embodiments, the height of the chamber is about 3.times. to
about 2.5.times., about 3.times. to about 2.times., about 3.times.
to about 1.9.times., about 3.times. to about 1.8.times., about
3.times. to about 1.7.times., about 3.times. to about 1.6.times.,
about 3.times. to about 1.5.times., about 3.times. to about
1.4.times., about 3.times. to about 1.3.times., about 3.times. to
about 1.2.times., about 3.times. to about 1.1.times., about
2.5.times. to about 2.times., about 2.5.times. to about 1.9.times.,
about 2.5.times. to about 1.8.times., about 2.5.times. to about
1.7.times., about 2.5.times. to about 1.6.times., about 2.5.times.
to about 1.5.times., about 2.5.times. to about 1.4.times., about
2.5.times. to about 1.3.times., about 2.5.times. to about
1.2.times., about 2.5.times. to about 1.1.times., about 2.times. to
about 1.9.times., about 2.times. to about 1.8.times., about
2.times. to about 1.7.times., about 2.times. to about 1.6.times.,
about 2.times. to about 1.5.times., about 2.times. to about
1.4.times., about 2.times. to about 1.3.times., about 2.times. to
about 1.2.times., about 2.times. to about 1.1.times., about
1.9.times. to about 1.8.times., about 1.9.times. to about
1.7.times., about 1.9.times. to about 1.6.times., about 1.9.times.
to about 1.5.times., about 1.9.times. to about 1.4.times., about
1.9.times. to about 1.3.times., about 1.9.times. to about
1.2.times., about 1.9.times. to about 1.1.times., about 1.8.times.
to about 1.7.times., about 1.8.times. to about 1.6.times., about
1.8.times. to about 1.5.times., about 1.8.times. to about
1.4.times., about 1.8.times. to about 1.3.times., about 1.8.times.
to about 1.2.times., about 1.8.times. to about 1.1.times., about
1.7.times. to about 1.6.times., about 1.7.times. to about
1.5.times., about 1.7.times. to about 1.4.times., about 1.7.times.
to about 1.3.times., about 1.7.times. to about 1.2.times., about
1.7.times. to about 1.1.times., about 1.6.times. to about
1.5.times., about 1.6.times. to about 1.4.times., about 1.6.times.
to about 1.3.times., about 1.6.times. to about 1.2.times., about
1.6.times. to about 1.1.times., about 1.5.times. to about
1.4.times., about 1.5.times. to about 1.3.times., about 1.5.times.
to about 1.2.times., about 1.5.times. to about 1.1.times., about
1.4.times. to about 1.3.times., about 1.4.times. to about
1.2.times., about 1.4.times. to about 1.1.times., about 1.3.times.
to about 1.2.times., about 1.3.times. to about 1.1.times., or about
1.2.times. to about 1.1.times. greater than the height of the
connecting channel. In some embodiments, the height of the chamber
is about 3.times., about 2.5.times., about 2.times., about
1.9.times., about 1.8.times., about 1.7.times., about 1.6.times.,
about 1.5.times., about 1.4.times., about 1.3.times., about
1.2.times., or about 1.1.times.. In some embodiments, the height of
the chamber is at least about 3.times., about 2.5.times., about
2.times., about 1.9.times., about 1.8.times., about 1.7.times.,
about 1.6.times., about 1.5.times., about 1.4.times., about
1.3.times., or about 1.2.times. greater than the height of the
connecting channel. In some embodiments, the height of the chamber
is at most about 2.5.times., about 2.times., about 1.9.times.,
about 1.8.times., about 1.7.times., about 1.6.times., about
1.5.times., about 1.4.times., about 1.3.times., about 1.2.times.,
or about 1.1.times. greater than the height of the connecting
channel. In some embodiments, the height of the chamber is from
about 1.1.times. to about 1.8.times. greater than the height of the
connecting channel. In some embodiments, the height of the chamber
is from about 1.4.times. to about 1.8.times. greater than the
height of the connecting channel. In some embodiments, the height
of the chamber is about 1.1.times. greater than the height of the
connecting channel. In some embodiments, the height of the chamber
is about 1.2.times. greater than the height of the connecting
channel. In some embodiments, the height of the chamber is about
1.3.times. greater than the height of the connecting channel. In
some embodiments, the height of the chamber is about 1.4.times.
greater than the height of the connecting channel. In some
embodiments, the height of the chamber is about 1.5.times. greater
than the height of the connecting channel. In some embodiments, the
height of the chamber is about 1.6.times. greater than the height
of the connecting channel. In some embodiments, the height of the
chamber is about 1.7.times. greater than the height of the
connecting channel. In some embodiments, the height of the chamber
is about 1.8.times. greater than the height of the connecting
channel. In some embodiments, the height of the chamber is about
1.9.times. greater than the height of the connecting channel. In
some embodiments, the height of the chamber is about 2.times.
greater than the height of the connecting channel.
[0311] In some embodiments, the height of the chamber is about 50
microns to about 120 microns. In some embodiments, the height of
the chamber is about 120 microns to about 100 microns, about 120
microns to about 90 microns, about 120 microns to about 80 microns,
about 120 microns to about 70 microns, about 120 microns to about
60 microns, about 120 microns to about 50 microns, about 100
microns to about 90 microns, about 100 microns to about 80 microns,
about 100 microns to about 70 microns, about 100 microns to about
60 microns, about 100 microns to about 50 microns, about 90 microns
to about 80 microns, about 90 microns to about 70 microns, about 90
microns to about 60 microns, about 90 microns to about 50 microns,
about 80 microns to about 70 microns, about 80 microns to about 60
microns, about 80 microns to about 50 microns, about 70 microns to
about 60 microns, about 70 microns to about 50 microns, or about 60
microns to about 50 microns. In some embodiments, the height of the
chamber is about 120 microns, about 100 microns, about 90 microns,
about 80 microns, about 70 microns, about 60 microns, or about 50
microns. In some embodiments, the height of the chamber is at least
about 120 microns, about 100 microns, about 90 microns, about 80
microns, about 70 microns, or about 60 microns. In some
embodiments, the height of the chamber is at most about 100
microns, about 90 microns, about 80 microns, about 70 microns,
about 60 microns, or about 50 microns. In some embodiments, the
height of the chamber is about 80 microns.
[0312] In some embodiments, the height of the chamber is about 300
microns to about 1,000 microns. In some embodiments, the height of
the chamber is about 1,000 microns to about 750 microns, about
1,000 microns to about 600 microns, about 1,000 microns to about
500 microns, about 1,000 microns to about 450 microns, about 1,000
microns to about 400 microns, about 1,000 microns to about 350
microns, about 1,000 microns to about 300 microns, about 750
microns to about 600 microns, about 750 microns to about 500
microns, about 750 microns to about 450 microns, about 750 microns
to about 400 microns, about 750 microns to about 350 microns, about
750 microns to about 300 microns, about 600 microns to about 500
microns, about 600 microns to about 450 microns, about 600 microns
to about 400 microns, about 600 microns to about 350 microns, about
600 microns to about 300 microns, about 500 microns to about 450
microns, about 500 microns to about 400 microns, about 500 microns
to about 350 microns, about 500 microns to about 300 microns, about
450 microns to about 400 microns, about 450 microns to about 350
microns, about 450 microns to about 300 microns, about 400 microns
to about 350 microns, about 400 microns to about 300 microns, or
about 350 microns to about 300 microns. In some embodiments, the
height of the chamber is about 1,000 microns, about 750 microns,
about 600 microns, about 500 microns, about 450 microns, about 400
microns, about 350 microns, or about 300 microns. In some
embodiments, the height of the chamber is at least about 1,000
microns, about 750 microns, about 600 microns, about 500 microns,
about 450 microns, about 400 microns, or about 350 microns. In some
embodiments, the height of the chamber is at most about 750
microns, about 600 microns, about 500 microns, about 450 microns,
about 400 microns, about 350 microns, or about 300 microns. In some
embodiments, the height of the chamber is about 500 microns.
[0313] In some embodiments, the width of the connecting channel is
about 50 microns to about 120 microns. In some embodiments, the
width of the connecting channel is about 120 microns to about 100
microns, about 120 microns to about 90 microns, about 120 microns
to about 80 microns, about 120 microns to about 70 microns, about
120 microns to about 60 microns, about 120 microns to about 50
microns, about 100 microns to about 90 microns, about 100 microns
to about 80 microns, about 100 microns to about 70 microns, about
100 microns to about 60 microns, about 100 microns to about 50
microns, about 90 microns to about 80 microns, about 90 microns to
about 70 microns, about 90 microns to about 60 microns, about 90
microns to about 50 microns, about 80 microns to about 70 microns,
about 80 microns to about 60 microns, about 80 microns to about 50
microns, about 70 microns to about 60 microns, about 70 microns to
about 50 microns, or about 60 microns to about 50 microns. In some
embodiments, the width of the connecting channel is about 120
microns, about 100 microns, about 90 microns, about 80 microns,
about 70 microns, about 60 microns, or about 50 microns. In some
embodiments, the width of the connecting channel is at least about
120 microns, about 100 microns, about 90 microns, about 80 microns,
about 70 microns, or about 60 microns. In some embodiments, the
width of the connecting channel is at most about 100 microns, about
90 microns, about 80 microns, about 70 microns, about 60 microns,
or about 50 microns. In some embodiments, the width of the
connecting channel is about 80 microns.
[0314] In some embodiments, the height of the connecting channel is
about 35 microns to about 75 microns. In some embodiments, the
height of the connecting channel is about 75 microns to about 65
microns, about 75 microns to about 55 microns, about 75 microns to
about 50 microns, about 75 microns to about 45 microns, about 75
microns to about 40 microns, about 75 microns to about 35 microns,
about 65 microns to about 55 microns, about 65 microns to about 50
microns, about 65 microns to about 45 microns, about 65 microns to
about 40 microns, about 65 microns to about 35 microns, about 55
microns to about 50 microns, about 55 microns to about 45 microns,
about 55 microns to about 40 microns, about 55 microns to about 35
microns, about 50 microns to about 45 microns, about 50 microns to
about 40 microns, about 50 microns to about 35 microns, about 45
microns to about 40 microns, about 45 microns to about 35 microns,
or about 40 microns to about 35 microns. In some embodiments, the
height of the connecting channel is about 75 microns, about 65
microns, about 55 microns, about 50 microns, about 45 microns,
about 40 microns, or about 35 microns. In some embodiments, the
height of the connecting channel is at least about 75 microns,
about 65 microns, about 55 microns, about 50 microns, about 45
microns, or about 40 microns. In some embodiments, the height of
the connecting channel is at most about 65 microns, about 55
microns, about 50 microns, about 45 microns, about 40 microns, or
about 35 microns. In some embodiments, the height of the connecting
channel is about 50 microns.
[0315] In some embodiments, the chambers are configured to reduce
the flow rate of the droplets as the droplets travel through the
device. In some embodiments, the flow rate is reduced due to an
increase in the cross-sectional area of the chamber relative to the
microfluidic channel upstream of the chamber. For example, a
chamber having 10.times. the cross-sectional area compared to the
microfluidic channel upstream of the chamber would have a flow rate
through the chamber of 10% of the flow rate compared to the flow
rate through the upstream microfluidic channel. In some
embodiments, the flow rate through the chambers is about 1% to
about 25% of the flow rate through the microfluidic channel
upstream of the chambers. In some embodiments, the flow rate
through the chambers is about 25% to about 20%, about 25% to about
15%, about 25% to about 12%, about 25% to about 10%, about 25% to
about 8%, about 25% to about 5%, about 25% to about 3%, about 25%
to about 1%, about 20% to about 15%, about 20% to about 12%, about
20% to about 10%, about 20% to about 8%, about 20% to about 5%,
about 20% to about 3%, about 20% to about 1%, about 15% to about
12%, about 15% to about 10%, about 15% to about 8%, about 15% to
about 5%, about 15% to about 3%, about 15% to about 1%, about 12%
to about 10%, about 12% to about 8%, about 12% to about 5%, about
12% to about 3%, about 12% to about 1%, about 10% to about 8%,
about 10% to about 5%, about 10% to about 3%, about 10% to about
1%, about 8% to about 5%, about 8% to about 3%, about 8% to about
1%, about 5% to about 3%, about 5% to about 1%, or about 3% to
about 1% of the flow rate through the microfluidic channel upstream
of the chambers. In some embodiments, the flow rate through the
chambers is about 25%, about 20%, about 15%, about 12%, about 10%,
about 8%, about 5%, about 3%, or about 1% of the flow rate through
the microfluidic channel upstream of the chambers. In some
embodiments, the flow rate through the chambers is at least about
25%, about 20%, about 15%, about 12%, about 10%, about 8%, about
5%, or about 3%. In some embodiments, the flow rate through the
chambers is at most about 20%, about 15%, about 12%, about 10%,
about 8%, about 5%, about 3%, or about 1% of the flow rate through
the microfluidic channel upstream of the chambers. In some
embodiments, the flow rate through the chambers is about 10% of the
flow rate through the microfluidic channel upstream of the
chambers. In some embodiments, the flow rate through the
microfluidic channel upstream of the chambers varies at different
points of the device. In such embodiments, the flow rate used for
the flow rate comparison to the chambers is the fastest flow rate
after the droplet formation junction (e.g. the microfluidic channel
portion with the smallest cross-sectional area).
[0316] In some embodiments, the chambers are configured such that
the droplets formed on the microfluidic device have substantially
the same residence time travelling through the device. In some
embodiments, the microfluidic device is configured such that the
droplets form on the device have substantially the same residence
time travelling through the device. In some embodiments, this is
measured by the dispersion ratio. The dispersion ratio is
calculated according to the following formula: 6.sigma./T.sub.avg;
wherein T.sub.avg is the average residence time of a droplet
travelling through the device and .sigma. is the standard deviation
of residence time of droplets travelling through the device. In
some embodiments, the device has a dispersion ratio of at most
about 10%, about 8%, about 6%, about 5%, about 4%, about 3%, about
2%, or about 1%. In some embodiments, the device has a dispersion
ratio of at most about 10%. In some embodiments, the device has a
dispersion ratio of at most about 8%. In some embodiments, the
device has a dispersion ratio of at most about 6%. In some
embodiments, the device has a dispersion ratio of at most about 5%.
In some embodiments, the device has a dispersion ratio of at most
about 4%. In some embodiments, the device has a dispersion ratio of
at most about 3%. In some embodiments, the device has a dispersion
ratio of at most about 2%. In some embodiments, the device has a
dispersion ratio of at most about 1%.
[0317] FIG. 18. provides an exemplary data set depicting a level of
uniformity for incubation period using a microfluidic device as
depicted FIGS. 9A and 10. Specifically, two aqueous inputs were
provided to inlets 101, 102, wherein one aqueous input contained a
buffer solution and fluorophore ("fluorophore solution"), and the
other aqueous input contained just the buffer solution ("buffer
solution"). The fluorophore solution and buffer solution were
provided with setpoint pressures that were offset by about 3%, such
that one solution would flow through the assay flow channel with a
higher concentration than the other. Initially, the buffer solution
was provided with the higher pressure, after which the setpoint
pressures were switched such that the fluorophore solution was
provided at a higher pressure. As depicted in FIG. 18, the PMT
count for a first period of time is less than 100 rfu after which
there is a sudden increase. The dispersion amongst the data set was
calculated to be only 1.7%, with a sigma of 3.19. As such, this
displays a level of uniform incubation period as the fluorophore
solution provided detection signals within 2% dispersion, and
without significant lag when switching the concentrations. As such,
this correlates to encapsulations moving along the assay flow path
at a relatively uniform rate. FIG. 19 provides a similar analysis
using the microfluidic device from FIG. 11, wherein the fluorophore
solution was provided with a higher pressure initially, before
being switched to a lower pressure. The dispersion amongst the data
point was calculated to be slightly higher at 4.52%, with a sigma
of 7.25. As such, this similarly displays a level of uniformity for
the incubation period as the fluorophore solution provided
detection signals with less than 5% dispersion.
[0318] In some embodiments, the device further comprises one or
more collection chambers. In some embodiments, the one or more
collection chambers are configured to receive a subset of the
plurality of droplets passing through the assay flow path. In some
embodiments, the collection chambers are configured to incubate the
subset for an extended period of time. In some embodiments, the
collection chambers are configured to lengthen the residence time
for the subset of plurality of droplets.
[0319] In some embodiments, the device further comprises one or
more shunts positioned along the flow path of the device. A shunt
may be positioned at any location of the device. The shunt may be
used for a variety of purposes. In some embodiments, a shunt is
used to insert additional immiscible carrier fluid into the
microfluidic channel in order to affect droplet spacing. In some
embodiments, a shunt is used to divert droplets of carrier fluid
off of the microfluidic device. In some embodiments, a shunt is
used in initiation of the device. In some embodiments, a shunt is
used in equilibration of the device. In some embodiments, the
device is equilibrated
[0320] In some embodiments, the assay flow path comprises a first
shunt. In some embodiments, the first shunt is positioned in an
upstream area of the assay flow path. In some embodiments, the
first shunt is positioned upstream of the serpentine area of the
assay flow path. In some embodiments, the first shunt is positioned
upstream of the one or more chambers. In some embodiments, the
first shunt is opened during an equilibration phase of using the
device. In some embodiments, carrier fluid is run through the
device in a reverse direction from normal operation during an
equilibration stage of the device and allowed to exit the device
through the first shunt. In some embodiments, aqueous droplets are
simultaneously introduced into the microfluidic device upstream of
the first shunt and allowed to exit the device through the first
shunt. In some embodiments, the shunt is closed once pressures of
input fluids on the device have been adjusted to desired levels in
order to run the system as desired (e.g. flow rates, pressures,
droplet size, droplet spacing, etc.).
[0321] In some embodiments, the first shunt configured to allow
droplets to bypass at least a portion of the assay flow path. In
some embodiments, an alternate flow path is coupled to the first
shunt. The alternate flow path can have any property and can be
used to affect the assay flow path in any manner. For example, the
alternate flow path can be used to change the incubation time or
residence time of droplets on the microfluidic device, add an
additional reagent steam (e.g. a droplet merging junction or
pico-injection site), or to incubate droplets off the device
entirely.
[0322] The cleavage region may comprise a mechanism for liberating
an effector that is linked to a bead by a cleavable linker. In some
embodiments, the cleavage region comprises a pico-injection site or
droplet merging site to introduce reagents to cleave the effector
from a scaffold. In some embodiments, the cleavage region comprises
a light source configured to cleave effectors from scaffolds
disposed within the assay flow path. In some embodiments, the light
source is a source of UV light. In some embodiments, the light
source is a waveguide. In some embodiments, the light source is a
fiberoptic cable. In some embodiments, the light source is a light
source configured to cleave effectors from scaffolds disposed
within the assay flow path. In some embodiments, the light source
is configured to have an optical axis substantially parallel with
the device plane. In some embodiments, the light source illuminates
a passing droplet at a curve in the assay flow path. In some
embodiments, the light source is configured to have an optical axis
substantially perpendicular to the device plane. In some
embodiments, the light source is aligned with the microfluidic
channel of the cleavage region by pillars mounted on the device. In
some embodiments, the light source is configured to emit light over
an area covering multiple portions of the microfluidic channel
passing through the cleavage region. In some embodiments, the
cleavage region comprises a serpentine flow path.
[0323] The cleavage region can be at any point along the
microfluidic device depending upon the needs of the assay being
employed on the device. In some embodiments, the cleavage region is
upstream of the detection region, the sorting region, and the
stimulation region. In some embodiments, the cleavage region is
upstream of the detection region. In some embodiments, the cleavage
region is upstream of the sorting region. In some embodiments, the
cleavage region is upstream of the stimulation region. In some
embodiments, the cleavage region is upstream of the detection
region and the sorting region.
[0324] In some embodiments, the device comprises an additional
inlet and outlet positioned on the microfluidic channel upstream
and downstream of the cleavage region. In some embodiments, the
inlet and outlets are positioned immediately before and immediately
after the cleavage region.
[0325] In some embodiments, these inlets and outlets are configured
to allow for a calibration of the cleavage region. The calibration
allows for control over device-to-device variability in how much
light the samples passing through the cleavage region are exposed
to. Such variability can come from small changes to a variety of
parameters of the device, including the coupling of the light
source to the device. Variability in exposure intensity time and
duration can lead to variability in amount of compound released
from beads, which can cause errors in ultimate screening assay
readouts.
[0326] In some embodiments, the inlets and outlets are used for the
calibration procedure. In some embodiments, the calibration
procedure comprises flowing a solution comprising a fluorescent dye
through the cleavage region. FIG. 12D provides an exemplary
depiction of the cleavage region for a microfluidic device
described herein, wherein the calibration inlet and UV waveguide
for exposing the encapsulations (e.g., droplets) to light are
shown. In some embodiments, the calibrant channel is filled with
UV-sensitive fluorophore to measure the UV intensity in the
cleavage region. In some embodiments, the UV waveguide directs
light from a UV LED coupled fiberoptic into a confined area. In
some embodiments, the UV LED power is then set, based on a
calibrant dye being measured. FIG. 12E provides exemplary data
correlating a calibrant dye with a given light exposure (BD
Horizon.TM. BV510).
[0327] In some embodiments, encoded effector-fluorophore beads are
introduced into encapsulations (e.g., droplets) using a
microfluidic device as described herein. FIG. 12A provides an
exemplary solution of beads with an encoded-effector modified with
a fluorophore, wherein the solution can comprise a library of
beads. As shown in FIG. 12B, the effector-fluorophore may be
connected by a photo-cleavable, or pro-photo-cleavable linker. In
some embodiments, the encoded-fluorophore beads are introduced into
droplets at approximately 200 pL in volume. In some embodiments,
the droplets are introduced into the cleavage region and exposed to
the UV light. As shown in FIG. 12B, when exposed to the UV light,
the effector is liberated (i.e. cleaving the photocleavable
linker), such that the effector is released from the bead (FIG.
12C). The droplets then continue to flow through the microfluidic
device, as described herein, until reaching an "interrogation
region" of the microfluidic device, wherein the droplets are
subject to laser excitation (e.g., confocal laser excitation, FIG.
12F), thereby exciting the released effector-fluorophore. The
emission from the encoded effector-fluorophore is then collected by
PMT detectors, as shown in FIGS. 13A and 14A (represented by PMT 2
Smooth), which represent the effector release based on exposure
from 100 mV and 600 mV light respectively, thereby measuring the
released effector-fluorophore concentration. FIGS. 13B and 14B
provide the peak emission measured for each droplet based on
exposure from 100 mV and 600 mV light respectively, plotted as a
heat-map over time to observe the stability of the signal. As
shown, increasing the UV LED power increases the exposure, thereby
enabling the ability to control the final concentration of released
effector-fluorophore. FIG. 15B provides a histogram with compressed
droplet maps (e.g., from FIGS. 13B and 14B), so as to depict
normally distributed intensity values. The median value is
correlated to known Fluorophore concentration calibrations (e.g.,
FIG. 15A), so as to determine the final concentration of the
effector-fluorophore after UV release. As such, the emission
intensity, as measured with a calibrant fluid, can be correlated to
a resultant effector-release concentration, thereby providing a
predictive quantitative release.
[0328] The detection region is configured with a detector capable
of detecting any desired readout of an assay to be performed on the
device. In some embodiments, the detection region comprises a
fluorometer. In some embodiments, the fluorometer comprises a
photomultiplier tube detector, a light source, an excitation filter
and an emission filter. In some embodiments, the fluorometer is
configured to have an optical axis substantially parallel to the
device plane. In some embodiments, the fluorometer is configured to
have an optical axis substantially perpendicular to the device
plane. In some embodiments, the fluorometer illuminates a passing
droplet at a curve in the assay flow path. In some embodiments, the
detection region comprises confocal detection and laser scanning.
In some embodiments, the detection region comprises a confocal
laser scanning device, as shown in FIGS. 22A-B (providing a top
view of the device). FIG. 12F provides an exemplary schematic of
encapsulation detection via confocal laser scanning. In some
embodiments, the detection region comprises laser scanning. In some
embodiments, the detection region comprises fluorescence. In some
embodiments, the detection region comprises any combination of
detection means described herein.
[0329] In some embodiments, the detection region comprises an
objective or fiber for emitting an excitation light into the
detection region. In some embodiments, the detection region
comprises an objective, fiber, or charged coupled device configured
to collect emission from the detection region. In some embodiments,
a single objective is configured to direct excitation and collect
emission from the detection region. In some embodiments, the
objective configured to collect emission from the detection region
(which may be the same as the excitation objective) is an inverted
objective lens. In some embodiments, the objective configured to
collect emission from the detection region (which may be the same
as the excitation objective) is configured to collect, collimate,
and direct the emitted light through optical fibers. In some
embodiments, the optical fibers are coupled to a detector
configured to quantify the emission. In some embodiments, the
detector configured to quantify the emission is a photomultiplier
tube, charged coupled device, or photodiode.
[0330] In some embodiments, the detection region is capable of
being moved on the chip. In some embodiments, the detection region
comprises an excitation light source that is not coupled to the
device. In some embodiments, the detection region comprises an
objective that is not coupled to the device. In some embodiments,
having a light source or detector for the detection region not
coupled to the device allows for the system to be adjusted based on
assay need. For example, the system can be adjusted to increase or
decrease the time between detection and sorting. Additionally, the
system can be adjusted so that a single light source may be used
for calibration and initialization of the device prior to
performing a screening assay on the device.
[0331] In some embodiments, the detection region is configured to
detect two or more wavelengths of fluorescence. This allows for the
detection of the abundance of a plurality of fluorescent probes. In
some embodiments, the droplet being assayed may comprise a control
fluorophore and an assay fluorophore. The assay fluorophore gives a
readout of the assay, e.g. a positive or negative result of the
assay. The control fluorophore, if present, may be detected and
quantified. In some embodiments, the control fluorophore is placed
into the aqueous fluid of the first microfluidic channel at a known
concentration. When the droplet comprising the aqueous fluid of the
first microfluidic channel reaches the detection region, the amount
of control fluorophore fluorescence detected can be used to
quantify the size of the droplet. This can be used to normalize the
results of the assay fluorophore readout. In some embodiments, the
detection region is configured to measure two or more assay
fluorophores.
[0332] In some embodiments, the device comprises a single detection
region. In some embodiments, the detection region is downstream of
the cleavage region. In some embodiments, the detection region is
downstream of the stimulation region. In some embodiments, the
detection region is upstream of the sorting region.
[0333] In some embodiments, the device comprises multiple detection
regions. When the device comprises multiple detection regions, they
may be placed anywhere on the device. In some embodiments, the
detection region is configured to be in communication with another
region. For example, the detection region may be in communication
with the sorting region to allow sorting to occur based on the
detection of a signal. In some embodiments, a detection region is
configured to be in communication with a pico-injector. When a
detection region is in communication with a pico-injector, reagents
or other assay components can be selectively added only when
certain conditions are met, such as the presence or absence of a
signal.
[0334] In some embodiments, the device comprises a stimulation
region. In some embodiments, the stimulation region comprises one
or more actuators for stimulating an ion channel. Any method of
stimulating an ion channel may be employed by the actuators when
the device is configured to perform an ion channel modulation
assay. In some embodiments, the stimulation region comprises one or
more actuators for stimulating an ion channel. In some embodiments,
the one or more actuators for stimulating the ion channel comprises
at least one light source, at least one electrode, or at least one
pico-injection site equipped with an ion channel toxin. In some
embodiments, the one or more actuators comprises at least one light
source. In some embodiments, the one or more actuators comprises at
least one electrode. In some embodiments, the one or more actuators
comprises an injection site for an ion channel toxin.
[0335] In some embodiments, the one or more actuators comprises at
least one electrode. Any type of electrode capable of delivering an
electromagnetic current to the encapsulation may be employed. In
some embodiments, the electrode lies along a wall of the assay flow
path and delivers an electric field to the passing stream. In some
embodiments, the electric field is pulsed to match the frequency at
which droplets pass the electrode.
[0336] In some embodiments, the one or more actuators comprises a
pair of electrodes on opposite walls of the assay flow path such
that when a droplet passes the pair of electrodes the droplet
contacts the electrodes, thereby allowing a current to flow through
the droplet. In some embodiments, the device comprises multiple
pairs of electrodes so configured. In some embodiments, the
stimulation region comprises about 1 pair to about 20 pairs of
electrodes so configured. In some embodiments, the stimulation
region comprises about 1 pair to about 2 pairs, about 1 pair to
about 3 pairs, about 1 pair to about 5 pairs, about 1 pair to about
7 pairs, about 1 pair to about 10 pairs, about 1 pair to about 20
pairs, about 2 pairs to about 3 pairs, about 2 pairs to about 5
pairs, about 2 pairs to about 7 pairs, about 2 pairs to about 10
pairs, about 2 pairs to about 20 pairs, about 3 pairs to about 5
pairs, about 3 pairs to about 7 pairs, about 3 pairs to about 10
pairs, about 3 pairs to about 20 pairs, about 5 pairs to about 7
pairs, about 5 pairs to about 10 pairs, about 5 pairs to about 20
pairs, about 7 pairs to about 10 pairs, about 7 pairs to about 20
pairs, or about 10 pairs to about 20 pairs of electrodes so
configured. In some embodiments, the stimulation region comprises
about 1 pair, about 2 pairs, about 3 pairs, about 5 pairs, about 7
pairs, about 10 pairs, or about 20 pairs of electrodes so
configured. In some embodiments, the stimulation region comprises
at least about 1 pair, about 2 pairs, about 3 pairs, about 5 pairs,
about 7 pairs, or about 10 pairs of electrodes so configured. In
some embodiments, the stimulation region comprises at most about 2
pairs, about 3 pairs, about 5 pairs, about 7 pairs, about 10 pairs,
or about 20 pairs of electrodes so configured.
[0337] Any number of actuators may be employed on the microfluidic
device. In some embodiments, the stimulation region comprises about
1 actuator to about 20 actuators. In some embodiments, the
stimulation region comprises about 1 actuator to about 2 actuators,
about 1 actuator to about 3 actuators, about 1 actuator to about 5
actuators, about 1 actuator to about 7 actuators, about 1 actuator
to about 10 actuators, about 1 actuator to about 20 actuators,
about 2 actuators to about 3 actuators, about 2 actuators to about
5 actuators, about 2 actuators to about 7 actuators, about 2
actuators to about 10 actuators, about 2 actuators to about 20
actuators, about 3 actuators to about 5 actuators, about 3
actuators to about 7 actuators, about 3 actuators to about 10
actuators, about 3 actuators to about 20 actuators, about 5
actuators to about 7 actuators, about 5 actuators to about 10
actuators, about 5 actuators to about 20 actuators, about 7
actuators to about 10 actuators, about 7 actuators to about 20
actuators, or about 10 actuators to about 20 actuators. In some
embodiments, the stimulation region comprises about 1 actuator,
about 2 actuators, about 3 actuators, about 5 actuators, about 7
actuators, about 10 actuators, or about 20 actuators. In some
embodiments, the stimulation region comprises at least about 1
actuator, about 2 actuators, about 3 actuators, about 5 actuators,
about 7 actuators, or about 10 actuators. In some embodiments, the
stimulation region comprises at most about 2 actuators, about 3
actuators, about 5 actuators, about 7 actuators, about 10
actuators, or about 20 actuators.
