U.S. patent application number 12/174601 was filed with the patent office on 2009-02-05 for arrays, substrates, devices, methods and systems for detecting target molecules.
Invention is credited to Habib Ahmad, Rong Fan, James R. Heath.
Application Number | 20090036324 12/174601 |
Document ID | / |
Family ID | 40260363 |
Filed Date | 2009-02-05 |
United States Patent
Application |
20090036324 |
Kind Code |
A1 |
Fan; Rong ; et al. |
February 5, 2009 |
ARRAYS, SUBSTRATES, DEVICES, METHODS AND SYSTEMS FOR DETECTING
TARGET MOLECULES
Abstract
Arrays and substrates comprising a material, in particular
capture agents and/or detectable targets, attached to the
substrates along substantially parallel lines forming a barcoded
pattern and related methods and systems.
Inventors: |
Fan; Rong; (Pasadena,
CA) ; Ahmad; Habib; (Los Angeles, CA) ; Heath;
James R.; (South Pasadena, CA) |
Correspondence
Address: |
Steinfl & Bruno
301 N Lake Ave Ste 810
Pasadena
CA
91101
US
|
Family ID: |
40260363 |
Appl. No.: |
12/174601 |
Filed: |
July 16, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60959666 |
Jul 16, 2007 |
|
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60998981 |
Oct 15, 2007 |
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Current U.S.
Class: |
506/9 ; 506/13;
506/16; 506/17; 506/30; 506/39; 506/40 |
Current CPC
Class: |
B01L 2300/0864 20130101;
B01J 2219/00547 20130101; B01J 2219/0074 20130101; B01L 3/502753
20130101; G01N 33/54366 20130101; B01J 19/0046 20130101; B01J
2219/00725 20130101; B01L 3/5025 20130101; B01L 3/502746 20130101;
B01L 2300/0867 20130101; B01J 2219/00621 20130101; B01L 2400/0487
20130101; B01L 2400/084 20130101; G01N 33/54393 20130101; G01N
33/582 20130101; B01J 2219/00596 20130101; B01L 2300/0636 20130101;
B01L 3/502715 20130101; B01J 2219/00605 20130101; G01N 2458/10
20130101; B01J 2219/00722 20130101 |
Class at
Publication: |
506/9 ; 506/13;
506/16; 506/17; 506/39; 506/30; 506/40 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/00 20060101 C40B040/00; C40B 40/06 20060101
C40B040/06; C40B 50/14 20060101 C40B050/14; C40B 60/14 20060101
C40B060/14; C40B 40/08 20060101 C40B040/08; C40B 60/12 20060101
C40B060/12 |
Goverment Interests
STATEMENT OF GOVERNMENT GRANT
[0002] The U.S. Government has certain rights in this disclosure
pursuant to Grant No. CA119347 awarded by the National Institutes
of Health.
Claims
1. An array for detecting at least one target in a sample, the
array comprising: at least one capture agent or component thereof
attached to a substrate, the at least one capture agent capable of
specifically binding the at least one target to form a capture
agent target binding complex, the at least one capture agent or
component thereof arranged on the array so that capture agent
target binding complexes are detectable along substantially
parallel lines forming a barcoded pattern.
2. The array of claim 1, wherein the at least one target is a
plurality of targets, and the at least one capture agent or
component thereof is a plurality of capture agents or components
thereof, each capture agent of the plurality of capture agents
bindingly distinguishable and positionally distinguishable from
another, each capture agent of the plurality of capture agents
capable of specifically binding each target of the plurality of
targets to form a capture agent target binding complex.
3. The array of claim 2, wherein the plurality of targets comprises
a plurality of biomarkers.
4. The array of claim 3, wherein the barcoded pattern is associated
with a biological profile.
5. The array of claim 4, wherein the biological profile provides a
diagnostic indication upon comparison with a predetermined
biological profile associated with a disease.
6. The array of claim 1, wherein said substantially parallel lines
are formed by microfluidic channels or portions thereof, the
microfluidic channels being microfluidic channels of the array.
7. The array of claim 2, wherein the plurality of capture agents or
components thereof comprise: a plurality of array polynucleotides
attached to the array, each polynucleotide of the plurality of
array polynucleotides attached to the array being sequence specific
and positionally distinguishable from another.
8. The array of claim 7, wherein the plurality of capture agents or
components thereof further comprise a plurality of
polynucleotide-encoded proteins, each polynucleotide-encoded
protein comprising a protein and an encoding polynucleotide
attached to the protein, wherein the protein specifically binds to
a predetermined target of a plurality of targets and the encoding
polynucleotide specifically binds to a sequence-specific and
positionally distinguishable polynucleotide of the plurality of
polynucleotides attached to the array, each protein and encoding
polynucleotide being bindingly distinguishable from another.
9. A microfluidic device comprising the array of claim 1.
10. The microfluidic device of claim 9, further comprising a
separating unit for separating a fluidic component of a fluid
sample, the separating unit comprising a flowing microfluidic
channel in fluidic communication with the inlet, the flowing
microfluidic channel having a flowing channel resistance, and an
assaying microfluidic channel in fluidic communication with the
flowing channel, the assaying microfluidic channel having an
assaying channel resistance, wherein the flowing microfluidic
channel resistance and the assaying microfluidic channel resistance
are adapted to control flowing of the fluidic component from the
flowing microfluidic channel to the assaying microfluidic channel
and wherein the array is located on the assaying microfluidic.
11. The microfluidic device of claim 9, wherein the at least one
target is a plurality of targets, and the at least one capture
agent or component thereof is a plurality of capture agents or
components thereof, each capture agent of the plurality of capture
agents bindingly distinguishable and positionally distinguishable
from another, each capture agent of the plurality of capture agents
capable of specifically binding each target of the plurality of
targets to form a capture agent target binding complex.
12. The microfluidic device of claim 11, wherein the plurality of
capture agents or components thereof comprise a plurality of array
polynucleotides attached to the array, each polynucleotide of the
plurality of array polynucleotides attached to the array being
sequence specific and positionally distinguishable from
another.
