U.S. patent application number 15/388103 was filed with the patent office on 2017-08-10 for rare earth spatial/spectral barcodes for multiplexed biochemical testing.
The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Paul Bisso, Patrick S. Doyle, Jiseok Lee, Albert Swiston.
Application Number | 20170226417 15/388103 |
Document ID | / |
Family ID | 50686174 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170226417 |
Kind Code |
A1 |
Bisso; Paul ; et
al. |
August 10, 2017 |
RARE EARTH SPATIAL/SPECTRAL BARCODES FOR MULTIPLEXED BIOCHEMICAL
TESTING
Abstract
Hydrogel microparticles spatially and spectrally encoded using
upconverting phosphor nanoparticles are described for use in
biochemical testing. In each microparticle, upconversion
nanocrystals having spectrally distinguishable emission spectra are
disposed in different partions of an encoding region of the
microparticle.
Inventors: |
Bisso; Paul; (Belmont,
MA) ; Swiston; Albert; (Baltimore, MD) ; Lee;
Jiseok; (Melrose, MA) ; Doyle; Patrick S.;
(Sudbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Family ID: |
50686174 |
Appl. No.: |
15/388103 |
Filed: |
December 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14214594 |
Mar 14, 2014 |
9528145 |
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15388103 |
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61800995 |
Mar 15, 2013 |
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61801351 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09K 11/02 20130101;
Y10T 436/13 20150115; C12Q 1/6825 20130101; G07D 7/0043 20170501;
G01N 21/6486 20130101; G07D 7/2033 20130101; G01N 2021/6439
20130101; C09K 11/7773 20130101; C09K 11/025 20130101; G07D 7/1205
20170501; G01N 21/6428 20130101; Y10T 436/143333 20150115 |
International
Class: |
C09K 11/77 20060101
C09K011/77; C12Q 1/68 20060101 C12Q001/68; G01N 21/64 20060101
G01N021/64; C09K 11/02 20060101 C09K011/02 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under
Contract no. FA8721-05-C-0002 awarded by the U.S. Air Force and
under Grant Nos. DMR-1006147 and CMMI-1120724 awarded by the
National Science Foundation. The government has certain rights in
the invention.
Claims
1. A microparticle for use in a biochemical or chemical assay, the
microparticle comprising: a body comprising a hydrogel, the body
including a probe region and an encoded region; a first plurality
of upconversion nanocrystals disposed in a first portion of the
encoded region, the first plurality of upconversion nanocrystals
having a first spectral signature; and a second plurality of
upconversion nanocrystals disposed in a second portion of the
encoded region spatially separated from the first portion of the
encoded region, the second plurality of upconversion nanocrystals
having a second spectral signature different than the first
spectral signature.
2. The microparticle of claim 1, wherein the second spectral
signature is different than the first spectral signature.
3. The microparticle of claim 1, wherein the first plurality of
upconversion nanocrystals includes a first material doped with one
or more rare earth elements and the second plurality of
upconversion nanocrystals includes a second material doped with one
or more rare earth elements.
4. The microparticle of claim 1, wherein the body is asymmetric in
shape.
5. The microparticle of claim 1, wherein the upconversion
nanocrystals are covalently bound to the hydrogel material.
6. The microparticle of claim 1, wherein the upconversion
nanocrystals are bound to the hydrogel material at the time of
particle synthesis through an acrylate group.
7. The microparticle of claim 1, wherein for each portion of the
encoded region, the plurality of upconversion nanocrystals are
dispersed without aggregation.
8. The microparticle of claim 1, further comprising a third
plurality of upconversion nanocrystals disposed in a third portion
of the encoded region spatially separated from the first portion of
the encoded region and spatially separated from the second portion
of the encoded region, the third plurality of upconversion
nanocrystals having a third spectral signature different than the
first spectral signature.
9. The microparticle of claim 8, wherein the encoded region
includes at least five different portions.
10. The microparticle of claim 1, wherein each spectral signature
includes luminescence in multiple distinct bands within a range of
400-800 nm.
11. The microparticle of claim 1, wherein the probe region
comprises polyethylene glycol diacrylate (PEG-DA).
12. The microparticle of claim 1, wherein the encoded region
comprises di-acrylated monomer.
13. The microparticle of claim 1, wherein the probe region
comprises a first polymer material and the encoding region
comprises a second polymer material different than the first
polymer material.
14. The microparticle of claim 1, wherein the probe region includes
one or more molecular recognition elements.
15. The microparticle of claim 1, wherein the upconversion
nanocrystals are paramagnetic or ferromagnetic.
16. A method of making a hydrogel microparticle for use in a
biochemical or chemical assay, the method comprising: providing a
first encoded region source material including a hydrogel and a
first plurality of upconversion nanocrystals having a first
spectral signature; providing a second encoded region source
material including a hydrogel and a second plurality of
upconversion nanocrystals having a second spectral signature;
providing a probe region source material including a hydrogel;
cross-linking the first encoded region source material, the second
encoded region source material and the probe region source material
forming a first portion of an encoded region, a second portion of
the encoded region and a probe region with the probe region
crosslinked with one or both of the first portion and the second
portion of the encoded region to form a contiguous
microparticle.
17. The method of claim 16, wherein the second spectral signature
is different than the first spectral signature.
18. The method of claim 16, wherein each of the first plurality of
upconversion nanocrystals and each of the second plurality of
upconversion nanocrystals has a hydrophilic surface.
19. The method of claim 16, wherein the probe region source
material includes one or more molecular recognition elements.
20-25. (canceled)
26. A method of performing a biochemical or chemical assay
comprising: exposing a sample to a plurality of microparticles,
each microparticle comprising: a body comprising a hydrogel, the
body including a probe region with one or more molecular
recognition elements and an encoded region; a first plurality of
upconversion nanocrystals disposed in a first portion of the
encoded region, the first plurality of upconversion nanocrystals
having a first spectral signature; and a second plurality of
upconversion nanocrystals disposed in a second portion of the
encoded region spatially separated from the first portion of the
encoded region, the second plurality of upconversion nanocrystals
having a second spectral signature different than the first
spectral signature; for each microparticle, illuminating the
microparticle with an excitation light source; for each
microparticle, detecting light emitted from the illuminated
microparticle, the detected light including upconverted luminescent
light from the first plurality of upconversion nanocrystals and the
second plurality of upconversion nanocrystals and light associated
with the one or more molecular recognition elements; and
identifying each microparticle based on the detected light.
27-30. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application claims benefit of, and priority to
U.S. patent application Ser. No. 14/214,594, filed Mar. 14, 2014,
which claims the benefit of U.S. Provisional Patent Application No.
61/801,351, filed Mar. 15, 2013, and U.S. Provisional Patent
Application No. 61/800,995, filed Mar. 15, 2013, all of the above
applications being herein incorporated by reference in their
entirety.
BACKGROUND
[0003] There are many different approaches currently being employed
for performing multiplexed assays (e.g., two dimensional surface
adsorbed arrays, fluorophore-bead systems, spatially-labeled
microparticle systems). The multiplexing capabilities of these
techniques may be insufficient to simultaneously probe some
complex, heterogeneous biological systems.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0005] FIG. 1 schematically depicts an exemplary microparticle, in
accordance with an embodiment.
[0006] FIG. 2 is a graph of an emission spectrum of exemplary
upconversion nanocrystals (UCNs) labeled "UCN1", in accordance with
an embodiment.
[0007] FIG. 3 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN2", in accordance with an embodiment.
[0008] FIG. 4 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN3", in accordance with an embodiment.
[0009] FIG. 5 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN4", in accordance with an embodiment.
[0010] FIG. 6 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN5", in accordance with an embodiment.
[0011] FIG. 7 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN6", in accordance with an embodiment.
[0012] FIG. 8 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN7", in accordance with an embodiment.
[0013] FIG. 9 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN8", in accordance with an embodiment.
[0014] FIG. 10 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN9", in accordance with an embodiment.
[0015] FIG. 11 is a graph of an emission spectrum of exemplary UCNs
labeled "UCN10", in accordance with an embodiment.
[0016] FIG. 12 is a graph of spectral responsivity of RGB channels
of a CCD image sensor with UCN emission bands overlaid, in
accordance with an embodiment.
[0017] FIG. 13 is a graph of the emission spectrum of UCN6
overlaying the spectral responsivity of RGB channels of a CCD image
sensor, in accordance with an embodiment.
[0018] FIG. 14 is a graph showing unique upconversion emission
spectra produced by varying dopant concentrations, in accordance
with an embodiment.
[0019] FIG. 15 is an image of different types of UCNs under NIR
illumination, in accordance with an embodiment.
[0020] FIG. 16 is a transmission electron micrograph of different
types of UCNs, in accordance with an embodiment.
[0021] FIG. 17 includes graphs of emission spectra for different
batches of UCNs, in accordance with an embodiment.
[0022] FIG. 18 includes luminescence images of UCNs in liquid with
and without an applied external magnetic field, in accordance with
an embodiment.
[0023] FIG. 19 is a graph of magnetization versus applied magnetic
field for UCN4, in accordance with an embodiment.
[0024] FIG. 20 is a block diagram schematically representing a
method of forming a microparticle, in accordance with an
embodiment.
[0025] FIG. 21 schematically depicts a stop flow lithographic
method of forming a contiguous microparticle, in accordance with an
embodiment.
[0026] FIG. 22 is a luminescence image of microparticles having
different numbers of encoded regions, in accordance with some
embodiments.
[0027] FIG. 23 includes graphs of integrated intensity values for
microparticles, each including a different type of UCNs, in
accordance with some embodiments.