[0338] In some embodiments, the device comprises multiple
stimulation regions. Stimulation regions may be distributed in any
orientation throughout the microfluidic device. In some
embodiments, the stimulation region is downstream of the cleavage
region. In some embodiments, the stimulation region is upstream of
the detection region. In some embodiments, the stimulation region
is upstream of the sorting region.
[0339] In some embodiments, the device comprises an additional
inlet configured to insert carrier fluid into the flow path of the
microfluidic device. Optimal spacing of droplets is an important
consideration in order to accurately sort desired droplets. Factors
which can affect accurate sorting of droplets include droplet size,
average separation of droplets, total oil fraction of the flow,
ionic strength of the droplets, and contents of the droplets.
Individual assays performed on the devices provided herein may
require optimization of spacing, which is allowed by the presence
of the additional inlet. In some embodiments, the additional inlet
inserts additional carrier fluid into the flow path of the
microfluidic device to increase spacing of the droplets. In some
embodiments, the additional inlet inserts additional carrier fluid
into the flow path of the microfluidic device to focus the
droplets. In some embodiments, the additional carrier fluid is the
immiscible fluid from the second microfluidic channel. In some
embodiments, the additional carrier fluid is different from the
immiscible fluid from the second microfluidic channel. In some
embodiments, the additional inlet operates at a constant flow. In
some embodiments, the additional inlet operates at a variable flow.
In preferred embodiments, the additional inlet is positioned
shortly upstream of the detection region. In some embodiments, the
additional inlet operates at a flow rate selected to optimally
space the droplets. In some embodiments, the device comprises two
additional inlets. In some embodiments, the device comprises a
first additional inlet configured to deliver spacing oil and a
second additional inlet configured to deliver focusing oil.
[0340] In some embodiments, the devices comprise a sorting region.
Any method of sorting the droplets in the device may be used. In
some embodiments, the sorting region is in communication with the
detection region. In some embodiments, the sorting region comprises
a sorting apparatus that sorts the droplets based on the detection
of the presence, absence, or level of a signal detected by the
detection region. In some embodiments, the sorting region comprises
a sorting electrode. In some embodiments, the sorting electrode is
an electrophoresis electrode. In some embodiments, the sorting
electrode is a dielectrophoresis electrode. In some embodiments,
the sorting region comprises a valve configured for sorting. In
some embodiments, the sorting region comprises a deflectable
membrane configured for sorting. In some embodiments, the sorting
region comprises an acoustic wave generator configured for sorting.
In some embodiments, the sorting region comprises an inlet for
fluid configured to guide a passing droplet down a sorted path.
[0341] In some embodiments, the device comprises microfluidic
channels which are fully enclosed. In some embodiments, the device
comprises microfluidic channels encompassed on all sides of the
microfluidic channel, except for any inlets and outlets into the
device. In some embodiments, the device comprises a cover slip
configured to enclose the channels. In some embodiments, the cover
slip is coated with a hydrophobic material (e.g. PDMS). The cover
slip may be of any size (e.g. 5 micron, 10 micron, 15 micron, 20
micron, 30 micron, 40 micron, 50 micron or greater).
[0342] Control of flow of fluids through the device may be
accomplished in any manner. In preferred embodiments, the flow of
fluids is controlled by a device capable of delivering fluid
through the device for a prolonged period of time and/or in a
continuous fashion (e.g. a pneumatic pump or a peristaltic pump).
Such pumps have several advantages over other pumps, such as
syringe pumps, including the ability to run the system for a
prolonged period of time at constant pressure, thus allowing for
continuous feed of material through the device and control over
residence time of droplets travelling through the device. In some
embodiments, the flow of fluids is controlled by a continuous pump.
In some embodiments, the flow of fluids is controlled by a
pneumatic pump. In some embodiments, the fluids are delivered to
the device from a reservoir of fluid off of the device. This allows
the device to draw a much larger amount of fluid than would be
possible from an on-device reservoir. Any of the sample fluids,
immiscible fluids, spacing oil, focusing oil, or other fluid
delivered onto the chip can be delivered in this manner.
[0343] In some embodiments, the pump is configured to deliver
fluids through the device for a continuous period of at least 4
hours, at least 8 hours, at least 12 hours, at least 16 hours, or
at least 24 hours. In some embodiments, the pump is configured to
deliver fluids through the device for a continuous period of at
least 12 hours. In some embodiments, the pump is configured to
deliver fluids through the device for a continuous period of at
least 24 hours.
[0344] A non-limiting, exemplary microfluidic device is shown in
FIG. 9A. The exemplary microfluidic device contains a first inlet
101. The first inlet 101 is configured to accept an aqueous fluid,
such as an aqueous assay reagent. The exemplary microfluidic device
also contains a second inlet 102. In this example, the second inlet
102 is configured to accept another aqueous fluid. This may be the
same or different as the aqueous fluid added to the first inlet
101. The second inlet 102 may be configured to accept beads as
provided herein, or the first inlet 101 may be so configured. In
other examples of a microfluidic device, there may only be a single
inlet stream. The exemplary microfluidic device shown in FIG. 9A
further comprises an inlet 103 for carrier fluid (e.g. an oil
immiscible with an aqueous fluid) in fluid connection with a
droplet formation junction or extrusion junction 104. The inlet 103
in this example is connected to the droplet formation junction 104
by two channels, each reaching an aqueous stream channel at the
same point on opposite sides of the aqueous stream channel. The
droplet formation junction 104 comprises a microfluidic channel
that continues down the flow path towards cleavage region 106. Near
cleavage region 106 is a fiberoptic waveguide 105a configured to
deliver light into the microfluidic channel of the cleavage region
106. The fiberoptic waveguide 5a is embedded in the plane of the
device such that the light emitted enters the microfluidic channel
of cleavage region 106 from the device plane. Also near cleavage
region 106 is a pillar 105b configured to fix a fiberoptic manifold
which can be configured to emit light from above the plane of the
device into the microfluidic channel of cleavage region 106. The
light sources of 105a and 105b can be used alternatively or in
combination. The device also comprises an inlet for calibration
fluid 107a in fluid connection with the cleavage region 106 and an
outlet for calibration fluid 107b. The inlet for calibration fluid
107a is configured to receive and deliver to the cleavage region
106 a fluid configured to normalize photon exposure within the
cleavage region. After passing through the cleavage region 106, the
calibration fluid exits through the outlet for calibration fluid
107b. The cleavage region 106 is in fluid communication via a
microfluidic channel to an incubation region 109. In the example of
FIG. 9A, the incubation region 109 contains a series of widened
chambers, each chamber connected to the next chamber in the series
by a microfluidic channel. The configuration of these chambers
affect the flow rate and residence time of the droplets formed at
droplet formation region 104 through the device. In some
embodiments, the chambers are configured to prevent trapping of
droplets as they pass through incubation region 109. Such
configuration of the chambers is particularly important when using
a carrier fluid that is denser than the aqueous droplets (e.g.
3-ethoxyperfluoro(2-methylhexane)). In some embodiments, this
desired configuration is achieved by configuring the chambers and
connecting channels to have only small difference in channel height
between the chambers and the connecting channels. In some
embodiments, the height of the chamber is about 80 .mu.m and the
height of the connecting channel is about 50 .mu.m. As an
additional design feature to aid in prevention of trapping of
bubbles within the device, the height of the flow path does not
change between the width of the chamber has been narrowed as the
droplet approaches the connecting channel, thus facilitating the
smooth transition of droplets from chamber to chamber without
trapping. Configured on either end of incubation region 109 are
bypass shunts 108a and 108b. The bypass shunts 108a and 108b are
configured to allow a fluid coupled to the shunt to flow in or out
of the main microfluidic channel. If fluid is diverted out of the
main microfluidic channel at bypass shunt 108a, the material will
not pass through incubation region 109. Positioned downstream of
incubation region 109 is inlet for carrier fluid 110. Inlet for
carrier fluid 110 is in fluid communication with the main
microfluidic channel of the device and is configured to deliver
additional immiscible carrier fluid into the main microfluidic
channel in order to space droplets as desired. Also in fluid
communication with the main microfluidic channel is inlet for
carrier fluid 111, which is configured to deliver droplet focusing
oil into the main microfluidic channel. Downstream of inlets for
carrier fluid 110 and 111 is detection position 116. The detection
position 116 indicates the point on the device that the desired
signal from the assay being run on the chip is detected. The
detection position 116 may be based on an alignment of an objective
or fiber that directs an excitation light at the sample passing
detection position 116 and an additional objective or fiber coupled
to a detector configured to detect an emission from detection
position 116. Alternatively, the objective for the excitation light
may be configured to also collect the emission. In some
embodiments, the excitation source is reflected from detection
position 116 through an inverted objective lens, where the emission
is collected, columnated, and directed through optical fibers for
quantification by a photomultiplier tube or other detector. In some
embodiments, the objective or fiber aligned at detection position
116 is not coupled to the device. When not coupled to the device,
the detector or emission objective or fiber can be moved to adjust
the detection positions 116 on the device in order to adjust the
time between detection and sorting. When not coupled to the device,
the detector or emission objective may also be moved for use in
calibration of the device or initiation of the device, thus
allowing a single light source to be used for multiple functions.
Downstream of inlets for carrier fluid 110 and 111 and detection
position 116 is discrimination junction electrode 112. The
discrimination junction electrode 112 may be a dielectrophoresis
electrode configured to propel droplets down outlet 114 if the
droplet is determined to display a desired signal or to outlet 115
if the droplet is determined to lack a desired signal. The
discrimination junction electrode 112 is connected to a
discrimination junction ground circuit, which is connected to the
device at circuit connection points 113a and 113b. A zoomed in
drawing of the sorting and detection region of the exemplary device
is shown in FIG. 9B. FIG. 9C shows a picture of a microfluidic
device substantially as described in this example. FIG. 10 provides
another exemplary depiction of the microfluidic device from FIG.
9A, wherein an Optical Glue is displayed within the fiberoptic
waveguide. In some embodiments, the Optical Glue helps to minimize
scattering of the light from the fiberoptic wave guide.
[0345] FIG. 11 provides another exemplary microfluidic device that
can be used for the methods and systems described herein. The
exemplary microfluidic device contains a first inlet 201. The first
inlet 201 is configured to accept an aqueous fluid, such as an
aqueous assay reagent. The exemplary microfluidic device also
contains a second inlet 202. In this example, the second inlet 202
is configured to accept another aqueous fluid. This may be the same
or different as the aqueous fluid added to the first inlet 201. The
second inlet 202 may be configured to accept beads as provided
herein, or the first inlet 201 may be so configured. In some
embodiments, the exemplary microfluidic device also contains a
third inlet 218. In this example, the third inlet 218 is configured
to accept another aqueous fluid. This may be the same or different
as the aqueous fluid added to the first inlet 201 and/or the second
inlet 202. The third inlet 218 may be configured to accept beads as
provided herein. In other examples of a microfluidic device, there
may only be a single inlet stream. In some embodiments of a
microfluidic device, there are four or more inlets. In some
embodiments the four or more inlets may be aqueous inlets. The
exemplary microfluidic device shown in FIG. 11 further comprises an
inlet 203 for carrier fluid (e.g. an oil immiscible with an aqueous
fluid) in fluid connection with a droplet formation junction or
extrusion junction 204. The inlet 203 in this example is connected
to the droplet formation junction 204 by two channels, each
reaching an aqueous stream channel at the same point on opposite
sides of the aqueous stream channel. The droplet formation junction
204 comprises a microfluidic channel that continues down the flow
path towards cleavage region 206. Near cleavage region 206 is a UV
waveguide 205 configured to deliver light into the microfluidic
channel of the cleavage region 206. In some embodiments, the UV
waveguide is a fiberoptic wave guide. The UV waveguide 205 is
embedded in the plane of the device such that the light emitted
enters the microfluidic channel of cleavage region 206 from the
device plane. In some embodiments, the UV waveguide comprises a
parabolic lens at an end closest to the cleavage region. In some
embodiments, the parabolic lens is configured to columnate light
inside the cleavage region. In some embodiments, the parabolic
lens, or a curved lens, minimizes the tendency for the light from
the UV waveguide to be scattered. In some embodiments, the cleavage
region is exposed to UV light projected normal to the circuit
plane, exposing a defined area to UV where the compound is cleaved.
In some embodiments, an Optical Glue 217 is provided with the UV
waveguide. In some embodiments, the Optical Glue 217 helps to
minimize light being scattered by UV waveguide. Also near cleavage
region 206 may be a pillar (not shown) configured to fix a
fiberoptic manifold which can be configured to emit light from
above the plane of the device into the microfluidic channel of
cleavage region 206. The device also comprises an inlet for
calibration fluid 207a in fluid connection with the cleavage region
206 and an outlet for calibration fluid 207b. The inlet for
calibration fluid 207a is configured to receive and deliver to the
cleavage region 206 a fluid configured to normalize photon exposure
within the cleavage region. In some embodiments, the cleavage
region 206 comprises a serpentine flow path. After passing through
the cleavage region 206, the calibration fluid exits through the
outlet for calibration fluid 207b. The cleavage region 206 is in
fluid communication via a microfluidic channel to an incubation
region 209. In the example of FIG. 11, the incubation region 209
contains a series of widened chambers, each chamber connected to
the next chamber in the series by a microfluidic channel. The
configuration of these chambers affect the flow rate and residence
time of the droplets formed at droplet formation region 204 through
the device. In some embodiments, the chambers are configured to
prevent trapping of droplets as they pass through incubation region
209. Such configuration of the chambers is particularly important
when using a carrier fluid that is denser than the aqueous droplets
(e.g. 3-ethoxyperfluoro(2-methylhexane)). In some embodiments, the
height of the chamber is about 30 .mu.m to about 1,000 .mu.m. In
some embodiments, the height of the chamber is about 50 .mu.m to
about 500 .mu.m. In some embodiments, the depth of the chambers of
this exemplary microfluidic device (FIG. 11) is larger than the
depth of the chambers in the device from FIG. 9A. As such, in some
embodiments, this exemplary device (FIG. 11) provides for a longer
incubation region since a larger depth would result in faster
moving droplets, and thereby a decreased residence time if the same
length of incubation region as compared to the device in FIG. 9A
was used. In some embodiments, collection chambers 219 are
optionally provided with this exemplary microfluidic device.
Configured on either end of incubation region 209 are bypass shunts
208a and 208b. The bypass shunts 208a and 208b are configured to
allow a fluid coupled to the shunt to flow in or out of the main
microfluidic channel. If fluid is diverted out of the main
microfluidic channel at bypass shunt 208a, the material will not
pass through incubation region 209. Positioned downstream of
incubation region 209 is inlet for carrier fluid 210. Inlet for
carrier fluid 210 is in fluid communication with the main
microfluidic channel of the device and is configured to deliver
additional immiscible carrier fluid into the main microfluidic
channel in order to space droplets as desired. Also in fluid
communication with the main microfluidic channel is inlet for
carrier fluid 211, which is configured to deliver droplet focusing
oil into the main microfluidic channel. In some embodiments,
downstream of inlets for carrier fluid 210 and 211 is detection
position 216. The detection position 216 indicates the point on the
device that the desired signal from the assay being run on the chip
is detected. The detection position 216 may be based on an
alignment of an objective or fiber that directs an excitation light
at the sample passing detection position 216 and an additional
objective or fiber coupled to a detector configured to detect an
emission from detection position 216. Alternatively, the objective
for the excitation light may be configured to also collect the
emission. In some embodiments, the excitation source is reflected
from detection position 216 through an inverted objective lens,
where the emission is collected, columnated, and directed through
optical fibers for quantification by a photomultiplier tube or
other detector. In some embodiments, the objective or fiber aligned
at detection position 216 is not coupled to the device. When not
coupled to the device, the detector or emission objective or fiber
can be moved to adjust the detection positions 216 on the device in
order to adjust the time between detection and sorting. When not
coupled to the device, the detector or emission objective may also
be moved for use in calibration of the device or initiation of the
device, thus allowing a single light source to be used for multiple
functions. Downstream of inlets for carrier fluid 210 and 211 and
detection position 216 is discrimination junction electrode 212.
The discrimination junction electrode 212 may be a
dielectrophoresis electrode configured to propel droplets down
outlet 214 if the droplet is determined to display a desired signal
or to outlet 215 if the droplet is determined to lack a desired
signal. The discrimination junction electrode 212 is connected to a
discrimination junction ground circuit, which is connected to the
device at circuit connection points 213a and 213b.
[0346] FIG. 16 provides a data set indicating confinement of the UV
light emitted to a cleavage region of a microfluidic device
described herein. As such, UV light is not scattered throughout a
microfluidic device that results in additional encoded effectors
from being released while along an assay flow path. In some
embodiments, targeting and confining the UV light onto a specific
region of an assay flow path helps ensure a predetermined amount of
encoded effector is released. As an exemplary method to confirm
such confined UV emission, an assay flow path may be pre-filled
with an assay comprising encapsulations having a fluorophore dye.
Thus, a number of encapsulations are located downstream of a UV
exposure region (e.g., cleavage region), and would not be expected
to provide any detectable signals at a detection point of a
microfluidic device. As depicted in FIG. 16, an incubation delay
period of 1218 seconds is shown wherein there are minimal
encapsulations exhibiting detectable signals, followed by a
distinct number of encapsulations having detectable signals. As
such, the UV light emitted was generally confined to the cleavage
region of a microfluidic, such that the encapsulations passing
through the cleavage region was exposed to the UV light, within
minimal scattered light being exposed to encapsulations further
along the assay flow path.
[0347] FIG. 20 depicts an example of performing fluorescence assay
kinetics using the microfluidic device from FIGS. 9A and 10 is
provided. In some embodiments, the fluorescence is measured at
various locations within the assay flow path, so as to measure the
progression of interaction between an encoded effector and sample
as it is incubated. FIG. 21 provides a depiction for positioning a
laser spot in a given channel position so as to measure the PMT
emission. FIGS. 23A-24B provide a graphical output of the intensity
measured by an assay at different incubation times (PMT 1). For
example, FIGS. 23A-B depicts a raw signal and real-time smoothing
intensity measured at the outset of the incubation period (T=0 s),
wherein a very low count is measured (e.g., 25 counts at peak). By
contrast, FIGS. 24A-B depicts a raw signal and real-time smoothing
intensity (PMT 1) measured at the pre-sort junction of the
incubation period (T=1333 s), wherein a significantly higher count
is measured (approximately 380 counts at the peak). FIG. 25
provides a comparison of data quality between standard microplate
assay and a microfluidic device as described herein. The Figure
traces show the kinetic activity of a protease within a microplate
(left) and droplet compartment (right) on a microfluidic device.
The uniform incubation time provides high reproducibility and
uniformity at each time-point, reducing variance and proving strong
statistical significance compared to negative control better than a
microplate
Screen Normalization Methods
[0348] Provided herein are methods for improving the output results
of screens utilizing encoded effectors. Other methods suffer from
high rates of false negatives or positives due to variable loading
of effectors or encodings on scaffolds. The variations in amounts
of effector or encoding loaded on a scaffold may be due to either
low concentration of encoding/effector on scaffolds, or due to
degradation of the encoding/effector during synthesis, the
screening process, or storage. In some embodiments, the methods
described herein overcome this limitation by amplifying the level
of encodings on scaffolds to uniform levels. Thus, all the
encodings are present at substantially the same level and none are
drowned out by higher signals from more abundant encodings.
[0349] Additionally, in some embodiments, the methods provided
herein provide a means for determining the concentration of
effectors bound to scaffolds. In some embodiments, effector loading
in an entire library can be determined. Having knowledge of the
effector load on a scaffold can allow for determination if an
effector that displays a positive result in a screen is due to high
potency of the effector of interest, or if that particular effector
was present at a high concentration within the encapsulation which
was screened. Thus, the methods provided herein give the user a way
to readily ascertain how potent a particular effector is and can
help remove false positives from an effector screening set.
[0350] Further provided herein are methods for amplifying primers
for linking nucleic acids from the samples to the encoding for
optimal detection of nucleic acids. In methods without this
amplification step, incomplete capture of nucleic acids released by
the sample may occur due to low levels of encodings present on the
scaffolds. Lower levels of nucleic acid capture could be
interpreted as a lack of potency. By amplifying the primers within
the encapsulation during or after the screen is completed, all of
the sample nucleic acids can be captured. Thus, the method improves
the readout of nucleic acid levels in a screen. In some instances,
this results in improved yield and knowledge of the expression
levels of various sample components or other knowledge
ascertainable from capturing sample nucleic acids.
Barcode Normalization Method
[0351] Provided herein are methods for normalization of nucleic
acid encoding levels across a library after performing a screen.
During a library screen of nucleic acid encoded effectors, the
levels of nucleic acid encodings bound to beads can vary
substantially from bead to bead. This can be due to low synthesis
yields during synthesis of the bead, or due to damage to the
encoding itself during the screen or during storage. Some beads may
have concentrations of encodings bound to beads far in excess of
other beads. Consequently, when sequencing the resulting "hit"
beads after performing a screen to determine which effectors were
efficacious, effectors whose encodings are low in concentration are
difficult to detect. This is due to the amplification reactions
that occur during sequencing, which results in a much higher
presence of encodings whose concentrations start higher. For
example, amplifying the encodings from a pooled collection of
encoded effectors can generate noise (e.g., background signal)
during sequencing analysis, arising from template switching, or
mis-hybridization, which generate chimeric sequences, which are
misrepresentative of the true effector population. Therefore, it
would take a prohibitively high number of reads to detect encodings
which are present in substantially lower concentrations than
others. For this reason, a method to normalize the levels of
nucleic acid encodings after a screen is highly desirable and
advantageous, as it allows detection of substantially all effectors
that had a positive result in the screen. In an exemplary method,
isolated amplification of each encoding from a collection of
encoded effectors helps to prevent templates from different
encodings being formed from the mechanisms leading to chimeric
sequences.
[0352] In some embodiments, a plurality of screened encoded
effectors and corresponding scaffolds are provided in a plurality
of corresponding encapsulations, wherein each scaffold is bound to
one or more nucleic acid encodings that encode a corresponding
screened encoded effector. In some embodiments, the plurality of
encapsulations are lysed. In some embodiments, contents within the
plurality of encapsulations that were unbound to a scaffold are
removed. In some embodiments, the plurality of scaffolds are then
suspended in a liquid medium. In some embodiments, the plurality of
scaffolds are then encapsulated in a plurality of new
encapsulations, wherein each new encapsulation encapsulates one or
more scaffolds. In some embodiments, the nucleic acid encodings of
the beads. In some embodiments, the nucleic acids of each bead are
amplified to form corresponding amplified nucleic acid encodings.
In some embodiments, the amplified nucleic acid encodings within
the plurality of new encapsulations are limited to the nucleic acid
encodings and reagents within the respective new encapsulation,
thereby improving uniformity of the number of amplicons
representing each encoding. In some embodiments, the amplified
nucleic acid encodings are amplified, such that the concentration
of the amplified nucleic acid encodings for each scaffold are
within a minimum level of uniformity to each other.
[0353] The nucleic acid encoded library can be subjected to a
screen. Any type of screen can work with the methods and systems
provided herein. In some embodiments, the screen previously
performed is one of the screening methods provided herein. In some
embodiments, the screened encoded effectors have been sorted in the
previous screen. In some embodiments, only the "hit" effector beads
from the library screen are included in the present method. In some
embodiments, providing the screened encoded effectors and
corresponding scaffolds comprises performing a screen of the
nucleic acid encoded library. In some embodiments, the screen
comprises a sorting step to separate nucleic acid encoded effectors
that displayed a positive result in the screen.
[0354] In some embodiments, the screened encoded effectors and
corresponding scaffolds are provided in an emulsion, within a
plurality of encapsulations. The provided encapsulations containing
the screened encoded effectors and scaffolds may be lysed by a
variety of methods. In some embodiments, lysing the encapsulations
comprises introducing a demulsifying reagent, filtration, or
sonication to the emulsion. In some embodiments, lysing the
encapsulations comprises introducing a demulsifying reagent to the
emulsion. In some embodiments, lysing the encapsulations comprises
filtering the emulsion. In some embodiments, lysing the
encapsulations comprises introducing a demulsifying reagent to the
emulsion.
[0355] Any demulsifying reagent can be used with the methods and
systems provided herein. In some embodiments, the demulsifying
reagent is an acid or a salt. In some embodiments, the demulsifying
reagent is an acid. In some embodiments, the demulsifying reagent
is sulfuric acid or hydrochloric acid. In some embodiments, the
demulsifying reagent is an organic acid. In some embodiments, the
demulsifying reagent is a salt. In some embodiments, the salt is
sodium chloride, potassium pyrophosphate, or sodium sulfate. In
some embodiments, the salt is sodium chloride. In some embodiments,
the salt is potassium pyrophosphate. In some embodiments, the salt
is sodium sulfate.
[0356] In some embodiments, the scaffolds with the encapsulations
are washed to remove unbound contents. In some embodiments, washing
the scaffolds comprises rinsing the scaffolds with a wash buffer.
In some embodiments, the wash buffer is an aqueous buffer, an
organic solution, or a mixture thereof. In some embodiments, the
wash buffer is an aqueous buffer. In some embodiments, the buffer
is from pH 4 to pH 10. In some embodiments, the buffer is from pH 5
to 9. In some embodiments, the buffer is from pH 6 to pH 8. In some
embodiments, the pH is about pH 7. In some embodiments, the wash
buffer is a phosphate buffer. In some embodiments, the wash buffer
is an isotonic buffer. In some embodiments, the wash buffer is an
organic solution. In some embodiments, the organic solution
comprises methanol, ethanol, isopropyl alcohol, acetonitrile,
benzene, toluene, dichloromethane, ethyl acetate, hexanes, any
other organic solvent, or any combination thereof. In some
embodiments, the wash buffer comprises a denaturing agent.
[0357] Washing the scaffolds may remove unbound content from the
scaffolds and/or that were located within the corresponding
encapsulation. In some embodiments, at least 50%, at least 60%, at
least 70%, at least 80%, or at least 90% of unbound contents are
removed from the scaffolds during one or more was steps. In some
embodiments, at least 90%, at least 95%, at least 97%, at least
98%, or at least 99% of unbound contents are removed from the
scaffolds during one or more wash steps.
[0358] Multiple washes may be performed. In some embodiments, the
scaffolds are subject to multiple wash and collection steps. In
some embodiments, the scaffolds are collected by centrifugation or
filtration after each wash step. In some embodiments, the scaffolds
are collected by centrifugation after each wash step. In some
embodiments, the scaffolds are collected by filtration after each
wash step. In some embodiments, there is a single wash step. In
some embodiments, there are 2 wash steps. In some embodiments, the
was step is repeated 3, 4, 5, 6, 7, 8, 9, 10 or more times.
[0359] After the wash step, in some embodiments, the scaffolds are
suspended in a liquid medium. In some embodiments, the liquid
medium is an aqueous solution. In some embodiments, the liquid
medium comprises an organic solvent. In some embodiments, the
liquid medium is compatible with nucleic acid amplification. In
some embodiments, the liquid medium comprises the amplification
mix.
[0360] In some embodiments, the scaffolds are then encapsulated in
a plurality of encapsulations ("new encapsulations"). In some
embodiments, the scaffolds are encapsulated into a plurality of
droplets. In some embodiments, the scaffolds are reintroduced into
an emulsion. In some embodiments, each new encapsulation comprises
one or more scaffolds. In some embodiments, the scaffolds are
encapsulated such that a majority of the new encapsulations
comprise one or more single scaffolds. In embodiments, each droplet
comprises an amplification mix.
[0361] In some embodiments, encapsulating the scaffolds or
re-introducing the scaffold into an emulsion comprises placing the
scaffolds through a microfluidic device. In some embodiments, the
microfluidic device is a microfluidic chip. In some embodiments,
the scaffolds are reintroduced into an emulsion by placing the
scaffolds into a one-pot emulsifier.
[0362] As described herein, in some embodiments, the scaffold is a
solid support. In some embodiments, the scaffold is a bead, a
fiber, nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly. In some embodiments, the scaffold
is a bead. In some embodiments, the bead is a polymer bead, a glass
bead, a metal bead, or a magnetic bead. In some embodiments, the
bead is a polymer bead. In some embodiments, the bead is a glass
bead. In some embodiments, the bead is a metal bead. In some
embodiments, the bead is a magnetic bead. Beads for use in the
systems and methods as described herein can be any size. In some
embodiments, the beads are at most 10 nm, at most 100 nm, at most 1
.mu.m, at most 10 .mu.m, or at most 100 .mu.m in diameter. In some
embodiments, the beads are at least 10 nm, at least 100 nm, at
least 1 .mu.m, at least 10 .mu.m, or at least 100 .mu.m in
diameter. In some embodiments, the beads are about 10 .mu.m to
about 100 .mu.m in diameter.
[0363] In some embodiments, the amplification mix can be added to
the new encapsulations in a separate step. In some embodiments, the
amplification mix is added after the plurality of encapsulations
are formed. In some embodiments, the amplification mix is
encapsulated at the same time the scaffolds are being encapsulated.
In some embodiments, the amplification mix is added after
reintroducing the scaffolds into an emulsion. In some embodiments,
the amplification mix is added by pico-injection. In some
embodiments, the amplification mix is added by droplet merging. In
some embodiments, the amplification mix is added at the
encapsulation step.
[0364] The amplification mix is capable of amplifying the nucleic
acids in the new encapsulations. In some embodiments, the
amplification mix comprises PCR reagents. In some embodiments, the
amplification mix comprises reagents for room temperature
amplification.
[0365] In some embodiments, the nucleic acid encodings of each
scaffold are amplified to form amplified nucleic acid encodings,
such that the concentration of the amplified nucleic acid encodings
for each scaffold are within a minimum level of uniformity to each
other. In some embodiments, the minimum level of uniformity
comprises a concentration of nucleic acid encodings in each new
encapsulation, wherein about 90% of the new encapsulations have a
concentration of amplified nucleic acid encodings within about 10%
of an average concentration of amplified nucleic acid encodings in
the plurality of new encapsulations. In some embodiments, the
minimum level of uniformity comprises a concentration of nucleic
acid encodings in each new encapsulation, wherein about 80% of the
new encapsulations have a concentration of amplified nucleic acid
encodings within about 20% of an average concentration of amplified
nucleic acid encodings in the plurality of new encapsulations. In
some embodiments, the minimum level of uniformity comprises a
concentration of nucleic acid encodings in each new encapsulation,
wherein about 75% of the new encapsulations have a concentration of
amplified nucleic acid encodings within 25% of an average
concentration of amplified nucleic acid encodings in the plurality
of new encapsulations. In some embodiments, the minimum level of
uniformity comprises a concentration of nucleic acid encodings in
each new encapsulation, wherein about 70% to about 90% of the new
encapsulations have a concentration of amplified nucleic acid
encodings within about 10% to about 30% of an average concentration
of amplified nucleic acid encodings in the plurality of new
encapsulations. In some embodiments, the minimum level of
uniformity comprises a concentration of nucleic acid encodings in
each new encapsulation, wherein about 70% to about 90% of the new
encapsulations containing scaffolds have a concentration of
amplified nucleic acid encodings within 10-fold, 15-fold, 20-fold,
50-fold, or 100-fold of each other.