13. The microfluidic device of claim 12, wherein the plurality of
capture agents or components thereof comprise a plurality of
polynucleotide-encoded proteins, each polynucleotide-encoded
protein comprising a protein and an encoding polynucleotide
attached to the protein, wherein the protein specifically binds to
a predetermined target of a plurality of targets and the encoding
polynucleotide specifically binds to a sequence-specific and
positionally distinguishable polynucleotide of the plurality of
polynucleotides attached to the array, each protein and encoding
polynucleotide being bindingly distinguishable from another.
14. A system for the detection of at least one target in a sample,
the system comprising the array of claim 1; and a device for
detecting the barcoded pattern on the array.
15. The system of claim 14, wherein the at least one target is a
plurality of targets, and the at least one capture agent or
component thereof is a plurality of capture agents or components
thereof, each capture agent of the plurality of capture agents
bindingly distinguishable and positionally distinguishable from
another, each capture agent of the plurality of capture agents
capable of specifically binding each target of the plurality of
targets to form a capture agent target binding complex.
16. The system of claim 15, wherein the barcoded pattern of the
array is associated with a biological profile and the device for
the detecting the barcoded pattern comprises provides a visual
indication of the biological profile
17. The system of claim 15, wherein the barcoded pattern of the
array is associated with a diagnostic indication and the device for
the detecting the barcoded pattern comprises provides a visual
indication of the diagnostic indication.
18. A system for the detection of a plurality of targets in a
sample, the system comprising the array of claim 7; and a plurality
of polynucleotide-encoded proteins, each polynucleotide-encoded
protein comprising a protein and an encoding polynucleotide
attached to the protein, wherein the protein specifically binds to
a predetermined target of the plurality of targets and the encoding
polynucleotide specifically binds to a sequence-specific and
positionally distinguishable polynucleotide of the plurality of
polynucleotides attached to the array, each protein and encoding
polynucleotide being bindingly distinguishable from another
19. The system of claim 18, the system further comprising a
plurality of labeled molecules, each labeled molecule comprising a
component that specifically binds one target of the plurality of
targets and a label compound attached to said component, the label
compound providing a labeling signal, each labeled molecule being
detectably distinguishable from another.
20. A method for detecting a plurality of targets in a sample, the
method comprising contacting said sample with the array of claim 2,
for a time and under conditions to allow binding of said plurality
of targets with said plurality of capture agents to form capture
agent target binding complexes; and detecting said capture agent
target binding complexes.
21. A substrate for detecting at least one detectable target, the
substrate configured to allow attachment of said at least one
detectable target on the substrate along substantially parallel
lines, the substantially parallel lines forming a barcoded
pattern.
22. A microfluidic device comprising the substrate according to
claim 21.
23. A system for detecting a plurality of detectable targets, the
system comprising: the substrate according to claim 21 and a device
for detecting the barcoded pattern.
24. A method for detecting a plurality of targets, in a sample, the
method comprising: contacting said sample with the substrate
according to claim 21 for a time and under conditions to allow
binding of said plurality of targets with said substrate; and
detecting said plurality of targets attached to the substrate.
25. A method to attach a molecule on a fluidic support along a
predetermined microfluidic pattern, the method comprising:
providing a mold, the mold comprising fluidic channels, each
microfluidic channel having an inlet and an outlet, each of the
outlets of the channels configured to form part of the
predetermined pattern, providing the support, said support suitable
to be coupled with the mold, coupling the mold with the support,
providing the molecule in the fluidic channels for a time and under
conditions to allow attachment of the molecule on the support; and
decoupling the mold from the support.
26. The method of claim 25, wherein said pattern comprises
substantially parallel lines forming a barcoded pattern.
27. The method of claim 26, wherein said support is the substrate
according to claim 21.
28. The method of claim 26, wherein said support comprises the
array of claim 1.
29. The method according to claim 25, wherein the fluidic support
is a microfluidic support and the fluidic channels are microfluidic
channels.
30. A system to attach a molecule on a fluidic support along a
predetermined microfluidic pattern, the system comprising: a mold,
the mold comprising microfluidic channels, the microfluidic
channels having an inlet and an outlet, the outlets of the channels
configured to form part of the predetermined pattern, and a support
suitable to be coupled with the mold.
31. The system of claim 30, wherein said pattern comprises
substantially parallel lines forming a barcoded pattern.
32. The system of claim 31, wherein said support is the substrate
according to claim 21.
33. The system of claim 31, wherein said support comprises the
array of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application entitled "An Integrated Blood Platform for Blood
Separation and Protein Detection" Ser. No. 60/959,666, filed on
Jul. 16, 2007 Docket No. CIT4943-P, and to U.S. Provisional
Application entitled "High-Density Bar-code Array: A Generic
Patterning Technique and Biodetection Devices Fabricated Therefrom"
Ser. No. 60/998,981 filed on Oct. 15, 2007 Docket No. CIT-5017, the
disclosures of both of which are incorporated herein by reference
in their entirety. The Application is also related to the U.S.
application entitled "Methods and Systems for Detecting and/or
Sorting Targets" Ser. No. 11/888,502 filed on Aug. 1, 2007, Docket
Number P017-US, and to U.S. application entitled "Microfluidic
Devices, Methods and Systems for Detecting Target Molecules" Serial
No. to be assigned filed on Jul. 16, 2008, Docket Number P235-US,
the disclosures of both of which are also incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0003] The present disclosure relates to patterning of materials,
performance of assays and in particular detection of target
molecules in a sample. More specifically, it relates to arrays,
devices, methods and systems for detecting a plurality of target
molecules in a sample.
BACKGROUND
[0004] Detection of target molecules and in particular of
biomarkers has been a challenge in the field of biological molecule
analysis. In particular, qualitative and quantitative detection of
biomarkers is often a critical step in several applications ranging
from diagnostics to fundamental biology studies.
[0005] In particular, qualitative and quantitative detection of
multiple biomarkers has become increasingly important in several
applications, such as clinical diagnostic wherein accurate
detection of a plurality of biomarkers is desired. More
particularly, in some of those applications detection of the
multiple biomarkers is directed to identify a biological profile
(e.g. proteome and/or metabolome) which can be associated to an
indication of interest (e.g. a diagnostic indication).