[0028] FIG. 24 is a scatter plot of integrated intensity data for
microparticles including different types of nanocrystals, in
accordance with some embodiments.
[0029] FIG. 25 is a plot of mean measured integrated intensity data
and expected integrated intensity date for the red channel versus
the green channel showing five-sigma confidence contours, in
accordance with an embodiment.
[0030] FIG. 26 shows integrated intensity data for different
batches of microparticles, in accordance with some embodiments.
[0031] FIG. 27 is a graph of emission spectra of UCN4 after each
step in surface chemical modification of the UCNs, in accordance
with some embodiments.
[0032] FIG. 28 shows microparticle emission intensity as a function
of time during intense sustained NIR irradiation, in accordance
with some embodiments.
[0033] FIG. 29 shows graphs of intensity versus microparticle age
for microparticles with carboxyl-terminated UCN and for
microparticles with acrylated UCN, in accordance with some
embodiments.
[0034] FIG. 30 is a graph of integrated intensity for different
color channels for microparticles, in accordance with an
embodiment.
[0035] FIG. 31 schematically depicts detection of a nucleic acid of
interest by a molecular recognition element in the probe region, in
accordance with some embodiments.
[0036] FIG. 32 includes images of microparticles having two
different codes under NIR illumination, in accordance with an
embodiment.
[0037] FIG. 33 includes a bright field image and a fluorescence
image of a non-encoded standard microparticle, in accordance with
an embodiment.
[0038] FIG. 34 is a graph of fluorescence intensity for encoded
microparticles and for standard (non-encoded) microparticles, in
accordance with an embodiment.
[0039] FIG. 35 is a table with images of encoded microparticles
used for multiplexed assays, in accordance with an embodiment.
[0040] FIG. 36 is a graph of integrated intensity data in red
versus green channels for portions or stripes of encoded PEG
microparticles.
[0041] FIG. 37 is a graph of integrated intensity data in red
versus green channels for portions or stripes of encoded PUA
microparticles and encoded PEG microparticles.
[0042] FIG. 38 is an image of microparticles with a PUA encoded
region and a PEG-DA probe region under NIR illumination, in
accordance with an embodiment.
[0043] FIG. 39 is a block diagram of a method for performing
biochemical or chemical assay, in accordance with an
embodiment.
[0044] FIG. 40 includes images of a process of reading out spectral
codes from a luminescence image of a microparticle, in accordance
with some embodiments.
[0045] FIG. 41 includes images used to distinguish two different
codes of microparticles, in accordance with an embodiment.
[0046] FIG. 42 schematically depicts a flow lithography and
decoding system for particle synthesis, in accordance with some
embodiments.
[0047] FIG. 43 is an image of the system for particle synthesis of
FIG. 42.
[0048] Additional features, functions and benefits of the disclosed
methods, systems and media will be apparent from the description
which follows, particularly when read in conjunction with the
appended figures.
DETAILED DESCRIPTION
[0049] Embodiments include hydrogel microparticles for use in
biochemical or chemical assays, methods of producing the
microparticles, and methods of performing biochemical or chemical
assays using the microparticles. Each hydrogel microparticle has a
probe region including one or more molecular recognition elements
and an encoded region. The encoded region includes multiple
portions, with each portion including an associated plurality of
upconversion nanocrystals (UCNs) with a distinct spectral
signature. The multiple portions of the encoding region enable
spatial encoding of the microparticle. The associated plurality of
UCN for each region are selected from a set of spectrally
distinguishable UCN, which enables spectral encoding for each
portion of the microparticle. By combining spatial and spectral
encoding, the microparticles have massive multiplexing capabilities
with superior scaling capability.
[0050] The coding scales exponentially as C.sup.S for asymmetric
particles and as C.sup.S/2 for symmetric particles, where C is the
number of distinguishable spectral signatures (UCN `colors`) and S
is the number of spatial features (e.g., microparticle `stripes`).
For example, for a symmetric microparticle with S encoding portions
and a set of C different spectrally distinguishable UCNs, the
following equation lists the number of codes or unique identifiers
that would be available:
x = 0 S - 1 C ( S - x ) ##EQU00001##
[0051] For example, about 20,000 unique identifiers/codes can be
generated for a system in which the encoding region of symmetric
microparticles has six portions and each portion includes a
plurality of UCN selected from a set of five different types of
spectrally distinct UCNs. As another example, about 500,000 unique
identifiers/codes can be generated for a system in which the
encoding region of the symmetric microparticle has six portions and
each portion includes a plurality of nanocrystals selected from a
set of nine different types of spectrally distinct nanocrystals.
Thus, a modest number of colors may be coupled with a similarly
modest number of stripes to yield considerable encoding capacities
that scale rapidly with incremental changes to either quantity. To
increase the encoding capacity, asymmetric microparticles could be
employed. For example, an asymmetric microparticle with six
portions with each portion including one of nine different types of
spectrally distinct nanocrystals would produce over a million
unique identifiers/codes.
[0052] Some embodiments combine spatial patterning with rare-earth
upconversion nanocrystals (UCNs), single wavelength near-infrared
excitation and portable charge-coupled device (CCD)-based decoding
to distinguish particles synthesized by means of flow lithography.
Some embodiments exhibit large, exponentially scalable encoding
capacities (>10.sup.6), an ultralow decoding false-alarm rate
(<10.sup.-9), the ability to manipulate particles by applying
magnetic fields, and dramatic insensitivity to both particle
chemistry.
[0053] Some embodiments employ a robust encoding method for
compatibility with high-throughput particle synthesis and portable
CCD-based decoding. In some embodiments, the resulting particles
and decoding system exhibit dramatic insensitivity to particle
chemistry--enabling tuning of encoding capacity and decoding error
rate independently of particle material properties--as well as the
capacity for straightforward magnetic manipulation. In the examples
described below, the inventors demonstrate quantitatively
predictable decoding of both biocompatible particles in
challenging, realistic environments. With single-particle encoding
capacities in excess of 1 million and error rates of less than 1
part per billion (ppb), some embodiments expand the practically
accessible number of codes for applications like multiplexed
bioassays by orders of magnitude.
[0054] FIG. 1 schematically depicts an exemplary microparticle 10
for use in a biochemical or chemical assay, in accordance with an
embodiment. The microparticle 10 has a body 12 including a
hydrogel. The body 12 includes a probe region 20 and an encoded
region 30. The probe region 20 includes one or more molecular
recognition elements. The encoded region 30 includes multiple
different portions (e.g., portions 31, 32, 33, 34, 34, 35) with
each portion (31-35) having an associated plurality of upconversion
nanocrystals (UCNs) (e.g., UCN 41) selected from a set of
spectrally distinguishable UCN (see discussion accompanying FIGS.
2-11 below). In some embodiments, one or more portions may not
include any nanocrystals and may serve as a "blank" or null portion
for encoding. In some embodiments, the hydrogel body material is
mesoporous to allow the diffusion of large (>10 nm) biomolecules
though the hydrogel material).
[0055] For example, in some embodiments, a first plurality of UCNs
with a first spectral signature is disposed in a first portion 31
of the encoded region. A second portion 32 of the encoded region
includes a second plurality of UCNs with a second spectral
signature different than the first spectral signature. In some
embodiments, the encoded region of the microparticle also includes
a third portion 33 having a third plurality of UCNs. In some
embodiments, the encoded region of the microparticle also includes
a fourth portion 34 having a fourth plurality of UCNs. In some
embodiments, the encoded region of the microparticle also includes
a fifth portion 35 having a fifth plurality of UCNs. The plurality
of microparticles in each portion (31-35) of the encoded region is
selected from a set of spectrally distinguishable UCNs.
[0056] One of ordinary skill in the art in view of the present
disclosure would recognize that each microparticle may include an
encoding region with fewer than five portions and associated
pluralities of UCNs (e.g., four portions, three portions, two
portions) or more than five portions and associated pluralities of
UCNs (e.g., six portions, seven portions, eight portions, nine
portions, ten portions, etc.).
[0057] The spectral signature associated with a plurality of UCN
disposed in a portion of the encoded region is also referred to
herein as the spectral signature of the portion of the encoded
region. In some embodiments, two or more portions of the encoded
region may have the same spectral signature. In some embodiments,
two or more portions of the encoded region with the same spectral
signature may be adjacent to each other. In some embodiments, any
portions of the encoded region with the same spectral signature
must be separated from each other by one or more portions of the
encoded region having different spectral signature(s). In some
embodiments, each portion of the encoded region must have a
spectral signature different from that of every other portion of
the encoded region. In some embodiments, one or more portions of
the encoded region do not include UCNs so that the portion or
portions is "blank" without a spectral signature.
[0058] The spectral signature of a UCN includes information
associated with the emission spectrum of the UCN that distinguishes
it from another type of UCN. In some embodiments, the spectral
signature of a UCN or of a plurality UCNs of the same type may
include the integrated intensity of emission of one spectral band
(or emission in one spectral range) versus another spectral band
(or emission in another spectral range). A spectral signature or
information regarding a spectral signature may be referred to
herein as a spectral code.
[0059] FIGS. 2-10 show emission spectra for an example set of nine
spectrally distinguishable types of UCNs, labeled UCN1-UCN9
respectively, when excited with near infrared (NIR) light (e.g.,
980 nm light from an NIR diode laser). UCNs in the example set
luminesce in multiple narrow bands (e.g., bands less than 70 nm
wide at full width half maximum (FWHM)) in the visible range when
exposed to lower frequency (e.g., near infrared (NIR)) light.