[0366] In some embodiments, sequencing the amplified nucleic acid
encodings results in lower background signal than a nucleic acid
encoded library that has not been subjected to the method. In some
embodiments, the background signal is reduced by at least 10%, at
least 20%, at least 30%, at least 40%, or at least 50%. In some
embodiments, the background signal is reduced by at least 75%. In
some embodiments, the background signal is reduced by at least 90%.
In some embodiments, the background signal is reduced by at least
95%.
[0367] In some embodiments, the lower background signal allows for
detection of nucleic acid encoded effectors whose encoding
concentrations before the screen are 100.times., 1000.times.,
10000.times., 100000.times., or 1000000.times. lower in
concentration than the average encoding concentration in the
library. In some embodiments, the lower background signal allows
for detection of nucleic acid encoded effectors whose encoding
concentrations before the screen are 100.times. lower in
concentration than the average encoding concentration in the
library. In some embodiments, the lower background signal allows
for detection of nucleic acid encoded effectors whose encoding
concentrations before the screen are 1000.times. lower in
concentration than the average encoding concentration in the
library. In some embodiments, the lower background signal allows
for detection of nucleic acid encoded effectors whose encoding
concentrations before the screen are 10000.times. lower in
concentration than the average encoding concentration in the
library. In some embodiments, the lower background signal allows
for detection of nucleic acid encoded effectors whose encoding
concentrations before the screen are 100000.times. lower in
concentration than the average encoding concentration in the
library. In some embodiments, the lower background signal allows
for detection of nucleic acid encoded effectors whose encoding
concentrations before the screen are 1000000.times. lower in
concentration than the average encoding concentration in the
library.
[0368] Primer Amplification Method
[0369] Provided herein is a method for amplifying a primer to
maximize cellular nucleic acid capture. In some screening methods
provided herein, nucleic acid contents of cells are transferred to
the nucleic acid encodings of various effectors. The nucleic acid
encodings are sometimes linked to scaffolds, such as beads.
However, a library of beads may comprise individual beads that may
have dramatically different levels of nucleic acids encodings on
the beads. Consequently, such beads are unable to attach
significant levels of cellular nucleic acids, or other beads are
able to attach substantially more levels of cellular nucleic acids.
Such discrepancies make it difficult to determine if the cellular
nucleic acid level differences are due to the differential effects
of various effectors, or if there were simply less capture sites
available to gather the cellular nucleic acids. Therefore, a method
of producing additional primers to label the cellular nucleic acids
with the nucleic acid encoding would have substantial benefits.
[0370] In one aspect, provided herein, is a method for amplifying a
primer to maximize cellular nucleic acid capture. In some
embodiments, the primer is a copy of a nucleic acid encoding
(encoded nucleic acid primer). In some embodiments, the method
comprises encapsulating a nucleic acid encoded scaffold with one or
more cells, an amplification mix, and a nicking enzyme. In some
embodiments, the nicking enzyme targets a specific nucleotide
sequence. As described herein, a nucleic acid encoded scaffold is
bound to one or more nucleic acid encodings. In some embodiments,
the one or more nucleic acid encodings comprise a specific
nucleotide sequence. In some embodiments, the cell is lysed to
release one or more cellular nucleic acids. In some embodiments,
the nucleic acid encoding is nicked with the nicking enzyme,
thereby creating an encoded nucleic acid primer. In some
embodiments, nicking refers to a single strand of a an encoding
being displaced. In some embodiments, the nicking enzyme targets a
specific site in the nucleic acid encoding. In some embodiments,
the specific site comprises the specific nucleotide sequence. In
some embodiments, nicking the nucleic acid encoding creates an
encoded nucleic acid primer. In some embodiments, the encoded
nucleic acid primer is amplified. In some embodiments, the encoded
nucleic acid primer is amplified via interaction between the
nicking site and the amplification mix. In some embodiments, a
released cellular nucleic acid is labeled with an encoded nucleic
acid primer.
[0371] In some embodiments, amplifying the encoded nucleic acid
primer comprises first creating a copy of the nucleic acid
encoding, which is extended from the nicking site, followed by
nicking the nucleic acid encoding copy. In some embodiments,
amplifying the encoded nucleic acid primer comprises simultaneously
1) creating a copy of the nucleic acid encoding, which extends from
the nicking site, and 2) displacing the nucleic acid encoding
copy.
[0372] In some embodiments, the amplification mix comprises an
amplification enzyme. In some embodiments, the amplification enzyme
enables for the creation of a nucleic acid encoding copy, and then
the subsequent nicking. In some embodiments, the nicking enzyme
enables the nicking of the copy of the nucleic acid encoding copy.
In some embodiments, the amplification enzyme enables for a copy of
the nucleic acid encoding to be simultaneously created and
displaced. In some embodiments, the amplification enzyme is a
polymerase. In some embodiments, the creation of nucleic acid
encoding copies and nicking, or the simultaneous creation and
displacement of the nucleic acid encoding copies, repeats to
generate a population of single stranded nucleic acid encodings
that serve as a primer (encoded nucleic acid primer) for labeling
cellular nucleic acids. In some embodiments, the encoded nucleic
acid primers are generated isothermally.
[0373] In some embodiments, each encoded nucleic acid primer
comprises a capture site that prescribes a target cellular nucleic
acid to label a specific released cellular nucleic acid. In some
embodiments, the target nucleic acid is a target mRNA. In some
embodiments, the target mRNA encodes a protein of interest. In some
embodiments, the nicking enzyme enables an increase in target mRNA
capture and labeling with the nucleic acid encoding. In some
embodiments, the target mRNA capture is increased by at least 10%,
25%, 50%, 100%, or 200%.
[0374] In some embodiments, a plurality of cellular nucleic acids
are labeled with an respective encoded nucleic acid primer. In some
embodiments, the nucleic acid encoded scaffold comprises a bead,
and the encoded nucleic acid primer comprises a unique bead barcode
and an effector encoding.
[0375] FIG. 3 illustrates an exemplary method for amplifying a
primer to maximize cellular nucleic acid capture, as described
herein. As shown in FIG. 3, in step 1, a nucleic acid encoded
scaffold is shown with the nucleic acid encoding bound thereto,
wherein a plurality of cellular encodings (e.g., nucleic acid) are
also shown to have been released from a lysed cell. In some
embodiments, the nucleic acid encoded scaffold and cellular
encodings are provided within an encapsulation. The nicking site is
identified on the nucleic acid encoding, along with a capture site.
In some embodiments, the nicking site corresponds to a specific
nucleotide sequence in the nucleic acid encoding. As shown in step
2, the nucleic acid encoding is nicked at the nicking site. As
shown, in some embodiments, nicking herein refers to a single
strand of the encoding being displaced from the nucleic acid
encoded scaffold. As shown in steps 3-4 of FIG. 3, an amplification
enzyme may interact with the nicking site, thereby creating a new
copy of the nucleic acid encoding (step 4), while the previously
nicked nucleic acid encoding copy (encoded nucleic acid primer) is
unbound and moves within the encapsulation, such that the encoded
nucleic acid primer may interact with a released cellular encoding
(e.g., cellular nucleic acid), as shown in step 5. In some
embodiments, the encoded nucleic acid primer labels the cellular
encoding. In some embodiments, the capture site of the encoded
nucleic acid primer prescribes a targeted cellular nucleic acid. In
some embodiments, an enzyme enables such labeling. As shown in step
6, the encoded cell encoding is labeled with the encoded nucleic
acid primer, while a created copy of the nucleic acid encoding is
displaced from the scaffold, wherein the process returns to step
3.
[0376] The cell may be lysed in order to release the desired
nucleic acids and to make the desired nucleic acids available for
amplification. In some embodiments, the encapsulation further
comprises a cell lysis buffer. In some embodiments, the lysis
buffer is added by pico-injection. In some embodiments, the lysis
buffer comprises a salt. In some embodiments, the lysis buffer
comprises a detergent. In some embodiments, the detergent is SDS,
Triton, or Tween. In some embodiments, the lysis buffer comprises a
chemical which causes cell lysis. In some embodiments, cell lysis
buffer is added to the encapsulation. In some embodiments, the cell
lysis buffer is added to the encapsulation by pico-injection.
[0377] In some embodiments, the encapsulation is a droplet, an
emulsion, a macrowell, a microwell, a bubble, or a microfluidic
confinement. Once an encapsulation is formed, any component inside
the encapsulation may remain in the encapsulation until the
encapsulation is destroyed or broken down. In some embodiments, the
encapsulations used in herein remain stable for at least 4 hours,
at least 12 hours, at least 1 day, at least 2 days, at least 3
days, or at least 1 week. In some embodiments, the encapsulations
are stable for the duration of the screen to be performed so that
no intermingling of reagents between encapsulations occurs.
[0378] In some embodiments, the encapsulation is a droplet. In some
embodiments, the droplet is at most 1 picoliter, at most 10
picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10
nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. In some embodiments, the droplet is at least 1 picoliter,
at least 10 picoliters, at least 100 picoliters, at least 1
nanoliter, at least 10 nanoliters, at least 100 nanoliters, or at
least 1 microliter in volume. In some embodiments, the droplet is
between about 200 picoliters and about 10 nanoliters.
[0379] In some embodiments, the droplet is an aqueous droplet in a
larger body of oil. In some embodiments, the oil acts as an
immiscible carrier fluid. In some embodiments, the droplets are
placed in an oil emulsion. In some embodiments, the oil comprises a
silicone oil, a fluorosilicone oil, a hydrocarbon oil, a mineral
oil, a paraffin oil, a halogenated oil, a fluorocarbon oil or any
combination thereof. In some embodiments, the oil comprises a
silicone oil. In some embodiments, the oil comprises a
fluorosilicone oil. In some embodiments, the oil comprises a
hydrocarbon oil. In some embodiments, the oil comprises a mineral
oil. In some embodiments, the oil comprises a paraffin oil. In some
embodiments, the oil comprises a halogenated oil. In some
embodiments, the oil is a fluorocarbon oil.
[0380] In some embodiments, an amplification mix is used to amplify
nucleic acid encodings to create additional primers for labeling
cellular nucleic acids of interest in a screen. In some
embodiments, the amplification mix is an isothermal amplification
mix. In some embodiments, the isothermal amplification mix
comprises reagents for loop-mediated isothermal amplification
(LAMP), strand displacement amplification (SDA), helicase-dependent
amplification (HAD), recombinase polymerase amplification (RPA),
rolling circle replication (RCA), or nicking enzyme amplification
reaction (NEAR). In some embodiments, the encapsulation further
comprises reagents for isothermal amplification of the target
nucleic acid. In some embodiments, the method comprises adding
reagents for isothermal amplification to the encapsulation. In some
embodiments, the reagents for isothermal amplification are targeted
to the specific nucleic acid sequence. In some embodiments, the
amplification mix comprises a nicking enzyme. In some embodiments,
the amplification mix comprises a nicking-enzyme amplification
mixture. In some embodiments, the nicking enzyme is an
endonuclease. In some embodiments, the nicking enzyme is a
restriction enzyme. In some embodiments, the amplification mix
comprises a reverse transcriptase. In some embodiments, the
amplification mix comprises an amplification enzyme. In some
embodiments, the amplification enzyme comprises a polymerase.
[0381] In some embodiments, the specific nucleotide sequence of
interest can be amplified within the encapsulation. In some
embodiments, the method comprises amplifying the cellular nucleic
acid comprising the specific nucleotide sequence to produce
amplified cellular nucleic acids. In some embodiments, amplifying
the cellular nucleic acids is accomplished by PCR. In some
embodiments, amplifying the cellular nucleic acids is accomplished
by isothermal amplification. In some embodiments, cellular nucleic
acids comprising the specific nucleotide sequence are amplified. In
some embodiments, the amplified cellular nucleic acid is barcoded
with the nucleic acid encoding the scaffold.
[0382] Any type of scaffold may be utilized in this method. In some
embodiments, the scaffold acts as a solid support and keeps the
nucleic acid encoding the scaffold linked in space to the scaffold.
In some embodiments, the scaffold is a structure with a plurality
of attachment points that allow linkage of one or more molecules.
In some embodiments, the nucleic acid encoding the scaffold is
bound to the scaffold. In some embodiments, the scaffold is a solid
support. In some embodiments, the scaffold is a bead, a fiber,
nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly.
[0383] In some embodiments, the scaffold is a bead. In some
embodiments, the bead is a polymer bead, a glass bead, a metal
bead, or a magnetic bead. In some embodiments, the bead is a
polymer bead. In some embodiments, the bead is a glass bead. In
some embodiments, the bead is a metal bead. In some embodiments,
the bead is a magnetic bead.
[0384] Beads for use in the systems and methods as described herein
can be any size. In some embodiments, the beads are at most 10 nm,
at most 100 nm, at most 1 .mu.m, at most 10 .mu.m, or at most 100
.mu.m in diameter. In some embodiments, the beads are at least 10
nm, at least 100 nm, at least 1 .mu.m, at least 10 .mu.m, or at
least 100 .mu.m in diameter. In some embodiments, the beads are
about 10 .mu.m to about 100 .mu.m in diameter.
[0385] The scaffolds may comprise effectors attached to the
scaffold. In some embodiments, the effectors are attached to the
scaffold by the cleavable linkers described herein. In some
embodiments, the cleavable linker is cleaved by electromagnetic
radiation, an enzyme, chemical reagent, heat, pH adjustment, sound
or electrochemical reactivity. In some embodiments, the cleavable
linker is cleaved from the scaffold using electromagnetic
radiation. In some embodiments, the amount of effector cleaved is
controlled by the intensity or duration of exposure to
electromagnetic radiation. In some embodiments, the cleavable
linker is cleaved using a cleavage reagent. In some embodiments,
the amount of effector cleaved is controlled by the concentration
of the cleavage reagent in the encapsulation. In some embodiments,
the effector is cleaved from the scaffold using an enzyme. In some
embodiments, the enzyme is a protease, a nuclease, or a hydrolase.
In some embodiments, the rate of effector cleavage is controlled by
the amount of enzyme in the encapsulation.
[0386] In some embodiments, the encoded nucleic acid primers
amplified in the present methods are utilized to detect and
quantify the amount of a target nucleic acid in the one or more
cells being screened with an effector utilizing the nucleic acid
encoded scaffold. In some embodiments, the encoded nucleic acid
primer hybridizes with a target nucleic acid.
[0387] In some embodiments, the specific nucleotide sequence acts
as an amplification primer with the target nucleic acid. In some
embodiments, the target nucleic acid is barcoded with the nucleic
acid encoding the scaffold using the specific nucleotide sequence.
In some embodiments, the target nucleic acid is barcoded with the
nucleic acid encoding the scaffold using the specific nucleotide
sequence which has been extended with the nucleic acid encoding the
scaffold.
[0388] The target nucleic acid can by any type of nucleic acid from
a cell. In some embodiments, the target nucleic acid is a target
mRNA. In some embodiments, the target mRNA encodes a protein of
interest. In some embodiments, the target nucleic acid comprises a
plurality of target mRNAs. In some embodiments, barcoding the
plurality of target mRNAs creates an expression fingerprint of the
cell treated with an effector. In some embodiments, the target
nucleic acid is genomic DNA. In some embodiments, the target
nucleic acid is mitochondrial DNA.
[0389] The methods provided herein increase target nucleic acid
capture and labeling with the nucleic acid encoding the scaffold.
In some embodiments, target nucleic acid capture is increased by at
least 10%, 25%, 50%, 100%, or 200% compared to a method without the
nicking enzyme that targets the specific nucleotide sequence. In
some embodiments, target nucleic acid labeling is increased by at
least 10%, 25%, 50%, 100%, or 200% compared to a method without the
nicking enzyme that targets the specific nucleotide sequence. In
some embodiments, target nucleic acid capture is increased by at
least 5-fold, at least 10-fold, at least 50-fold, or at least
100-fold compared to a method without the nicking enzyme that
targets the specific nucleotide sequence. In some embodiments,
target nucleic acid barcoding is increased by at least 5-fold, at
least 10-fold, at least 50-fold, or at least 100-fold compared to a
method without the nicking enzyme that targets the specific
nucleotide sequence.
[0390] In some embodiments, labeling the cellular nucleic acids
with encoded nucleic acid primers, as described herein, comprises
barcoding the cellular nucleic acids. The encapsulation can further
comprise barcoding reagents. In some embodiments, the encapsulation
further comprises barcoding reagents. In some embodiments, the
encapsulation further comprises barcoding reagents to effectuate
the barcoding of the cellular nucleic acids with the encoded
nucleic acid primers. In some embodiments, the encapsulation
further comprises barcoding reagents to effectuate the barcoding of
the nucleic acid encoding the scaffold with amplified nucleic
acids.
[0391] The barcoding reagents can be any set of reagents that allow
the joining of different nucleic acids. In some embodiments, the
barcoding reagents comprise an enzyme or chemical cross-linking
reagent. In some embodiments, the enzyme is a polymerase, a ligase,
a restriction enzyme, or a recombinase. In some embodiments, the
enzyme is a polymerase. In some embodiments, the additional
reagents comprise a chemical cross-linking reagent. In some
embodiments, the chemical cross-linking reagent is psoralen.
[0392] The amplification of primers described herein can be
performed at any time. In some embodiments, the above methods can
be performed at the same time as an effector screen. In some
embodiments, the cell is being screened against the effector. In
some embodiments, an effector screen occurs concomitantly with the
primer amplification method. In some embodiments, the primer
amplification method described herein occurs prior to an effector
screen. In some embodiments, the method is used to prepare the
nucleic acid encoded scaffold for a screen. In some embodiments,
the cell is used to prepare the nucleic acid encoded scaffold for a
screen.
Effector Load Normalization Method
[0393] Provided herein are methods of measuring effector loading
onto scaffolds and libraries of scaffolds. Generally, when a
library of encoded effectors bound to scaffolds is prepared, the
final concentration of effectors bound to the scaffolds varies
considerably among individual scaffolds. This is due to differences
in yield of each synthesis step of the effector built onto the
scaffold. Consequently, when ultimately used in a screen, different
samples may receive different dosages of effectors. This can skew
the results of the screen, as low potency, high abundance effectors
may drown out the signal of higher potency, low abundance
effectors. Thus, a method of determining effector loading onto
scaffolds in a library can help avoid this skewing of results.
[0394] Provided herein are methods of measuring effector loading on
scaffolds. In some embodiments, the method comprises (a) attaching
an effector subunit to effector attachment sites on a plurality of
scaffolds. In some embodiments, the method comprises (b) attaching
a detectable label to any remaining free effector attachment sites
on the plurality of scaffolds after the step of attaching an
effector subunit. In some embodiments, the method comprises (c)
removing a subset of scaffolds from the plurality. In some
embodiments, the method comprises (d) measuring the amount of
detectable label attached to the subset of scaffolds to determine
the amount of effector subunits successfully attached to the
effector attachment sites. In some embodiments, the method
comprises (e) optionally activating the attached effector subunits
to create new effector attachment sites. In some embodiments, the
listed steps are repeated until a desired effector is assembled. In
some embodiments, the scaffold further comprises a nucleic acid
encoding the effector. In some embodiments, the method further
comprises attaching nucleic acid encoding subunits to the scaffold
corresponding to the effector subunits as the effector subunits are
added to the scaffold. In some embodiments, there is no activating
step after the last effector subunit is attached.
[0395] In some embodiments, each effector subunit attached to the
scaffold is independently an amino acid, a small molecule fragment,
a nucleotide, or a compound. In some embodiments, each effector
subunit attached to the scaffold is an amino acid. In some
embodiments, each effector subunit attached to the scaffold is a
compound. In some embodiments, each effector subunit attached to
the scaffold is a small molecule fragment. In some embodiments,
each effector subunit attached to the scaffold is a nucleotide.
[0396] The effector attachment sites may have any group capable of
performing a chemical reaction. In some embodiments, the effector
attachment sites comprise reactive functionalities. In some
embodiments, the effector attachment sites comprise amino groups,
carboxylate groups, alcohol groups, phenol groups, alkyne groups,
aldehyde groups, or ketone groups. In some embodiments, the
effector attachment sites comprise amino or carboxylate groups. IN
some embodiments, the effector attachment sites comprise
biorthogonal or CLICK chemistry reactive groups.
[0397] The encoding subunits can comprise functional groups that
may react with the reactive functionalities on the effector
attachment site. In some embodiments, the encoding subunits form a
covalent bond with the reactive functionalities. In some
embodiments, the effector subunits comprise reactive groups
complementary to the effector attachment sites.
[0398] The detectable labels, in some embodiments, comprise
functional groups that may react with the reactive functionalities
on the effector attachment site. In some embodiments, the
detectable labels form a covalent bond with the reactive
functionalities. In some embodiments, the detectable labels
comprise reactive groups complementary to the effector attachment
sites.
[0399] The detectable label may any label that can produce a signal
that can be detected and quantified. In some embodiments, the
detectable label is a fluorophore.
[0400] In some embodiments, there is a yield associated with each
effector attachment step. In some embodiments, the yield is
measured a percentage of free effector attachment sites after the
step of attaching an effector subunit. In some embodiments, at most
10%, at most 20%, at most 30%, at most 40%, or at most 50% of the
effector attachment sites are free after the step of attaching the
effector subunit.
[0401] A subset of beads may be removed in order to quantify the
loading at each step of the synthesis of the desired effector. In
some embodiments, removing a subset of the plurality of scaffolds
comprises removing no more than 1%, no more than 2%, no more than
3%, no more than 5%, or no more than 10% of the remaining
scaffolds. In some embodiments, removing a subset of the plurality
of scaffolds comprises removing no more than 1% of the remaining
scaffolds. In some embodiments, removing a subset of the plurality
of scaffolds comprises removing no more than 2% of the remaining
scaffolds. In some embodiments, removing a subset of the plurality
of scaffolds comprises removing no more than 3% of the remaining
scaffolds. In some embodiments, removing a subset of the plurality
of scaffolds comprises removing no more than 5% of the remaining
scaffolds. In some embodiments, removing a subset of the plurality
of scaffolds comprises removing no more than 10% of the remaining
scaffolds.
[0402] In some embodiments, wherein measuring the amount of
detectable label attached to the subset of scaffolds to determine
the amount of effector subunits successfully attached to the
effector attachment sites comprises comparing the measurement of
the detectable label to the measurement of detectable label on a
scaffold without any effector subunits attached. In some
embodiments, the amount of effector subunits successfully attached
to the subset of scaffolds is expressed as a percentage of total
attachment sites occupied by the effector subunits.
[0403] To begin a new step of attaching effector subunits, a
previously attached effector subunit may need to be activated. In
some embodiments, activation reveals the presence of a new effector
attachment site. In some embodiments, optionally activating the
attached effector subunits to create a new effector attachment site
comprises removing a protecting group from the attached effector
subunit. In some embodiments, the protecting group is an amino
protecting group, a carboxylate protecting group, an alcohol
protecting group, a phenol protecting group, an alkyne protecting
group, an aldehyde protecting group, or a ketone protecting group.
In some embodiments, the protecting group is an amino protecting
group. In some embodiments, the amino protecting group is
9-fluorenylmethyloxcarbonyl (Fmoc), tert-butyloxycarbonyl (BOC),
carbobenzyloxy (Cbz), benzyl (Bz), tosyl (Ts) or trichloroethyl
chloroformate (Troc). In some embodiments, the protecting group is
a carboxylate protecting group. In some embodiments, the
carboxylate protecting group is a methyl ester, a benzyl ester, a
tert-butyl ester, a 2,6-disubstituted phenolic ester, a silyl
ester, or an orthoester. In some embodiments, the protecting group
is an alcohol protecting group. In some embodiments, the protecting
group is a phenol protecting group. In some embodiments, the
protecting group is an alkyne protecting group. In some
embodiments, the protecting group is an aldehyde protecting group.
In some embodiments, the protecting group is a ketone protecting
group.
[0404] The new effector attachment site can be any suitable
reactive functionality. In some embodiments, the new effector
attachment site is the same functionality as the previous effector
attachment site. In some embodiments, the new effector attachment
site is a different functionality from the previous effector
attachment site.
[0405] The desired effectors can be synthesized using any number of
steps and use any number of effector subunits. In some embodiments,
steps (a)-(e) are repeated at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 10, or at least 20 times.
In some embodiments, the desired effector is comprised of at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 10, or at least 20 subunits.
[0406] Any type of scaffold may be used with the methods and
systems provided herein. In some embodiments, the scaffold is a
bead, a fiber, a nanofibrous scaffold, a molecular cage, a
dendrimer, or a multi-valent molecular assembly. In some
embodiments, the scaffold is a bead. In some embodiments, the bead
is a polymer bead, a glass bead, a metal bead, or a magnetic bead.
In some embodiments, the bead is a polymer bead. In some
embodiments, the bead is a glass bead. In some embodiments, the
bead is a metal bead. In some embodiments, the bead is a magnetic
bead.
[0407] The beads utilized in the methods provided herein may be
made of any material. In some embodiments, the bead is a polymer
bead. In some embodiments, the bead comprises a polystyrene core.
In some embodiments, the beads are derivatized with polyethylene
glycol. In some embodiments, the beads are grafted with
polyethylene glycol. In some embodiments, the polyethylene glycol
contains reactive groups for the attachment of other
functionalities, such as effectors or encodings. In some
embodiments, the reactive group is an amino or carboxylate group.
In some embodiments, the reactive group is at the terminal end of
the polyethylene glycol chain. In some embodiments, the bead is a
TentaGel.RTM. bead.
[0408] The polyethylene glycol (PEG) attached to the beads may be
any size. In some embodiments, the PEG is up to 20 kDa. In some
embodiments, the PEG is up to 5 kDa. In some embodiments, the PEG
is about 3 kDa. In some embodiments, the PEG is about 2 to 3
kDa.
[0409] In some embodiments, the PEG group is attached to the bead
by an alkyl linkage. In some embodiments, the PEG group is attached
to a polystyrene bead by an alkyl linkage. In some embodiments, the
bead is a TentaGel.RTM. M resin.
[0410] In some embodiments, the bead comprises a PEG attached to a
bead through an alkyl linkage and the bead comprises two bifunction
species. In some embodiments, the beads comprise surface
modification on the outer surface of the beads that are
orthogonally protected to reactive sites in the internal section of
the beads. In some embodiments the beads comprise both cleavable
and non-cleavable ligands. In some embodiments, the bead is a
TentaGel.RTM. B resin.
[0411] Beads for use in the systems and methods as described herein
can be any size. In some embodiments, the beads are at most 10 nm,
at most 100 nm, at most 1 .mu.m, at most 10 .mu.m, or at most 100
.mu.m in diameter. In some embodiments, the beads are at least 10
nm, at least 100 nm, at least 1 .mu.m, at least 10 .mu.m, or at
least 100 .mu.m in diameter. In some embodiments, the beads are
about 10 .mu.m to about 100 .mu.m in diameter.
[0412] Nucleic acids encoding the effector are utilized in the
described method. The nucleic acids encoding the effector may be
bound to the scaffold as a pre-synthesized nucleic acid,
synthesized concomitantly with the effector, or synthesized on the
scaffold prior to synthesis of the effector. In some embodiments, a
nucleic acid encoding the effector is attached to the scaffold. In
some embodiments, the method further comprises attaching nucleic
acid encoding subunits to the scaffold corresponding to the
effector subunits as the effector subunits are added to the
scaffold.
[0413] The methods described herein are especially useful when
applied to libraries of effectors on scaffolds. In some
embodiments, libraries of effectors are synthesized in parallel. In
some embodiments, libraries of effectors are synthesized in
individual wells. In some embodiments, libraries of effectors are
synthesized using high-throughput synthesis techniques. In some
embodiments, a library of effector loaded scaffolds are synthesized
concurrently. The library of effector loaded scaffolds can be any
size. In some embodiments, the library comprises at least 10.sup.3,
10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, 10.sup.15,
or 10.sup.16 effector loaded scaffolds. In some embodiments, each
effector loaded scaffold comprises a unique effector. In some
embodiments, some effector loaded scaffolds are repeated in the
library.
[0414] In some embodiments, subsets of beads from an effector
attachment step from the library are pooled prior to detection of
the detectable label. In some embodiments, subsets of beads from
all scaffolds in the library are pooled together. In some
embodiments, a portion of the subset of beads from the scaffolds in
the library are pooled together.
[0415] The pooled subsets of beads are placed into encapsulations
for further analysis. An encapsulation refers to the formation of a
compartment within a larger system. In some embodiments, the
encapsulation is a droplet, an emulsion, a macrowell, a microwell,
a bubble, or a microfluidic confinement. In some embodiments, a
majority of the encapsulations comprise a single scaffold.
[0416] In some embodiments, the encapsulation is a droplet. In some
embodiments, the droplet is at most 1 picoliter, at most 10
picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10
nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. In some embodiments, the droplet is at least 1 picoliter,
at least 10 picoliters, at least 100 picoliters, at least 1
nanoliter, at least 10 nanoliters, at least 100 nanoliters, or at
least 1 microliter in volume. In some embodiments, the droplet is
between about 200 picoliters and about 10 nanoliters.
[0417] In some embodiments, the droplet is an aqueous droplet in a
larger body of oil. In some embodiments, the droplets are placed in
an oil emulsion. In some embodiments, the oil comprises a silicone
oil, a fluorosilicone oil, a hydrocarbon oil, a mineral oil, a
paraffin oil, a halogenated oil, or any combination thereof. In
some embodiments, the oil comprises a silicone oil. In some
embodiments, the oil comprises a fluorosilicone oil. In some
embodiments, the oil comprises a hydrocarbon oil. In some
embodiments, the oil comprises a mineral oil. In some embodiments,
the oil comprises a paraffin oil. In some embodiments, the oil
comprises a halogenated oil.
[0418] After the scaffolds are placed into encapsulations, the
level of fluorophore bound to the scaffolds may be assessed. In
some embodiments, scaffolds from the subset of scaffolds are binned
according to the amount of detectable label detected. In some
embodiments, each bin comprises a unique range of detectable label
detected. In some embodiments, the bins correspond to 0-25%,
25-50%, 50-75%, and 75-100% loading of detectable label detected
compared to scaffolds where no effector subunit was loaded. In some
embodiments, the bins correspond to 0-20%, 20-40%, 40-60%, 60-80%,
and 80-100% loading of detectable label detected compared to
scaffolds where no effector subunit was loaded. In some
embodiments, the bins correspond to 0-10%, 10-20%, 20-30%, 30-40%,
40-50%, 50-60%, 60-70%, 70-80%, 80-90%, and 90-100% loading of
detectable label detected compared to scaffolds where no effector
subunit was loaded. Any combination of bins is acceptable to use
with the methods and systems provided herein.
[0419] The bins may then be sequenced to reveal which effectors had
particular yields in the attachment step. In some embodiments, the
method further comprises the step of sequencing encoding nucleic
acids or encoding nucleic acid subunits of the pools to reveal
which effector subunits correspond to a particular yield in a step
of attaching effector subunits to effector attachment sites. In
some embodiments, the sequencing step is performed each time steps
(a)-(e) are repeated. In some embodiments, yields of each step
(a)-(e) for each unique scaffold are collected to create a dataset
which reveals the loading of the complete desired effector on each
scaffold. In some embodiments, yields of attachment of each encoder
subunit for each unique scaffold are collected to create a dataset
which reveals the loading of the complete desired effector on each
scaffold. In some embodiments, the loading of desired effector on
each unique scaffold is calculated.