[0006] Detection of multiple biomarkers is performed by several
surface-bound assays known in the art. In those assays capture
agents (e.g. primary antibodies) attached to a surface (e.g. a
substrate surface) bind the targets of interest in capture agent
binding complexes. The capture agent binding complexes are then
detected, typically through further binding of the targets with
labeling molecules (e.g. secondary antibodies coupled with
fluorescent dyes).
[0007] A number of critical parameters is associated with
successful execution of a surface-bound assay and include: a)
sensitivity of the assay, or minimum concentration, of the
biomolecule that can be detected, b) concentration range over which
that biomolecule can be detected, c) numbers of different
biomolecules that can simultaneously be detected, d) variability
from measurement to measurement, e) numbers of different types of
biomolecules (e.g. mRNAs, proteins, etc.) that can simultaneously
be detected, f) minimum sample size required for the measurement,
and g) speed at which the measurement can be performed.
[0008] A number of those assays are typically performed in a
microfluidic environment. Microfluidics-based assays are
particularly attractive for applications where minimum sample size
and short time of execution are desired, because they require only
small amounts of biological materials and small amounts of capture
agents, materials and associated reagents.
SUMMARY
[0009] Provided herein, are devices, methods and systems for
detection of a plurality of targets that allow a fast and sensitive
detection of a large number of multiple targets in a sample and/or
provide results in an easily readable fashion.
[0010] According to a first aspect, an array for detecting at least
one target in a sample, and in particular a plurality of targets in
a sample is disclosed. The array comprises, at least one capture
agent or component thereof attached to a substrate, the at least
one capture agent capable of specifically binding the at least one
target to form a capture agent target binding complex. In the
array, the at least one capture agent or component thereof arranged
on the array so that capture agent target binding complexes are
detectable along substantially parallel lines forming a barcoded
pattern. The at least one target can be a plurality of targets, the
capture agent can be a plurality of capture agents, with each
capture agent of the plurality of capture agents bindingly
distinguishable and positionally distinguishable from another and
capable of specifically binding each target of the plurality of
targets to form a capture agent target binding complex.
[0011] According to a second aspect, a microfluidic device is
disclosed that comprises an array according to the present
disclosure.
[0012] According to a third aspect, a system for the detection of a
plurality of targets in a sample is disclosed. The system comprises
an array disclosed herein and a device for detecting the barcoded
pattern on the array.
[0013] According to a fourth aspect, a method for detecting a
plurality of targets in a sample is disclosed. The method
comprises: contacting said sample with an array herein disclosed
for a time and under conditions to allow binding of said plurality
of targets with said plurality of capture agents to form capture
agent target binding complexes; and detecting said capture agent
target binding complexes.
[0014] According to a fifth aspect, a substrate is disclosed, the
substrate for detecting a target, and in particular a plurality of
targets, in a sample. The substrate is configured to allow
attachment of the target on the substrate so that said target is
detectable along substantially parallel lines forming a barcoded
pattern.
[0015] According to a sixth aspect, a microfluidic device is
disclosed that comprises a substrate according to the present
disclosure.
[0016] According to a seventh aspect, a system for the detection of
a target, and in particular a plurality of targets, in a sample is
disclosed. The system comprises a substrate disclosed herein and a
device for detecting the barcoded pattern on the substrate.
[0017] According to an eighth aspect, a method for detecting a
target and, in particular, a plurality of targets, in a sample is
disclosed. The method comprises: contacting said sample with a
substrate herein disclosed for a time and under conditions to allow
binding of said target with said substrate; and detecting said
target attached to the substrate.
[0018] According to a ninth aspect, a method to attach a molecule
on a microfluidic support along a predetermined microfluidic
pattern is disclosed. The method comprises: providing a mold
comprising microfluidic channels, the microfluidic channels having
an inlet and an outlet, the outlets of the channels configured to
form part of the predetermined pattern, providing the support, said
support suitable to be coupled with the mold, coupling the mold
with the support, providing the molecule in the microfluidic
channels for a time and under conditions to allow attachment of the
molecule on the support; and decoupling the mold from the
support.
[0019] According to a tenth aspect a system to attach a molecule on
a microfluidic support along a predetermined microfluidic pattern
is disclosed. The system comprises: a mold comprising microfluidic
channels, the microfluidic channels having an inlet and an outlet,
the outlets of the channels configured to form part of the
predetermined pattern, and a support suitable to be coupled with
the mold.
[0020] The methods and systems for attaching a molecule on a
support on a microfluidic support along a predetermined
microfluidic pattern can be used to manufacture an array and/or a
substrate according to the present disclosure, in embodiments
wherein the pattern is composed of substantially parallel lines
forming a barcoded pattern.
[0021] Arrays, substrates, devices, methods and systems herein
disclosed provide information in a one-dimensional fashion which
can be detected with a single line scan (line profile)
perpendicular to the strip direction to complete reading all
information. In this way, is possible to obtain all the necessary
information without need of a precise move of a reader (e.g. a scan
head) which is instead required in imaging 2D array of the art.
This feature can allow, in certain embodiments, the reading of
barcode DNA array as easy as scanning the product barcode in
supermarket.
[0022] Arrays, substrates, devices, methods and systems herein
disclosed can provide an increased concentration of capture agents
suitable to bind the target and, therefore, increased detection
sensitivity (e.g. up to 0.1 picomolar) when compared to prior art
techniques.
[0023] Arrays, substrates, devices, methods and systems herein
disclosed can allow an increased number of locations for a specific
capture agent on a surface (herein also indicated as spots).
Accordingly, the arrays, devices methods and systems herein
disclosed also allow detection of an increased number of targets or
target related parameters (e.g. 50 targets or more) in comparison
with the ones detectable with prior art techniques.
[0024] Arrays, substrates, devices, methods and systems herein
disclosed are also compatible with microfluidic fabrication
techniques, since the spots can be placed in positions that can be
defined not only with respect to each other, but also with respect
to microfluidic channels and/or other structure on the surface.