Specifically, the example set of spectrally distinguishable UCNs
(e.g., UCN1-UCN10) emit in two or more bands centered around 470 nm
(e.g., 445-500 nm), centered around 550 nm (e.g., 520-560 nm), and
centered around 650 nm (e.g., 650-670 nm). For simplicity, the
445-500 nm band is referred to herein as the blue band, the 520-560
nm band is referred to herein as the green band, and the 640-670 nm
band is referred to herein as the red band.
[0060] One of ordinary skill in the art in view of the present
disclosure would recognize that the set of UCN may include fewer
than nine (e.g., eight, seven, six, five, four, three, two) or more
than nine (e.g., ten, nine, ten, eleven, twelve, etc.) different
types of spectrally distinguishable UCNs. Further, one of skill in
the art in view of the present disclosure would recognize that UCNs
having different spectra than those shown, and UCNs than emit in
different bands than those shown, also fall within the scope of
embodiments. For example, FIG. 11 shows an emission spectrum for a
UCN labeled UCN10 that may be used in the set as an alternative to
any of UCN1-UCN9, or in addition to UCN1-UCN9. To augment encoding
capacity, the palette of spectrally distinct UCNs may be further
expanded by adjusting Yb--Er--Tm ratios with negligible impact on
the decoding error rate.
[0061] The spectral signature of a plurality of UCNs may include
information related to the ratio or ratios of the integrated
intensities emitted in various bands (e.g., the ratio of the red
band to the green band or vice versa, the ratio of the red band to
the blue band or vice versa, the ratio of the blue band to the
green band or vice versa, or any combination of the
aforementioned). These ratios can be defined with respect to the
emission spectra of the UCNs. However, in some embodiments, the
spectral signature of a plurality of UCNs may include both
information regarding the intensity of light emitted in various
bands and include information regarding the responsivity of the
image sensor to be used. Any detector, image sensor, or imaging
device may be employed. For example, the detector or imaging device
may be a charge-coupled device (CCD), a photomultiplier tube-based
device (PMT), a complementary metal-oxide-semiconductor (CMOS)
imaging sensor, an avalanche photodiode array (APD) imaging device,
etc In some embodiments, an imaging sensor with more than one color
channel may be employed.
[0062] FIG. 12 shows the spectral responsivity of red 61, green 62
and blue 63 channels for a typical RGB CCD device that may be used
as a detector in some embodiments. As shown, the red 71, green 72,
and blue 73 emission bands of the exemplary set of UCNs overlap the
spectral responsivities of the respective red 61, green 62, and
blue 63 channel responsivity curves. For example, FIG. 13 shows the
emission spectrum of UCN6 overlaying the spectral responsivity of
channels of a typical RGB device. A convolution of the emission
spectrum with the expected spectral responsivity for each image
sensor channel yields curves corresponding to the expected spectral
response of each channel of the CCD image sensor to each type of
UCN. The spectral signature for a type of UCNs can include
information regarding the expected spectral response of an image
sensor to a specific UCN emission spectrum, such as a ratio of the
expected integrated intensity detected for two color channels.
[0063] For example, the Table 1 below shows the expected spectral
response of a CCD device to the emission spectra of the UCN3-UCN7
and UCN10 types of UCNs (see FIGS. 4-8 and 11 for emission
spectra). The expected spectral response is a convolution of the
emission spectrum for type of UCN with the image sensor channel
spectral responsivity shown in FIG. 12. Specifically, Table 1 shows
the expected integrated total intensity for each color channel due
to emission of the UCNs. Table 1 also includes ratios for the
expected total intensity for the green channel to the red channel,
for the blue channel to the red channel, and for the blue cannel to
the green channel. Expressing the integrated intensities as ratios
for different color channels reduces or eliminates the need for
calibration to determine the absolute intensity for any particular
color channel or emission band.
TABLE-US-00001 TABLE 1 Expected Expected Expected Integrated
Integrated Integrated Channel Channel Channel Intensity* Intensity
Intensity Ratio Ratio Ratio Type R Channel G Channel B Channel G/R
B/R B/G UCN3 163.4 86.3 0 0.528 0 0 UCN4 225.4 197.5 0 0.876 0 0
UCN5 91.9 164.5 0 1.790 0 0 UCN7 24.7 52.1 219.9 2.109 8.9 4.220
UCN6 138.5 158.1 120.4 1.141 0.869 0.7609 UCN10 161.6 131.5 0 0.814
0 0
[0064] The inventors have found that employing UCN for identifying
each encoded region of a particle has many benefits when compared
with other techniques currently used for encoding particles. For
example, some other techniques employ one-dimensional or
two-dimensional thickness variations or holes in a fluorescently
labeled coded region of a microparticle for identification.
[0065] In contrast with UCNs having multiple narrow emission bands,
commonly used fluorescent labeling molecules (e.g., fluorophores)
each tend to emit in a single broad band (e.g., DAPI fluorescent
dye has a single emission band that is about 100 nm wide FWHM). In
microparticles using fluorophores for encoding, the broad emission
bands of the fluorophores limits the number of different
fluorophores that may be employed without having significant
overlap between emission bands and resulting ambiguity in
identification. In addition, the absence of multiple emission bands
for a single fluorophore may require the use of an external
calibration standard. In contrast, UCNs have multiple narrow
emission bands in different portions of the visible spectrum (e.g.,
separated by tens to hundreds of nm). The ratio of intensity of
emission in various bands can be used to distinguish between
different UCNs, and also acts as an internal calibration standard,
obviating the need for external calibration.
[0066] Microparticles using UCNs for encoding may experience less
reduction of the signal to noise ratio due to autoluminescence than
microparticles using fluorophores for encoding. Luminescent UCNs
absorb light in one range of wavelengths and emit light in a
shorter range of wavelengths (e.g., absorb in the NIR range and
emit in the visible range). In contrast, commonly used fluorophores
and quantum dots usually absorb light in a wavelength range and
emit light in a longer wavelength range (e.g., absorbing in the
ultraviolet range and emitting in the visible range). For example,
the commonly used fluorophore 4',6-diamidino-2-phenylindole (DAPI)
has absorption maximum around 370 nm (UV) and an emission maximum
around 450 nm (blue). Illumination of the fluorophores for
identification (e.g., with UV light) may result in unintended
autofluorescence of materials and solvents in the visible
wavelengths that decreases the signal to noise ratio, which can be
a significant problem with biological samples. Because the UCNs
described herein are upconverting, the NIR light used to excite the
UCNs generally does not cause autoluminescence in the shorter
wavelengths of the visible range. Thus, the use of UCN may improve
the signal to noise ratio for an encoded region.
[0067] Microparticles using different types of UCNs for encoding
may require only a single narrow band excitation source as opposed
to microparticles using different types of fluorophores, which may
require multiple light sources to provide excitation in different
wavelength bands. For example, a 980 nm light source with a power
density of less than 10 W/cm.sup.2 (e.g., an near infra-red (NIR)
laser diode) may be used as a single excitation source for multiple
different types of UCNs. In contrast, microparticles using common
fluorophores for parts of the visual light spectrum, such as DAPI
(blue), Oregon green 500 (green) and ALEXA FLUOR 633 (red) with
absorption maximums at 350 nm, 503 nm and 632 nm, respectively, may
require multiple different excitation sources such as a UV laser,
an argon-ion laser, and a red helium-neon laser.
[0068] In some embodiments, the UCNs are rare-earth nanocrystals,
which are bright anti-Stokes emitters with tunable spectral
properties. Individual UCNs absorb continuous-wave (CW) NIR light
at a single wavelength and emit in multiple narrow bands of the
visible spectrum. Large anti-Stokes shifts reduce spectral
interference from sample autogluorescence and lead to enhanced
signal-to-noise ratios. In contrast to M-ink (an optically active
dye in which nanostructured magnetic materials reflect different
wavelengths of light) or quantum dots, these benefits persist even
in the presence of obscurants or a complex reflective background.
Tuning of emission intensities in multiple bands by adjusting
relative stoichiometries of lanthanide dopants permits
ratiometrically unique spectral encoding, in which the ratio of
integrated intensities in two or more bands serve as the code,
rather than absolute intensity. In some embodiments, external
spectral standards (e.g., as required by porous silicon crystals),
precise dye loading (e.g., as used with quantum dots and luminex),
sensitive instrumentations (e.g. as required by M-Ink), and
extensive calibration may be unnecessary for readout, enabling the
use of standard CCD imaging for decoding.
Example Synthesis of UCNs
[0069] Lanthanide-doped NaYF.sub.4 UCNs were made via a scalable
batch hydrothermal synthesis, which is only one of numerous known
protocols for synthesis of NaYF.sub.4 UCNs.
[0070] Aqueous rare-earth chloride salts, sodium hydroxide,
ammonium fluoride, ethanol and oleic acid were heated in a
TEFLON-coated stainless steel pressure vessel. Specifically, 2 ml
of ReCl.sub.3 (0.4 M, RE=Y, Yb, Er, Gd, Tm) and 2 ml of NH.sub.4F
(2 M) were added to a mixture of 3 ml of NaOH (0.6 M), 10 ml of
ethanol and 10 ml of oleic acid. The solution was transferred to a
50 ml TEFLON-lined autoclave and heated at 200.degree. C. for 2
hours. The resulting products were centrifuged to collect the UCNs,
which were then repeatedly washed with ethanol and deionized water
and then re-dispersed in cyclohexane.
[0071] During synthesis, the inventors used the concentration of
various lanthanide dopants and the reaction time and temperature to
improve the luminescence intensity of the UCNs and to alter the
upconversion spectrum of the nanocrystals.