Screening Devices and Methods of Use
[0420] Further provided herein are devices for use in screening
encoded effectors and methods of use. In some embodiments, the
devices provided herein lock an encoded effector into a location.
In some embodiments, the sample being screened is similarly fixed
in a position. By locking the two in place, the risk of
encapsulations breaking down or merging with other encapsulations
may be minimized. In some embodiments, the need for encapsulation
is eliminated entirely. Additionally, knowledge of the structure of
the effector at particular locations of the device may allow a user
to easily determine which effectors had a desired effect on a
sample. The devices described below are compatible with any of the
methods described elsewhere herein.
[0421] Nucleic Acid Patch Array
[0422] Provided herein is an array for screening encoded beads. The
array can comprise nucleic acid patches interspersed on a
hydrophobic surface. The positioning of the nucleic acid patches on
the hydrophobic patch can be such that when a liquid media is added
to the device, droplets form encapsulating the nucleic acid
patches, but the hydrophobic surfaces remain free of media. In some
embodiments, each nucleic acid patch is encapsulated in its own
droplet. In some embodiments, there is no liquid or fluid
connection between the different nucleic acid patches after the
media is added. The nucleic acid patches may be able to bind beads,
cells, or both. Additionally, the array may further comprise
channels beneath the surface. The channels can have terminal ends
that allow for fluids to flow through the channels to the nucleic
acid patches. Such channels can allow for the addition of reagents
to the nucleic acid patches.
[0423] In one aspect, provided herein, is an array device for
screening encoded beads. In some embodiments, the device comprises
a hydrophobic surface. In some embodiments, the device comprises
nucleic acid patches. In some embodiments, the nucleic acid patches
are interspersed on the hydrophobic surface. In some embodiments,
the hydrophobic surface and nucleic acid patches are configured
such that when a proscribed amount of media is deployed across the
surface each nucleic acid patch is covered with media. In some
embodiments, the hydrophobic surface between the nucleic acid
patches does not contain media.
[0424] The array device may comprise channels. In some embodiments,
the device comprises one or more channels beneath the hydrophobic
surface. In some embodiments, the channels from a network. In some
embodiments, the channels are microfluidic channels. In some
embodiments, the channels are branched. In some embodiments, the
channels comprise terminal ends within nucleic acid patches. In
some embodiments, the channels comprise terminal ends within each
nucleic acid patch of the array.
[0425] The channels may be configured to deliver liquid solutions
to the nucleic acid patches. In some embodiments, the channels are
configured to deliver reagents to the nucleic acid patches. In some
embodiments, the reagents are delivered as a liquid solution. In
some embodiments, the liquid solution is an aqueous solution.
[0426] The channels may be any size. In some embodiments, the
channels have a diameter of about 0.1 .mu.m, about 0.5 .mu.m, about
1 .mu.m, about 5 .mu.m, about 10 .mu.m, or about 20 .mu.m. In some
embodiments, the channels have a diameter of up to about 0.1 .mu.m,
up to about 0.5 .mu.m, up to about 1 .mu.m, up to about 5 .mu.m, up
to about 10 .mu.m, or up to about 20 .mu.m. In some embodiments,
the channels have a diameter of at least about 0.1 .mu.m, at least
about 0.5 .mu.m, at least about 1 .mu.m, at least about 5 .mu.m, at
least about 10 .mu.m, or at least about 20 .mu.m.
[0427] The hydrophobic surface may be made of any suitable
hydrophobic material. In some embodiments, the hydrophobic surface
is comprised of a hydrophobic polymer. In some embodiments, the
hydrophobic surface comprises a hydrophobic polymer. In some
embodiments, the hydrophobic polymer comprises a polyacrylic, a
polyamide, a polycarbonate, a polydiene, a polyester, a polyether,
a polyfluorocarbon, a polyolefin, a polystyrene, a polyvinyl
acetal, a polyvinyl chloride, a polyvinyl ester, a polyvinyl ether,
a polyvinyl ketone, a polyvinyl pyridine, a polyvinylpyrrolidone, a
polysilane, a polyfluorosilane, a poly perfluorosilane or a
combination thereof. In some embodiments, the hydrophobic polymer
comprises a polyfluorocarbon. In some embodiments, the hydrophobic
polymer comprises a polyperfluorocarbon. In some embodiments, the
hydrophobic polymer is fluorinated.
[0428] The hydrophobic surface may be a surface functionalized with
groups having hydrophobic properties. In some embodiments, the
hydrophobic surface is a surface functionalized with hydrophobic
groups. In some embodiments, the hydrophobic groups are fatty
acids, alkyl groups, alkoxy groups, aromatic groups, alkyl silanes,
fluorosilanes, perfluorosilanes, or combinations thereof. In some
embodiments, the hydrophobic groups are perfluorosilanes. In some
embodiments, the hydrophobic groups are fatty acids. In some
embodiments, the hydrophobic groups are fluorinated fatty acids. In
some embodiments, the hydrophobic groups are perfluorinated fatty
acids. In some embodiments, the hydrophobic groups are
fluorinated.
[0429] The hydrophobic surface may exhibit desired binding
properties. In some embodiments, cells do not bind to the
hydrophobic surface. In some embodiments, cells do not grow on the
hydrophobic surface.
[0430] The nucleic acid patches may exhibit desired binding
properties. In some embodiments, the nucleic acid patches bind
cells. In some embodiments, the nucleic acid patches bind cells
through non-specific interaction. In some embodiments, the nucleic
acid patches bind cells through specific interaction. In some
embodiments, the nucleic acid patches are configured to attract
media. In some embodiments, single nucleic acid patches
encapsulated within single droplets of the media. In some
embodiments, the nucleic acid patches are capable of binding beads.
In some embodiments, the beads are nucleic acid encoded beads. In
some embodiments, the nucleic acid patches bind beads. In some
embodiments, the nucleic acid patches comprise nucleic acids
capable of binding nucleic acid encoded beads. In some embodiments,
the nucleic acids bind beads non-specifically, by binding a
complementary nucleic acid on the bead, or by binding another group
on the bead. In some embodiments, the nucleic acids bind nucleic
acid encoded beads non-specifically, by binding a complementary
nucleic acid on the bead, or by binding another group on the
bead.
[0431] The nucleic acid patches may comprise any type of nucleic
acid. In some embodiments, the nucleic acid patches comprise DNA,
RNA, combinations thereof. In some embodiments, the nucleic acid
patches comprise DNA. In some embodiments, the nucleic acid patches
comprise double-stranded DNA. In some embodiments, the nucleic acid
patches comprise single-stranded DNA. In some embodiments, the
nucleic acid patches comprise RNA. In some embodiments, the nucleic
acid patches comprise single-stranded RNA. In some embodiments, the
nucleic acid patches comprise double-stranded RNA.
[0432] The nucleic acid patches may be any size. In some
embodiments, the nucleic acid patches are up to about 1 .mu.m.sup.2
in size, up to about 10 .mu.m.sup.2 in size, up to about 100
.mu.m.sup.2 in size, up to about 1000 .mu.m.sup.2 in size, or up to
about 10000 .mu.m.sup.2 in size. In some embodiments, the nucleic
acid patches are at least about 1 .mu.m.sup.2 in size, at least
about 10 .mu.m.sup.2 in size, at least about 100 .mu.m.sup.2 in
size, at least about 1000 .mu.m.sup.2 in size, or at least about
10000 .mu.m.sup.2 in size. In some embodiments, the nucleic acid
patches are about 1 .mu.m.sup.2 in size, about 10 .mu.m.sup.2 in
size, about 100 .mu.m.sup.2 in size, about 1000 .mu.m.sup.2 in
size, or about 10000 .mu.m.sup.2 in size.
[0433] The nucleic acid patches may be separated by a defined
distance. In some embodiments, the nucleic acid patches are
separated by up to about 1 .mu.m, up to about 10 .mu.m, up to about
100 .mu.m, up to about 1000 .mu.m, or up to about 10000 .mu.m. In
some embodiments, the nucleic acid patches are separated by at
least about 1 .mu.m, at least about 10 .mu.m, at least about 100
.mu.m, at least about 1000 .mu.m, or at least about 10000 .mu.m. In
some embodiments, the nucleic acid patches are separated by about 1
.mu.m, about 10 .mu.m, about 100 .mu.m, about 1000 .mu.m, or about
10000 .mu.m.
[0434] The nucleic acid patches may be arranged on the surface in
any configuration. In some embodiments, the nucleic acid patches
are arranged in a grid pattern. In some embodiments, the nucleic
acid patches are distributed randomly. In some embodiments, the
nucleic acid patches are arranged in a circular configuration.
[0435] The nucleic acid patches may be of any density on the
surface. In some embodiments, the density of nucleic acid patches
is at least 100 patches/cm.sup.2, at least 1000 patches/cm.sup.2,
at least 10000 patches/cm.sup.2, at least 100000 patches/cm.sup.2,
at least 1000000 patches/cm.sup.2, or at least 10000000
patches/cm.sup.2. In some embodiments, the density of nucleic acid
patches is about 100 patches/cm.sup.2, about 1000 patches/cm.sup.2,
about 10000 patches/cm.sup.2, about 100000 patches/cm.sup.2, about
1000000 patches/cm.sup.2, or about 10000000 patches/cm.sup.2.
[0436] The array device may be any size. In some embodiments, the
surface area of the device is at least 1 cm.sup.2, at least 5
cm.sup.2, at least 10 cm.sup.2, at least 25 cm.sup.2, at least 50
cm.sup.2, at least 100 cm.sup.2, at least 500 cm.sup.2, or at least
1000 cm.sup.2. In some embodiments, the surface area of the device
is about 1 cm.sup.2, about 5 cm.sup.2, about 10 cm.sup.2, about 25
cm.sup.2, about 50 cm.sup.2, about 100 cm.sup.2, about 500
cm.sup.2, or about 1000 cm.sup.2. In some embodiments, the surface
area of the device is at most 1 cm.sup.2, at most 5 cm.sup.2, at
most 10 cm.sup.2, at most 25 cm.sup.2, at most 50 cm.sup.2, at most
100 cm.sup.2, at most 500 cm.sup.2, or at most 1000 cm.sup.2.
[0437] In one aspect, provided herein, is a method of performing a
screen using the arrays described herein. In some embodiments, the
method comprises binding nucleic acid encoded beads to the nucleic
acid patches of the array. In some embodiments, the method
comprises sequencing the nucleic acid encoded beads. In some
embodiments, cells are bound to the nucleic acid patches. In some
embodiments, an assay is performed on the array.
[0438] The beads may contain an effector. In some embodiments the
beads comprise encoded effectors. In some embodiments, the beads
comprise nucleic acid encoded effectors. In some embodiments, the
effectors are released from the beads. In some embodiments, the
effectors are released by cleaving a cleavable linker. In some
embodiments, the cleavable linker is cleaved by electromagnetic
radiation. In some embodiments, the cleavable linker is cleaved by
a cleaving reagent. In some embodiments, the method comprises
adding a cleaving reagent to the nucleic acid patches.
[0439] In some embodiments, reagents are added through the channels
beneath the surface. In some embodiments, the cleaving reagent is
added through the channels. In some embodiments, detection reagents
are added through the channels.
[0440] Sequencing the beads allows the locations of encoded beads
in space to be determined. In some embodiments, sequencing the
beads allows determination of the physical location of specific
nucleic acid encoded beads.
[0441] Any assay may be performed on the array. In some
embodiments, the assay produces a detectable signal. In some
embodiments, the detectable signal is electromagnetic radiation. In
some embodiments, the signal is fluorescence or luminescence.
[0442] The nucleic acid patches can bind any amount of cells or
beads. In some embodiments, each nucleic acid patch binds a single
bead. In some embodiments, each nucleic acid patch binds a single
cell. In some embodiments, each nucleic acid patch binds a single
bead and a single cell. In some embodiments, each nucleic acid
patch binds a plurality of beads. In some embodiments, each nucleic
acid patch binds a plurality of cells.
Numbered Embodiments
[0443] The following embodiments recite nonlimiting permutations of
combinations of features disclosed herein. Other permutations of
combinations of features are also contemplated. In particular, each
of these numbered embodiments is contemplated as depending from or
relating to every previous or subsequent numbered embodiment,
independent of their order as listed.
[0444] Embodiment 1: A method for screening an encoded effector,
the method comprising: a) providing a sample, an encoded effector,
and an encoding in an encapsulation; wherein the encoded effector
is bound to a scaffold by a cleavable linker; b) activating the
cleavable linker using an activating reagent; c) cleaving the
cleavable linker so as to release a predetermined amount of the
encoded effector; d) detecting a signal from the encapsulation,
wherein the signal results from an interaction of the encoded
effector and the sample; and e) sorting the encapsulation based on
the detection of the signal. Embodiment 2: The method of Embodiment
1, wherein the activating reagent is provided with the
encapsulation in step (a). Embodiment 3: The method of Embodiment
1, wherein the activating reagent is added into the encapsulation.
Embodiment 4: The method of Embodiment 3, wherein the activating
reagent is added into the encapsulation by pico-injection.
Embodiment 5: The method of Embodiment 1, wherein the activating
reagent is added to the encapsulation by droplet merging, wherein
the encapsulation is a droplet. Embodiment 6: The method of
Embodiment 1, wherein the activating reagent is a disulfide
reducing reagent. Embodiment 7: The method of Embodiment 1, wherein
the activating reagent is a tetrazine. Embodiment 8: The method of
Embodiment 1, wherein the concentration of the activating reagent
used to activate the cleavable linker is at most 100 picomolar
(pM), at most 500 pM, at most 1 nanomolar (nM), at most 10 nM, at
most 100 nM, at most 1 micromolar (.mu.M), at most 10 .mu.M, at
most 100 .mu.M, at most 1 millimolar (mM), at most 10 mM, at most
100 mM, or at most 500 mM. Embodiment 9: The method of Embodiment
1, wherein the activate reagent is added from a stock solution at
least 2.times., 5.times., 10.times., 20.times., 30.times.,
50.times., 100.times., 500.times., or 1000.times. more concentrated
than the desired final concentration in the encapsulation.
Embodiment 10: The method of Embodiment 1, wherein the
predetermined amount of effector released from the scaffold is to a
concentration of at least 100 pM, at least 500 pM, at least 1 nM,
at least 10 nM, at least 100 nM, at least 1 .mu.M, at least 10
.mu.M, at least 100 .mu.M, at least 1 mM, at least 10 mM, at least
50 mM, at least 100 mM, or at least 250 mM. Embodiment 11: The
method of Embodiment 1, wherein the cleavable linker is a disulfide
or substituted trans-cyclooctene. Embodiment 12: The method of
Embodiment 1, wherein the sample comprises at least one cell, a
protein, an enzyme, a nucleic acid, a cellular lysate, a tissue
extract, or combinations thereof. Embodiment 13: The method of
Embodiment 12, wherein the sample is one or more cells, a protein,
or an enzyme. Embodiment 14: The method of Embodiment 1, wherein
the scaffold is a bead, a fiber, a nanofibrous scaffold, a
molecular cage, a dendrimer, or a multi-valent molecular assembly.
Embodiment 15: The method of Embodiment 14, wherein the scaffold is
polymer-bead, a glass bead, a metal bead, or a magnetic bead.
Embodiment 16: The method of Embodiment 15, wherein the bead is
about 1 .mu.m to about 100 .mu.m in diameter. Embodiment 17: The
method of Embodiment 15, wherein the bead is about 1 .mu.m to about
20 .mu.m in diameter. Embodiment 18: The method of Embodiment 1,
wherein the encoded effector is a peptide, a compound, protein, an
enzyme, a macrocycle compound, or a nucleic acid. Embodiment 19:
The method of Embodiment 18, wherein the encoded effector is a
non-natural peptide. Embodiment 20: The method of Embodiment 18,
wherein the encoded effector is a polymer. Embodiment 21: The
method of Embodiment 18, wherein the compound is a drug-like small
molecule. Embodiment 22: The method of Embodiment 1, wherein the
encapsulation is a droplet. Embodiment 23: The method of Embodiment
22, wherein the droplet is at most 1 picoliter, at most 10
picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10
nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. Embodiment 24: The method of Embodiment 1, wherein the
signal comprises electromagnetic radiation, thermal radiation, a
visual change in the sample, or combinations thereof. Embodiment
25: The method of Embodiment 24, wherein the electromagnetic
radiation is in the visible spectrum. Embodiment 26: The method of
Embodiment 24, wherein the electromagnetic radiation is
fluorescence or luminescence. Embodiment 27: The method of
Embodiment 26, wherein the signal is fluorescence emitted by a
TaqMan probe or a molecular beacon. Embodiment 28: The method of
Embodiment 24, wherein the signal comprises thermal radiation
detected with an infrared camera. Embodiment 29: The method of
Embodiment 24, wherein the signal comprises a morphological or
visual change in the sample measured by recording a series of
images of the encapsulation. Embodiment 30: The method of
Embodiment 1, further comprising incubating the encapsulation for a
period of time to allow the effector and the sample to interact.
Embodiment 31: The method of Embodiment 30, wherein the period of
time is controlled by a residence time as the encapsulation travels
through a microfluidic channel, wherein the residence time of each
encapsulation is within a maximum dispersion ratio of the
incubation period of time for the plurality of encapsulations,
wherein the dispersion ratio is based on a deviation about an
average residence time of the plurality of encapsulations passing
through a region of the microfluidic device. Embodiment 32: The
method of Embodiment 31, wherein the maximum dispersion is at most
from about 3% to about 10%. Embodiment 33: The method of Embodiment
1, wherein sorting the encapsulation comprises placing the droplet
into a first collection tube if the signal is at or above a
predetermined threshold or placing the droplet into a second
collection tube if the signal is below a predetermined threshold.
Embodiment 34: The method of Embodiment 1, wherein sorting the
encapsulation comprises using a waveform pulse generator to move
the encapsulation to a collection tube by an electrical field
gradient, by sound, by a diaphragm, by modifying geometry of the
microfluidic channel, or by changing the pressure of the
microfluidic channel. Embodiment 35: The method of Embodiment 1,
wherein the encapsulation is an emulsion in an oil. Embodiment 36:
The method of Embodiment 1, wherein the encoding is a nucleic acid
and the method further comprises the step of sequencing the
encoding nucleic acid. Embodiment 37: The method of Embodiment 36,
wherein the encoding is cleaved from the scaffold prior to
sequencing. Embodiment 38: The method of Embodiment 37, wherein
cleaving the nucleic acid encoding from the scaffold comprises
cleaving a cleavable linker with a cleaving reagent or through
electromagnetic radiation.
[0445] Embodiment 39: A method for screening an encoded effector,
the method comprising: a) providing a sample, an encoded effector,
and an encoding in an encapsulation; wherein the encoded effector
is bound to a scaffold by a cleavable linker; b) cleaving the
cleavable linker with a cleaving reagent, wherein the cleaving
reagent is added at a concentration configured to release a
predetermined amount of the encoded effector; c) detecting a signal
from the encapsulation, wherein the signal results from an
interaction of the encoded effector and the sample; and d) sorting
the encapsulation based on the detection of the signal. Embodiment
40: The method of Embodiment 39, wherein the cleaving reagent is
added to the encapsulation by pico-injection. Embodiment 41: The
method of Embodiment 39, wherein the cleaving reagent is added to
the encapsulation at a step separate from forming the
encapsulation. Embodiment 42: The method of Embodiment 39, wherein
the cleaving reagent is added to the encapsulation using a solution
comprising the cleaving reagent and the sample prior to formation
of the encapsulation. Embodiment 43: The method of Embodiment 39,
wherein the concentration of cleaving reagent used to cleave the
cleavable linker is at most 100 picomolar (pM), at most 500 pM, at
most 1 nanomolar (nM), at most 10 nM, at most 100 nM, at most 1
micromolar (.mu.M), at most 10 .mu.M, at most 100 .mu.M, at most 1
millimolar (mM), at most 10 mM, at most 100 mM, or at most 500 mM.
Embodiment 44: The method of Embodiment 39, wherein the cleaving
reagent is added from a stock solution at least 2.times., 5.times.,
10.times., 20.times., 30.times., 50.times., 100.times., 500.times.,
or 1000.times. more concentrated than the desired final
concentration in the encapsulation. Embodiment 45: The method of
Embodiment 39, wherein the predetermined amount of effector
released from the scaffold is to a concentration of at least 100
pM, at least 500 pM, at least 1 nM, at least 10 nM, at least 100
nM, at least 1 .mu.M, at least 10 .mu.M, at least 100 .mu.M, at
least 1 mM, at least 10 mM, at least 50 mM, at least 100 mM, or at
least 250 mM. Embodiment 46: The method of Embodiment 39, wherein
the cleavable linker is a disulfide or substituted
trans-cyclooctene. Embodiment 47: The method of Embodiment 39,
wherein the cleaving reagent is a disulfide reducing reagent.
Embodiment 48: The method of Embodiment 39, wherein the cleaving
reagent is a tetrazine. Embodiment 49: The method of Embodiment 39,
wherein the sample comprises at least one cell, a protein, an
enzyme, a nucleic acid, a cellular lysate, a tissue extract, or
combinations thereof. Embodiment 50: The method of Embodiment 49,
wherein the sample is one or more cells, a protein, or an enzyme.
Embodiment 51: The method of Embodiment 49, wherein the scaffold is
a bead, a fiber, a nanofibrous scaffold, a molecular cage, a
dendrimer, or a multi-valent molecular assembly. Embodiment 52: The
method of Embodiment 51, wherein the scaffold is polymer-bead, a
glass bead, a metal bead, or a magnetic bead. Embodiment 53: The
method of Embodiment 52, wherein the bead is about 1 .mu.m to about
100 .mu.m in diameter. Embodiment 54: The method of Embodiment 52,
wherein the bead is about 1 .mu.m to about 20 .mu.m in diameter.
Embodiment 55: The method of Embodiment 39, wherein the encoded
effector is a peptide, a compound, protein, an enzyme, a macrocycle
compound, or a nucleic acid. Embodiment 56: The method of
Embodiment 55, wherein the encoded effector is a non-natural
peptide. Embodiment 57: The method of Embodiment 55, wherein the
encoded effector is a polymer. Embodiment 58: The method of
Embodiment 55, wherein the compound is a drug-like small molecule.
Embodiment 59: The method of Embodiment 39, wherein the
encapsulation is a droplet. Embodiment 60: The method of Embodiment
59, wherein the droplet is at most 1 picoliter, at most 10
picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10
nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. Embodiment 61: The method of Embodiment 39, wherein the
signal comprises electromagnetic radiation, thermal radiation, a
visual change in the sample, or combinations thereof. Embodiment
62: The method of Embodiment 61, wherein the electromagnetic
radiation is in the visible spectrum. Embodiment 63: The method of
Embodiment 61, wherein the electromagnetic radiation is
fluorescence or luminescence. Embodiment 64: The method of
Embodiment 63, wherein the signal is fluorescence emitted by a
TaqMan probe or a molecular beacon. Embodiment 65: The method of
Embodiment 61, wherein the signal comprises thermal radiation
detected with an infrared camera. Embodiment 66: The method of
Embodiment 61, wherein the signal comprises a morphological or
visual change in the sample measured by recording a series of
images of the encapsulation. Embodiment 67: The method of
Embodiment 39, further comprising incubating the encapsulation for
a period of time to allow the effector and the sample to interact.
Embodiment 68: The method of Embodiment 67, wherein the period of
time is controlled by a residence time as the encapsulation travels
through a microfluidic channel, wherein the residence time of each
encapsulation is within a maximum dispersion ratio of the
incubation period of time for the plurality of encapsulations,
wherein the dispersion ratio is based on a deviation about an
average residence time of the plurality of encapsulations passing
through a region of the microfluidic device. Embodiment 69: The
method of Embodiment 68, wherein the maximum dispersion is at most
from about 3% to about 10%. Embodiment 70: The method of Embodiment
39, wherein sorting the encapsulation comprises placing the droplet
into a first collection tube if the signal is at or above a
predetermined threshold or placing the droplet into a second
collection tube if the signal is below a predetermined threshold.
Embodiment 71: The method of Embodiment 39, wherein sorting the
encapsulation comprises using a waveform pulse generator to move
the encapsulation to a collection tube by an electrical field
gradient, by sound, by a diaphragm, by modifying geometry of the
microfluidic channel, or by changing the pressure of the
microfluidic channel. Embodiment 72: The method of Embodiment 39,
wherein the encapsulation is an emulsion in an oil. Embodiment 73:
The method of Embodiment 39, wherein the encoding is a nucleic acid
and the method further comprises the step of sequencing the
encoding nucleic acid. Embodiment 74: The method of Embodiment 73,
wherein the encoding is cleaved from the scaffold prior to
sequencing. Embodiment 75: The method of Embodiment 74, wherein
cleaving the nucleic acid encoding from the scaffold comprises
cleaving a cleavable linker with a cleaving reagent or through
electromagnetic radiation.
[0446] Embodiment 76: A method for screening an encoded effector,
the method comprising: a) providing at least one cell and a
scaffold in an encapsulation, wherein the scaffold comprises an
encoded effector bound to the scaffold by a photocleavable linker
and a nucleic acid encoding the effector; b) cleaving the
photocleavable linker to release the encoded effector from the
scaffold; and c) detecting a signal from the droplet, wherein the
signal results from an interaction between the encoded effector and
the at least one cell. Embodiment 77: The method of Embodiment 76,
further comprising sorting the encapsulation based on the detection
of the signal. Embodiment 78: The method of Embodiment 77, wherein
sorting the droplet comprises using a waveform pulse generator to
move the droplet to a collection tube by an electrical field
gradient, by sound, by a diaphragm, by modifying geometry of the
microfluidic channel, or by changing the pressure of the
microfluidic channel. Embodiment 79: The method of Embodiment 77,
further comprising identifying the encoded effector by sequencing
the nucleic acid encoding the effector. Embodiment 80: The method
of Embodiment 76, further comprising barcoding the nucleic acid
encoding the effector. Embodiment 81: The method of Embodiment 80,
wherein the barcoding is via the addition of a reagent into the
droplet. Embodiment 82: The method of Embodiment 76, wherein
cleaving the photocleavable linker releases a pre-determined amount
of the encoded effector into the droplet. Embodiment 83: The method
of Embodiment 76, wherein the photocleavable linker is cleaved
using electromagnetic radiation. Embodiment 84: The method of
Embodiment 76, wherein cleaving the photocleavable linker comprises
exposing the encapsulation to a light from a light source.
Embodiment 85: The method of Embodiment 84, wherein the light is a
calibrated amount of light. Embodiment 86: The method of Embodiment
84, wherein the light is UV light. Embodiment 87: The method of
Embodiment 84, wherein the light intensity of a light is from about
0.01 J/cm.sup.2 to about 200 J/cm.sup.2. Embodiment 88: The method
of Embodiment 76, wherein detecting the signal comprises detecting
morphological changes in the sample measured by recording a series
of images of the droplet or detecting fluorescence emitted by a
molecular beacon or probe. Embodiment 89: The method of Embodiment
76, wherein the interaction between the encoded effector and the
cell comprises inhibition of one or more cellular components.
Embodiment 90: The method of Embodiment 76, further comprising
identifying the encoded effector by sequencing the nucleic acid
encoding the effector. Embodiment 91: The method of Embodiment 76,
wherein two or more cells are provided with the scaffold.
Embodiment 92: The method of Embodiment 76, further comprising
providing an activating reagent to activate the photocleavable
linker, so as to enable the photocleavable linker to be cleaved
from the encoded effector. Embodiment 93: The method of Embodiment
92, wherein the activating reagent is provided with the
encapsulation. Embodiment 94: The method of Embodiment 92, wherein
the activating reagent is added into the encapsulation through pico
injection or droplet merging. Embodiment 95: The method Embodiment
76, further comprising the step of lysing the one or more cells.
Embodiment 96: The method of Embodiment 95, wherein lysing the one
or more cells comprises adding lysis buffer to the encapsulation.
Embodiment 97: The method of Embodiment 96, wherein the lysis
buffer is added to the encapsulation by pico-injection. Embodiment
98: The method of Embodiment 76, wherein the scaffold is a bead, a
fiber, a nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly. Embodiment 99: The method of
Embodiment 98, wherein the scaffold is polymer-bead, a glass bead,
a metal bead, or a magnetic bead. Embodiment 100: The method of
Embodiment 98, wherein the bead is about 1 .mu.m to about 100 .mu.m
in diameter. Embodiment 101: The method of Embodiment 98, wherein
the bead is about 1 .mu.m to about 20 .mu.m in diameter. Embodiment
102: The method of Embodiment 76, wherein the encoded effector is a
peptide, a compound, protein, an enzyme, a macrocycle compound, or
a nucleic acid. Embodiment 103: The method of Embodiment 102,
wherein the encoded effector is a non-natural peptide. Embodiment
104: The method of Embodiment 102, wherein the encoded effector is
a polymer. Embodiment 105: The method of Embodiment 102, wherein
the compound is a drug-like small molecule. Embodiment 106: The
method of Embodiment 76, wherein the encapsulation is a droplet.
Embodiment 107: The method of Embodiment 106, wherein the droplet
is at most 1 picoliter, at most 10 picoliters, at most 100
picoliters, at most 1 nanoliter, at most 10 nanoliters, at most 100
nanoliters, or at most 1 microliter in volume. Embodiment 108: The
method of Embodiment 76, further comprising incubating the droplet
for a period of time to allow the effector and the at least one
cell to interact. Embodiment 109: The method of Embodiment 108,
wherein the period of time is controlled by a residence time as the
encapsulation travels through a microfluidic channel, wherein the
residence time of each encapsulation is within a maximum dispersion
ratio of the incubation period of time for the plurality of
encapsulations, wherein the dispersion ratio is based on a
deviation about an average residence time of the plurality of
encapsulations passing through a region of the microfluidic device.
Embodiment 110: The method of Embodiment 109, wherein the maximum
dispersion is at most from about 3% to about 10%. The method of
Embodiment 33, wherein sorting the droplet comprises placing the
droplet into a first collection tube if the signal is at or above a
predetermined threshold or placing the droplet into a second
collection tube if the signal is below a predetermined threshold.
Embodiment 111: The method of Embodiment 76, wherein the signal
comprises electromagnetic radiation, thermal radiation, a visual
change in the sample, or combinations thereof. Embodiment 112: The
method of Embodiment 111, wherein the electromagnetic radiation is
in the visible spectrum. Embodiment 113: The method of Embodiment
111, wherein the electromagnetic radiation is fluorescence or
luminescence. Embodiment 114: The method of Embodiment 113, wherein
the signal is fluorescence emitted by a TaqMan probe or a molecular
beacon. Embodiment 115: The method of Embodiment 111, wherein the
signal comprises thermal radiation detected with an infrared
camera. Embodiment 116: The method of Embodiment 111, wherein the
signal comprises a morphological or visual change in the sample
measured by recording a series of images of the encapsulation.