[0025] Arrays, substrates, devices, methods and systems herein
disclosed allow providing high density capture agents on a
substrate, with a decreased level of impurities in comparison to
prior art techniques.
[0026] Arrays, substrates, devices, methods and systems herein
disclosed also allow detection of a larger number of biomarkers in
a reduced time (e.g. about 9 minutes) with respect prior art
techniques, in particular in embodiments wherein the array is
integrated with microfluidics.
[0027] Arrays, substrates, devices, methods and systems herein
disclosed allow detection from a sample reduced in size (e.g. 500
nano liter per barcode and/or protein sections from only one cell)
in comparison to the samples analyzed with prior art techniques, in
particular in embodiments wherein the array is integrated with
microfluidics
[0028] Additionally, since the arrays, substrates, devices, systems
and methods herein disclosed allow detection of multiple biomarkers
within the same environment, and in particular the same
microfluidics environment, using a single assay technique, the
relative error associated with measurements of different biomarkers
from the same sample is minimized.
[0029] The arrays, substrates, devices, methods and systems herein
disclosed are applicable to performance of the detection of various
types of target molecules that can bind to immobilized capture
agents. Suitable target molecules include, but are not limited to,
proteins, peptide, polypeptide, ligands, metabolites, nucleic acid,
polynucleotide, carbohydrate, amino acid, hormone, steroid,
vitamin, drug, drug candidate, virus, bacteria, cells,
microorganisms, fragments, portions, components, products, epitopes
of virus, bacteria, microorganisms and/or cells, polysaccharides,
lipids, lipopolysaccharides, glycoproteins, cell surface markers,
receptors, immunoglobulins, albumin, hemoglobin, coagulation
factors, volatile gas molecules, particles, metal ions and the
antibodies to any of the above substrates.
[0030] The arrays, substrates, devices, methods and systems herein
disclosed are applicable to performance of assays including
diagnostic assays, environmental monitoring assays, heath/drug
response monitoring assays and assays performed for research
purposes. Exemplary assays that can be performed include but are
not limited to detection of cancer biomarkers (e.g. prostate cancer
antigen (PSA), and human chorionic gonadotropin (hCG)), detection
of liver toxicity biomarker C-reactive protein (CRP) and
plasminogen, detection of immuno complement proteins like C3,
detection of cytokines such as interferon gamma (IFN-gamma), tumor
necrosis factor alpha (TNF-a), interleukin 1 alpha (IL-1 alpha),
interleukin 1 beta (IL-1 beta), transforming growth factor beta
(TGF beta), interleukin 6 (IL-6), interleukin 10 (IL-10),
interleukin 12 (IL-12), granulocyte macrophage colony stimulating
factor (GM-CSF) etc, detection of chemokines: CCL2 (also called
monocyte chemoattractive protein-1, MCP--1), and demonstration of
detection of complementary DNA molecules.
[0031] Additional applications of the arrays, substrates, devices,
methods and systems herein disclosed include but are not limited to
use the patterning technique to make a barcode array of gas
selective polymers as gas sensors; patterning liquid crystal film
for LCD, and assemble magnetic particle array using DNA-iron oxide
nanoparticle conjugates (just like the antibody-DNA conjugates) for
magnetic barcodes (product ID).
[0032] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The accompanying drawings, which are incorporated into and
constitute a part of this specification, illustrate one or more
embodiments of the present disclosure and, together with the
detailed description, serve to explain the principles and
implementations of the disclosure.
[0034] FIG. 1 shows a schematic representation of the method to
manufacture a reversed or inversed phase barcoded array according
to an embodiment herein disclosed. Panel A shows a barcode pattern
including a number of stripes or bars corresponding to immobilized
serum molecules from various patients. Panel B shows a barcode
pattern wherein the bars are provided by microfluidic channels
formed on top of the array of Panel A.
[0035] FIG. 2 shows a schematic representation of a method and
equipment to detect a barcoded array according to an embodiment
herein disclosed.
[0036] FIG. 3 shows a schematic representation of a comparative
detection of a spot array and of a barcoded array according to an
embodiment herein disclosed.
[0037] FIG. 4 shows a schematic representation of an exemplary
passage in the pattering methods and systems for producing a
barcoded array according to an embodiment herein disclosed.
[0038] FIG. 5 shows a schematic representation of the method to
manufacture a patterned substrate, using a multi-layer fluidic
channel device according to an embodiment herein disclosed.
[0039] FIG. 6 shows an exemplary array according to an embodiment
herein disclosed.
[0040] FIG. 7 shows two images corresponding to an exemplary
molecular detection using a 20 .mu.m barcoded array (panel A) and a
2 .mu.m barcoded array (panel B) according to an embodiment herein
disclosed.
[0041] FIG. 8 shows a computer-aided design of the barcode array
according to an embodiment herein disclosed and a related use. The
panel on the bottom shows thirteen different capture agents (A-M)
flowed into a set of parallel fluidic channels each channel having
a width of 20 .mu.m. The top panel is the enlarged view of a
selected area.
[0042] FIG. 9 shows the execution of multiple assays in twelve
isolated wells using a barcoded array according to an embodiment
herein disclosed. Panel A shows a barcoded array manufactured on a
supporting glass slide. Panel B shows protein detection from the
array of Panel A visualized by fluorescence imaging.
[0043] FIG. 10 shows a schematic representation of the method to
detect target molecules using a group of distinct capture agents
that are directly patterned into a barcoded array according to an
embodiment herein disclosed.
[0044] FIG. 11 shows a schematic representation of the method to
detect target molecules using a group of distinct capture agents
that are immobilized onto the specific location of a pre-determined
barcoded array via a set of linkers according to an embodiment
herein disclosed. This is exemplified by the detection of target
antigen using captured antibodies encoded by a set of complementary
DNA molecules.
[0045] FIG. 12 shows a schematic representation of the method to
vary the loading of capture agents and consequently the sensitivity
and concentration range for the detection of targets using a
barcoded array according to an embodiment herein disclosed.
[0046] FIG. 13 shows a schematic representation of a method to
manufacture a device including a barcoded array according to an
embodiment herein disclosed and a related use.