[0072] The synthesis procedure described above can produce
NaYF.sub.4 UCNs in two different phases having different crystal
structures: an .alpha.-phase with a cubic crystal structure and a
.beta.-phase with a hexagonal crystal structure. Generally
speaking, luminescence intensity is significantly higher in
.beta.-phase crystals than in .alpha.-phase crystals due to the
lower ratio of surface defects to crystal volume in the
.beta.-phase. Without high levels of gadolinium doping, relatively
high temperatures must be maintained for relatively long times
(e.g., 350.degree. C. for 24 hours) to induce the
.alpha..fwdarw..beta. phase transition in the UCNs. In contrast,
the inventors doped with 30 mol % gadolinium (Gd) to induce the
.alpha..fwdarw..beta. phase transition at a lower temperature
(200.degree. C.) held for a shorter time (2 hours). The Gd has
little to no effect on the shape of the upconversion emission
spectrum generated due to the presence of the other dopants.
[0073] Increasing reaction time and increasing reaction temperature
tended to increase the luminescence intensity of the UCNs due to
increased nanocrystal size. Increasing the UCN size decreases the
ratio of surface area to volume for the UCNs, thereby decreasing
the ratio of surface defects to crystal volume. Further,
luminescence for larger UCNs was less likely to be red-shifted due
to preferential quenching of high frequency emission, which can
occur in smaller UCNs.
[0074] The concentrations of dopants other than Gd were used to
change the upconversion emission spectrum. Spectrally distinct UCNs
were produced by adjusting the relative stoichiometries of the
lanthanide ions Yb.sup.3+, Er.sup.3+ and Tm.sup.3+ in the UCN
reaction premix. The lanthanide dopant stoichiometries have
relatively little impact on the UCN nanostructure and surface
chemistry, decoupling control of the emission spectrum from the
particle chemistry and resulting material properties. Ytterbium
(Yb.sup.3+) is an important dopant for bright multicolor emission,
because it acts as a high-NIR absorption cross-section and energy
transfer agent for upconverting emission. Increasing the Yb
percentage tends to `red-shift` the upconversion spectrum,
increasing the ratio of the emission intensity in the red band
(640-670 nm) relative to the emission intensity in the green band
(520-560 nm) in Erbium (Er.sup.3+) co-doped crystals. FIG. 14
illustrates how increasing the Yb concentration shifts the emission
spectrum and shifts overall emission color from green to orange.
Doping with Er.sup.3+ at low levels (2% or less) leads to narrow
peaks centered at 550 nm and 650 nm. Overall perceived emission
color for materials doped with Yb.sup.3+ and Er.sup.3+ can range
from green to red, depending on the Yb concentration. Doping with
Thulium (Tm.sup.3+) at very low levels (.about.0.2%) leads to
emission in the blue band (445-500 nm) and a more intense peak at
800 nm.
[0075] The inventors produced ten different types of spectrally
distinguishable lanthanide-doped NaYF.sub.4 UCNs labeled
UCN1-UCN10, whose spectra appear in FIGS. 2-11. The overall colors
of the UCN1-UCN9 types when irradiated with an NIR laser diode are
shown in FIG. 15, which includes a luminescence image of
suspensions of UCN1-UCN9 in cyclohexane upon 980 nm near infra-red
(NIR) excitation. As illustrated by FIG. 15, the colors of the UCNs
can be readily distinguished by the naked eye. The composition of
the dopant used for each type of UCNs is listed in Table 2 below.
The Y concentration, which makes up the balance of each dopant
concentration, is in square brackets because it is not an active
dopant.
TABLE-US-00002 TABLE 2 Description Gd Yb Er Tm [Y of Label (mol %)
(mol %) (mol %) (mol %) (mol %)] overall color UCN1 30 69.7 0.1 0.2
[0] Violet UCN2 30 69.9 0.1 -- [0] Red UCN3 30 68 2 -- [0] Orange
UCN10 30 40 2 -- [28] Dark Yellow UCN4 30 30 2 -- [38] Yellow UCN5
30 18 2 -- [50] Green UCN6 30 20 0.1 0.2 [49.7] Cobalt UCN7 30 18
-- 0.2 [51.8] Blue UCN8 30 18 0.03 0.2 [51.77] Sky Blue UCN9 30
31.7 0.1 0.2 [38] Grey
[0076] FIG. 16 shows transmission electron microscopy (TEM) images
of the UCN1-UCN9 types of UCNs produced by the process described
above, as well as an enlarged image of the UCN6 nanocrystals. In
FIG. 16, the scale bars are 100 nm. The TEM samples were prepared
by placing a drop of UCNs in cyclohexane onto the surface of a
copper grid. Overall, the UCNs produced were rod-shaped with an
average size of 250-450 nm in length and 40-60 nm in width.
[0077] The inventors made several different batches of the same
type of UCNs to confirm that the emission spectra were consistent
from batch to batch. Upconversion luminescence spectra of UCNs were
measured in a poly (urethane acrylate) (PUA) prepolymer solution
(9/1 PUA/PI (v/v)) with a fluorescence spectrometer with a 1 W CW
diode laser (980 nm) used as the excitation source. FIG. 17 shows
the normalized emission spectra for three different batches of UCN7
type nanocrystal. As shown, emission spectra for the three
different batches are practically indistinguishable on the combined
graph.
[0078] The high Gd content of UCN1-UCN10 makes the UCNs
paramagnetic and subject to physical manipulation through external
magnetic fields. The inventors confirmed this by manipulating the
nanocrystals suspended in vials using external ferromagnets. FIG.
18 includes luminescence images of UCNs in liquid in a vial (a)
settled to the bottom of the vial with no applied magnetic field,
and (b) with an applied magnetic field from a ferromagnet drawing
the UCNs to the left side of the vial. FIG. 19 is a graph of data
for magnetization as a function of applied magnetic field for UCN4,
which was obtained using a superconducting quantum interference
device (SQUID).
Example Surface Modifications of UCN
[0079] The synthesis process described above produced UCNs capped
with oleic acid, a fatty acid with a 17-carbon hydrocarbon tail. As
a result of the oleic acid capping, the resulting UCNs were
insoluble in aqueous media, which created problems with dispersing
the UCNs in aqueous or hydrophilic source materials. Furthermore,
the UCNs with oleic acid tails luminesced brightly only in
hydrophobic media. Exposure of the oleic acid capped UCNs to water
caused significant aggregation and a high degree of reversible
luminescence attenuation due to surface defect-mediated
quenching.
[0080] The inventors utilized a method of modifying the oleic acid
tail on the UCNs to improve their solubility in water and increase
their luminescence in hydrophilic media. The oleic acid double bond
was oxidized to form an alcohol, and then cleaved, thereby
releasing the outward-facing hydrophobic part of the oleic acid
chain and forming a carboxylic acid group.
[0081] The specific procedure employed to modify the oleic acid
tail of the UCNs involved adding 0.1 gram of UCNs to a mixture of
cyclohexane (100 mL), tert-butanol (70 mL), water (10 mL) and 5 wt
% K.sub.2CO.sub.3 solution (5 mL) and stirring for about 20 minutes
at room temperature. Then, 20 mL of Lemieux-von Rudloff reagent
(5.7 mM KMnO.sub.4 and 0.1 M NaIO.sub.4 aqueous solution) was added
dropwise to the solution. The resulting mixture was stirred for 48
hours. The product was centrifuged and washed with deionized water,
acetone, and ethanol. Subsequently, the UCNs were dispersed in
hydrochloric acid (50 mL) of pH 4, and stirred for 1 hour forming
carboxyl-terminated UCNs, which were washed 5 times with deionized
water and collected by centrifugation. The resulting
carboxyl-terminated UCNs dispersed without aggregation in aqueous
media and luminesced strongly in hydrophilic media.
[0082] The inventors developed a method for modifying the
carboxyl-terminated UCN to form acrylate-terminated UCN that could
be cross-linked with the hydrogel material of the microparticle.
The method included mixing 200 .mu.l of EDC (20 mg/ml) and 200
.mu.l of sulfo-N-hydroxysuccinimide (sulfo-NHS) (20 mg/ml) with 200
.mu.l of carboxy-terminated UCNs in 2-(N-morpholino) ethanesufonic
acid (MES) buffer (0.1 M, pH 6.0, 40 mg/ml) and stirring for two
hours at room temperature to activate the surface as carboxylic
acid groups. The NHS-activated UCNs were centrifuged and washed
with water. The precipitate was re-dispersed in 200 .mu.l of PBS
buffer (0.1 M, 5 ml, pH 7.2) containing 200 .mu.l of
2-hydroxyethylacrylate (20 mg/ml). The mixture was then stirred for
24 hours at room temperature. The resulting acrylated UCN were
purified by repeated centrifugation (3000 rpm, 5 min, 5 times) and
resuspended in deionized water.
[0083] FIG. 20 is a flow diagram 110 of a method of making a
hydrogel microparticle for use in a biochemical or chemical assay.
A first encoded region source material is provided (112). The first
encoded region source material includes a hydrogel and a first
plurality of UCNs having a first spectral signature. For example,
the first plurality of UCNs may be the nanocrystals described above
and labeled UCN3. The spectral signature of the first plurality of
the UCNs (type UCN3) may be described as the spectrum shown in FIG.
4, or may be described by the ratio of the integrated intensity in
one detection channel relative to another detection channel (e.g.,
the ratio of the green detection channel integrated intensity the
red detection channel integrated intensity as shown in Table 1), or
by multiple different integrated intensity ratios (e.g., green to
red, blue to red, red to green). A second encoded region source
material is also provided (114). The second encoded region source
material includes a second plurality of UCNs having a second
spectral signature different than the first spectral signature. The
second plurality of UCNs may be the nanocrystals described above
and labeled UCN4. The spectral signature of the second plurality of
the UCN (type UCN4) may be described as the spectrum shown in FIG.