[0447] Embodiment 117: A method for screening an encoded effector,
the method comprising: a) providing a sample, an encoded effector,
and an encoding in an encapsulation; b) detecting a signal
resulting from an interaction between the effector and sample,
wherein detecting the signal comprises 1) detecting morphological
changes in the sample measured by recording a series of images of
the encapsulation, 2) detecting fluorescence emitted by a molecular
beacon or probe, or 3) combinations thereof, and c) sorting the
encapsulation based on the detection of the signal. Embodiment 118:
The method of Embodiment 117, wherein the signal comprises
detecting a morphological or visual change in the sample measured
by recording a series of images of the encapsulation. Embodiment
119: The method of Embodiment 118, wherein the encapsulation
further comprises a detection reagent. Embodiment 120: The method
of Embodiment 119, wherein the detection reagent comprises an
intercalation dye. Embodiment 121: The method of Embodiment 120,
wherein the intercalation dye comprises ethidium bromide, propidium
iodide, crystal violet, a dUTP-conjugated probe, DAPI
(4',6-diamidino-2-phenylindole), 7-AAD (7-aminoactinomycin D),
Hoechst 33258, Hoechst 33342, Hoechst 34580, combinations thereof,
or derivatives thereof. Embodiment 122: The method of Embodiment
118, further comprising superimposing the series of images of the
sample in the encapsulation. Embodiment 123: The method of
Embodiment 117, wherein the signal comprises detecting fluorescence
emitted by a molecular beacon or TaqMan probe. Embodiment 124: The
method of Embodiment 123, wherein the signal comprises detecting
fluorescence emitted by a molecular beacon, wherein molecular
beacon is complementary to a portion of a target nucleic acid
sequence of the sample. Embodiment 125: The method of Embodiment
123, wherein the signal comprises detecting fluorescence emitted by
a TaqMan probe, wherein the TaqMan probe is complementary to a
portion of a target nucleic acid sequence. Embodiment 126: The
method of Embodiment 123, wherein the encapsulation further
comprises a Taq polymerase. Embodiment 127: The method of
Embodiment 123, wherein the TaqMan probe or molecular beacon is
added to the encapsulation by pico-injection. Embodiment 128: The
method of Embodiment 117, wherein the encoded effector is attached
to a scaffold. Embodiment 129: The method of Embodiment 128,
wherein the scaffold is a bead, a fiber, a nanofibrous scaffold, a
molecular cage, a dendrimer, or a multi-valent molecular assembly.
Embodiment 130: The method of Embodiment 129, wherein the scaffold
is polymer-bead, a glass bead, a metal bead, or a magnetic bead.
Embodiment 131: The method of Embodiment 128, wherein the encoded
effector is covalently bound to the scaffold. Embodiment 132: The
method of Embodiment 128, wherein the encoded effector is bound to
the scaffold by a cleavable linker. Embodiment 133: The method of
Embodiment 132, wherein the cleavable linker is a disulfide or
substituted trans-cyclooctene. Embodiment 134: The method of
Embodiment 132, further comprising cleaving the cleavable linker.
Embodiment 135: The method of Embodiment 134, wherein the number of
encoded effectors cleaved from the scaffold is controlled by the
intensity or duration of exposure to electromagnetic radiation.
Embodiment 136: The method of Embodiment 134, wherein the number of
encoded effectors cleaved from the scaffold is controlled by
controlling the concentration of a cleaving reagent in the
encapsulation. Embodiment 137: The method of Embodiment 136,
wherein the cleaving reagent is added by pico-injection. Embodiment
138: The method of Embodiment 134, wherein the cleavable linker is
activated through interaction with an activating reagent, thereby
enabling the cleavable linker to be cleaved. Embodiment 139: The
method of Embodiment 134, wherein the encoded effectors are
released to a desired concentration within the encapsulation.
Embodiment 140: The method of Embodiment 117, further comprising
incubating the encapsulation for a period of time to allow the
encoded effector and the sample to interact. Embodiment 141: The
method of Embodiment 140, wherein the period of time is at least 1
minute, at least 10 minutes, at least 1 hour, at least 4 hours, or
at least 1 day. Embodiment 142: The method of Embodiment 140,
wherein the period of time is controlled by a residence time as the
encapsulation travels through a microfluidic channel, wherein the
residence time of each encapsulation is within a maximum dispersion
ratio of the incubation period of time for the plurality of
encapsulations, wherein the dispersion ratio is based on a
deviation about an average residence time of the plurality of
encapsulations passing through a region of the microfluidic device.
Embodiment 143: The method of Embodiment 142, wherein the maximum
dispersion is at most from about 3% to about 10%. Embodiment 144:
The method of Embodiment 142, wherein the residence time is
controlled by a flow rate through the microfluidic channel, a
geometry of the microfluidic channel, a valve in the microfluidic
channel, or by removing the one or more droplets from the
microfluidic channel and transferring the one or more droplets to a
separate vessel. Embodiment 145: The method of Embodiment 117,
wherein the encoded effector comprises a compound, a peptide, a
protein, an enzyme, a macrocycle compound, or a nucleic acid.
Embodiment 146: The method of Embodiment 145, wherein the encoded
effector is a non-natural peptide. Embodiment 147: The method of
Embodiment 145, wherein the encoded effector is a polymer.
Embodiment 148: The method of Embodiment 145, wherein the compound
is a drug-like small molecule. Embodiment 149: The method of
Embodiment 117, wherein the sample comprises one or more cells.
Embodiment 150: The method of Embodiment 149, further comprising
the step of lysing the one or more cells. Embodiment 151: The
method of Embodiment 117, wherein detecting the signal comprises
providing one or more droplets through a microfluidic channel
comprising a detector. Embodiment 152: The method of Embodiment
117, wherein sorting the encapsulation comprises placing the
encapsulation into a first collection tube if the signal is at or
above a predetermined threshold or placing the encapsulation into a
second collection tube if the signal is below a predetermined
threshold. Embodiment 153: The method of Embodiment 117, wherein
sorting the encapsulation comprises using a waveform pulse
generator to move encapsulation to a collection tube by an
electrical field gradient, by sound, by a diaphragm, by modifying
geometry of a microfluidic channel, or by changing the pressure of
the microfluidic channel. Embodiment 154: The method of Embodiment
117, wherein the encoding comprises a nucleic acid. Embodiment 155:
The method of Embodiment 154, further comprising sequencing the
encoding nucleic acid. Embodiment 156: The method of Embodiment
155, wherein the encoding is cleaved from the scaffold prior to
sequencing. Embodiment 157: The method of Embodiment 156, wherein
cleaving the nucleic acid encoding from the scaffold comprises
cleaving a cleavable linker with a cleaving reagent or through
electromagnetic radiation. Embodiment 158: The method of Embodiment
117, wherein the encapsulation is a droplet. Embodiment 159: The
method of Embodiment 158, wherein the droplet the is at most 1
picoliter, at most 10 picoliters, at most 100 picoliters, at most 1
nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at
most 1 microliter in volume. Embodiment 160: The method of
Embodiment 117, wherein the encapsulation is an emulsion in an oil.
Embodiment 161: The method of Embodiment 160, wherein the oil is a
silicone oil, fluorosilicone oil, hydrocarbon oil, mineral oil,
paraffin oil, halogenated oil, or any combination thereof.
Embodiment 162: The method of Embodiment 117, further comprising
injecting one or more reagents into one or more encapsulations.
Embodiment 163: The method of Embodiment 162, wherein the one or
more reagents are injected by pico-injection or droplet merging.
Embodiment 164: The method of Embodiment 162, wherein injecting the
one or more reagents further comprises monitoring the one or more
encapsulations in flow, wherein the one or more reagents are
injected at the same frequency at which the one or more
encapsulations are passing an injection site. Embodiment 165: The
method of Embodiment 140, wherein a rate of injection of the one or
more reagents is determined by a flow rate of the one or more
encapsulations.
[0448] Embodiment 166: A method for detecting one or more cellular
nucleic acid using a nucleic acid encoded effector screen, the
method comprising: a) providing an encoded effector, a nucleic acid
encoding the encoded effector, and one or more cells comprising the
one or more cellular nucleic acids, wherein the encoded effector,
nucleic acid encoding and the one or more cells are provided in an
encapsulation; b) incubating the encapsulation for a period of time
to allow for the encoded effector and the one or more cells to
interact, thereby producing a signal; c) transferring at least one
cellular nucleic acid of the one or more cellular nucleic acids to
the nucleic acid encoding; d) detecting the signal; and e) sorting
the encapsulation based on the detection of the signal.
[0449] Embodiment 167: The method Embodiment 166, further
comprising the step of lysing the one or more cells. Embodiment
168: The method of Embodiment 167, wherein lysing the one or more
cells comprises adding lysis buffer to the encapsulation.
Embodiment 169: The method of Embodiment 168, wherein the lysis
buffer is added to the encapsulation by pico-injection. Embodiment
170: The method of Embodiment 166, wherein the one or more cellular
nucleic acids comprise DNA, RNA, or combinations thereof.
Embodiment 171: The method of Embodiment 166, wherein the one or
more cellular nucleic acids comprise mRNA. Embodiment 172: The
method of Embodiment 664, wherein the one or more cellular nucleic
acids are added to the nucleic acid encoding as antibody-DNA
constructs, proximity ligation products, or proximity extension
products. Embodiment 173: The method of Embodiment 166, wherein
transferring the at least one cellular nucleic acid to the nucleic
acid encoding comprises annealing, ligating, amplifying, or
chemically crosslinking the at least one cellular nucleic acid to
the nucleic acid encoding. Embodiment 174: The method Embodiment
166, wherein transferring the at least one cellular nucleic acid to
the nucleic acid encoding allows for quantification of the amount
of the one or more cellular nucleic acids encapsulated with the
nucleic acid encoded effector. Embodiment 175: The method of
Embodiment 166, wherein a plurality of different cellular nucleic
acids are transferred to the nucleic acid encoding. Embodiment 176:
The method of Embodiment 166, further comprising adding one or more
reagents for transferring the at least one cellular nucleic acid to
the nucleic acid encoding. Embodiment 177: The method of Embodiment
176, wherein the one or more reagents are provided in the
encapsulation in step (a). Embodiment 178: The method of Embodiment
176, wherein the one or more reagents are added during the
incubation step or after the incubation step. Embodiment 179: The
method of Embodiment 176, wherein the one or more reagents are
added by droplet merging, pico-injection, or interaction with
solid-phase particles comprising the one or more reagents.
Embodiment 180: The method of Embodiment 176, wherein the one or
more reagents comprises an enzyme. Embodiment 181: The method of
Embodiment 180, wherein the enzyme is a ligase, a polymerase, a
restriction enzyme, or a recombinase. Embodiment 182: The method of
Embodiment 176, wherein the one or more reagents comprises assay
detection reagents, labelling reagents, antibodies, target
effectors, cell lysis reagents, nucleic acid ligation reagents,
amplification reagents, or combinations thereof. Embodiment 183:
The method of Embodiment 176, wherein the one or more reagents are
only added if a signal is detected. Embodiment 184: The method of
Embodiment 166, wherein the signal is electromagnetic radiation,
thermal radiation, or a visual change in the sample. Embodiment
185: The method of Embodiment 166, wherein detecting the signal
comprises providing the encapsulation through a microfluidic
channel equipped with a detector. Embodiment 186: The method of
Embodiment 166, wherein sorting the encapsulation is based on the
level, presence, or absence of the signal. Embodiment 187: The
method of Embodiment 166, wherein the period of time is controlled
by a residence time as the encapsulation travels through a
microfluidic channel, wherein the residence time of each
encapsulation is within a maximum dispersion ratio of the
incubation period of time for the plurality of encapsulations,
wherein the dispersion ratio is based on a deviation about an
average residence time of the plurality of encapsulations passing
through a region of the microfluidic device. Embodiment 188: The
method of Embodiment 187, wherein the maximum dispersion is at most
from about 3% to about 10%. Embodiment 189: The method of
Embodiment 166, wherein the period of time is at least 1 minute, at
least 10 minutes, at least 1 hour, at least 4 hours, or at least 1
day. Embodiment 190: The method of Embodiment 166, wherein the
encapsulation is a droplet, an emulsion, a picowell, a microwell, a
bubble, or a microfluidic confinement. Embodiment 191: The method
of Embodiment 166, wherein the encapsulation is a droplet.
Embodiment 192: The method of Embodiment 191, wherein the droplet
is at most 1 picoliter, at most 10 picoliters, at most 100
picoliters, at most 1 nanoliter, at most 10 nanoliters, at most 100
nanoliters, or at most 1 microliter in volume. Embodiment 193: The
method of Embodiment 191, wherein the droplet is suspended in an
emulsion. Embodiment 194: The method of Embodiment 1166, wherein
the effector comprises a compound, a peptide, a protein, an enzyme,
a macrocycle compound, or a nucleic acid. Embodiment 195: The
method of Embodiment 166, further comprising 1) amplifying the
nucleic acid encoding the encoded effector with the transferred at
least one cellular nucleic acid, 2) sequencing the nucleic acid
encoding with the transferred at least one cellular nucleic acid,
3) quantifying the at least one cellular nucleic acid, or any
combination thereof. Embodiment 196: The method of Embodiment 166,
wherein the encoded effector is attached to a scaffold. Embodiment
197: The method of Embodiment 196, wherein the scaffold is a bead.
Embodiment 198: The method of Embodiment 196, wherein the scaffold
is a polymer-bead, a glass bead, a metal bead, a molecular cage, or
a multi-valent molecular assembly. Embodiment 199: The method of
Embodiment 196, wherein the encoded effector is attached to the
scaffold by a cleavable linker. Embodiment 200: The method of
Embodiment 199, wherein the cleavable linker is a photocleavable
linker. Embodiment 201: The method of Embodiment 199, wherein the
encoded effector is covalently attached to the cleavable linker.
Embodiment 201: The method of Embodiment 199, further comprising
cleaving the cleavable linker. Embodiment 202: The method of
Embodiment 199, wherein the nucleic acid encoding is attached to
the scaffold by a second cleavable linker. Embodiment 203: The
method of Embodiment 203, further comprising cleaving the second
cleavable linker.
[0450] Embodiment 205: A method for screening a nucleic acid
encoded protein, the method comprising: a) providing an
encapsulation comprising: i) a nucleic acid encoding attached to a
scaffold, the nucleic acid encoding comprises an encoding barcode
and a coding section for the expression of an encoded effector
protein, and ii) an expression system for the production of the
encoded effector protein; b) expressing the encoded effector
protein within the encapsulation; c) detecting the signal produced
from an interaction with the encoded effector protein and one or
more detection reagents disposed within the encapsulation; and d)
sorting the encapsulation based on the signal. Embodiment 206: The
method of Embodiment 205, further comprising the step of sequencing
the nucleic acid encoding based on the sorted encapsulation.
Embodiment 207: The method of Embodiment 205, wherein the encoded
effector protein is a signaling protein, an enzyme, a binding
protein, an antibody or antibody fragment, a structural protein, a
storage protein, or a transport protein, or any mutant thereof.
Embodiment 208: The method of Embodiment 205, wherein the encoded
effector protein is an enzyme or enzyme mutant being screened for
an enzymatic activity. Embodiment 209: The method of Embodiment
208, wherein the enzymatic activity comprises oxidation, reduction,
ligation, polymerization, bond cleavage, bond formation, or
isomerization. Embodiment 210: The method of Embodiment 205,
wherein the encoded effector protein is an amino acid
dehydrogenase, a natural amine dehydrogenase, an opine
dehydrogenase, or an imine reductase. Embodiment 211: The method of
Embodiment 205, wherein the interaction between the encoded
effector protein and the one or more detection reagents comprises
forming a bond between 1) a first molecular probe from a first
detection reagent and second molecular probe from a second
detection reagent of the one or more reagents, or 2) one or more
chemical compounds for a first detection reagent and one or more
chemical compounds from a second detection reagent. Embodiment 212:
The method of Embodiment 211, wherein the bond is a covalent bond.
Embodiment 213: The method of Embodiment 211, wherein the bond is
an irreversible covalent bond. Embodiment 214: The method of
Embodiment 211, wherein the first reagent and the second reagent
exhibit a fluorescent signal when the first and second molecular
probes are bound together. Embodiment 215: The method of Embodiment
214, wherein the fluorescent signal is due to fluorescence
resonance energy transfer (FRET), bioluminescence resonance energy
transfer (BRET), lanthanide chelate excite time resolved
fluorescence resonance energy transfer (LANCE TR-FRET), or an
amplified luminescent proximity homogeneous assay. Embodiment 216:
The method of Embodiment 211, wherein the first and second
detection reagents comprise chemical compounds. Embodiment 217: The
method of Embodiment 211, wherein the first and second reagents
comprise a FRET pair or a fluorophore/quencher pair. Embodiment
218: The method of Embodiment 217, wherein the first and second
detection reagents comprise fluorophores or quenchers independently
selected from 4-(4-dimethylaminophenyl azo),
5-((3-aminoethyl)amino)-1-napthalene sulfonic acid,
5-((2-aminoethyl)amino)-1-napthalene sulfonic acid (EDANS),
4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL), and
fluorescein-isothiocyanate (FITC), or derivatives thereof.
Embodiment 219: The method of Embodiment 217, wherein the FRET pair
or fluorophore/quencher pair comprise different fluorophores.
Embodiment 220: The method of Embodiment 217, wherein the FRET
pairing is duplicate copies of the same fluorophore. Embodiment
221: The method of Embodiment 211, wherein forming of the bond
comprising an imine reduction. Embodiment 222: The method of
Embodiment 221, wherein the imine reduction is enantiospecific.
Embodiment 223: The method of Embodiment 211, wherein the
encapsulation further comprises a reporter enzyme. Embodiment 224:
The method of Embodiment 223, wherein the reporter enzyme reacts
with another reagent to produce a functional readout. Embodiment
225: The method of Embodiment 223, wherein the bond between the
first and second molecular probes creates a new molecule that
inhibits the reporter enzyme. Embodiment 226: The method of
Embodiment 205, wherein the scaffold is a bead, a fiber, a
nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly. Embodiment 227: The method of
Embodiment 226, wherein the scaffold is a bead. Embodiment 228: The
method of Embodiment 226, wherein the scaffold is polymer-bead, a
glass bead, a metal bead, or a magnetic bead. Embodiment 229: The
method of Embodiment 205, wherein the encapsulation is a droplet,
an emulsion, a picowell, a macrowell, a microwell, a bubble, or a
microfluidic confinement. Embodiment 230: The method of any
Embodiment 229, wherein the encapsulation is a droplet. Embodiment
231: The method of Embodiment 230, wherein the droplet is at most 1
picoliter, at most 10 picoliters, at most 100 picoliters, at most 1
nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at
most 1 microliter in volume. Embodiment 232: The method of
Embodiment 205, wherein the expression system comprises an in vitro
transcription/translation system. Embodiment 233: The method of
Embodiment 205, wherein the one or more detection reagents are
added through pico-injection or droplet merging. Embodiment 234:
The method of Embodiment 205, further comprising incubating the
encapsulation for a period of time after the one or more detection
reagents have been added. Embodiment 235: The method of Embodiment
205, wherein detecting the signal comprises providing the
encapsulation through a microfluidic channel equipped with a
detector. Embodiment 236: A method of screening a library of
nucleic acid encoded proteins, the method comprising performing the
screen of any of Embodiments 205-235 against a library of nucleic
acid encoded proteins, wherein the library of nucleic acid encoded
proteins comprises a plurality of different mutant versions of the
nucleic acid encoded protein. Embodiment 237: The method of
Embodiment 236, wherein each mutant version of the nucleic acid
encoded protein is encoded by a unique barcode.
[0451] Embodiment 238: A method for normalizing the results of a
nucleic acid encoded library screen comprising: a) providing a
plurality of screened encoded effectors and corresponding scaffolds
in a plurality of encapsulations, wherein each scaffold is bound to
one or more nucleic acid encodings that encode a corresponding
screened encoded effector; b) lysing the plurality of
encapsulations; c) removing contents unbound to the plurality of
scaffolds; d) suspending the plurality of scaffolds in a liquid
medium; e) encapsulating the plurality of scaffolds in a plurality
of new encapsulations, wherein each new encapsulation encapsulates
one or more scaffolds of the plurality of scaffolds; and f)
amplifying the one or more nucleic acid encodings of each scaffold
to form corresponding amplified nucleic acid encodings, such that
the amplified nucleic encodings within the plurality of new
encapsulations are limited to the contained encoding scaffold(s)
and the reagent(s) within the plurality of new encapsulations.
Embodiment 239: The method of Embodiment 238, wherein 90% of the
plurality of new encapsulations have a concentration of amplified
nucleic acid encodings within 10% of an average concentration of
the amplified nucleic acid encodings in the plurality of new
encapsulations. Embodiment 240: The method of Embodiment 238,
wherein providing a plurality of screened encoded effectors
comprises performing a screen of a pre-screened nucleic acid
encoded library. Embodiment 241: The method of Embodiment 240,
wherein performing the screen comprises a sorting step to separate
nucleic acid encoded effectors from the pre-screened nucleic acid
encoded library that displayed a positive result in the screen.
Embodiment 242: The method of Embodiment 240, wherein the plurality
of screened encoded effectors comprises the nucleic acid encoded
effectors that displayed a positive result in the screen of the
pre-screened nucleic acid encoded library. Embodiment 243: The
method of Embodiment 238, wherein lysing the plurality of
encapsulations comprises introducing a demulsifying reagent,
filtration, centrifugation, or sonication to an emulsion containing
the plurality of encapsulations. Embodiment 244: The method of
Embodiment 243, wherein the demulsifying reagent is an acid or a
salt. Embodiment 245: The method of Embodiment 243, wherein the
demulsifying reagent is sulfuric acid or hydrochloric acid.
Embodiment 246: The method of Embodiment 243, wherein the
demulsifying reagent is sodium chloride, potassium pyrophosphate,
or sodium sulfate. Embodiment 247: The method of Embodiment 238,
wherein the removing of unbound contents from the plurality of
scaffolds comprises washing the plurality of scaffolds. Embodiment
248: The method of Embodiment 247, wherein washing the plurality of
scaffolds comprises rinsing the plurality of scaffolds with a wash
buffer. Embodiment 249: The method of Embodiment 248, wherein the
wash buffer is an aqueous buffer, an organic solution, or a mixture
thereof. Embodiment 250: The method of Embodiment 247, wherein the
plurality of scaffolds are subject to multiple wash and collection
steps, wherein each wash step comprises rinsing the plurality of
scaffolds with a wash buffer, and each collection step comprises
centrifugation or filtration of the plurality of scaffolds.
Embodiment 251: The method of Embodiment 238, wherein the liquid
medium is an aqueous solution. Embodiment 252: The method of
Embodiment 238, wherein the liquid medium comprises an organic
solvent. Embodiment 253: The method of Embodiment 238, wherein each
scaffold of the plurality of scaffolds is a bead, a fiber, a
nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly. Embodiment 254: The method of
Embodiment 238, wherein each scaffold of the plurality of scaffolds
is polymer-bead, a glass bead, a metal bead, or a magnetic bead.
Embodiment 255: The method of Embodiment 238, wherein the liquid
medium comprises an amplification mix. Embodiment 256: The method
of Embodiment 238, wherein each new encapsulation is a droplet.
Embodiment 257: The method of Embodiment 238, wherein encapsulating
the plurality of scaffolds in new encapsulations comprises
introducing the plurality of scaffolds into an emulsion. Embodiment
258: The method of Embodiment 257, wherein introducing the
plurality of scaffolds into an emulsion comprises placing the
plurality of scaffolds through a microfluidic device. Embodiment
259: The method of Embodiment 258, wherein the microfluidic device
is a microfluidic chip. Embodiment 260: The method of Embodiment
257, wherein introducing the plurality of scaffolds into an
emulsion comprises placing the plurality of scaffolds into a
one-pot emulsifier. Embodiment 261: The method of Embodiments 238,
wherein an amplification mix is encapsulated with the plurality of
scaffolds in the plurality of new encapsulations. Embodiment 262:
The method of Embodiment 238, wherein an amplification mix is added
to the plurality of new encapsulations. Embodiment 263: The method
of Embodiment 262, wherein the amplification mix is added by
pico-injection. Embodiment 264: The method of embodiment 262,
wherein the amplification mix is added by droplet merging, wherein
each encapsulation is a droplet. Embodiment 265: The method of
Embodiment 261 or 262, wherein the amplification mix comprises PCR
reagents. Embodiment 266: The method of Embodiment 238, further
comprising sequencing the amplified nucleic acid encodings of each
scaffold. Embodiment 267: The method of Embodiment 238, wherein the
method results in a lower background signal than a nucleic acid
encoded library that has not been subjected to the method.
Embodiment 268: The method of Embodiment 267, wherein the
background signal is reduced by at least 10%, at least 20%, at
least 30%, at least 40%, or at least 50%. Embodiment 269: The
method of Embodiment 267, wherein the lower background signal
allows for detection of nucleic acid encoded effectors whose
encoding concentrations before the screen are 100.times.,
1000.times., 10000.times., 100000.times., or 1000000.times. lower
in concentration than the average encoding concentration of the
provided screened encoded effectors and corresponding
scaffolds.
[0452] Embodiment 270: A system for screening an encoded effector,
the system comprising: a) a sample; b) a scaffold, wherein an
encoded effector is bound to the scaffold by a cleavable linker,
wherein a nucleic acid encoding the effector is bound to the
scaffold; and c) a microfluidic device configured to: i) receive
the sample and scaffold; ii) encapsulate the sample and scaffold
within an encapsulation; iii) cleave the cleavable linker from the
encoded effector to release a predetermined amount of the encoded
effector within the encapsulation; iv) incubate the encoded
effector with the sample for an incubation period of time; v)
detect a signal from the encapsulation, wherein the signal results
from an interaction between the encoded effector and the sample;
and vi) sort the encapsulation based on the detection of the
signal. Embodiment 271: The system of Embodiment 270, wherein the
cleavable linker is a photocleavable linker. Embodiment 272: The
system of Embodiment 271, wherein cleaving the photocleavable
linker comprises exposing the droplet to a light from a light
source. Embodiment 273: The system of Embodiment 272, wherein the
light is UV light. Embodiment 274: The system of Embodiment 272,
wherein the light intensity of the light is from about 0.01
J/cm.sup.2 to about 200 J/cm.sup.2. Embodiment 275: The system of
Embodiment 271, wherein the encapsulation further encapsulates a
reagent configured to activate the photocleavable linker so as to
enable the photocleavable linker to be cleaved from the encoded
effector. Embodiment 276: The system of Embodiment 275, wherein the
microfluidic device is configured to introduce the reagent within
the encapsulation. Embodiment 277: The system of Embodiment 270,
wherein the signal is detected based on detecting morphological
changes in the sample measured by recording a series of images of
the droplet or detecting fluorescence emitted by a molecular beacon
or probe. Embodiment 278: The system of Embodiment 270, wherein the
interaction between the encoded effector and the cell comprises
inhibition of one or more cellular components. Embodiment 279: The
system of Embodiment 270, further comprising a sequencing apparatus
configured to identify the encoded effector by sequencing the
nucleic acid encoding the effector. Embodiment 280: The system of
Embodiment 270, wherein the scaffold is a bead, a fiber, a
nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly. Embodiment 281: The system of
Embodiment 280, wherein the scaffold is polymer-bead, a glass bead,
a metal bead, or a magnetic bead. Embodiment 282: The system of
Embodiment 280, wherein the bead is about 1 .mu.m to about 100
.mu.m in diameter. Embodiment 283: The system of Embodiment 280,
wherein the bead is about 1 .mu.m to about 20 .mu.m in diameter.
Embodiment 284: The system of Embodiment 270, wherein the encoded
effector is a peptide, a compound, protein, an enzyme, a macrocycle
compound, or a nucleic acid. Embodiment 285: The system of
Embodiment 284, wherein the encoded effector is a non-natural
peptide or a polymer. Embodiment 286: The system of Embodiment 284,
wherein the encoded effector is a small molecule or macromolecule.
Embodiment 287: The system of Embodiment 284, wherein the compound
is a drug-like small molecule. Embodiment 288: The system of
Embodiment 270, wherein the encapsulation is a droplet. Embodiment
289: The system of Embodiment 288, wherein the droplet is at most 1
picoliter, at most 10 picoliters, at most 100 picoliters, at most 1
nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at
most 1 microliter in volume. Embodiment 290: The system of
Embodiment 270, wherein the sample comprises at least one cell, a
protein, or an enzyme. Embodiment 291: The system of Embodiment
270, wherein the period of time is controlled by residence time as
the encapsulation travels through a microfluidic channel of the
microfluidic device. Embodiment 292: The system of Embodiment 270,
wherein the microfluidic device further comprises a first
collection tube and second collection tube for sorting the
encapsulation, wherein the encapsulation is placed in 1) the first
collection tube if the signal is at or above a predetermined
threshold or 2) the second collection tube if the signal is below a
predetermined threshold. Embodiment 293: The system of Embodiment
292, further comprising a waveform pulse generator to move the
encapsulation to the first or second collection tube by an
electrical field gradient, by sound, by a diaphragm, by modifying
geometry of the microfluidic channel, or by changing the pressure
of a microfluidic channel of the microfluidic device. Embodiment
294: The system of Embodiment 270, wherein the microfluidic device
further comprises: a) a first microfluidic channel comprising an
aqueous fluid comprising the sample and scaffold; b) a second
microfluidic channel comprising a fluid immiscible with the aqueous
fluid; c) a junction at which the first microfluidic channel is in
fluid communication with the second microfluidic channel, wherein
the junction of the first and second microfluidic channels defines
a device plane, wherein the junction is configured to form the
encapsulations of the aqueous fluid within the fluid from the
second microfluidic channel, wherein the fluid from the second
microfluidic channel with the encapsulations therein moves past the
junction in a third microfluidic channel that defines an assay flow
path; d) a cleavage region to cleave the cleavable linker within
the encapsulation disposed in the assay flow path; e) a detection
region to detect the signal; and f) a sorting region to sort the
encapsulation. Embodiment 295: The system of Embodiment 294,
wherein the third microfluidic channel is a continuation of the
second microfluidic channel. Embodiment 296: The system of
Embodiment 294, wherein a plurality of encapsulations are disposed
within the assay flow path. Embodiment 297: The system of
Embodiment 294, wherein cleavage region is configured to expose
each encapsulation to a light from a light source so as to cleave
the encoded effector from the scaffold disposed within the assay
flow path. Embodiment 298: The system of Embodiment 297, wherein
the light intensity of the light is from about 0.01 J/cm.sup.2 to
about 200 J/cm.sup.2. Embodiment 299: The system of Embodiment 297,
wherein the plurality of encapsulations are exposed to a uniform
intensity or duration of the light. Embodiment 300: The system of
Embodiment 297, wherein the intensity or duration of the light that
each encapsulation is exposed to within about 0.1% to about 10% of
each other. Embodiment 301: The system of Embodiment 270, wherein
the incubation period of time for each encapsulation is within a
maximum dispersion ratio of the incubation period of time for the
plurality of encapsulations, wherein the dispersion ratio is based
on a deviation about an average residence time of the plurality of
encapsulations passing through a region of the microfluidic device.