[0047] FIG. 14 shows an exemplary detection of protein targets
according to an embodiment herein disclosed.
[0048] FIG. 15 shows an exemplary protein detection using a
barcoded array according to an embodiment herein disclosed and
comparison with the protein detection using a conventional
pin-spotted array.
[0049] FIG. 16 shows an exemplary detection of target
polynucleotides according to an embodiment herein disclosed.
[0050] FIG. 17 shows an exemplary multiplexed detection of multiple
protein targets in a sample using a barcoded array according to an
embodiment herein disclosed.
[0051] FIG. 18 shows an exemplary detection of a protein target
according to an embodiment herein disclosed.
[0052] FIG. 19 shows an exemplary detection of multiple targets in
a sample using a barcoded array according to an embodiment herein
disclosed, and its comparison to the conventional array.
[0053] FIG. 20 shows a schematic representation of a method and
system to detect targets according to an embodiment herein
disclosed.
[0054] FIG. 21 shows an exemplary detection of a target in a series
of samples according to an embodiment herein disclosed.
[0055] FIG. 22 an exemplary detection of a protein target in a
series of samples over a large concentration range according to an
embodiment herein disclosed.
[0056] FIG. 23 shows an exemplary detection of a biological profile
according to an embodiment herein disclosed.
[0057] FIG. 24 shows an exemplary detection of a target at
different concentration ranges according to an embodiment herein
disclosed.
[0058] FIG. 25 shows data concerning the exemplary detection of a
biological profile of FIG. 20A.
[0059] FIG. 26 shows detection of a protein profiling in a time
span according to an embodiment herein disclosed.
[0060] FIG. 27 shows an exemplary quantitative detection according
to an embodiment herein disclosed.
[0061] FIG. 28 shows an exemplary elaboration of biological
profiles detected according to the exemplary embodiment illustrated
in FIG. 21(A) embodiment herein disclosed.
[0062] FIG. 29 shows an exemplary detection of target proteins in a
drop of fresh human blood.
[0063] FIG. 30 shows an exemplary detection of a human plasma
proteome according to an embodiment herein disclosed.
[0064] FIG. 31 shows a schematic representation of the method to
manufacture a patterned substrate according to an embodiment herein
disclosed.
DETAILED DESCRIPTION
[0065] Arrays, substrates, devices, methods and systems for
detecting a target, and in particular, a plurality of target
molecules in a sample are herein disclosed.
[0066] The term "detect" or "detection" as used herein indicates
the determination of the existence, presence or fact of a target or
signal in a limited portion of space, including but not limited to
a sample, a reaction mixture, a molecular complex and a substrate.
A detection is "quantitative" when it refers, relates to, or
involves the measurement of quantity or amount of the target or
signal (also referred as quantitation), which includes but is not
limited to any analysis designed to determine the amounts or
proportions of the target or signal. A detection is "qualitative"
when it refers, relates to, or involves identification of a quality
or kind of the target or signal in terms of relative abundance to
another target or signal, which is not quantified.
[0067] The term "target" or "target molecule" as used herein
indicates an analyte of interest. The term "analyte" refers to a
substance, compound or component whose presence or absence in a
sample has to be detected. Analytes include but are not limited to
biomolecules and in particular biomarkers. The term "biomolecule"
as used herein indicates a substance compound or component
associated to a biological environment including but not limited to
sugars, amino acids, peptides proteins, oligonucleotides,
polynucleotides, polypeptides, organic molecules, haptens,
epitopes, biological cells, parts of biological cells, vitamins,
hormones and the like. The term "biomarker" indicates a biomolecule
that is associated with a specific state of a biological
environment including but not limited to a phase of cellular cycle,
health and disease state. The presence, absence, reduction,
upregulation of the biomarker is associated with and is indicative
of a particular state. Exemplary biomarkers include breast cancer
marker HER2, ovarian cancer marker CA125, and heart disease marker
thrombin.
[0068] The term "sample" as used herein indicates a limited
quantity of something that is indicative of a larger quantity of
that something, including but not limited to fluids from a
biological environment, specimen, cultures, tissues, commercial
recombinant proteins, synthetic compounds or portions thereof.
[0069] In some embodiments, arrays, substrates, methods and systems
are herein disclosed for the detection of multiple, distinct
targets, such as biomolecules, or a panel of biomarkers. In the
arrays, substrates, devices methods and systems herein disclosed
each target is detected in a particular location on a surface, and
the collection of detected biomolecules forms a pattern, or a
barcode. In particular, the arrays, devices, methods and systems
herein disclosed can apply to the detection of the biomarker panel
within a micro fluidics environment.
[0070] In some embodiments of the arrays, substrates devices
methods and systems herein disclosed a plurality of capture agents
attached to a substrate.
[0071] The wording "capture agents" as used herein indicate a
molecule capable of specific binding with a predetermined binding.
Exemplary capture agents include but are not limited to
polynucleotides and proteins, and in particular antibodies.
[0072] The term "polynucleotide" as used herein indicates an
organic polymer composed of two or more monomers including
nucleotides, nucleosides or analogs thereof. The term "nucleotide"
refers to any of several compounds that consist of a ribose or
deoxyribose sugar, joined to a purine or pyrimidine base and to a
phosphate group and that are the basic structural units of nucleic
acids. The term "nucleoside" refers to a compound (as guanosine or
adenosine) that consists of a purine or pyrimidine base combined
with deoxyribose or ribose and is found especially in nucleic
acids. The term "nucleotide analog" or "nucleoside analog" refers
respectively to a nucleotide or nucleoside in which one or more
individual atoms have been replaced with a different atom or a with
a different functional group. Accordingly, the term polynucleotide
includes nucleic acids of any length DNA RNA analogs and fragments
thereof. A polynucleotide of three or more nucleotides is also
called nucleotidic oligomers or oligonucleotide.