5, or may be described by the ratio of the integrated intensity in
one detection channel relative to another detection channel (e.g.,
the ratio of the green detection channel integrated intensity the
red detection channel integrated intensity as shown in Table 1), or
by multiple different integrated intensity ratios (e.g., green to
red, blue to red, red to green). Although the flow chart only
specifies a first encoded region source material and a second
encoded region source material, the number of encoded region source
materials required corresponds to the number of portions of the
encoded region desired in the resulting microparticle. A probe
region source material including a hydrogel material is also
provided (116).
[0084] The first encoded region source material, the second encoded
region source material, and the probe region source material are
cross-linked forming the first portion of an encoded region 31, the
second portion of the encoded region 32, and the probe region 20.
The probe region 20 is cross-linked with one or both of the first
portion 31 and the second portion 32 of the encoding region to form
a contiguous microparticle. In embodiments with more than two
portions of the encoded region, each portion is cross-linked with
one or more other portions of the encoded region and/or with the
probe region.
[0085] In some embodiments, the UCNs for at least some of the
portions of the encoded region have a hydrophilic surface. In some
embodiments, the UCNs for at least some of the portions of the
encoded region have a hydrophilic ligand. In some embodiments,
providing the first encoded region source material and providing
the second encoded region source material may include modifying the
first plurality of nanocrystals and the second plurality of
nanocrystals to have a hydrophilic surface and/or a hydrophilic
ligand. Having a hydrophilic surface and/or a hydrophilic ligand
may aid in dispersing the UCNs in the respective source
material.
[0086] In some embodiments, the UCNs for at least some of the
portions of the encoded region have acrylated ligands for
cross-linking with the polymers of the hydrogel matrix. In some
embodiments, providing the first encoded region source material and
providing the second encoded region source material may include
modifying the first plurality of nanocrystals and the second
plurality of nanocrystals to include acrylated ligands. In some
embodiments, the plurality of UCNs is bound to the polymer material
at the time of particle synthesis through an acrylate group.
[0087] In other embodiments, another type of covalent linkage could
be made between the UCNs and the hydrogel matrix. The UCNs can be
bound to the hydrogel matrix using any number of covalent
attachment mechanisms (e.g., amide linkages, disulfides, esters,
ethers, aldehydes/ketones, cycloadditions, click chemistry, azides,
and carbamates).
[0088] In some embodiments, at least some of the UCNs are doped
with rare-earth metals. In some embodiments, at least some of the
UCNs are doped with a composition including at least 30 mol % Gd.
In some embodiments, at least some of the UCNs are
paramagnetic.
[0089] In some embodiments, the material for each portion of the
encoded region and for the probe region is the same material. In
some embodiments, the material for the portions of the encoded
region is different than the material for the probe region.
[0090] As noted above, in some embodiments, the UCNs have a
hydrophilic surface. In some embodiments, the UCNs have a
hydrophilic ligand. Having a hydrophilic surface and/or a
hydrophilic ligand may aid in dispersing the UCNs in the source
material.
[0091] In some embodiments, the method also includes co-flowing the
source material for each encoded region and the source material for
the probe region to an area for cross-linking. For example, a
stop-flow lithography (SFL) technique may be employed for forming
the microparticles. In SFL, viscous UV-sensitive pre-polymer
solutions (which may be referred to herein as source materials)
undergo laminar co-flow into a small microfluidic device, which may
be made of polydimethylsiloxane (PDMS). For organic synthesis, the
microfluidic device may be made from perfluoropolyether (PFPE). The
flow of the pre-polymer solutions is stopped for a brief period in
which the pre-polymer solutions in the device are exposed to
photomask-patterned ultraviolet light. The UV light causes
cross-linking, polymerization, or both within milliseconds in the
region delineated by the photomask forming micro-sized polymeric
particles. The shape of each particle is defined by the photomask.
The composition of each striped portion of the particle is
determined by the composition of the laminar co-flowing streams
(e.g., the source materials). The SFL technique is particularly
well suited for spatial and spectral encoding of microparticles
using nanocrystals because of the ability to control both overall
microparticle particle shape and the composition of different
striped portions of the microparticle.
[0092] FIG. 15 schematically depicts SFL being used to make a
hydrogel microparticle with a probe region and an encoding region
with different portions of the encoding region including UCNs with
distinguishable spectral signatures. In the diagram the encoded
region source materials are labeled ERSM1-ESRM5, and the probe
region source material is labeled PRSM. Each of the encoded region
source materials includes a pre-polymer 142 and a plurality of
UCNs, which may be acrylated UCN 144 in some embodiments. As used
herein, the term pre-polymer includes monomers, and polymer chains
that can be cross-linked. As used herein, the term cross-linking
refers broadly to forming links between polymer chains, to forming
links between a polymer and a nanoparticle, and to polymerization
of monomers. The one or more encoded region source materials
ERSM1-ERSM5 and one or more probe region source materials PRSM are
flowed to an area 150 within a microfluidic device. When the
co-flows are briefly stopped, a light source 160 (e.g., a 350 nm UV
light source) a photomask 162 and a focusing optic (e.g., objective
lens 164) provide patterned and focused light at the area 150 for
cross-linking/polymerization of the pre-polymer 142. Cross-linking
146 of the pre-polymer source materials forms the microparticle 170
by creating a hydrogel polymer network. As shown, the UCNs 144 may
include acrylated ligands, which allows the UCNs 144 to crosslink
146 with the hydrogel polymer network 148. Each encoded region
source material ERSM1-ERSM5 forms a corresponding portion 171-175
of the encoded region and the probe region source material PRSM
forms the probe region 180 of the microparticle 170. In some
embodiments, the UCNs are not cross-linked with the hydrogel
polymer network, but instead are physically entrained by the matrix
pore size of the hydrogel polymer network.
[0093] Although photomask 162 is shown having a pattern that only
forms one microparticle at a time, in some embodiments, the
photomask may have a pattern for forming multiple microparticles
simultaneously. In some embodiments, a photomask may have a pattern
that produces microparticles having different shapes
simultaneously. In some embodiments, the photomask may produce
asymmetric particles and/or particles having nonrectangular
shapes.
[0094] Although microparticle 170 is shown with five encoded
regions, in other embodiments, there may be more or fewer than six
encoded regions. For example, FIG. 22 shows luminescence images of
various microparticles each having between two to six encoded
regions. Microparticles with an additional encoding region would
boost the encoding capacity while requiring little more than an
additional input port on the microfluidic synthesis device.
[0095] For further details regarding the SFL technique for forming
hydrogel microparticles, see U.S. Patent Application Publication
No. US 2012/0316082 A1, published Dec. 13, 2012, and U.S. Patent
Application Publication No. US 2012/0003755 A1, published Jan. 5,
2012, each of which is incorporated by reference herein in its
entirety. An exemplary flow lithography system is described below
with respect to FIGS. 42 and 43.
Example Production of PEG-DA Hydrogel Microparticles with UCNs
[0096] The inventors produced polyethylene glycol diacrylate
(PEG-DA) polymer microparticles by stop flow lithography.
Initially, the inventors made sets of microparticles, with each set
including only one type of nanocrystal to determine whether
incorporating the nanocrystals into microparticles changes the
emission spectral of the nanocrystals. For each of the nanocrystal
types UCN1-UCN10, fifty PEG-DA hydrogel microparticles were
produced. A CCD device was used to obtain a three color image (red
channel, green channel and blue channel) of each microparticle
while illuminated by NIR light producing a red channel image, a
green channel image and a blue channel image. For each channel
image, the intensity (pixel value) within the boundaries of each
microparticle was integrated yielding a "pixel value" for each
channel for each microparticle. FIG. 23 includes histograms of the
integrated "pixel values" for the red, green and blue channels from
fifty microparticles for the UCN1-UCN9 types. The histograms for
some of the types also include an inset image of a representative
NIR-illuminated microparticle. As shown by the inset images, a stop
flow lithography process can be used to make different
microparticle shapes.
[0097] The mean measured integrated intensity values from fifty
microparticles for each type of UCNs were then compared with the
expected integrated intensity data obtained from a convolution of
the UCN emission data and the image sensor response curves. Table 3
below includes measured mean integrated intensity data, the
standard deviation and the coefficient of variability for UCNs in
microparticles. Expected integrated intensity data based on
emission spectra from UCNs in solution are also included for
comparison. As shown in the table, the mean integrated intensity
and the expected integrated intensity values are consistent. The
average coefficient of variation across all particles and UCN
colors was 2%. This corresponds to an average standard deviation of
2.1 RGB units (on a scale of 255) for separately acquired images of
separately synthesized particles, indicating outstanding
particle-to-particle reproducibility. In addition, error ellipses
are non-overlapping to better than 6 sigma, indicating that
decoding error rates of less than 1 ppb are to be expected. Thus,
if the emission spectrum of a type of nanocrystals is known, the
integrated intensity for detection in a color channel can be
reliably predicted.
TABLE-US-00003 TABLE 3 Mean Expected Mean Integrated Integrated
Integrated Expected Intensity .+-. Expected Mean Intensity .+-.