Embodiment 302: The system of Embodiment 301, wherein the region of
the microfluidic device is the assay flow path. Embodiment 303: The
system of Embodiment 301, wherein the maximum dispersion ratio is
at most about 10%. Embodiment 304: The system of Embodiment 301,
wherein the maximum dispersion ratio is at most about 5%.
Embodiment 305: The system of Embodiment 270, the incubation period
of time for each encapsulation is within about 0.1% to about 10% of
each other. Embodiment 306: The system of Embodiment 294, wherein
the detection region comprises a fluorometer. Embodiment 307: The
system of Embodiment 294, wherein the detection region comprises a
confocal detection, laser scanning, or fluorescence, or
combinations thereof. Embodiment 308: The system of Embodiment 294,
wherein the sorting region comprises a sorter configured to sort
the encapsulations based on a signal detected in the detection
region.
[0453] Embodiment 309: A system for screening an encoded effector,
the system comprising: a) one or more cells; b) a scaffold, wherein
an encoded effector is bound to the scaffold by a cleavable linker,
wherein a nucleic acid encoding the effector is bound to the
scaffold; and c) a microfluidic device configured to: i) receive
the one or more cells and scaffold; ii) encapsulate the one or more
cells and scaffold within an encapsulation; iii) cleave the
cleavable linker from the encoded effector to release a
predetermined amount of the encoded effector within the
encapsulation; iv) incubate the encoded effector with the one or
more cells for an incubation period of time; v) detect a signal
from the encapsulation, wherein the signal results from an
interaction between the encoded effector and one or more cells; and
vi) sort the encapsulation based on the detection of the signal.
Embodiment 310: The system of Embodiment 309, wherein the cleavable
linker is a photocleavable linker. Embodiment 311: The system of
Embodiment 310, wherein cleaving the photocleavable linker
comprises exposing the droplet to a light from a light source.
Embodiment 312: The system of Embodiment 311, wherein the light is
UV light. Embodiment 313: The system of Embodiment 311, wherein the
light intensity of the light is from about 0.01 J/cm.sup.2 to about
200 J/cm.sup.2. Embodiment 314: The system of Embodiment 310,
wherein the encapsulation further encapsulates a reagent configured
to activate the photocleavable linker so as to enable the
photocleavable linker to be cleaved from the encoded effector.
Embodiment 315: The system of Embodiment 314, wherein the
microfluidic device is configured to introduce the reagent within
the encapsulation. Embodiment 316: The system of Embodiment 309,
wherein the signal is detected based on detecting morphological
changes in the one or more cells measured by recording a series of
images of the droplet or detecting fluorescence emitted by a
molecular beacon or probe. Embodiment 317: The system of Embodiment
309, wherein the interaction between the encoded effector and the
one or more cells comprises inhibition of one or more cellular
components. Embodiment 318: The system of Embodiment 309, further
comprising a sequencing apparatus configured to identify the
encoded effector by sequencing the nucleic acid encoding the
effector. Embodiment 319: The system of Embodiment 309, wherein the
scaffold is a bead, a fiber, a nanofibrous scaffold, a molecular
cage, a dendrimer, or a multi-valent molecular assembly. Embodiment
320: The system of Embodiment 319, wherein the scaffold is
polymer-bead, a glass bead, a metal bead, or a magnetic bead.
Embodiment 321: The system of Embodiment 320, wherein the bead is
about 1 .mu.m to about 100 .mu.m in diameter. Embodiment 322: The
system of Embodiment 320, wherein the bead is about 1 .mu.m to
about 20 .mu.m in diameter. Embodiment 323: The system of
Embodiment 309, wherein the encoded effector is a peptide, a
compound, protein, an enzyme, a macrocycle compound, or a nucleic
acid. Embodiment 324: The system of Embodiment 323, wherein the
encoded effector is a non-natural peptide or a polymer. Embodiment
325: The system of Embodiment 323, wherein the encoded effector is
a small molecule or macromolecule. Embodiment 326: The system of
Embodiment 323, wherein the compound is a drug-like small molecule.
Embodiment 327: The system of Embodiment 309, wherein the
encapsulation is a droplet. Embodiment 328: The system of
Embodiment 327, wherein the droplet is at most 1 picoliter, at most
10 picoliters, at most 100 picoliters, at most 1 nanoliter, at most
10 nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. Embodiment 329: The system of Embodiment 309, wherein the
period of time is controlled by residence time as the encapsulation
travels through a microfluidic channel of the microfluidic device.
Embodiment 330: The system of Embodiment 309, wherein the
microfluidic device further comprises a first collection tube and
second collection tube for sorting the encapsulation, wherein the
encapsulation is placed in 1) the first collection tube if the
signal is at or above a predetermined threshold or 2) the second
collection tube if the signal is below a predetermined threshold.
Embodiment 331: The system of Embodiment 330, further comprising a
waveform pulse generator to move the encapsulation to the first or
second collection tube by an electrical field gradient, by sound,
by a diaphragm, by modifying geometry of the microfluidic channel,
or by changing the pressure of a microfluidic channel of the
microfluidic device. Embodiment 332: The system of Embodiment 309,
wherein the microfluidic device further comprises: a) a first
microfluidic channel comprising an aqueous fluid comprising the one
or more cells and scaffold; b) a second microfluidic channel
comprising a fluid immiscible with the aqueous fluid; c) a junction
at which the first microfluidic channel is in fluid communication
with the second microfluidic channel, wherein the junction of the
first and second microfluidic channels defines a device plane,
wherein the junction is configured to form the encapsulations of
the aqueous fluid within the fluid from the second microfluidic
channel, wherein the fluid from the second microfluidic channel
with the encapsulations therein moves past the junction in a third
microfluidic channel that defines an assay flow path; d) a cleavage
region to cleave the cleavable linker within the encapsulation
disposed in the assay flow path; e) a detection region to detect
the signal; and f) a sorting region to sort the encapsulation.
Embodiment 333: The system of Embodiment 332, wherein the third
microfluidic channel is a continuation of the second microfluidic
channel. Embodiment 334: The system of Embodiment 332, wherein a
plurality of encapsulations are disposed within the assay flow
path. Embodiment 335: The system of Embodiment 332, wherein
cleavage region is configured to expose each encapsulation to a
light from a light source so as to cleave the encoded effector from
the scaffold disposed within the assay flow path. Embodiment 336:
The system of Embodiment 335, wherein the light intensity of the
light is from about 0.01 J/cm.sup.2 to about 200 J/cm.sup.2.
Embodiment 337: The system of Embodiment 335, wherein the plurality
of encapsulations are exposed to a uniform intensity or duration of
the light. Embodiment 338: The system of Embodiment 335, wherein
the intensity or duration of the light that each encapsulation is
exposed to is within about 0.1% to about 10% of each other.
Embodiment 339: The system of Embodiment 332, wherein the
incubation period of time for each encapsulation is within a
maximum dispersion ratio of the incubation period of time for the
plurality of encapsulations, wherein the dispersion ratio is based
on a deviation about an average residence time of the plurality of
droplets passing through a region of the microfluidic device.
Embodiment 340: The system of Embodiment 339, wherein the region of
the microfluidic device is the assay flow path. Embodiment 341: The
system of Embodiment 339, wherein the maximum dispersion ratio is
at most about 10%. Embodiment 342: The system of Embodiment 339,
wherein the maximum dispersion ratio is at most about 5%.
Embodiment 343: The system of Embodiment 309, the incubation period
of time for each encapsulation is within about 0.1% to about 10% of
each other. Embodiment 344: The system of Embodiment 332, wherein
the detection region comprises a fluorometer. Embodiment 345: The
system of Embodiment 332, wherein the detection region comprises a
confocal detection, laser scanning, or fluorescence, or
combinations thereof. Embodiment 346: The system of Embodiment 332,
wherein the sorting region comprises a sorter configured to sort
the encapsulations based on a signal detected in the detection
region.
[0454] Embodiment 347: A microfluidic device for droplet based
encoded library screening comprising: a) a first microfluidic
channel comprising an aqueous fluid; b) a second microfluidic
channel comprising a fluid immiscible with the aqueous fluid; c) a
junction at which the first microfluidic channel is in fluid
communication with the second microfluidic channel, wherein the
junction of the first and second microfluidic channels defines a
device plane, wherein the junction is configured to form
encapsulations of the aqueous fluid within the fluid from the
second microfluidic channel, wherein the fluid from the second
microfluidic channel with the encapsulations therein moves past the
junction in a third microfluidic channel that defines an assay flow
path; d) a cleavage region for cleaving effectors bound to
scaffolds disposed within the assay flow path; e) a detection
region; and f) a sorting region; g) wherein the device is
configured for a droplet generation frequency of at least about 80
Hz. Embodiment 348: The microfluidic device of Embodiment 347,
wherein the third microfluidic channel is a continuation of the
second microfluidic channel. Embodiment 349: The microfluidic
device of Embodiment 347, wherein the cleavage region is upstream
of the detection region and the sorting region. Embodiment 350: The
microfluidic device of Embodiment 347, wherein the cleavage region
is downstream of the junction. Embodiment 351: The microfluidic
device of Embodiment 347, wherein the assay flow path comprises a
serpentine flow path region. Embodiment 352: The microfluidic
device of Embodiment 351, wherein the serpentine flow path region
comprises at least 10, at least 20, at least 30, at least 40, at
least 50, or at least 100 curves. Embodiment 353: The microfluidic
device of Embodiment 347, wherein the detection region comprises a
fluorometer. Embodiment 354: The microfluidic device of Embodiment
353, wherein the fluorometer is configured to have an optical axis
substantially parallel to the device plane. Embodiment 355: The
microfluidic device of Embodiment 353, wherein the fluorometer
illuminates a passing droplet at a curve in the assay flow path.
Embodiment 356: The microfluidic device of Embodiment 353, wherein
the fluorometer is configured to detect two or more wavelengths of
fluorescence. Embodiment 357: The microfluid device of Embodiment
347, wherein the detection region comprises a confocal detection,
laser scanning, or fluorescence, or combinations thereof.
Embodiment 358: The microfluidic device of Embodiment 347, wherein
the device comprises two or more channels comprising an aqueous
fluid. Embodiment 359: The microfluidic device of Embodiment 347,
wherein the detection region is upstream of the sorting region.
Embodiment 360: The microfluidic device of Embodiment 347, wherein
the sorting region comprises a sorter configured to sort droplets
based on a signal detected in the detection region. Embodiment 361:
The microfluidic device of Embodiment 347, wherein the assay flow
path comprises one or more chambers disposed within the assay flow
path. Embodiment 362: The microfluidic device of Embodiment 347,
wherein the assay flow path comprises a plurality of chambers
disposed within the assay flow path, wherein the chambers are
connected by connecting channels. Embodiment 363: The microfluidic
device of Embodiment 362, wherein the height of a chamber of the
plurality of chambers is at most about 2.times. greater than the
height of a connecting channel of the plurality of connecting
channels. Embodiment 364: The microfluidic device of Embodiment
362, wherein the height of the chamber does not decrease until the
width of the channel has been narrowed to substantially match the
width of the connecting channel. Embodiment 365: The microfluidic
device of Embodiment 361, wherein the flow rate through the
chambers is about 10% of the flow rate of the flow rate through the
assay flow path upstream of the chambers. Embodiment 366: The
microfluidic device of Embodiment 347, wherein the device has a
dispersion ratio of at most about 10%. Embodiment 367: The
microfluidic device of Embodiment 347, wherein the device is
configured to incubate the encapsulations for an incubation period
of time, wherein the incubation period of time for each
encapsulation is within a maximum dispersion ratio of the
incubation period of time for the plurality of encapsulations,
wherein the dispersion ratio is based on a deviation about an
average residence time of the plurality of droplets passing through
a region of the microfluidic device. Embodiment 368: The system of
Embodiment 367, wherein the region of the microfluidic device is
the assay flow path. Embodiment 369: The system of Embodiment 368,
wherein the maximum dispersion ratio is at most about 10%.
Embodiment 370: The system of Embodiment 368, wherein the maximum
dispersion ratio is at most about 5%. Embodiment 371: The system of
Embodiment 367, the incubation period of time for each
encapsulation is within about 0.1% to about 10% of each other.
[0455] Embodiment 372: A method for amplifying a primer to maximize
cellular nucleic acid capture comprising: a) providing an
encapsulation comprising a nucleic acid encoded scaffold with one
or more cells, an amplification mix, and a nicking enzyme, wherein
a nucleic acid encoding is bound to the nucleic acid encoded
scaffold; b) lysing the one or more cells to release one or more
cellular nucleic acids; c) nicking the nucleic acid encoding with
the nicking enzyme, thereby creating an encoded nucleic acid
primer; d) amplifying the encoded nucleic acid primer via the
nicking site and amplification mix; and e) labeling a released
cellular nucleic acid with the encoded nucleic acid primer.
Embodiment 373: The method of Embodiment 372, wherein the nicking
enzyme targets a specific site in the nucleic acid encoding.
Embodiment 374: The method of Embodiment 373, wherein the specific
site comprises a specific nucleotide sequence. Embodiment 375: The
method of Embodiment of Embodiment 372, wherein amplifying the
encoded nucleic acid primer comprises 1) creating a copy of the
nucleic acid encoding that extends from the nicking site, and 2)
nicking the nucleic acid encoding copy to create another encoded
nucleic acid primer. Embodiment 376: The method of Embodiment of
Embodiment 372, wherein amplifying the encoded nucleic acid primer
comprises simultaneously 1) creating a copy of the nucleic acid
encoding that extends from the nicking site, and 2) displacing the
nucleic acid encoding copy to create another encoded nucleic acid
primer. Embodiment 377: The method of Embodiment 376, wherein the
amplification mix comprises an amplification enzyme, such that the
amplification enzyme enables for a copy of the nucleic acid
encoding to be simultaneously created and displaced. Embodiment
378: The method of Embodiment 377, wherein the amplification enzyme
comprises a polymerase. Embodiment 379: The method of Embodiment
372, wherein each nucleic acid encoding comprises a capture site
that prescribes a target cellular coding or a target cellular
nucleic acid to label a released cellular nucleic acid. Embodiment
380: The method of Embodiment 379, wherein the target nucleic acid
is a target mRNA. Embodiment 381: The method of Embodiment 380,
wherein the target mRNA encodes a protein of interest. Embodiment
382: The method of Embodiment 380, wherein the nicking enzyme
enables an increase in target mRNA capture and labeling with the
nucleic acid encoding. Embodiment 383: The method of Embodiment
380, wherein target mRNA capture is increased by at least 10%, 25%,
50%, 100%, or 200%. Embodiment 384: The method of Embodiment 372,
wherein a plurality of cellular nucleic acids are labeled with an
respective encoded nucleic acid primer. Embodiment 385: The method
of Embodiment 372, wherein the nucleic acid encoded scaffold
comprises a bead, and the encoded nucleic acid primer comprises a
unique bead barcode and an effector encoding. Embodiment 386: The
method of Embodiment 372, wherein the encapsulation further
comprises a cell lysis buffer. Embodiment 387: The method of
Embodiment 372, wherein the encapsulation is a droplet, an
emulsion, a picowell, a macrowell, a microwell, a bubble, or a
microfluidic confinement. Embodiment 388: The method of Embodiment
372, wherein the encapsulation is a droplet. Embodiment 389: The
method of Embodiment 388, wherein the droplet is at most 1
picoliter, at most 10 picoliters, at most 100 picoliters, at most 1
nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at
most 1 microliter in volume. Embodiment 390: The method of
Embodiment 372, wherein the amplification mix is an isothermal
amplification mix. Embodiment 391: The method of Embodiment 372,
wherein the amplification mix comprises a
nicking-enzyme-amplification mixture. Embodiment 392: The method of
Embodiment 372, wherein the amplification mix comprises a reverse
transcriptase. Embodiment 393: The method of Embodiment 372,
wherein the nucleic acid encoded scaffold is a bead, a fiber, a
nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly. Embodiment 394: The method of
Embodiment 393, wherein the scaffold is polymer-bead, a glass bead,
a metal bead, or a magnetic bead. Embodiment 395: The method of
Embodiment 372, wherein the nucleic acid encoded scaffold comprises
an effector attached thereto. Embodiment 396: The method of
Embodiment 395, wherein the effector comprises a compound, a
peptide, a protein, an enzyme, or a nucleic acid. Embodiment 397:
The method of Embodiment 395, wherein effector is attached to the
scaffold by a cleavable linker. Embodiment 398: The method of
Embodiment 397, wherein the cleavable linker is cleaved by
electromagnetic radiation, an enzyme, chemical reagent, heat, pH
adjustment, sound or electrochemical reactivity. Embodiment 399:
The method of Embodiment 398, wherein the effector is cleaved from
the scaffold using electromagnetic radiation. Embodiment 400: The
method of Embodiment 398, wherein the amount of effector cleaved is
controlled by the intensity or duration of exposure to
electromagnetic radiation. Embodiment 401: The method of Embodiment
398, wherein the cleavable linker is cleaved using a cleavage
reagent. Embodiment 402: The method of Embodiment 401, wherein the
amount of effector cleaved is controlled by the concentration of
the cleavage reagent in the encapsulation. Embodiment 403: The
method of Embodiment 398, wherein the rate of effector cleavage is
controlled by the concentration of the cleavage reagent in the
encapsulation. Embodiment 404: The method of Embodiment 398,
wherein the effector is cleaved from the scaffold using an enzyme.
Embodiment 405: The method of Embodiment 404, wherein the enzyme is
a protease, a nuclease, or a hydrolase. Embodiment 406: The method
of Embodiment 404, wherein the rate of effector cleavage is
controlled by the amount of enzyme in the encapsulation. Embodiment
407: The method of Embodiment 372, wherein labeling a released
cellular nucleic acids with the encoded nucleic acid primer
comprises barcoding the released cellular nucleic acid. Embodiment
408: The method of Embodiment 407, wherein the encapsulation
further comprises barcoding reagents. Embodiment 409: The method of
Embodiment 407, wherein barcoding the encoded nucleic acid primer
comprises adding barcoding reagents to the encapsulation.
Embodiment 410: The method of Embodiment 408 or 409, wherein the
barcoding reagents comprise an enzyme or chemical cross-linking
reagent. Embodiment 411: The method of Embodiment 410, wherein the
barcoding reagents comprise an enzyme. Embodiment 412: The method
of Embodiment 411, wherein the enzyme is polymerase, a ligase, a
restriction enzyme, or a recombinase. Embodiment 413: The method of
Embodiment 410, wherein the barcoding reagent is a chemical
cross-linking reagent. Embodiment 414: The method of Embodiment
413, wherein the chemical cross-linking reagent is psoralen.
Embodiment 415: The method of Embodiment 372, further comprising
performing an effector screen, wherein the one or more cells are
being screened against an encoded effector. Embodiment 416: The
method of Embodiment 372, wherein the one or more cells are used to
prepare the nucleic acid encoded scaffold for a screen.
[0456] Embodiment 417: A method for screening an encoded effector,
the method comprising: a) providing an encapsulation comprising a
sample and one or more scaffolds, wherein the scaffold comprises:
i) an encoded effector bound to the scaffold by a cleavable linker
and a nucleic acid encoding the effector; b) adding one or more
reagents to the encapsulation through pico-injection or by droplet
merging; c) cleaving the cleavable linker to release a
pre-determined amount of the effector; d) detecting one or more
signals from the encapsulation, wherein the signal results from an
interaction between the encoded effector and the sample; and e)
sorting the encapsulation based on the detection of the signal.
Embodiment 418: The method of Embodiment 417, wherein the reagent
is added after a pre-determined amount of the effector has been
released. Embodiment 419: The method of Embodiment 417, wherein the
one or more reagents are added to the encapsulation by
pico-injection. Embodiment 420: The method of Embodiment 417,
wherein the concentration a reagent of the one or more reagents is
at most 100 picomolar (pM), at most 500 pM, at most 1 nanomolar
(nM), at most 10 nM, at most 100 nM, at most 1 micromolar (.mu.M),
at most 10 at most 100 at most 1 millimolar (mM), at most 10 mM, at
most 100 mM, or at most 500 mM. Embodiment 421: The method of
Embodiment 417, wherein at least one reagent comprises antibodies.
Embodiment 422: The method of Embodiment 417, wherein the
predetermined amount of effector released from the scaffold is to a
concentration of at least 100 pM, at least 500 pM, at least 1 nM,
at least 10 nM, at least 100 nM, at least 1 .mu.M, at least 10 at
least 100 .mu.M, at least 1 mM, at least 10 mM, at least 50 mM, at
least 100 mM, or at least 250 mM. Embodiment 423: The method of
Embodiment 417, wherein the sample comprises at least one cell, a
protein, an enzyme, a nucleic acid, a cellular lysate, a tissue
extract, or combinations thereof. Embodiment 424: The method of
Embodiment 417, at least one reagent comprises one or more
fluorophores. Embodiment 425: The method of Embodiment 417, further
comprising barcoding the nucleic acid encoding the effector.
Embodiment 426: The method of Embodiment 425, wherein the barcoding
is via the one or more reagents added to the encapsulation.
Embodiment 427: The method of Embodiment 417, wherein the cleavable
linker is a photocleavable linker. Embodiment 428: The method of
Embodiment 427, wherein the photocleavable linker is cleaved using
electromagnetic radiation. Embodiment 429: The method of Embodiment
427, wherein cleaving the photocleavable linker comprises exposing
the encapsulation to a light from a light source. Embodiment 430:
The method of Embodiment 429, wherein the light intensity of the
light is from about 0.01 J/cm.sup.2 to about 200 J/cm.sup.2.
Embodiment 431: The method of Embodiment 427, wherein the one or
more reagents are configured to activate the photocleavable linker,
so as to enable the photocleavable linker to be cleaved from the
encoded effector. Embodiment 432: The method of Embodiment 431,
wherein at least one reagent is a disulfide reducing reagent.
Embodiment 433: The method of Embodiment 431, wherein at least one
reagent is a tetrazine. Embodiment 434: The method of Embodiment
417, wherein detecting the signal comprises detecting morphological
changes in the sample measured by recording a series of images of
the droplet or detecting fluorescence emitted by a molecular beacon
or probe. Embodiment 435: The method of Embodiment 417, wherein the
scaffold is a bead, a fiber, a nanofibrous scaffold, a molecular
cage, a dendrimer, or a multi-valent molecular assembly. Embodiment
436: The method of Embodiment 417, wherein the scaffold is
polymer-bead, a glass bead, a metal bead, or a magnetic bead.
Embodiment 437: The method of Embodiment 435, wherein the bead is
about 1 .mu.m to about 100 .mu.m in diameter. Embodiment 438: The
method of Embodiment 435, wherein the bead is about 1 .mu.m to
about 20 .mu.m in diameter. Embodiment 439: The method of
Embodiment 417, wherein the encoded effector is a peptide, a
compound, protein, an enzyme, a macrocycle compound, or a nucleic
acid. Embodiment 440: The method of Embodiment 417, wherein the
encapsulation is a droplet. Embodiment 441: The method of
Embodiment 440, wherein the droplet is at most 1 picoliter, at most
10 picoliters, at most 100 picoliters, at most 1 nanoliter, at most
10 nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. Embodiment 442: The method of Embodiment 440, further
comprising incubating the droplet for a period of time to allow the
effector and the at least one cell to interact. Embodiment 443: The
method of Embodiment 417, wherein the signal comprises
electromagnetic radiation, thermal radiation, a visual change in
the sample, or combinations thereof.
[0457] Embodiment 444: A method for screening a library of encoded
effectors, the method comprising: (a) encapsulating a plurality of
beads into a plurality of droplets in a microfluidic channel with a
sample, wherein the plurality of beads are bound to a library of
unique encoded effectors, wherein each bead of the plurality of
beads is bound to one or more encoded effectors, wherein the
library of unique encoded effectors comprise at least about 250,000
unique effectors, wherein each unique encoded effector is encoded
with a unique nucleic acid encoding, wherein each droplet comprises
one or more beads, (b) cleaving the photocleavable linker between
at least one encoded effector and corresponding bead; (c) detecting
a signal from one or more droplets of the plurality of droplets,
wherein each signal results from an interaction between a
respective encoded effector and sample within the corresponding
droplet; and (d) sorting the plurality of droplets based on the
detection of a corresponding signal. Embodiment 445: The method of
Embodiment 444, wherein cleaving the photocleavable linker releases
a predetermined amount of an encoded effector. Embodiment 446: The
method of Embodiment 445, wherein the predetermined amount of an
encoded effector released from the bead is to a concentration of at
least 100 pM, at least 500 pM, at least 1 nM, at least 10 nM, at
least 100 nM, at least 1 .mu.M, at least 10 .mu.M, at least 100
.mu.M, at least 1 mM, at least 10 mM, at least 50 mM, at least 100
mM, or at least 250 mM. Embodiment 447: The method of Embodiment
444, wherein the sample comprises at least one cell, a protein, an
enzyme, a nucleic acid, a cellular lysate, a tissue extract, or
combinations thereof. Embodiment 448: The method of Embodiment 447,
wherein the sample is one or more cells, a protein, or an enzyme.
Embodiment 449: The method of Embodiment 447, further comprising
barcoding a nucleic acid encoding a respective effector. Embodiment
450: The method of Embodiment 447, wherein the barcoding is via
adding one or more reagents to a droplet. Embodiment 451: The
method of Embodiment 447, wherein the photocleavable linker is
cleaved using electromagnetic radiation. Embodiment 452: The method
of Embodiment 451, wherein cleaving the photocleavable linker
comprises exposing the encapsulation to a light from a light
source. Embodiment 453: The method of Embodiment 452, wherein the
light intensity of the light is from about 0.01 J/cm.sup.2 to about
200 J/cm.sup.2. Embodiment 454: The method of Embodiment 447,
wherein one or more reagents are added to a droplet, wherein the
one or more reagents are configured to activate the photocleavable
linker of a respective encoded effector, so as to enable the
photocleavable linker to be cleaved from said encoded effector.
Embodiment 455: The method of Embodiment 454, wherein the
activating reagent is a disulfide reducing reagent. Embodiment 456:
The method of Embodiment 454, wherein the activating reagent is a
tetrazine. Embodiment 457: The method of Embodiment 447, wherein
detecting the signal comprises detecting morphological changes in
the sample measured by recording a series of images of the droplet
or detecting fluorescence emitted by a molecular beacon or probe.
Embodiment 458: The method of Embodiment 447, wherein one or more
beads is a polymer-bead, a glass bead, a metal bead, or a magnetic
bead. Embodiment 459: The method of Embodiment 458, wherein one or
more beads is about 1 .mu.m to about 100 .mu.m in diameter.
Embodiment 460: The method of Embodiment 458, wherein one or more
beads is about 1 .mu.m to about 20 .mu.m in diameter. Embodiment
461: The method of Embodiment 447, wherein an encoded effector is a
peptide, a compound, protein, an enzyme, a macrocycle compound, or
a nucleic acid. Embodiment 462: The method of Embodiment 447,
wherein one or more droplets is at most 1 picoliter, at most 10
picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10
nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. Embodiment 463: The method of Embodiment 447, further
comprising incubating a droplet for a period of time to allow the
respective effector and the corresponding sample to interact.
Embodiment 464: The method of Embodiment 447, wherein the signal
comprises electromagnetic radiation, thermal radiation, a visual
change in the sample, or combinations thereof.
[0458] Disclosed herein, in some embodiments, is a method for
screening combinations of encoded effectors against a sample, the
method comprising: (a) amplifying a target protein within an
encapsulation, wherein the encapsulation comprises: (i) a nucleic
acid coding the expression of the target protein, wherein the
nucleic acid comprises a barcode region; and (ii) an in vitro
transcription/translation system; (b) introducing two or more
nucleic acid encoded effectors into the encapsulation, wherein the
two or more nucleic acid encoded effectors comprise nucleic acid
encodings; (c) barcoding the nucleic acid encodings of the two or
more encoded effectors using the barcode on the nucleic acid
encoding the target protein; (d) incubating the encapsulation for a
period of time to allow the two or more effectors to interact with
the target protein; and (e) measuring a signal produced by the
interaction between the two or more effectors and the target
protein. In some embodiments, the method further comprising the
step (f) sorting the encapsulation based on the measurement of the
signal as compared to a predetermined threshold. In some
embodiments, the method further comprising the step (g) sequencing
the nucleic acid encoding the effector which now comprises the
barcode from the nucleic acid coding for the target protein. In
some embodiments, the method further comprising the step of (h)
identifying combinations of effectors that conferred efficacy
against the target protein. In some embodiments, wherein amplifying
the target protein comprises activating expression of the target
protein. In some embodiments, wherein amplifying the target protein
comprises expressing the protein to a desired concentration. In
some embodiments, the target protein is a signaling protein, an
enzyme, a binding protein, an antibody or antibody fragment, a
structural protein, a storage protein, or a transport protein In
some embodiments, the target protein is an enzyme. 99. In some
embodiments, the encapsulation is a droplet. In some embodiments,
the droplet is at most 1 picoliter, at most 10 picoliters, at most
100 picoliters, at most 1 nanoliter, at most 10 nanoliters, at most
100 nanoliters, or at most 1 microliter in volume. In some
embodiments, the barcoded nucleic acid encoding the target protein
comprises a primer sequence complementary to a sequence on the one
or more nucleic acids encoding the one or more effectors. In some
embodiments, the barcoded nucleic acid coding the expression of the
target protein comprises a promoter sequence. In some embodiments,
wherein introducing two or more nucleic acid encoded effectors to
the droplet comprises pico-injection or droplet merging. In some
embodiments, wherein two or more nucleic acid encoded effectors are
introduced into the encapsulation. In some embodiments, wherein at
least two nucleic acid encoded effectors are introduced into the
droplet. In some embodiments, wherein the nucleic acids encoding
the two or more effectors are least 10, 15, 20, 25, 50, 75, or 100
nucleotides in length. In some embodiments, wherein each nucleic
acid encoding the one or more effectors comprises a primer sequence
complementary to a sequence encoded on the barcoded nucleic acid
coding the expression of the target protein. In some embodiments,
wherein each effector is a chemical compound. In some embodiments,
wherein each effector is a chemical fragment. In some embodiments,
wherein at least one of the nucleic acids encoded effectors is
attached to a scaffold. In some embodiments, the scaffold is a
bead, a fiber, a nanofibrous scaffold, a molecular cage, a
dendrimer, or a multi-valent molecular assembly. In some
embodiments, the scaffold is polymer-bead, a glass bead, a metal
bead, or a magnetic bead. In some embodiments, the nucleic acid
encoding the effector is attached to the scaffold. In some
embodiments, the attachment to the scaffold is through a cleavable
linker. In some embodiments, the cleavable linker is cleavable by
electromagnetic radiation, an enzyme, chemical reagent, heat, pH
adjustment, sound or electrochemical reactivity. In some
embodiments, the cleavable linker is cleavable by electromagnetic
radiation. In some embodiments, the amount of effector, nucleic
acid, or molecular weight barcode released can be controlled by the
intensity or duration of exposure to electromagnetic radiation. In
some embodiments, the cleavable linker is cleavable by a cleaving
reagent. In some embodiments, the cleavable linker is a disulfide
bond or a substituted trans-cyclooctene, and the cleaving reagent
is a phosphine or a tetrazine. In some embodiments, the amount of
effector, nucleic acid, or molecular weight barcode released is
controlled by the concentration of the chemical reagent in the
encapsulation. In some embodiments, the rate of effector, nucleic
acid, or molecular weight barcode released is controlled by the
concentration of the chemical reagent in the droplet. In some
embodiments, the cleavable linker is cleavable by an enzyme. In
some embodiments, the enzyme is a protease, a nuclease, or a
hydrolase. In some embodiments, the rate of effector, nucleic acid,
or molecular weight barcode released is controlled by the amount of
enzyme in the droplet. In some embodiments, wherein barcoding the
nucleic acids encoding the two or more effectors with the barcode
on the nucleic acid coding the target protein comprises hybridizing
the one or more nucleic acids encoding the effector with mRNA
transcribed from the nucleic acid coding for the target protein and
extending the transcribed mRNA or the nucleic acid encoding the
effector with a polymerase enzyme. In some embodiments, the period
of time is at least 1 minute, at least 10 minutes, at least 1 hour,
at least 4 hours, or at least 1 day. In some embodiments, the
period of time is controlled by residence time as the droplet
travels through a microfluidic channel. In some embodiments, the
residence time is controlled by a flow rate through the
microfluidic channel, a geometry of the microfluidic channel, a
valve in the microfluidic channel, or by removing the droplet from
the microfluidic channel, or transferring the droplet to a separate
vessel. In some embodiments, the signal is electromagnetic
radiation, thermal radiation, or a visual change in the sample. In
some embodiments, the signal is electromagnetic radiation. In some
embodiments, the electromagnetic radiation is in the visible
spectrum. In some embodiments, the electromagnetic radiation is
fluorescence or luminescence. In some embodiments, the signal is
fluorescence emitted by a TaqMan probe or a molecular beacon. In
some embodiments, the signal is thermal radiation detected with an
infrared camera. In some embodiments, the signal is a morphological
of visual change in the sample measured by recording a series of
images of the encapsulation.