[0073] The term "polypeptide" as used herein indicates an organic
polymer composed of two or more amino acid monomers and/or analogs
thereof. The term "polypeptide" includes amino acid polymers of any
length including full length proteins and peptides, as well as
analogs and fragments thereof. A polypeptide of three or more amino
acids is also called a protein oligomer or oligopeptide. As used
herein the term "amino acid", "amino acidic monomer", or "amino
acid residue" refers to any of the twenty naturally occurring amino
acids including synthetic amino acids with unnatural side chains
and including both D and L optical isomers. The term "amino acid
analog" refers to an amino acid in which one or more individual
atoms have been replaced, either with a different atom, isotope, or
with a different functional group but is otherwise identical to its
natural amino acid analog.
[0074] The term "protein" as used herein indicates a polypeptide
with a particular secondary and tertiary structure that can
participate in, but not limited to, interactions with other
biomolecules including other proteins, DNA, RNA, lipids,
metabolites, hormones, chemokines, and small molecules.
[0075] The term "antibody" as used herein refers to a protein that
is produced by activated B cells after stimulation by an antigen
and binds specifically to the antigen promoting an immune response
in biological systems and that typically consists of four subunits
including two heavy chains and two light chains. The term antibody
includes natural and synthetic antibodies, including but not
limited to monoclonal antibodies, polyclonal antibodies or
fragments thereof. Exemplary antibodies include IgA, IgD, IgG1,
IgG2, IgG3, IgM and the like. Exemplary fragments include Fab Fv,
Fab' F(ab')2 and the like. A monoclonal antibody is an antibody
that specifically binds to and is thereby defined as complementary
to a single particular spatial and polar organization of another
biomolecule which is termed an "epitope". A polyclonal antibody
refers to a mixture of monoclonal antibodies with each monoclonal
antibody binding to a different antigenic epitope. Antibodies can
be prepared by techniques that are well known in the art, such as
immunization of a host and collection of sera (polyclonal) or by
preparing continuous hybridoma cell lines and collecting the
secreted protein (monoclonal).
[0076] The wording "specific" "specifically" or specificity" as
used herein with reference to the binding of a molecule to another
refers to the recognition, contact and formation of a stable
complex between the molecule and the another, together with
substantially less to no recognition, contact and formation of a
stable complex between each of the molecule and the another with
other molecules. Exemplary specific bindings are antibody-antigen
interaction, cellular receptor-ligand interactions, polynucleotide
hybridization, enzyme substrate interactions etc. The term
"specific" as used herein with reference to a molecular component
of a complex, refers to the unique association of that component to
the specific complex which the component is part of. The term
"specific" as used herein with reference to a sequence of a
polynucleotide refers to the unique association of the sequence
with a single polynucleotide which is the complementary
sequence.
[0077] The term "attach" or "attached" as used herein, refers to
connecting or uniting by a bond, link, force or tie in order to
keep two or more components together, which encompasses either
direct or indirect attachment such that for example where a first
molecule is directly bound to a second molecule or material, and
the embodiments wherein one or more intermediate molecules are
disposed between the first molecule and the second molecule or
material.
[0078] The term "substrate" as used herein indicates an underlying
support or substratum. Exemplary substrates include solid
substrates, such as glass plates, microtiter well plates, magnetic
beads, silicon wafers and additional substrates identifiable by a
skilled person upon reading of the present disclosure.
[0079] In some embodiments, the capture agents used in the arrays,
devices, methods and systems herein disclosed can be either
directly deposited onto substrate to form an array or immobilized
by linker molecules that are pre-deposited onto substrate and
capable to specific binding to capture agent for form an array.
Since they are functional to the attachment of capture agents to a
substrate, linker molecules can be considered as capture agent
components.
[0080] In the arrays, substrates, devices, methods and systems
herein disclosed, wherein multiple capture agents are used, each
capture agent can be bindingly distinguishable and/or positionally
distinguishable from another.
[0081] The wording "bindingly distinguishable" as used herein with
reference to molecules, indicates molecules that are
distinguishable based on their ability to specifically bind to, and
are thereby defined as complementary to a specific molecule.
Accordingly, a first molecule is bindingly distinguishable from a
second molecule if the first molecule specifically binds and is
thereby defined as complementary to a third molecule and the second
molecule specifically binds and is thereby defined as complementary
to a fourth molecule, with the fourth molecule distinct from the
third molecule.
[0082] The wording "positionally distinguishable" as used herein
refers to with reference to molecules, indicates molecules that are
distinguishable based on the point or area occupied by the
molecules. Accordingly, positionally distinguishable capture agents
are substrate polynucleotide that occupy different points or areas
on the assaying channel and are thereby positionally
distinguishable.
[0083] In arrays herein disclosed, each capture agent of the
plurality of capture agents is capable of specifically binding each
target of the plurality of targets to form a capture agent target
binding complex, and the plurality of capture agents arranged on
the array so that capture agent target binding complexes are
detectable along substantially parallel lines forming a barcoded
pattern.
[0084] In other embodiments, substrates systems and methods are
herein disclosed wherein the substrate is configured to allow
attachment of targets (herein also reverse barcode or
inversed-phase barcode), and in particular detectable targets,
along substantially parallel lines forming a barcoded pattern. An
exemplary illustration of reverse barcode is illustrated in FIG. 1,
wherein a barcoded pattern including a number of bars corresponding
to immobilized serum molecules from various patients and
microfluidic channels for providing various drugs to be contacted
with the serum of the patients for a bio-assay, are shown.
[0085] In some embodiments, detection of the attached target and/or
capture agent target complex is performed by providing a labeled
molecule, which includes any molecule that can specifically bind a
capture agent target complex to be detected (e.g. an antibody,
aptamers, peptides etc) and a label that provides a labeling
signal, the label compound attached to the molecule. The labeled
molecule is contacted with the attached target and/or capture agent
target complex and the labeling signal from the label compound
bound to attached target and/or the capture agent-target complex on
the substrate can then be detected, according to procedure
identifiable by a skilled upon reading of the present disclosure
and, in particular, of the Examples section.
[0086] In particular, the signal readout that is used in the
arrays, devices, methods and systems herein disclosed can be
realized using labels such as probes that transduce the capture
event of target molecule into optical, electrical or magnetic
signal. Exemplary probes include, but not limited to, fluorescent
dyes, gold nanoparticles, silver nanoparticles, semiconductor
nanoparticles (e.g. CdSe, ZnSe and/or their core-shell
nanoparticles), and iron oxide nanoparticles.