Intensity .+-. Integrated standard Integrated Integrated standard
standard Type Intensity deviation Cv Intensity Intensity Cv
deviation deviation Channel R R R G G G B B Cv UCN1 130.3 126.34
.+-. 1.43 0.02 68.5 65.30 .+-. 2.29 0.03 103.7 100.74 .+-. 2.48
0.02 UCN2 103.3 109.10 .+-. 1.87 0.01 44.8 42.70 .+-. 1.39 0.03
10.2 17.37 .+-. 1.43 0.08 UCN3 164.5 164.29 .+-. 2.26 0.01 91.9
91.73 .+-. 2.73 0.02 0 0 -- UCN10 161.6 160.86 .+-. 1.3 131.5
130.97 .+-. 1.3 0 0 -- UCN4 225.4 225.89 .+-. 2.29 0.01 197.5
194.71 .+-. 2.01 0.01 0 0 -- UCN5 91.9 86.10 .+-. 1.42 0.01 164.5
161.77 .+-. 1.89 0.01 0 0 -- UCN6 120.4 123.52 .+-. 2.15 0.01 158.1
163.40 .+-. 2.04 0.01 138.5 132.29 .+-. 2.54 0.02 UCN7 24.7 23.54
.+-. 2.02 0.08 55.1 63.22 .+-. 1.93 0.03 219.9 222.36 .+-. 2.9 0.01
UCN8 83.2 78.37 .+-. 2.59 0.01 132.6 128.58 .+-. 2.63 0.02 182.2
189.61 .+-. 1.89 0.01 UCN9 158.9 151.34 .+-. 2.02 0.01 131.1 127.62
.+-. 1.93 0.02 120.6 125.73 .+-. 2.92 0.02
[0098] FIG. 24 is a scatterplot showing the red channel, green
channel, and blue channel integrated intensity values for each
microparticles incorporating the UCN1-UCN9 type nanocrystals. All
of the UCN1-UCN9 types of nanocrystals have red channel and green
channel emission intensities. The UCN1, UCN2, UCN6, UCN7, UCN8 and
UCN9 types of nanocrystals have emission intensities in the blue
channel as well as the red and green channels. The ellipses around
each cluster of data points are the three-sigma, four-sigma and
five-sigma contours derived from fitting a Gaussian mixture model
to the data. As shown by separation between the tight clusters, the
UCN type for each microparticle can clearly be distinguished using
the red channel, green channel, and blue channel integrated
intensities for the microparticle. FIG. 25 shows a comparison of
the mean integrated intensity value (measured value squares) and
the expected integrated intensity value (convoluted value circles)
in the green channel versus the red channel for particles
integrating UCN1-UCN9 types of nanocrystals. The ellipses represent
the five-sigma confidence contours.
[0099] Thus, the inventors demonstrated noise-robust spectral
discrimination of six different types of UCNs in hydrogel particles
illuminated using an NIR diode laser and imaged using a standard
CCD camera. Further, as shown by the green channel vs. red channel
plot, the red channel integrated intensity and the green channel
integrated intensity are sufficient to distinguish between the six
different types of nanocrystals. The FIGS. 24 and 25 scatter plots
reveal that cluster overlap occurs only past six standard
deviations from the mean, implying an expected error rate of less
than 1 part per billion (ppb).
[0100] The inventors also compared different batches of hydrogel
microparticles produced at different times to determine the
reliability and the predictability of the integrated intensities of
microparticles from different batches. Five separate batches of
fifty microparticles were produced, each batch including the same
UCN4 type nanocrystals. The microparticles were illuminated with an
NIR light source and color images were obtained using a CCD camera.
Integrated intensity data was generated for microparticles in all
five batches and the average integrated intensity values for each
batch were compared. FIG. 26 is a graph comparing the average
integrated intensities for the green channel and for the red
channel for each batch of fifty microparticles. The integrated
intensities in the red and green channels were consistent across
the five batches. As expected, there was no detected signal the
blue channel. Table 4 below lists the measured red and green
channel integrated intensity values for each batch showing the
consistency and reproducibility of the spectral signature for
different batches of microparticles.
TABLE-US-00004 TABLE 4 Red Channel Green Channel Mean Integrated
Intensity .+-. Mean Integrated Intensity .+-. Type standard
deviation standard deviation 1 225.89 .+-. 2.29 194.71 .+-. 2.01 2
226.51 .+-. 2.97 195.46 .+-. 3.14 3 226.35 .+-. 3.42 195.36 .+-.
3.34 4 226.36 .+-. 3.01 194.22 .+-. 2.46 5 224.65 .+-. 2.05 194.68
.+-. 2.77
[0101] The inventors confirmed that the oxidation and acrylation
process does not change an emission spectrum of the UCNs. FIG. 27
is a graph of emission spectra of UCN4 type nanocrystals after each
step in the surface chemical modification of the UCNs (e.g., before
processing in cyclohexane, after oxidation, after acrylation, and
in PUA prepolymer solution). The spectra overlay each other
establishing that surface chemistry modifications of the UCNs
before incorporation into microparticles does not significantly
affect emission spectra of the resulting particles.
[0102] The inventors also confirmed that there was no attenuation
of the luminescence response of the UCNs integrated into hydrogel
microparticles upon prolonged intense NIR irradiation due to
photobleaching. FIG. 28 is a graph of intensity as a function of
time for hydrogel microparticles including UCN7 type nanocrystals
upon continuous exposure to a 980 nm NIR light from a 1 W laser.
This is in contrast to many commonly used fluorophores which
exhibit attenuation due to photobleaching.
[0103] The inventors also compared the stability of hydrogel
microparticles made with carboxyl-terminated UCNs, in which the
nanocrystals are trapped in pores in the hydrogel matrix, and
hydrogel particles made with acrylated UCNs, in which the
nanocrystals are bonded to the hydrogel matrix via acrylates. FIG.
29 includes graphs comparing intensity as a function of age of
microparticles including acrylated UCN7 type nanocrystals and
microparticles including carboxyl-terminated UCN7 type nanocrystals
without acrylation. As shown, there is a reduction in emission
intensity of the microparticles including carboxyl-terminated UCNs
without acrylation over 30 days, presumably due to the UCNs
diffusing out of the microparticles. In contrast, the
microparticles with acrylated UCNs showed no attenuation over 30
days of aging. Thus, acrylation of the UCNs and subsequent bonding
to the hydrogel matrix improves the luminescence stability (e.g.,
the shelf-life) of the microparticles.
Example Formation of PEG-DA Hydrogel Microparticles with Spectral
and Spatial Encoding
[0104] After establishing the predictability and reproducibility of
the method for forming UCNs and the predictability and
reproducibility of the spectra from hydrogel particles that each
include only one type of UCNs, the inventors produced PEG-DA
hydrogel microparticles with both spectral and spatial encoding.
PEG-DA microparticles are biocompatible and mesoporous allowing
diffusion of large biological macromolecules. Stable integration of
UCNs into microparticles involved use of hydrophilic surface
chemistry with a UV-active functional group on the UCNs for strong,
covalent incorporation as described above. In an embodiment in
which the hydrogel is more densely cross-linked covalent
incorporation of the UCNs may not be needed. In an embodiment in
which the UCNs are disposed in a non-hydrogel portion of the
particle, covalent incorporation of the UCNs may not be needed. For
example, in microparticles with a hydrogel probe region and a PUA
encoded region, the dense cross-linking of the PUA and the
hydrophobic surface chemistry large, rod-like UCN nanostructure may
enabled homogeneous and irreversible physical entrainment of the
UCNs in the PUA portion.
[0105] Specifically, elongated hydrogel microparticles were
produced that each included a probe region and an encoding region.
The encoding region was divided into five portions, (e.g., five
stripes) with each portion including a plurality of UCNs having
distinguishable spectral signature. Although the microparticles
produced included five portions of a encoded region, in some
embodiments, each microparticle may have an encoded region with
more than five portions or less than five portions. Although the
hydrogel microparticles produced were rectangular and elongated, in
some embodiments, the hydrogel microparticles may have a different
aspect ratio and/or a different shape. Further, the microparticles
produced may be symmetric or asymmetric.
[0106] The microparticles were produced by SFL using encoding
region source materials and a probe region source material.
Specifically, for each encoding region source material, acrylated
UCN were dispersed in a PEG-DA premixture solution yielding a
mixture of 45 vol % PEG-DA (Mn=700), 40 vol % UCNs (0.5 mg/.mu.l)
10 vol % poly(styrenesulfonate) PSS and 5 vol % DAROCUR 1173
photoinitiator (PI)). For microparticles with a PUA encoded
portion, the PUA microparticle source material comprised 150 mg of
UCNs dispersed in 300 .mu.l of a 9:1 volume ratio PUA/PI
solution.
[0107] The probe region source material employed a similar PEG-DA
premixture solution that also included molecular recognition
element, specifically a nucleic acid probe for miRNA target
molecules. The source materials were used to form microparticles
using SFL as described above with respect to FIG. 21.
[0108] A microfluidic device was fabricated from
poly-dimethylsiloxane (PDMS) for the SFL system. PDMS was mixed
with a curing agent in a 10:1 ratio and degassed under vacuum for
30 min. Degassed PDMS was poured onto an SU-8 master mold and cured
overnight at 65.degree. C. Channels were then cut out of the mold
and bonded with a glass slide coated with partially-cured PDMS in
order to assure oxygen permeability. The assembled device was fully
cured overnight at 65.degree. C. The microfluidic channel in the
microfluidic device of the SFL system was 300 .mu.m wide and 36
.mu.m high.
[0109] A photomask for the SFL was designed using a computer added
drafting program and printed with a high-resolution printer. The
mask was placed in the field-stop of a microscope before synthesis.
A microfluidic device was fabricated from poly-dimethylsiloxane
(PDMS) for the SFL system. PDMS was mixed with a curing agent in a
10:1 ratio and degassed under vacuum for 30 min. Degassed PDMS was
poured onto an SU-8 master mold.