[0459] Disclosed herein, in some embodiments, is a method for
screening an encoded effector without a physical sorting step, the
method comprising: (a) providing a sample, a nucleic acid encoded
effector, and a nucleic acid encoding in an encapsulation; (b)
detecting a signal in the encapsulation resulting from an
interaction between the effector and the sample; and (c) adding a
first capping mix to the droplet based on the detection, absence,
or level of the signal, wherein the first capping mix adds a first
nucleic acid cap to the nucleic acid encoding. In some embodiments,
the first nucleic acid cap comprises a first nucleic acid barcode.
In some embodiments, the first nucleic acid barcode indicates that
the effector has a desired activity. In some embodiments, the first
nucleic acid cap is added to the nucleic acid encoding by ligation,
hybridization, or extension of the nucleic acid encoding. In some
embodiments, the first capping mix further comprises additional
reagents to effectuate the adding of the first nucleic acid cap. In
some embodiments, the first nucleic acid cap is single-stranded
DNA, double-stranded DNA, single-stranded RNA, or double-stranded
RNA. In some embodiments, the method further comprising the step of
adding a second capping mix to the encapsulation if the first
capping mix is not added to the encapsulation, wherein the second
capping mix ads a second nucleic acid cap to the nucleic acid
encoding, wherein the first nucleic acid cap and the second nucleic
acid cap have different sequences. In some embodiments, the second
nucleic acid cap comprises a second nucleic acid barcode. In some
embodiments, the second nucleic acid barcode indicates that the
effector does not have a desired activity. In some embodiments, the
second nucleic acid cap is added to the nucleic acid encoding by
ligation, hybridization, or extension of the nucleic acid encoding.
In some embodiments, the second capping mix further comprises
additional reagents to effectuate the adding of the second nucleic
acid cap. In some embodiments, the second nucleic acid cap is
single-stranded DNA, double-stranded DNA, single-stranded RNA, or
double-stranded RNA. In some embodiments, the second capping mix is
added by pico-injection. In some embodiments, only the first
capping mix or only the second capping mix is added to the
encapsulation. In some embodiments, the first capping mix is added
by pico-injection. In some embodiments, the method does not
comprise a further physical sorting of the encapsulations. In some
embodiments, the sample is a biological sample. In some
embodiments, the sample is one or more cells, one or more proteins,
one or more enzymes, one or more nucleic acids, one or more
cellular lysates, or one or more tissue extracts. In some
embodiments, the sample is a single cell. In some embodiments, the
effector is a compound, a protein, a peptide, an enzyme, or a
nucleic acid. In some embodiments, the effector is a compound. In
some embodiments, the effector is a drug-like small molecule. In
some embodiments, the nucleic acid encoding comprises a terminal
capping site. In some embodiments, the terminal capping site
comprises a sequence complementary to a sequence on the first
nucleic acid cap. In some embodiments, the nucleic acid encoding
comprises single-stranded DNA, double-stranded DNA, single-stranded
RNA, or double-stranded RNA. In some embodiments, the encapsulation
is a droplet. In some embodiments, the droplet is at most 1
picoliter, at most 10 picoliters, at most 100 picoliters, at most 1
nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at
most 1 microliter in volume. In some embodiments, the encapsulation
is an emulsion in an oil. In some embodiments, the effector is
attached to a scaffold. In some embodiments, the scaffold is a
bead, a fiber, a nanofibrous scaffold, a molecular cage, a
dendrimer, or a multi-valent molecular assembly. In some
embodiments, the scaffold is polymer-bead, a glass bead, a metal
bead, or a magnetic bead. In some embodiments, the effector is
covalently attached to the scaffold by a first cleavable linker. In
some embodiments, the method further comprising cleaving the first
cleavable linker. In some embodiments, the nucleic acid encoding is
attached to the scaffold. In some embodiments, the nucleic acid
encoding is covalently attached to the scaffold by a second
cleavable linker. In some embodiments, the first and second
cleavable linkers are different. In some embodiments, the method
further comprising cleaving the second cleavable linker. In some
embodiments, the second cleavable linker is cleaved prior to adding
the first or second capping mix. In some embodiments, the signal is
electromagnetic radiation, thermal radiation, or a visual change in
the sample. In some embodiments, detecting the signal comprises
providing the encapsulation through a microfluidic channel equipped
with a detector. In some embodiments, the method further comprising
incubating the encapsulation for a period of time to allow the
effector and sample to interact. In some embodiments, the period of
time is controlled by a residence time as the encapsulation travels
through a microfluidic channel. In some embodiments, the method of
further comprising sequencing the nucleic acid encoding. In some
embodiments, the sequencing is next-generation sequencing. In some
embodiments, the method comprising performing the screen of any
embodiment described herein against a library of encoded effectors,
wherein the library of encoded effectors comprises a plurality of
different effectors.
[0460] In some embodiments, disclosed herein is a method of
measuring effector loading on scaffolds, the method comprising: (a)
attaching an effector subunit to effector attachment sites on a
plurality of scaffolds; (b) attaching a detectable label to any
remaining free effector attachment sites on the plurality of
scaffolds after the step of attaching an effector subunit; (c)
removing a subset of scaffolds from the plurality; (d) measuring
the amount of detectable label attached to the subset of scaffolds
to determine the amount of effector subunits successfully attached
to the effector attachment sites; (e) optionally activating the
attached effector subunits to create new effector attachment sites;
and (f) repeating steps (a)-(e) until a desired effector is
assembled; wherein the scaffold further comprises a nucleic acid
encoding the effector or wherein the method further comprises
attaching nucleic acid encoding subunits to the scaffold
corresponding to the effector subunits as the effector subunits are
added to the scaffold. In some embodiments, In some embodiments,
step (e) is omitted after the last effector subunit is attached. In
some embodiments, each effector subunit attached to the scaffold is
independently an amino acid, a small molecule fragment, a
nucleotide, or a compound. In some embodiments, each effector
subunit attached to the scaffold is an amino acid. In some
embodiments, each effector subunit attached to the scaffold is a
compound. In some embodiments, the effector attachment sites
comprise reactive functionalities. In some embodiments, the
effector attachment sites comprise amino or carboxylate groups. In
some embodiments, the effector attachment sites comprise
biorthogonal or CLICK chemistry reactive groups. In some
embodiments, the effector subunits comprise a reactive group
complementary to the effector attachment sites. In some
embodiments, the detectable label comprises a reactive group
complementary to the effector attachment sites. In some
embodiments, the detectable label comprises a reactive group which
is the same as a reactive group on the effector subunit whose
attachment is being measured by the detectable label. In some
embodiments, the detectable label is a fluorophore. In some
embodiments, at most 10%, at most 20%, at most 30%, at most 40%, or
at most 50% of the effector attachment sites are free after the
step of attaching the effector subunit. In some embodiments,
removing a subset of the plurality of scaffolds comprises removing
no more than 1%, no more than 2%, no more than 3%, no more than 5%,
or no more than 10% of the remaining scaffolds. In some
embodiments, measuring the amount of detectable label attached to
the subset of scaffolds to determine the amount of effector
subunits successfully attached to the effector attachment sites
comprises comparing the measurement of the detectable label to the
measurement of detectable label on a scaffold without any effector
subunits attached. In some embodiments, the amount of effector
subunits successfully attached to the subset of scaffolds is
expressed as a percentage of total attachment sites occupied by the
effector subunits. In some embodiments, optionally activating the
attached effector subunits to create a new effector attachment site
comprises removing a protecting group from the attached effector
subunit. In some embodiments, the protecting group is an amino
protecting group, a carboxylate protecting group, an alcohol
protecting group, a phenol protecting group, an alkyne protecting
group, an aldehyde protecting group, or a ketone protecting group.
In some embodiments, the protecting group is an amino protecting
group. In some embodiments, the amino protecting group is
9-fluorenylmethyloxcarbonyl (Fmoc), tert-butyloxycarbonyl (BOC),
carbobenzyloxy (Cbz), benzyl (Bz), tosyl (Ts) or trichloroethyl
chloroformate (Troc). In some embodiments, the protecting group is
a carboxylate protecting group. In some embodiments, the
carboxylate protecting group is a methyl ester, a benzyl ester, a
tert-butyl ester, a 2,6-disubstituted phenolic ester, a silyl
ester, or an orthoester. In some embodiments, the new effector
attachment site is the same functionality as the previous effector
attachment site. In some embodiments, the new effector attachment
site is a different functionality from the previous effector
attachment site. In some embodiments, steps (a)-(e) are repeated at
least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 10, or at least 20 times. 343. In some embodiments, the
scaffold is a bead, a fiber, a nanofibrous scaffold, a molecular
cage, a dendrimer, or a multi-valent molecular assembly. In some
embodiments, the scaffold is polymer-bead, a glass bead, a metal
bead, or a magnetic bead. In some embodiments, the scaffold
comprises a nucleic acid encoding the effector. In some
embodiments, the method further comprises attaching nucleic acid
encoding subunits to the scaffold corresponding to the effector
subunits as the effector subunits are added to the scaffold. In
some embodiments, a library of effector loaded scaffolds are
synthesized concurrently. In some embodiments, subsets of scaffolds
from an effector attachment step from the library are pooled prior
to detection of the detectable label In some embodiments, the
subsets of scaffolds are encapsulated in an encapsulation. In some
embodiments, the encapsulations are droplets. In some embodiments,
a majority of the encapsulations comprise a single scaffold. In
some embodiments, scaffolds from the subset of scaffolds are binned
according to the amount of detectable label detected. In some
embodiments, each bin comprises a unique range of detectable label
detected. In some embodiments, for any method described herein,
further comprising the step of sequencing encoding nucleic acids or
encoding nucleic acid subunits of the pools to reveal which
effector subunits correspond to a particular yield in a step of
attaching effector subunits to effector attachment sites. In some
embodiments, the sequencing step is performed each time steps
(a)-(e) are repeated. In some embodiments, yields of each step
(a)-(e) for each unique scaffold are collected to create a dataset
which reveals the loading of the complete desired effector on each
scaffold.
[0461] Disclosed herein, in some embodiments, is an array device
for screening encoded beads comprising: (a) a hydrophobic surface;
and (b) nucleic acid patches interspersed on the hydrophobic
surface; wherein the hydrophobic surface and nucleic acid patches
are configured such that when a proscribed amount of media is
deployed across the surface each nucleic acid patch is covered with
media and the hydrophobic surface between the nucleic acid patches
does not contain media. In some embodiments, an array device
described herein further comprising one or more channels beneath
the hydrophobic surface, wherein the channels comprise terminal
ends within nucleic acid patches. In some embodiments, the channels
are configured to deliver reagents to the nucleic acid patches. In
some embodiments, the reagents are delivered as a liquid solution.
In some embodiments, the hydrophobic surface comprises a
hydrophobic polymer. 3 In some embodiments, the hydrophobic polymer
comprises a polyacrylic, a polyamide, a polycarbonate, a polydiene,
a polyester, a polyether, a polyfluorocarbon, a polyolefin, a
polystyrene, a polyvinyl acetal, a polyvinyl chloride, a polyvinyl
ester, a polyvinyl ether, a polyvinyl ketone, a polyvinyl pyridine,
a polyvinylpyrrolidone, a polysilane, a polyfluorosilane, a poly
perfluorosilane or a combination thereof. In some embodiments, the
hydrophobic polymer comprises a polyfluorocarbon. In some
embodiments, the hydrophobic polymer is fluorinated. In some
embodiments, the hydrophobic surface is a surface functionalized
with hydrophobic groups. In some embodiments, the hydrophobic
groups are fatty acids, alkyl groups, alkoxy groups, aromatic
groups, alkyl silanes, fluorosilanes, perfluorosilanes, or
combinations thereof. In some embodiments, the hydrophobic groups
are fluorinated. In some embodiments, cells do not bind to the
hydrophobic surface. In some embodiments, the nucleic acid patches
bind cells. In some embodiments, single nucleic acid patches are
encapsulated within single droplets of the media. In some
embodiments, the nucleic acid patches comprise DNA, RNA,
combinations thereof. In some embodiments, the nucleic acid patches
comprise nucleic acids capable of binding nucleic acid encoded
beads. In some embodiments, the nucleic acids bind nucleic acid
encoded beads non-specifically, by binding a complementary nucleic
acid on the bead, or by binding another group on the bead. In some
embodiments, the nucleic acid patches are up to about 1 .mu.m2 in
size, up to about 10 .mu.m2 in size, up to about 100 .mu.m2 in
size, up to about 1000 .mu.m2 in size, or up to about 10000 .mu.m2
in size. In some embodiments, the nucleic acid patches are
separated by up to about 1 .mu.m, up to about 10 .mu.m, up to about
100 .mu.m, up to about 1000 .mu.m, or up to about 10000 .mu.m. In
some embodiments, the nucleic acid patches are arranged in a grid
pattern. In some embodiments, the media is an aqueous media. In
some embodiments, the density of nucleic acid patches is at least
100 patches/cm2, at least 1000 patches/cm2, at least 10000
patches/cm2, at least 100000 patches/cm2, at least 1000000
patches/cm2, or at least 10000000 patches/cm2. In some embodiments,
the surface area of the device is at least 1 cm2, at least 5 cm2,
at least 10 cm2, at least 25 cm2, at least 50 cm2, at least 100
cm2, at least 500 cm2, or at least 1000 cm2.
[0462] Disclosed herein, in some embodiments, is a method of
performing a screen, the method comprising: (a) binding nucleic
acid encoded beads to the nucleic acid patches of the array of any
one of embodiments described herein; (b) sequencing the nucleic
acid encoded beads (c) binding cells to the nucleic acid patches;
and (d) performing an assay on the array. In some embodiments, the
beads further comprise encoded effectors. In some embodiments, the
method further comprising the step of releasing the effectors from
the beads. In some embodiments, releasing the effectors from the
beads comprises adding a cleaving reagent to the nucleic acid
patches. In some embodiments, sequencing the beads allows
determination of the physical location of specific nucleic acid
encoded beads. In some embodiments, the assay produces a detectable
signal. 412. In some embodiments, each nucleic acid patch binds a
single bead and a single cell.
[0463] Disclosed herein, in some embodiments, is a method for
stimulating an ion channel, the method comprising: (a) providing a
cell in an encapsulation; (b) stimulating an ion channel of the
cell by electrostimulation, optical stimulation, or chemical
stimulation; and (c) detecting a signal from the cell by capturing
images of the cell in the encapsulation. In some embodiments, the
ion channel is stimulated by electrostimulation. In some
embodiments, the electrostimulation is performed by an electrode.
In some embodiments, the electrode is within a flow path of the
encapsulation. In some embodiments, the electrode is outside of a
flow path of the encapsulation. In some embodiments, the ion
channel is stimulated by optical stimulation. In some embodiments,
the ion channel of the cell comprises a mutation. In some
embodiments, the mutation sensitizes the ion channel to optical
stimulation. In some embodiments, the ion channel is stimulated by
chemical stimulation. In some embodiments, the chemical stimulation
comprises contacting the ion channel with a toxin. In some
embodiments, the toxin is added to the encapsulation by
pico-injection. In some embodiments, the pico-injection is
conditional pico-injection. 4 In some embodiments, the toxin is an
ion channel toxin. In some embodiments, the signal is a
morphological or visual change in the cell. In some embodiments,
capturing images of the cell comprises recording a series of images
of the encapsulation. In some embodiments, the method further
comprising superimposing the series of images of the sample in the
encapsulation. In some embodiments, the encapsulation further
comprises a detection reagent.
[0464] In one aspect, provided herein, is a method for stimulating
an ion channel, the method comprising: (a) providing a cell in an
encapsulation; (b) stimulating an ion channel of the cell by
electrostimulation, optical stimulation, or chemical stimulation;
and (c) detecting a signal from the cell by capturing images of the
cell in the encapsulation.
[0465] In one aspect, provided herein, is a method for screening
ion channel modulators, the method comprising: (a) providing an
encapsulation comprising: (i) a cell expressing an ion channel
protein; (ii) a set of voltage sensor probes; and (iii) an encoded
effector and its corresponding encoding; (b) stimulating an ion
channel of the cell; and (c) detecting a signal from at least one
member of the set of voltage sensor probes. In some embodiments,
the encapsulation is a droplet, an emulsion, a picowell, a
macrowell, a microwell, a bubble, or a microfluidic confinement. In
some embodiments, the encapsulation is a droplet. In some
embodiments, the droplet is at most 1 picoliter, at most 10
picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10
nanoliters, at most 100 nanoliters, or at most 1 microliter in
volume. In some embodiments, the cell comprises a mammalian cell.
In some embodiments, the cell comprises a human cell. In some
embodiments, the cell comprises a HEK293 cell. In some embodiments,
the ion channel protein comprises a sodium, calcium, chloride,
proton, or potassium ion channel protein. In some embodiments,
wherein the ion channel protein comprises a voltage gated ion
channel protein. In some embodiments, the ion channel protein
comprises an endogenous ion channel protein. In some embodiments,
the ion channel protein comprises an exogenous ion channel protein.
In some embodiments, the ion channel protein comprises a sodium,
calcium, chloride, proton, or potassium voltage gated ion channel
protein. In some embodiments, the ion channel protein comprises a
voltage gated calcium channel protein (VGCC). In some embodiments,
the ion channel protein comprises an L-type calcium channel, a
P-type calcium channel, an N-type calcium channel, an R-type
calcium channel, or a T-type calcium channel, or any mutant,
fragment, or conjugate thereof. In some embodiments, the ion
channel protein comprises a channelrhodopsin or any mutant,
fragment, or conjugate thereof. In some embodiments, the
channelrhodopsin is ChrimsonR or any mutant, fragment, or conjugate
thereof. In some embodiments, the ion channel protein is
overexpressed. In some embodiments, the set of voltage sensor
probes comprise a FRET pair. In some embodiments, the set of
voltage sensor probes comprises a voltage-sensitive oxonol, a
fluorescent coumarin, or both. In some embodiments, the set of
voltage sensor probes comprises a DiSBAC compound, a coumarin
phospholipid, or any combination or derivative thereof. In some
embodiments, the set of voltage sensors comprises a DiSBAC.sub.2,
DiSBAC.sub.4, DiSBAC.sub.6, CC1-DMPE, CC2-DMPE, or any combination
or derivative thereof. In some embodiments, the set of voltage
sensors comprises DiSBAC.sub.6 and CC2-DMPE. In some embodiments,
the encapsulation further comprises a voltage assay background
suppression compound. In some embodiments, the voltage assay
background suppression compound comprises VABSC-1. In some
embodiments, the effector and its corresponding encoding are bound
to a scaffold. In some embodiments, the scaffold is a bead, a
fiber, a nanofibrous scaffold, a molecular cage, a dendrimer, or a
multi-valent molecular assembly. In some embodiments, the scaffold
is polymer-bead, a glass bead, a metal bead, or a magnetic bead. In
some embodiments, the scaffold is a bead from 10 .mu.m to about 100
.mu.m in diameter. In some embodiments, the effector is bound to
the scaffold through a cleavable linker. In some embodiments, the
cleavable linker is a photocleavable linker. In some embodiments,
the method further comprises the step of cleaving the cleavable
linker. In some embodiments, the effector is a compound or a
peptide. In some embodiments, the effector is a small molecule. In
some embodiments, the encoding is a nucleic acid. In some
embodiments, stimulating the ion channel comprises
electrostimulation, optical stimulation, chemical stimulation, or
any combination thereof. In some embodiments, stimulating the ion
channel comprises electrostimulation. In some embodiments, wherein
stimulating the ion channel is performed by at least one electrode.
In some embodiments, the at least one electrode is in the flow path
of the encapsulation. In some embodiments, electrostimulation is
performed by non-contact electrodes to generate electric fields,
dielectrophoretic forces, or embedded metal-contact electrodes. In
some embodiments, electrostimulation is dictated by geometry of a
microfluidic device containing the encapsulation. In some
embodiments, the frequency of electrostimulation is about 10 Hz. In
some embodiments, stimulating the ion channel comprises optical
stimulation. In some embodiments, the optical stimulation is UV,
VIS, or near-infrared radiation. In some embodiments, the optical
stimulation is performed using an embedded fiber-optic wave guide
embedded in a microfluidic device containing the encapsulation. In
some embodiments, wherein the frequency of optical stimulation is
about 10 Hz. In some embodiments, the wavelength of light for
optical stimulation is about 660 nm. In some embodiments, the
intensity of light for optical stimulation is about 500
mJ/s/cm.sup.2. In some embodiments, stimulating the ion channel
comprises chemical stimulation. In some embodiments, chemical
stimulation comprises contacting the ion channel with an ion
channel toxin. In some embodiments, the ion channel toxin comprises
veratridine, OD-1, or another ion channel toxin, or any combination
thereof. In some embodiments, the ion channel toxin as added to the
encapsulation by pico-injection, droplet fusion, or through a
pre-arranged architecture of a microfluidic device which contains
the encapsulation. In some embodiments, the signal is
electromagnetic radiation. In some embodiments, the electromagnetic
radiation is luminescence or fluorescence. In some embodiments, the
electromagnetic radiation is fluorescence. In some embodiments, the
electromagnetic radiation is emitted due to a FRET interaction. In
some embodiments, the signal is an increase, decrease, or change in
electromagnetic radiation as compared to an identical encapsulation
without the encoded effector. In some embodiments, the signal is an
increase, decrease, or change in electromagnetic radiation as
compared to the encapsulation before the stimulation of the ion
channel. In some embodiments, the method further comprises the step
of sorting the encapsulation based on the presence, absence, level,
or change of the signal. In some embodiments, the method further
comprises measuring a property of the encoding to ascertain the
identity of the effector.
[0466] In one aspect, provided herein, is a microfluidic device for
droplet based encoded library screening comprising: (a) a first
microfluidic channel comprising an aqueous fluid; (b) a second
microfluidic channel comprising a fluid immiscible with the aqueous
fluid; (c) a junction at which the first microfluidic channel is in
fluid communication with the second microfluidic channel, wherein
the junction of the first and second microfluidic channels defines
a device plane, wherein the junction is configured to form droplets
of the aqueous fluid within the fluid from the second microfluidic
channel, wherein the second microfluidic channel is configured to
continue past the junction thereby defining an assay flow path; (d)
a cleavage region for cleaving effectors from scaffolds disposed
within the assay flow path; (e) a detection region; and (f) a
sorting region. In some embodiments, the device further comprises a
stimulation region. In some embodiments, the stimulation region
comprises one or more actuators for stimulating an ion channel. In
some embodiments, the one or more actuators for stimulating the ion
channel comprises at least one light source, at least one
electrode, or at least one pico-injection site equipped with an ion
channel toxin. In some embodiments, the one or more actuators
comprises at least one electrode. In some embodiments, the one or
more actuators comprises a pair of electrodes on opposite walls of
the assay flow path such that when a droplet passes the pair of
electrodes the droplet contacts the electrodes, thereby allowing a
current to flow through the droplet. In some embodiments, the
stimulation region comprises at least 1, at least 2, at least 3, at
least 5, at least 7, at least 10, or at least 20 actuators. In some
embodiments, at least one of the actuators for stimulating the ion
channel is substantially parallel with the device plane. In some
embodiments, at least one of the actuators for stimulating the ion
channel lies at a curve in the assay flow path. In some
embodiments, the stimulation region is upstream of the detection
region and downstream of the cleavage region. In some embodiments,
the cleavage region comprises a light source configured to cleave
effectors from scaffolds disposed within the assay flow path. In
some embodiments, the light source is a source of UV light. In some
embodiments, the light source is configured to have an optical axis
substantially parallel with the device plane. In some embodiments,
the light source illuminates a passing droplet at a curve in the
assay flow path. In some embodiments, the cleavage region is
upstream of the detection region and the sorting region. In some
embodiments, the cleavage region is downstream of the junction. In
some embodiments, the assay flow path comprises a serpentine flow
path region. In some embodiments, the serpentine flow path region
comprises at least 10, at least 20, at least 30, at least 40, at
least 50, or at least 100 curves. In some embodiments, the
detection region comprises a fluorometer. In some embodiments, the
fluorometer is configured to have an optical axis substantially
parallel to the device plane. In some embodiments, the fluorometer
illuminates a passing droplet at a curve in the assay flow path. In
some embodiments, the fluorometer is configured to detect two or
more wavelengths of fluorescence. In some embodiments, the
detection region is downstream of the cleavage region. In some
embodiments, the detection region is upstream of the sorting
region. In some embodiments, the sorting region comprises a sorter
configured to sort droplets based on a signal detected in the
detection region.
Definitions
[0467] Unless defined otherwise, all terms of art, notations and
other technical and scientific terms or terminology used herein are
intended to have the same meaning as is commonly understood by one
of ordinary skill in the art to which the claimed subject matter
pertains. In some cases, terms with commonly understood meanings
are defined herein for clarity and/or for ready reference, and the
inclusion of such definitions herein should not necessarily be
construed to represent a substantial difference over what is
generally understood in the art.
[0468] Throughout this application, various embodiments may be
presented in a range format. It should be understood that the
description in range format is merely for convenience and brevity
and should not be construed as an inflexible limitation on the
scope of the disclosure. Accordingly, the description of a range
should be considered to have specifically disclosed all the
possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the
breadth of the range.
[0469] As used in the specification and claims, the singular forms
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a sample"
includes a plurality of samples, including mixtures thereof.
[0470] The terms "determining," "measuring," "detecting,"
"evaluating," "assessing," "assaying," and "analyzing" are often
used interchangeably herein to refer to forms of measurement. The
terms include determining if an element is present or not (for
example, detection). These terms can include quantitative,
qualitative or quantitative and qualitative determinations.
Assessing can be relative or absolute. "Detecting the presence of"
can include determining the amount of something present in addition
to determining whether it is present or absent depending on the
context.
[0471] The term "in vivo" is used to describe an event that takes
place in a subject's body.
[0472] The term "ex vivo" is used to describe an event that takes
place outside of a subject's body. An ex vivo assay is not
performed on a subject. Rather, it is performed upon a sample
separate from a subject. An example of an ex vivo assay performed
on a sample is an "in vitro" assay.
[0473] The term "in vitro" is used to describe an event that takes
places contained in a container for holding laboratory reagent such
that it is separated from the biological source from which the
material is obtained. In vitro assays can encompass cell-based
assays in which living or dead cells are employed. In vitro assays
can also encompass a cell-free assay in which no intact cells are
employed.
[0474] The term "hit" refers to an effector that has been screened
against a sample and returned a positive result. The positive
result may depend upon the nature of the screen being employed, but
may include, without limitation, an indication of efficacy against
a target being interrogated.
[0475] The term "screen" as used herein refers to performing an
assay using a plurality of effectors in order to determine the
effect various effectors have on a particular sample.
[0476] The term "sequencing" refers to determining the nucleotide
sequence of a nucleic acid. Any suitable method for sequencing may
be employed with the methods and systems provided herein. The
sequencing may be accomplished by next generation sequencing. Next
generation sequencing encompasses many kinds of sequencing such as
pyrosequencing, sequencing-by-synthesis, single-molecule
sequencing, second-generation sequencing, nanopore sequencing,
sequencing by ligation, or sequencing by hybridization.
Next-generation sequencing platforms are those commercially
available from Illumina (RNA-Seq) and Helicos (Digital Gene
Expression or "DGE"). Next generation sequencing methods include,
but are not limited to those commercialized by: 1) 454/Roche
Lifesciences including but not limited to the methods and apparatus
described in Margulies et al., Nature (2005) 437:376-380 (2005);
and U.S. Pat. Nos. 7,244,559; 7,335,762; 7,211,390; 7,244,567;
7,264,929; 7,323,305; 2) Helicos Biosciences Corporation
(Cambridge, Mass.) as described in U.S. application Ser. No.
11/167,046, and U.S. Pat. Nos. 7,501,245; 7,491,498; 7,276,720; and
in U.S. Patent Application Publication Nos. US20090061439;
US20080087826; US20060286566; US20060024711; US20060024678;
US20080213770; and US20080103058; 3) Applied Biosystems (e.g. SOLiD
sequencing); 4) Dover Systems (e.g., Polonator G.007 sequencing);
5) lllumina, Inc. as described in U.S. Pat. Nos. 5,750,341;
6,306,597; and 5,969,119; and 6) Pacific Biosciences as described
in U.S. Pat. Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050;
7,462,468; 7,476,503; 7,315,019; 7,302,146; 7,313,308; and US
Application Publication Nos. US20090029385; US20090068655;
US20090024331; and US20080206764. Such methods and apparatuses are
provided here by way of example and are not intended to be
limiting.
[0477] The term "barcode" refers to a nucleic acid sequence that is
unique to a particular system. The barcode may be unique to a
particular method or to a particular effector. The nucleic acid
encodings of the methods and systems provided herein are analogous
to barcodes in that they are unique nucleic acid sequences that can
be used to identify the structure of a given effector. The length
of a barcode or nucleic acid encoding should be sufficient to
differentiate between all the effectors in a given library.
[0478] The term "flow" means any movement of liquid or solid
through a device or in a method of the disclosure, and encompasses
without limitation any fluid stream, and any material moving with,
within or against the stream, whether or not the material is
carried by the stream. For example, the movement of molecules,
cells or virions through a device or in a method of the disclosure,
e.g. through channels of a microfluidic chip of the disclosure,
comprises a flow. This is so, according to the disclosure, whether
or not the molecules, cells or virions are carried by a stream of
fluid also comprising a flow, or whether the molecules, cells or
virions are caused to move by some other direct or indirect force
or motivation, and whether or not the nature of any motivating
force is known or understood. The application of any force may be
used to provide a flow, including without limitation, pressure,
capillary action, electro-osmosis, electrophoresis,
dielectrophoresis, optical tweezers, and combinations thereof,
without regard for any particular theory or mechanism of action, so
long as molecules, cells or virions are directed for detection,
measurement or sorting according to the disclosure.