[0087] The terms "label" and "labeled molecule" as used herein as a
component of a complex or molecule refer to a molecule capable of
detection, including but not limited to radioactive isotopes,
fluorophores, chemoluminescent dyes, chromophores, enzymes, enzymes
substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions,
nanoparticles, metal sols, ligands (such as biotin, avidin,
streptavidin or haptens) and the like. The term "fluorophore"
refers to a substance or a portion thereof which is capable of
exhibiting fluorescence in a detectable image. As a consequence the
wording and "labeling signal" as used herein indicates the signal
emitted from the label that allows detection of the label,
including but not limited to radioactivity, fluorescence,
chemolumiescence, production of a compound in outcome of an
enzymatic reaction and the likes.
[0088] In embodiments wherein one or more targets and/or a
plurality of targets is detected described below in more details,
the labeled molecule can be formed of a plurality of labeled
molecules. Each labeled molecules comprises a molecule that
specifically binds one target of the one or more targets/plurality
of targets and a label compound attached to the molecule, the label
compound providing a labeling signal, each labeled molecule
detectably distinguishable from another.
[0089] The wording "detectably distinguishable" as used herein with
reference to labeled molecule indicates molecules that are
distinguishable on the basis of the labeling signal provided by the
label compound attached to the molecule. Exemplary label compounds
that can be use to provide detectably distinguishable labeled
molecules, include but are not limited to radioactive isotopes,
fluorophores, chemoluminescent dyes, chromophores, enzymes, enzymes
substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions,
nanoparticles, metal sols, ligands (such as biotin, avidin,
streptavidin or haptens) and additional compounds identifiable by a
skilled person upon reading of the present disclosure.
[0090] In embodiments, wherein bindingly distinguishable capture
agents are used different analytes can be detected by use of
detectably distinguishable labeled molecules each specific to a
separate analyte of interest.
[0091] In some embodiments, the detection method can be carried via
fluorescent based readouts, in which the labeled antibody is
labeled with fluorophore which includes but is not limited to small
molecular dyes, protein chromophores and quantum dots. In other
embodiments, on-chip detection can be performed with methods other
than fluorescence based techniques. Exemplary suitable techniques
include, calorimetric detection, enzyme-catalyzed production of
different colored or fluorescent dyes (with different colors being
associated with distinct analytes), microparticle/nanoparticle
based detection using electron microscopy, AFM, or dark-field
microscopy, magnetic detection using magnetic micro/nanoparticles,
electrical detection methods.
[0092] In some embodiments, detection can be performed by methods
that use signal amplification such as gold nanoparticle based
detection followed by gold or silver amplification. In particular,
in some embodiments, in any of the methods and systems herein
disclosed, detection can be carried out on gold
nanoparticle-labeled secondary detection systems in which a common
photographic development solution can amplify the gold
nanoparticles as further described below. Also, if the readout
comes from dark field scattering of gold particles, single molecule
digital proteomics is enabled.
[0093] The detection can be performed with the aid of suitable
equipments. In particular any equipment configured to read barcoded
pattern can be used as long as the relevant sensitivity is
applicable to the detection of choice.
[0094] For example, in some embodiments, reading the information of
the arrays herein disclosed can be performed using a simple
line-scan reader such as the laser line scanner schematically
illustrated in FIG. 2. The one-dimensional layout of the arrays
renders a higher reliability as compared to the conventional
circular spot arrays as schematically illustrated in FIG. 3. In the
illustration of FIG. 3, is shown how a scan reading from a same
line scanner (scan b) provides a higher reliability for a barcoded
pattern (panel B) if compared with a spotted array (Panel A).
[0095] Additional equipment suitable to detect the array herein
described can be identified by a skilled person upon reading of the
present disclosure. For example. when fluorescent probes are used
for signal readout, laser microarray scanner (such as. Axon Genepix
4000 series scanner, Affymetrix 300 scanner, etc), scanning laser
confocal microscope (e.g. Nikon Eclipse C1si microscope) can be
used to visualize the pattern. In particular, the parallel-stripe
pattern allows a single scan of laser to read out full information
with high fidelity and reliability as illustrated in FIGS. 2 and 3.
This feature opens the possibility of implementing a simple laser
line scanner similar as the barcode reader in supermarket for
reading the barcode array described herein.
[0096] In other embodiments, wherein gold nanoparticles are used,
light scattering microscope (such as Nikon.RTM. Eclipse LV100) can
be used. In other embodiments, wherein electroless metal plating is
used to enhance the nanoparticle signal, a flat bed scanner (such
as Nanosphere Verigene.RTM. reader) can be used besides light
scattering microscopes. In still other embodiments, wherein
magnetic particles are used as probes, a magnetoresistive sensor
similar to a scan head in a hard disk can be used to read out the
barcode information.
[0097] Additional techniques are identifiable by a skilled person
upon reading of the present disclosure and will not be further
discussed in details.
[0098] Arrays and substrates herein disclosed can be manufactured
using methods and systems to attach a material to a support along a
predetermined pattern herein also disclosed (herein also indicated
as patterning methods and systems). The methods and systems to
attach material can be used to manufacture arrays and substrate
according to any predetermined pattern. In embodiments, wherein the
patterned material is configured along substantially parallel lines
forming a barcoded pattern, the methods and systems herein
disclosed can be used to manufacture barcoded arrays and
substrates.
[0099] In some embodiments, the barcoded surface patterning can be
performed as described below in the exemplary procedure illustrated
with reference to microfluidics channels patterned from
polydimethylsiloxane (PDMS) that are weakly or strongly bonded to
glass substrates. A skilled person would understand that the
patterning method is not limited the specific microfluidic features
and materials used and that a different number of channels with
different dimensions as well other materials, such as injection
molded micro fluidics channels, semiconductor wafers, etc., all
identifiable by a skilled person upon reading of the present
disclosure, may all be utilized.