[0110] The microfluidic channel of the SFL system was loaded with
the composite monomer solution, aligned on a microscope stage, and
subjected to a pressure-driven flow. In every synthesis cycle, the
monomer flow was halted (350 ms) and particles were
photo-polymerized in the device using UV light filtered through a
dichroic filter set (365 nm wavelength light for 100 ns exposure
tine). The polymerized particles were then covected into a
collection tube for 500 ms. Synthesis occurred at a rate of
.about.5 particles per second. After synthesis the particles were
rinsed. The PEG particles were rinsed 3 times with 1.times.TET
(1.times.TE with 0.05% (v/v) Tween 20).
[0111] Although PEG-DA and PUA were used for the hydrogel
microparticles and partial hydrogel microparticles in the examples
described herein, any di-acrylated monomers that have been used in
stop-flow lithography may be used for the encoded region. Further,
any di-acrylated monomers into which UCNs (either nanocrystals with
modified surfaces or ligands or nanocrystals with unmodified
surfaces or ligands) may be well-dispersed can be employed.
[0112] In an initial batch of encoded hydrogel microparticles used
for testing, each portion of the encoded region included a
plurality of nanocrystals selected from the set of types UCN3,
UCN4, UCN5 and UCN7, whose characteristics are described above. As
used herein, encoded microparticles refers to microparticles that
each have one or more portions of the encoded region and that each
have one or more types of spectrally distinguishable UCNs. Eight
encoded microparticles were illuminated with the NIR diode laser
and imaged using a standard CCD image sensor. The integrated
intensity was calculated for the red and green channels of the
image sensor. FIG. 30 is a plot of the green channel integrated
intensity vs. the red channel integrated intensity for each portion
of the encoded region in the eight microparticles. As shown, the
integrated intensities for the portions of the encoded regions are
clumped into groups corresponding to the UCN3, UCN4, UCN5 and UCN7
nanocrystals types. The ellipses are the five-sigma Gaussian fits
to the data from the particles having only one type of
nanocrystals, which may be considered the "training data." All of
the data points for the encoded particles fell within the
five-sigma Gaussian fit for the training data.
[0113] FIG. 31 schematically depicts detection of a target nucleic
acid of interest by a molecular recognition element, specifically a
nucleic acid probe 182, in a probe region 180 of a microparticle
170. In some embodiments, the nucleic acid probe 182 includes a
capturing sequence 182c for binding a targeted nucleic acid of
interest 184 and an adjacent adapter sequence 182a for binding a
universal adaptor. Upon exposure to the target nucleic acid in a
sample solution, the target nucleic acid and the capturing sequence
hybridize as indicated by arrow A1. After exposure to the sample,
the microparticle is exposed to a universal adapter 186 (e.g., a
biotinylated universal adapter), which binds to the adapter
sequence of the nucleic acid probe and to the hybridized target
nucleic acid as indicated by arrow A2. The microparticle is then
exposed to a reporter molecule, such as a fluorescence reporter
(e.g., streptavidin-phycoerythrin (SA-PE)) that binds to the
universal adapter. For a more detailed explanation and other
examples of molecular recognition elements and detectable entities
that may be employed in the probe region see U.S. Patent
Application Publication No. US 2012/0316082 A1, published Dec. 13,
2012, which is incorporated by reference herein in its
entirety.
[0114] PEG-DA particles with distinct coding and bioassay regions
were synthesized, each including an encoding region with five
encoding regions (i.e., 5 stripes) yielding an encoding capacity on
the order of 10.sup.5. One set of the synthesized particles
contained a microRNA (MiRNA) probe for miR-210 and another
contained a probe for mi-R221.
[0115] The inventors produced microparticles having two different
codes for use in a multiplexed assay. Microparticles with the first
code (UCN4, UCN5, UCN3, UCN7, and UCN4 or 45734) included a probe
region with a molecular recognition element for 210 miRNA
(miR-210). Microparticles with the second code (47534) included a
molecular recognition element for 221 miRNA (miR-221). Images of
the two encoded microparticles under NIR illumination are shown in
FIG. 32.
[0116] In order to compare performance of spectrally-encoded
nanoparticle-containing hydrogel microparticles particles to
hydrogel particles without UCNs particle, a batch of "standard"
hydrogel particles including a miR-221 probe region flanked by two
control regions were synthesized. The standard particles had no
encoding region and no UCNs. Both the encoded hydrogel
microparticles and the standard hydrogel microparticles had probe
regions of identical dimensions to ensure similar mass transport
and reaction inside the gel network. FIG. 33 shows a bright field
image and a fluorescence image of the standard particle with no
encoding region and no UCNs after exposure to miR-221. FIG. 34
shows a graph comparing the fluorescence intensity in the probe
region for microparticles with the second code exposed to miR-221
and for the control particles exposed to miR-221 including data for
six particles of each. As shown by FIG. 34, the fluorescence
intensity of the probe region was not affected by the presence of
the encoded regions of UCNs.
[0117] The spectrally-encoded hydrogel particles functionalized
with DNA capture probes for miR-210 and miR-221 were used in a
microRNA assay to demonstrate specific and multiplexed detection of
the two targets. In the multiplexed assay, microparticles with the
two different codes were exposed to four different sample
solutions: one containing 500 amol of miR-210, one containing 500
amol of miR-221, one containing 500 amol of both 221 miR-210 and
miR-221, and one including neither. This enabled evaluation of
encoded microparticles both with regard to standard particles and
with regard to specificity. Post-target incubation, bound miRNA
targets were labeled using a biotinylated universal linker sequence
and a streptavidin-phycoerythrin (SA-PE) fluorophore, and imaged
under fluorescence.
[0118] Assay reactions were carried out in a final volume of 50
.mu.L inside a 0.65 mL Eppendorf tube. Each reaction contained a
total of 75 particles (25 particles of each type: (standard
miR-221, spectrally-encoded miR-221, spectrally-encoded miR-210)).
Target incubations were carried out in microRNA hybridization
buffer for 90 minutes at 55.degree. C. using a thermoshaker (1500
RPM). Post-incubation, particles were rinsed with three 500 .mu.l
volumes of microRNA rinse buffer (RB) using centrifugation. After
each rinse, supernatant was manually aspirated, leaving 50 .mu.L of
solution and particles in the reaction tube. A volume of 235 .mu.L
of a ligation mastermix, which was prepared using 100 .mu.L
10.times.NEB2, 900 .mu.L TET, 800 U/mL T4 DNA ligase, 40 nM
biotinylated universal linker sequence, and 250 nM ATP, was then
added to the reaction for a 30 minute incubation at 21.5.degree. C.
and 1500 RPM. Microparticles were rinsed three more times using
microRNA RB and incubated with streptavidin-phycoerythrin at a
final concentration of 2 .mu.g/mL for 45 minutes at 21.5.degree. C.
and 1500 RPM. After three more rinses with microRNA RB, particles
were exchanged into PTET (TET with 25% (v/v) PEG-200) for imaging.
DNA sequences for the two probes and the universal linker appear in
Table 5 below.
TABLE-US-00005 TABLE 5 DNA Sequences Target Probe Sequence SEQ ID
miR-210 5Acryd/GAT ATA TTT TAT CAG CCG No: 1 CTG TCA CAC GCA
CAG/3InvdT SEQ ID miR-221 5Acryd/GAT ATA TTT TAG AAA CCC No: 2 AGC
AGA CAA TGT AGC T/3InvdT SEQ ID Universal
/5Phos/TAAAATATATAAAAAAAAAAAA/ No: 3 Linker 3Bio/
[0119] FIG. 35 shows images of microparticles with the first code
and microparticles with the second upon exposure to the four
different sample solutions. Fluorescence images and images under
NIR illumination (1 W 980 nm NIR diode laser) were captured
separately. In the images in FIG. 35, the fluorescence image of
each microparticle overlays the image of the microparticle under
NIR illumination. As shown, the assay successfully discriminated
between the presence of miR-210 and miR-221 in the four sample
solutions.
[0120] FIG. 36 is a graph of integrated intensity of each portion
of each microparticle for the red and green color channels for the
encoded PEG-DA microparticles used for the bioassay. As shown, the
encoded PEG-DA microparticle data fits within the five-sigma
contours of the training data for all of the encoded PEG-DA
microparticles, which means that error rates of less than 1 part
per billion (ppb) may be achieved.
[0121] FIG. 37 is a graph of integrated intensity of each portion
of each microparticle for the red and green color channels for the
PEG-DA microparticles used for the bioassay and for PUA
microparticles used for labeling of a blister pack. As shown, the
data fits within the five-sigma contours for both types of
microparticles. Thus, the reliability of identification of encoded
regions applies across different microparticle materials.
[0122] The composite images shown in FIG. 35 and the data in FIGS.
34 and 36 demonstrate successful multiplexed miRNA detection, and
that the encoding strategy has negligible impact on the
fluorescence intensity observed in the probe region, which is an
important criterion for quantifying biomolecule concentrations.
[0123] Although the microparticles produced included a nucleic acid
probe for miRNA as a molecular recognition element, one of ordinary
skill in the art would recognize that many different types of
molecular recognition elements could be employed and incorporated
into the probe region source material of various embodiments. For
example, other types of molecular recognition elements that could
be employed include, but are not limited to various nucleic acids,
miRNA, ssDNA, proteins, receptor proteins, antibodies, enzymes,
peptides, aptamer, avimers, Fc domain fragments, phage, carbon
nanotube sensors, peptides, etc. Any existing molecular recognition
element, biological or not, compatible with the particle synthesis
process, may be incorporated.
[0124] Although the microparticles produced only included one probe
region, in some embodiments, each microparticle may include more
than one probe region. For embodiments with more than one probe
region, the different probe regions may have different types of
molecular recognition elements. In some embodiments, multiple types
of molecular recognition elements may be incorporated into one
probe region. Although the microparticles produced include a probe
region that is distinct from the encoded region, in some
embodiments, the probe region may partially or completely overlap
with one or more portions of the encoded region.