[0479] An "inlet region" is an area of a microfabricated chip that
receives molecules, cells or virions for detection measurement or
sorting. The inlet region may contain an inlet channel, a well or
reservoir, an opening, and other features which facilitate the
entry of molecules, cells or virions into the device. A chip may
contain more than one inlet region if desired. The inlet region is
in fluid communication with the main channel and is upstream
therefrom.
[0480] An "outlet region" is an area of a microfabricated chip that
collects or dispenses molecules, cells or virions after detection,
measurement or sorting. An outlet region is downstream from a
discrimination region, and may contain branch channels or outlet
channels. A chip may contain more than one outlet region if
desired.
[0481] An "analysis unit" is a microfabricated substrate, e.g., a
microfabricated chip, having at least one inlet region, at least
one main channel, at least one detection region and at least one
outlet region. Sorting embodiments of the analysis unit include a
discrimination region and/or a branch point, e.g. downstream of the
detection region, that forms at least two branch channels and two
outlet regions. A device according to the disclosure may comprise a
plurality of analysis units.
[0482] A "main channel" is a channel of the chip of the disclosure
which permits the flow of molecules, cells or virions past a
detection region for detection (identification), measurement, or
sorting. In a chip designed for sorting, the main channel also
comprises a discrimination region. The detection and discrimination
regions can be placed or fabricated into the main channel. The main
channel is typically in fluid communication with an inlet channel
or inlet region, which permits the flow of molecules, cells or
virions into the main channel. The main channel is also typically
in fluid communication with an outlet region and optionally with
branch channels, each of which may have an outlet channel or waste
channel. These channels permit the flow of cells out of the main
channel.
[0483] A "detection region" is a location within the chip,
typically within the main channel where molecules, cells or virions
to be identified, measured or sorted on the basis of a
predetermined characteristic. In an embodiment, molecules, cells or
virions are examined one at a time, and the characteristic is
detected or measured optically, for example, by testing for the
presence or amount of a reporter. For example, the detection region
is in communication with one or more microscopes, diodes, light
stimulating devices, (e.g., lasers), photo multiplier tubes, and
processors (e.g., computers and software), and combinations
thereof, which cooperate to detect a signal representative of a
characteristic, marker, or reporter, and to determine and direct
the measurement or the sorting action at the discrimination region.
In sorting embodiments, the detection region is in fluid
communication with a discrimination region and is at, proximate to,
or upstream of the discrimination region.
[0484] A "carrier fluid," "immiscible fluid," or "immiscible
carrier fluid" or similar term as used herein refers to a liquid in
which a sample or assay liquid is incapable of mixing and allows
formation of droplets of the sample or assay liquid within the
carrier fluid. These terms are used interchangeable herein and are
meant to encompass the same materials. Non-limiting examples of
such carrier fluids include silicon based oils, silicone oils,
hydrophobic oils (e.g. squalene, fluorinated oils, perfluorinated
oils), or any fluid capable of encapsulating another desired liquid
containing a sample to be analyzed.
[0485] An "extrusion region," "droplet extrusion region," or
"droplet formation region" is a junction between an inlet region
and the main channel of a chip of the disclosure, which permits the
introduction of a pressurized fluid to the main channel at an angle
perpendicular to the flow of fluid in the main channel. In some
embodiments, the fluid introduced to the main channel through the
extrusion region is "incompatible" (i.e., immiscible) with the
fluid in the main channel so that droplets of the fluid introduced
through the extrusion region are sheared off into the stream of
fluid in the main channel.
[0486] A "discrimination region" or "branch point" is a junction of
a channel where the flow of molecules, cells or virions can change
direction to enter one or more other channels, e.g., a branch
channel, depending on a signal received in connection with an
examination in the detection region. Typically, a discrimination
region is monitored and/or under the control of a detection region,
and therefore a discrimination region may "correspond" to such
detection region. The discrimination region is in communication
with and is influenced by one or more sorting techniques or flow
control systems, e.g., electric, electro-osmotic, (micro-) valve,
etc. A flow control system can employ a variety of sorting
techniques to change or direct the flow of molecules, cells or
virions into a predetermined branch channel.
[0487] A "branch channel" is a channel which is in communication
with a discrimination region and a main channel. Typically, a
branch channel receives molecules, cells or virions depending on
the molecule, cell or virion characteristic of interest as detected
by the detection region and sorted at the discrimination region. A
branch channel may be in communication with other channels to
permit additional sorting. Alternatively, a branch channel may also
have an outlet region and/or terminate with a well or reservoir to
allow collection or disposal of the molecules, cells or
virions.
[0488] The term "forward sorting" or flow describes a one-direction
flow of molecules, cells or virions, typically from an inlet region
(upstream) to an outlet region (downstream), and in some instances
without a change in direction, e.g., opposing the "forward" flow.
In some embodiments, molecules, cells or virions travel forward in
a linear fashion, i.e., in single file. A "forward" sorting
algorithm consists of running molecules, cells or virions from the
input channel to the waste channel, until a molecule, cell or
virion is identified to have an optically detectable signal (e.g.
fluorescence) that is above a pre-set threshold, at which point
voltages are temporarily changed to electro-osmotically divert the
molecule or to the collection channel.
[0489] The term "reversible sorting" or flow describes a movement
or flow that can change, i.e., reverse direction, for example, from
a forward direction to an opposing backwards direction. Stated
another way, reversible sorting permits a change in the direction
of flow from a downstream to an upstream direction. This may be
useful for more accurate sorting, for example, by allowing for
confirmation of a sorting decision, selection of particular branch
channel, or to correct an improperly selected channel.
[0490] Different "sorting algorithms" for sorting in the
microfluidic device can be implemented by different programs, for
example under the control of a personal computer. As an example,
consider a pressure-switched scheme instead of electro-osmotic
flow. Electro-osmotic switching is virtually instantaneous and
throughput is limited by the highest voltage that can be applied to
the sorter (which also affects the run time through ion depletion
effects). A pressure switched-scheme does not require high voltages
and is more robust for longer runs. However, mechanical compliance
in the system is likely to cause the fluid switching speed to
become rate-limiting with the "forward" sorting program. Since the
fluid is at low Reynolds number and is completely reversible, when
trying to separate rare molecules, cells or virions, one can
implement a sorting algorithm that is not limited by the intrinsic
switching speed of the device. The molecules, cells or virions flow
at the highest possible static (non-switching) speed from the input
to the waste. When an interesting molecule, cell or virion is
detected, the flow is stopped. By the time the flow stops, the
molecule, cell or virion may be past the junction and part way down
the waste channel. The system is then run backwards at a slow
(switchable) speed from waste to input, and the molecule, cell or
virion is switched to the collection channel when it passes through
the detection region. At that point, the molecule, cell or virion
is "saved" and the device can be run at high speed in the forward
direction again. Similarly, a device of the disclosure that is used
for analysis, without sorting, can be run in reverse to re-read or
verify the detection or analysis made for one or more molecules,
cells or virions in the detection region. This "reversible"
analysis or sorting method is not possible with standard gel
electrophoresis technologies (for molecules) nor with conventional
FACS machines (for cells). Reversible algorithms are particularly
useful for collecting rare molecules, cells or virions or making
multiple time course measurements of a molecule or single cell.
[0491] The term "emulsion" refers to a preparation of one liquid
distributed in small globules (also referred to herein as drops or
droplets) in the body of a second liquid. The first liquid, which
is dispersed in globules, is referred to as the discontinuous
phase, whereas the second liquid is referred to as the continuous
phase or the dispersion medium. In one embodiment, the continuous
phase is an aqueous solution and the discontinuous phase is a
hydrophobic fluid, such as an oil (e.g., decane, tetradecane, or
hexadecane). Such an emulsion is referred to here as an oil in
water emulsion. In another embodiment, an emulsion may be a water
in oil emulsion. In such an embodiment, the discontinuous phase is
an aqueous solution and the continuous phase is a hydrophobic fluid
such as an oil. The droplets or globules of oil in an oil in water
emulsion are also referred to herein as "micelles", whereas
globules of water in a water in oil emulsion may be referred to as
"reverse micelles".
[0492] As used herein, the term "about" a number refers to that
number plus or minus 10% of that number. The term "about" a range
refers to that range minus 10% of its lowest value and plus 10% of
its greatest value.
[0493] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
EXAMPLES
[0494] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
disclosure.
Example 1: Stoichiometric Cleavage of Encoded Effectors to Probe
Protease Inhibition
[0495] A library of beads containing nucleic acid encoded small
molecules is prepared, wherein the small molecules are linked to
the beads by a substituted trans-cyclooctene. In this example, the
library is being screened to detect small molecule inhibitors of
trypsin. A solution comprising the library of beads is placed in a
first reagent well 401 of a microfluidic device 400, as shown in
FIG. 4. A solution comprising trypsin is added to a reagent well
402, and an oil medium is added to reagent well 403. The contents
of reagent wells 401, 402, and 403 flow until they meet at a
junction 404, where the trypsin solution and bead solution form
droplets in an oil emulsion. The droplets then flow along flow path
405 until they reach pico-injection site 406. At pico-injection
site 406, pico-injector 407 adds a solution containing dimethyl
tetrazine and fluorescein isothiocyanate (FITC) labelled casein.
The pico-injection is configured such that each drop passing by
receives a uniform dose of the solution. The concentration of
dimethyl tetrazine in the solution is configured such that each
droplet comprising a bead releases substantially the same amount of
effector upon receiving the pico-injection. The droplet then
continues along flow path 405 until it reaches detector 408.
Detector 408 is a fluorimeter configured to measure the FITC FRET
emission (excitation 485 nm/emission 538 nm). Based on the
resulting fluorescence detected by detector 408, the sample is
sorted at junction 409 onto a path toward bin 410 if the FRET
emission is detected above a certain threshold and onto a path
toward bin 411 if the FRET emission is not detected above the
threshold. After the screen is completed, the nucleic acid
encodings in bin 410 are sequenced by next generation sequencing to
determine which small molecules had an inhibitory effect on
trypsin.
Example 2: Nucleic Acid Detection with Molecular Beacons
[0496] A library of beads containing nucleic acid encoded small
molecules is prepared, wherein the small molecules are linked to
the beads by a disulfide bond. In this example, the library is
being screened to detect an increase in cellular expression of a
protein of interest by measuring cellular mRNA using molecular
beacons. The molecular beacons used in this example contains a
sequence complementary to the mRNA which codes for the protein of
interest. The molecular beacons further comprises a Cyanine 5 dye
at one loop end and a DABCYL quencher at the other end. In this
example, the protein of interest is expressed by a sample cell, and
the desired outcome of the screen is an increase in the expression
of the protein of interest.
[0497] A solution comprising the library of beads is placed in a
first reagent well 501 of a microfluidic device 500, as shown in
FIG. 5. A solution comprising the cells that express the protein of
interest is added to a reagent well 502, and an oil medium is added
to reagent well 503. The contents of reagent wells 501, 502, and
503 flow until they meet at a junction 504, where the solution
containing the cells and the bead solution form droplets in an oil
emulsion. The device is configured such that a majority of the
encapsulations receive a single cell and a single bead. The
droplets then flow along flow path 505 until they reach
pico-injection site 506. At pico-injection site 506, pico-injector
507 adds a solution containing tris(2-carboxyethyl)phosphine
(TCEP). The pico-injection is configured such that each drop
passing by receives a uniform dose of the solution. The
concentration of TCEP in the solution is configured such that each
droplet comprising a bead releases substantially the same amount of
effector upon receiving the pico-injection. The droplet then
continues along flow path 505 until it reaches the second
pico-injection site 508, at which point the molecular beacon is
added to the encapsulation, along with lysis buffer to lyse the
cell, by pico-injector 509. The molecular beacons then hybridize
with any mRNA encoding the protein of interest, thereby allowing a
fluorescent emission from the Cyanine 5 moiety. The droplet
continues along flow path 505 until it reaches the detector 510.
Detector 510 is a fluorimeter configured to measure the Cyanine 5
fluorescent signal (excitation 646 nm/emission 669 nm). Based on
the resulting fluorescence detected by detector 510, the sample is
sorted at junction 511 onto a path toward bin 512 if the
fluorescence emission is detected above a certain threshold and
onto a path toward bin 513 if the fluorescence emission is not
detected above the threshold. After the screen is completed, the
nucleic acid encodings in bin 512 are sequenced by next generation
sequencing to determine which small molecules had the desired
effect of increasing production of the protein of interest.
Example 3: Screening of Mutant Imine Reductases
[0498] A library of beads containing nucleic acids coding for
different mutants of an imine reductase enzyme and a corresponding
barcode is provided. In this example, the library is being screened
to detect an enzyme that can effectuate an imine reduction between
Reagent 1
##STR00005##
and Reagent 2
##STR00006##
[0500] If the enzyme screened is capable of performing the imine
reduction, the Cyanine 3 and Cyanine 5 dyes will undergo a FRET
interaction and an emission at 680 nm will be observed after an
excitation at 540 nm.
[0501] A solution comprising the library of beads is placed in a
first reagent well 601 of a microfluidic device 600, as shown in
FIG. 6. A solution comprising an in vitro transcription/translation
system (IVTT) is then added to a reagent well 602, and an oil
medium is added to reagent well 603. The contents of reagent wells
601, 602, and 603 flow until they meet at a junction 604, where the
solution containing the IVTT and the bead solution form droplets in
an oil emulsion. The device is configured such that a majority of
the encapsulations receive a single bead. The IVTT then allows
expression of the mutant imine reductases within the droplets. The
droplets then flow along flow path 605 until they reach
pico-injection site 606. At pico-injection site 606, pico-injector
607 adds a solution containing Reagent 1 and Reagent 2. The
pico-injection is configured such that each drop passing by
receives a uniform dose of the solution. The droplet continues
along flow path 605 until it reaches the detector 508. Detector 510
is a fluorimeter configured to measure the Cyanine 5/Cyanine 3 FRET
emission (excitation 540 nm/emission 680 nm). Based on the
resulting fluorescence detected by detector 608, the sample is
sorted at junction 609 onto a path toward bin 610 if the
fluorescence emission is detected above a certain threshold and
onto a path toward bin 611 if the fluorescence emission is not
detected above the threshold. After the screen is completed, the
nucleic acid on the beads in bin 610 are sequenced by next
generation sequencing to determine which imine reductase mutants
had the desired effect of forming the amine bond between Reagent 1
and Reagent 2 during the screening
Example 4: Ion Channel Screening Using a Chip-Based
Spatio-Temporally Controlled Electrical Stimulation Assay
[0502] A library of nucleic acid encoded beads containing candidate
ion channel inhibitor molecules is prepared, wherein the inhibitor
molecules are linked to the beads by nitrobenzyl photocleavable
linker. The cell line used is the Human Embryonic Kidney (HEK) cell
line that expresses a sodium ion channel of interest. The cells are
treated with a FRET probe system, containing the dyes DiSBAC.sub.6
and CC2-DMPE which report a rapid change in fluorescence upon the
stimulation of an ion channel. Stimulation can occur by chemical,
optical and electrical means.
[0503] In this example, electrical stimulation is used. The bead
encoded library is placed in reagent well 702 of microfluidic
device 700, as shown in FIG. 7. The cell solution containing the
FRET probe system is added to reagent well 703, and an oil medium
is added to reagent well 701. From the reagent well 701, the oil
travels along flow path 705. The contents of reagent wells 702 and
703 flow along separate flow paths until they meet at junction 704.
The aqueous sample solution flows down the flow path channel until
it meets the oil at junction 706, where the solution containing the
cells and the bead solution form an emulsion stream of aqueous
droplets separated by the oil. The device can be configured such
that a majority of the droplets form containing a single cell and a
single bead, but this is not necessary. The droplets then flow
along the flow path 705 until they reach UV light exposure site
708, coupled to a UV source 707 by optical fiber, where the
inhibitor molecule is released into the droplet from the nucleic
acid encoded bead. As the droplet flows down the flow path 705, the
candidate inhibitor contacts the cell. The droplet continues along
the flow path 705, where multiple electrodes 709 are placed along
the flow path. The droplets are individually exposed to electrical
stimulation at each set of electrodes 709. The electrode spacing
and flow velocity defines the desired stimulation frequency, which
in this case is case 10 Hz. After stimulation, the droplets are
passed through a fluorescence detection region 711, coupled to a
light source and detector 710 by optical fiber. Droplets which
contain effective inhibitors will exhibit a distinctly different
fluorescence intensity, after electrical stimulation, relative to
droplets that contain ineffective inhibitors. Droplets are then
sorted at the sorting site 712 according to their distinctive
fluorescence signal and are directed to collection bin 713 if
designated as a hit. Misses are directed to collection bin 714.
Example 5: Development of a Chip Device for Screening Ion Channel
Modulators
Phase 1
Goals:
[0504] Phase 1: To determine the feasibility of deploying an
ultra-high-throughput microfluidic system, Ion Channel Chip (IC
Chip), to accommodate cell-based sodium ion channel activity
assays. Propose three different droplet microfluidic approaches to
trigger cell surface ion channel activities in a microfluidic chip
by spatio-temporally controlled electrical-stimulation (ES);
controlled optical-stimulation (OS), or by toxin-induced
stimulation (TS) subsequent to compound liberation and brief
incubation. These methods will be tested to demonstrate sodium ion
channel assay compatibility and screening feasibility in droplets.
The aforementioned three methods of triggering live cell sodium ion
channels may be executed. FIG. 8 shows an overview of the
development workflow for the design and evaluation of the devices
to accomplish the aforementioned methods.
[0505] The goal of Phase 1 is to demonstrate a proof-of-concept
system using known inhibitor control compounds photolytically
released from carrier beads in droplets. The released compound will
inhibit Na+ ion channel activity in cells with sufficient
discretion when compared to uninhibited cells from a model cell
line.
Objectives:
[0506] Stage 1: Detection of Droplet-Cell-Assay for Ion Channel
Inhibition
[0507] Summary: Demonstrate cell line compatibility, and cell assay
monitoring in microdroplets via DiSBAC.sub.6+CC2-DMPE FRET probe
emission. This early proof-of-concept will rely on merging cell
suspension with stimulatory toxin (Veratridine, etc.) just prior to
droplet-formation, followed by emission detection. The goal of this
stage is to optimize the optical interrogation techniques and
quantitate the confidence of discerning an inhibited ion channel
cell population from a control population, within droplets in flow.
Measuring fluorogenic probe emission in system is a basic
requirement for further development of in-droplet stimulation
methods within Stage 2. In addition, will initiate cloning and
selection of a channel rhodopsin expressing cell line, unless is
able to provide a cell line capable of optogenetic stimulation that
is compatible with the capabilities of for detection, to evaluate
optical stimulation methods in electrophysiology well-plates and
control assays.
[0508] Milestone: Satisfy a benchmark of .gtoreq.10% ratio
amplitude (+/- inhibitor) after toxin stimulation at peak or during
the tail-current, whichever is more sensitive. A Z'-score
calculation can also be applied to determine statistical confidence
in separating inhibited from control populations if ratio amplitude
is not relevant for toxin stimulation.
[0509] Stage 2: Stimulation of Droplet-Cell-Assay with
Spatio-Temporal Control and Design and Construction of a 10K Member
"Targeted Library" Against Desired Ion Channel
[0510] Summary: Demonstrate the ability to stimulate cells in
droplets, in flow, by one or more of three methods (ES, OS, or TS)
using appropriately designed IC chips (see FIG. 1). Subsequent to
stimulation, demonstrate detection of DiSBAC.sub.6+CC2-DMPE FRET
emission at an optimal time-point to segregate inhibited cell
population from a positive control. In addition, the design of a
10K member "targeted library" around chemotypes present in the
control molecules used in Stage 3 will be demonstrated using
chemoinformatic tools to maximize the structural and chemical
diversity of the 10K member targeted library. The initial synthetic
methodologies used to construct the library will be tested and the
chemical products of the methodologies will be validated with LC/MS
analysis. Lastly, the "targeted library" will be subjected to
UV-cleavage to demonstrate the release of library members from BELs
and the degree of cleavage will be quantified with LC/MS
analysis.
[0511] Milestone: Stimulation method and timing must satisfy a
qualifying Z'.gtoreq.0.4 beten +/- inhibitor cell populations. This
is a basic requirement for further microfluidic development to
include compound delivery, dosing, and sorting of inhibited cells
within Stage 3. The "targeted library" will be constructed with
yields >30% for each individual library member using the desired
synthetic methodologies and the library should demonstrate the
ability to be cleaved from beads using UV-cleavage methods
[0512] Stage 3: Complete Integrated Chip Design for POC Screen
Using Controls and a Subsequent Screen of a 10K Member Targeted
Library
[0513] Summary: Integrate the cell-in-droplet stimulation
architectures into a complete integrated device, including in-situ
release of control inhibitors, pre-stimulation mixing and
incubation, and post-stimulation sorting. Upon validation of a full
integrated chip and validation of control molecules for inhibiting
stimulation, a "targeted library" will be screened against a
desired ion channel to elucidate SAR around the controls from the
which the "targeted library" is derived. The "targeted library"
will be screened across 5 concentrations. Analytical tools will be
created to automate NGS analysis, decode "active" structures, rank
active members by potency, and provide insights into SAR. A
validation workflow will be established, to resynthesize the most
potent candidate molecules for analysis and profiling using 's
standard assays to verify EC.sub.50.
[0514] Milestone: Positive control inhibitor beads will be sampled,
and a dose-response measured across 6 concentrations. Data will be
used to determine a relative EC.sub.50 within system, which must be
within 3.times. of the known EC.sub.50. Sorting will also be
demonstrated, capable of isolating positive control beads from
negative controls with <10% false-sort events. The output from a
"targeted library" screen will up to 10 visualizations of the raw
data output from the screen.
Methodology and Project Design
Definitions
[0515] Probe=DiSBAC.sub.6 or analog, with CC2-DMPE unless otherwise
stated Toxin=Veratridine, OD-1 or other stimulatory molecule Model
cell line=HEK293 (Human Embryonic Kidney cells) unless otherwise
stated. Inhibitor=provided control compounds for system testing.
ChR=Channel rhodopsin or variant with tuned optical properties.
Fluorescent nuclear stains=DAPI, DRAQS, PicoGreen, etc. IC chip=Ion
Channel chip to initiate stimulation of sodium ion channels in
droplets. ICES=Ion Channel chip designed for electrical stimulation
of cells-in-droplet. ICTS=Ion Channel chip designed for toxin
stimulation of cells-in-droplet. ICOS=Ion Channel chip designed for
optogenetic stimulation of cells-in-droplet
[0516] Stage 1: Detection of Droplet-Cell-Assay for Ion Channel
Inhibition
[0517] 1A) A model cell-line expressing a relevant ion channel will
be used and a simple set of basic controls established to verify
all reagents and to understand the dynamic activity of toxin
stimulation by probe emission. [0518] 1) well-plate control assay
using model cell line, with probe, +/- inhibitor control using
toxin stimulation. [0519] 2) Fluorescence microscopy will determine
cell-line uniformity, and steady-state behavior for probe emission,
+/- inhibitor. [0520] 3) FRET emission profiles of bulk population
in plates collected to understand toxin kinetics, steady-state, and
EC.sub.50, +/- inhibitor.
[0521] 1B) A simple microfluidic droplet generator will be used to
introduce model cells with fluor-labels (Nuclear stain,
fluor-Anti-ion channel) or probe to test cell detection in droplets
in flow. [0522] 1) Fluor-Anti-ion channel or similar label will be
ideal to determine cell-expression uniformity, and to tune optical
detection in flow using photo-stable fluorophores to determine the
optical sensitivity for the system, without the variable of probe
leeching or photo-bleaching. [0523] 2) Membrane bound probe
emission (CC2-DMPE) detection in droplet will then finalize the
sensitivity required to observe probe emission in droplet in a flow
channel. [0524] 3) Model cell line biocompatibility and toxicity
measurements inside droplets using fluorogenic live or dead
stains.
[0525] 1C) Develop IC.sub.TS Chip v1.0) to merge cells+probe (+/-
inhibitor), with stimulatory toxin, just prior to droplet formation
then capture probe emission from cells at set time points [0526] 1)
Flow-velocity in addition to the spatial position of excitation and
detection dictates the time-delay post stimulation for assay
observation. [0527] 2) Toxin stimulation generates a depolarization
pulse followed by a persistent tail current. Detection position can
isolate specific points on this curve and can determine the best
location for differentiating +/- inhibitor cell populations. [0528]
3) Probe emission profiles for cells +/- inhibitor will be
compared, and a statistical confidence (Z'-score) determined at
various time points following toxin stimulation.
[0529] 1D) Channelrhodopsin expression cell line generation to
prove out optogenetic stimulation [0530] 1) Ion channel expression
in HEK cell line for electrical stim (or sced from) [0531] 2) Ion
Channel+ChR (ChrimsonR or other variant, DOI: 10.1038/nmeth.2836)
expression in model cell line for optical stimulation.
[0532] 1E) Cell stimulation control in electrophysiology
microplates, +/- inhibitor. [0533] 1) Electrode stimulation in
well-plate using ion channel expression cell line, observing
emission from fluorogenic probe (DiSBAC.sub.6+CC2-DMPE) as a
control for cell line quality. [0534] 2) Optical stimulation in
electrode well-plate using ion channel+ChR to detect current.
[0535] A) Require >99% spike occurrence from ChrimsonR
stimulation at 10 Hz using 660-nm light at 500 mJ/s/cm.sup.2.
[0536] 3) Optical stimulation in well-plate using ion
channel+ChR+probe to detect probe emission response. Stage 2:
Stimulation of Droplet-Cell-Assay with Spatio-Temporal Control and
Design and Construction of a 10K Member "Targeted Library" Against
Ion Channel
[0537] 2A) Covalent, photo-cleavable attachment of control
inhibitors to beads (positive control bead. [0538] 1) to
collaborate and provide control inhibitors to contain reactive
handles for attachment to photo-cleavable linker. [0539] a) Ideally
suited are free primary or secondary amine, carboxylic acid,
terminal amide, or phenol. [0540] 2) Full product cleavage and
LC-MS to verify photolabile-compound linkage. [0541] 3)
Photocleavage of inhibitor in well-plate to verify activity after
UV release.
[0542] 2B) Design and fabrication of IC chips for cell-in-droplet
stimulation, monitoring probe emission by PMT or imaging. This
stage is a significant engineering effort with multiple strategies
to prove out the most viable candidates for Stage 3. [0543] 1)
IC.sub.ES--Electrical stimulation in droplet will be examined using
two approaches, with frequency of stimulation dictated by geometry
(10 Hz). [0544] A) Non-contact electrodes to generate electric
fields or dielectrophoretic forces. [0545] 2) IC.sub.TS--Toxin
stimulation in droplet will be examined using a pico-injection,
droplet fusion, or a pre-injected architecture, which allows for
stimulatory toxin to be injected into droplet post compound dosing
in Stage 3. [0546] 3) IC.sub.OS--Optical stimulation in droplet
will be examined using an embedded fiber-optic waveguide
illuminating cells with either UV, VIS or NIR wavelengths at
geometry defined frequencies (10 Hz).
[0547] 2C) IC chip demonstrations showing clear differentiation
between cell populations+/- inhibitor. See FIG. 8 for strategy
overview. [0548] 1) IC.sub.ES and IC.sub.OS chips will be designed
for 10 Hz stimulation pulses, and probe-emission monitored at
spatially controlled time-delays post stimulation to evaluate the
assay Z'-score between +/- inhibitor cell populations at different
time-points. [0549] A) Inhibitor titration (5-point) and detection
will create an end-point dose-response profile to compare the
approximate potency to 's standard assays. [0550] 2) IC.sub.TS chip
design will be tested using the time-interval post stimulatory
toxin-injection determined in Stage 1C-2 to evaluate the assay
Z'-score between +/- inhibitor cell populations in dose response to
compare potency to 's standard assays. [0551] A) Inhibitor
titration (5-point) and detection will create an end-point
dose-response profile to compare the approximate potency to 's
standard assays.
[0552] 2D) Design of 10K member "targeted library" and validation
of synthetic methodologies used to construct the library. [0553] 1)
The design of the library will utilize chemistries, as desired to
permute the chemical structure of control compounds of known
activity. The library will be designed so as to maximize the
interpolation of structure-activity-relationships (SAR) of
individual library members. [0554] 2) The synthetic methodologies
used to construct the library will be validated with "building
blocks" representative of "building block" classes used to
construct the library. The yields of reactions with individual
building blocks will be quantified with LC/MS to validate the
reactivity of individual building blocks. [0555] 3) Individual
beads from the "targeted library" will be subjected to
photo-cleavage to verify that library members are cleaved from
beads in the library.
Stage 3: Complete Integrated Chip Design for POC Screen Using
Controls and a Subsequent Screen of a 10K Member "Targeted
Library
[0556] 3A) Candidate IC chip designs with qualifying Z'-scores will
be incorporated into a complete integrated system. [0557] 1)
IC.sub.xx chip 2.0 designed, fabricated, and tuned for
inhibitor-bead delivery, compound dosing, incubation,
cell-in-droplet stimulation, assay detection, and droplet sorting.
[0558] 2) POC of IC chip 2.0 devices using positive control beads,
releasing high-concentrations of inhibitor to optimize Z'-score
within the integrated system. [0559] 3) Demonstration of
inhibitor-bead isolation from negative-bead control by droplet
sorting with <10% false-sort events (droplets not containing
inhibitor-bead and cells.
[0560] 3B) Compound-release trio-calibration curve to enable
predictive compound dosing. [0561] 1) Fluorophore concentration vs
PMT detection calibration in droplet. [0562] 2) Fluorophore release
from bead by UV exposure vs PMT detection calibration in droplet.
[0563] 3) UV exposure vs calibrant dye emission calibration.
[0564] 3C) Control-bead titration with cell-population analysis to
showcase dose-response and approximation of EC.sub.50 for control
inhibitor [0565] 1) Bead-released inhibitor titration and assay
detection across 4Ln (i.e. 10 .mu.M, 3 .mu.M, 300 nM, <100 nM).
[0566] 2) The inferred IC.sub.50 of bead released compound in IC
chip needs to be <3.times. from that shown using standard plate
methods with the same model cell line.
[0567] 3D)) "Targeted Library" screen against ion channel using the
designed library from Stage 2. [0568] 1) The "targeted" library
will be screened, using 7 library equivalents, against ion channel
with "spiked-in" positive control compounds on beads used in 3C.
[0569] 2) Data will be analyzed with chemoinformatic tools and will
be presented at the conclusion of the screen within 1 month of the
screen being performed.
[0570] While preferred embodiments of the present disclosure have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
disclosure. It should be understood that various alternatives to
the embodiments of the disclosure described herein may be employed
in practicing the disclosure. It is intended that the following
claims define the scope of the disclosure and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
* * * * *