Example Formation of Hydrogel Microparticles with PEG-DA Probe
Region and PUA Encoded Region
[0125] As noted above, after the inventors selected polyethylene
glycol diacrylate PEG-DA as a suitable biocompatible polymer for
forming the hydrogel of the probe region, it was discovered that
oleic-acid capped UCNs do not disperse in the PEG-DA. Instead the
oleic-acid capped UCNs aggregated forming clumped distributions in
the PEG-DA pre-polymer solution, which led to clumped distributions
of UCNs in the microparticles. Before the inventors developed the
method of modifying the oleic acid group to form
carboxyl-terminated UCNs, the inventors initially employed a
hydrophobic polymer, polyurethane acrylate (PUA) (specifically
MINS-300 produced by Minuta Tech, Co. Ltd. of Gyeonggi-Do Korea)
for the encoded region in an attempt to address the problem of UCN
aggregation. The nanoparticles dispersed well in the MINS-300
hydrophobic PUA, but the MINS-300 hydrophobic PUA wouldn't form a
robust cross-linked interface with the PEG-DA of the probe region.
The inventors then selected another PUA that is only slightly
hydrophilic (specifically MINS-0311 produced by Minuta Tech, Co.
Ltd. of Gyeonggi-Do Korea) for the encoded region. The UCNs did not
disperse as well in the slightly hydrophilic second PUA MINS-0311;
however, the MINS-0311 second PUA formed a robust interface with
the PEG-DA of the probe region upon cross-linking. Using the
MINS-0311 second PUA as the polymer for the encoded region source
materials, the inventors were able to achieve some dispersion of
the UCNs in the portions of the encoded region and a robust
interface with the PEG-DA probe region.
[0126] FIG. 38 is a microscope image of microparticles 210 with a
PEG-DA probe region 220 (outlined in white) and a second PUA
(specifically, MINS-311) encoded region 230 excited by a NIR light
source. Each portion of the encoded region 231, 232, 233, 234, 235
included a plurality of UCN. The presence of color throughout each
portion of the encoded region 231-235 indicates that the UCNs were
distributed throughout the source materials. However, the
nonunifomity of the color in each encoded region 231-235 indicates
that the UCNs were not uniformly distributed in the source
materials. For comparison, see the images of microparticles in FIG.
26. The microparticles 210 formed with a PEG-DA probe region 220
and a PUA encoded region 230 experienced deformation due to
different amounts of swelling in aqueous solvents for the two
materials as shown by the white outline of the probe region 220.
Despite these shortcomings, the microparticles 210 could still be
employed in some bioassay applications. Further, the inventors
demonstrated that the UCN could be integrated into a microparticle
that has chemically distinct polymers in different portions of the
particle.
[0127] Any di-acrylated monomers that have been used in stop-flow
lithography may be used for the encoded region. Further, any
di-acrylated monomers into which UCN (either UCNs with modified
surfaces or ligands or UCNs with unmodified surfaces or ligands)
may be well-dispersed can be employed.
[0128] FIG. 39 schematically depicts a method of performing
biochemical or chemical assay 310. The method 310 includes exposing
a sample to a plurality of microparticles (312). Each microparticle
includes a hydrogel or partial-hydrogel body. The body includes a
probe region with one or more molecular recognition elements and an
encoded region. The body also includes a first plurality of UCN
disposed in the first portion of the encoded region and a second
plurality of UCN disposed in a second portion of the encoded region
spatially separated from the first portion of the encoded region. A
spectral signature of the second plurality of UCN is different than
a spectral signature of the first plurality of UCN. The method also
includes 314 illuminating each microparticle with an excitation
light source (e.g., an NIR light source) (314). The method further
includes detecting light emitted from the illuminated microparticle
(316). The detected light including upconverted luminescent light
from the first plurality of UCN and the second plurality of UCN and
light associated with the one or more molecular recognition
elements. The method also includes identifying each microparticle
based on the detected light.
[0129] A method of reading out the spectral codes of a
microparticle is described with respect to FIGS. 40 and 41, which
illustrate reading out a microparticle having six encoded regions
and no probe region. One of ordinary skill in the art in view of
the present disclosure would appreciate how the method may be
applied for reading out spectral codes of a microparticle having an
encoded region with multiple portions and a probe region.
Initially, a maximum or minimum is identified along the x or y axis
(step 1). A center and end points of the particle are identified
(step 2). A particle orientation is determined and, in the case of
an asymmetric particle, a direction of the particle is determined,
and the center of each stripe is identified (step 3). An average
RGB value is calculated within a sampled area around each stripe
(step 4).
[0130] Specifically, images of particles with 6 stripes were taken
via a CCD decoder and loaded into image processing and analysis
software (e.g., MATLAB by Mathworks of Natick, Mass.). Particle
boundaries were defined using a grayscale intensity-based edge
detection algorithm. Boundary pixel x and y values were averaged to
determine the particle centroid. Boundary pixels with minimum and
maximum x and y values (four points total) were noted, and
distances between adjacent points used to determine the particle
end point, or the pixel located on the 2nd shortest edge of the
particle boundary and its longitudinal axis. The end pixel and
centroid pixel were then used to determine both the code
orientation and a director for the particle's longitudinal axis.
The centroid of each striped region of the particle was determined
by segmenting the particle into six regions (the number of stripes
were presumed known a priori) along its longitudinal director. In
other embodiments, k-means image segmentation algorithms may be
employed to define regions of the particle based on color, without
a priori knowledge of the number of particle stripes. RGB values
were measured by averaging pixels within each of the six striped
regions of particles under test were compared against training RGB
values and standard deviations, as determined from a particle
training set. If an average set of RGB values fell within 3.5
standard deviations of a training RGB value, the values were
determined to match. In this way, `analog` RGB sequences were
translated into `digital` sequences of spectral signatures.
[0131] To test the identification, multiple microparticles were
generated with a "true code" and some with a different "false code"
as shown in FIG. 41. An automated decoding system employing the
process described above with respect to FIG. 40 correctly
distinguished the "true code" microparticles that matched a
provided "authentic code" from the "false code" microparticles that
did not match the provided authentic code, using luminescence
images. In FIG. 41, the identified "false code" images are
indicated with a box around the image.
[0132] Further details regarding an exemplary system of particle
synthesis are provided below. FIG. 42 schematically depicts a flow
lithography and decoding system for particle synthesis that
includes a flow lithography microscope setup, a decoding microscope
setup, and a spectrometer setup. FIG. 43 is an image of the flow
lithography and decoding system for particle synthesis. The flow
lithography microscope setup includes a UV LED light source, a
10.times. objective (Edmund optics), a CMOS camera, a dichroic cage
cube, a dichroic mirror, cage cube-mounted turning prism mirrors,
an XYZ sample stage, a mask holder, .phi.1'' lens tubes, an XY
translator, a high-precision zoom housing for 01'' optics, a 30 mm
cage, posts, an LED and valve control relay, which were controlled
with instrument control hardware and software, a camera adapter,
and a CCD camera. The decoding microscope setup included a 1 W 980
nm laser, a 950 nm cut-on filter, a collimator, a CCD camera
adapter, and a CCD camera. The spectrometer setup included a
spectrometer, a laser translation stage, an X,Y translating lens
mount, NIR achromatic doublet pairs, a collimator, a 950 nm cut-on
filter, a 30 mm cage, and posts.
[0133] The versatile, high-performance stop-flow lithography (SFL)
systems and techniques described herein are a high throughput
process for synthesizing particles. In a semicontinuous process,
multiple coflowing laminar streams--each containing a single
optically active UCN moiety or probe molecule--are convected into a
microchannel (e.g., formed from poly(dimethylsiloxane) (PDMS) or a
non-swelling thiolene-based resin for use with organic solvents),
stopped, and photopolymerized in place via mask-patterned
ultraviolet light (365 nm) to form barcoded particles at a rate of
18,000 particles/hr, which are then displaced when flow resumes.
This .about.10.sup.4 particles/hr synthesis rate is by no means
limiting; hydrodynamic flow focusing has been used to increase the
synthesis rate for similar particles to over 10.sup.5 particles/hr.
The synthesis platform may also be constructed using commercial
off-the-shelf parts and free-standing optics. Parallelization in an
industrial setting, with no further optimization, could readily
increase the facility-scale synthesis throughput by orders of
magnitude to meet industrial demand.
[0134] In describing exemplary embodiments, specific terminology is
used for the sake of clarity. For purposes of description, each
specific term is intended to at least include all technical and
functional equivalents that operate in a similar manner to
accomplish a similar purpose. Additionally, in some instances where
a particular exemplary embodiment includes a plurality of system
elements, device components or method steps, those elements,
components or steps may be replaced with a single element,
component or step. Likewise, a single element, component or step
may be replaced with a plurality of elements, components or steps
that serve the same purpose. Moreover, while exemplary embodiments
have been shown and described with references to particular
embodiments thereof, those of ordinary skill in the art will
understand that various substitutions and alterations in form and
detail may be made therein without departing from the scope of the
invention. Further still, other aspects, functions and advantages
are also within the scope of the invention.
[0135] Exemplary flowcharts are provided herein for illustrative
purposes and are non-limiting examples of methods. One of ordinary
skill in the art will recognize that exemplary methods may include
more or fewer steps than those illustrated in the exemplary
flowcharts, and that the steps in the exemplary flowcharts may be
performed in a different order than the order shown in the
illustrative flowcharts.
* * * * *