[0100] In some embodiments, a mold can be fabricated by molding a
polymer such as a PDMS elastomer from a master template, to include
microchannels each having an inlet and an outlet and each of the
outlets is such that it forms a portion of the desired pattern (in
particular a barcoded pattern). In some embodiments, the polymer is
molded using photolithography to create a photoresist pattern on a
silicon wafer. Those embodiments, allow a particularly rapid
prototyping. An exemplary illustration of a mold fabrication for
the patterning methods and systems herein disclosed is illustrated
in FIG. 4 wherein fabrication of a PDMS microchannel stamp for flow
patterning of a barcode array is disclosed.
[0101] In another embodiment, the mold can be manufactured by
providing a silicon "hard" master and by transferring the
photolithographically-defined pattern into the underlying silicon
wafer using a deep reactive ion etching (DRIE) process. Those
embodiments allow a robust and reusable mold for higher throughput
chip fabrication.
[0102] In some embodiments, the molded polymer can then be coupled
and in particular bonded onto a support, such as a glass surface,
which provides the floor for the channels of barcoded pattern. An
exemplary illustration of a design two-layer PDMS fluidic channel
device used for creating a multiple ring pattern (bull's eye) on a
glass slide is shown in FIG. 5.
[0103] In some of embodiments, the substrate can be pre-coated with
a material of interest. For example in embodiments wherein a
barcode is manufacture using the DEAL technology further illustrate
below, a polyamine polymer or poly-L-lysine polymer
(Sigma-Aldrich), can be pre-coated prior to bonding to increase DNA
loading of the final barcoded pattern (see below and in particular
Example 2).
[0104] The number of microfluidic channels determines the size of
the barcode array. In some exemplary embodiments the barcoded array
comprises 13 to 20 parallel microchannels that wind back and forth
to cover a large area (3 cm.times.2 cm) of the support with the DNA
barcode microarray.
[0105] In some embodiments, patterning can be performed by
contacting the capture agent or molecule of choice on the support
for a time and under conditions to allow attachment on the support.
More particularly, in some embodiments patterning can be performed
by providing solutions, each containing the molecule of choice
(e.g. a different strand of primary DNA oligomers prepared in
1.times.PBS buffer in embodiments wherein the array is coupled with
DEAL technology), can be flowed into each of the microfluidic
channels. Then, the solvent of the solution can be allowed to
evaporate, e.g. by placing the solution-filled chip in a dessicator
to allow solvent (e.g. water) to evaporate completely through the
gas-permeable PDMS, leaving the molecules to be attached (e.g. DNA
molecules) behind. In some embodiments, this process can take from
several hours to overnight to complete.
[0106] Following patterning of the molecules, the mold is usually
decoupled from the support. In some embodiments, once the mold is
removed from the support the patterned molecule can be subjected to
subsequent treatments (e.g. DNA molecule can be fixed to the glass
surface by thermal treatment at 80 C for 4 hours, or by UV
crosslinking; removal of salts or other precipitates that might
have formed during one or more of the previous operations which can
be removed, for example, by rapidly dipping the slide in deionized
water prior to bonding the blood-assay chip to the slide). An
exemplary procedure of the patterning method herein disclosed is
illustrated in Example 15.
[0107] In particular, in some specific embodiments, a series of
microfluidics channels is patterned into PDMS, and those channels
bonded onto a glass surface so that one out of the 4 channel walls
is the glass surface itself. The numbers of micro fluidics channels
determines the size of the barcoded array. In this way, a solution
flowing through the micro fluidics channel will come into contact
with the glass substrate. Typical dimensions of these micro
fluidics channels for barcoded used for biological assays are 10
micrometers or larger. In particular, in embodiments where material
is patterned to be subjected to a bio assay, the channel width
defines the width of an individual bio-assay measurement area
within the final bar code. In those embodiments, if the final
measurement of the biomolecule is done using optical methods, then
a 10 micrometer wide area constitutes a size that is readily imaged
using low-cost optics. Larger and smaller bars are also
possible.
[0108] A different material and in particular a different
biological species (or a different concentration of the same
biological species), such as DNA oligomers, can then be flowed in
to each of the individual micro fluidics channels.
[0109] The biological species or other patterned material can then
be attached to the glass surface areas within those microfluidics
channels using electrostatic or other chemical interactions. The
glass may be pre-coated with some molecular component to increase
the chemical interaction between the biological species and the
glass surface (see above and below in particular Example 2).
[0110] The solvent from the solution containing the patterned
material (e.g. the biological species) is then removed. If that
solution is water and the fluidics (e.g. microfluidics) is
fabricated from PDMS, then the water can be let naturally evaporate
through the PDMS, leaving the patterned material attached to the
substrate thus providing a the patterned array on the substrate. In
some embodiments, it may be desirable to introduce additional
channel (e.g. micro fluidics channels) at this point for handling
and introducing the biological sample of interest.
[0111] The microfluidic bar-code patterning chip may be made by
molding silicon elastomer from a master template. The master
template may be fabricated from many materials. One method is to
fabricate the master by using photolithography to expose an SU8
2015 photoresist. Regions of the photoresist are removed following
lithographic exposure, and the remaining material constitutes the
master. Alternatively, photolithographic patterning methods,
coupled with deep reactive ion etching (DRIE), can be utilized to
prepare a master from a silicon wafer. These various methods for
preparing microfluidics molds and microfluidics channels from those
molds are well known in the art. (Gael Thuillier and Chantal Khan
Malek, Microsys. Technol 12, 180, 2005.)
[0112] The patterned material can comprise any substance of
interest suitable to be attached to a support, including organic or
inorganic substances, Exemplary inorganic material that can be
patterned using the patterning methods and systems herein disclosed
include but are not limited to gold nanoparticles that can attach
to thiol functionalized substrate surface, iron oxide nanoparticles
that can be deposited onto the substrate using magnetic field, and
silica particles that can be immobilized by cationic polymer coated
substrate, and so on.
[0113] Exemplary organic that can be patterned using the patterning
methods and systems herein disclosed include but are not limited to
living species and their mixtures such as cells, virus, bacteria
and fungi, complex biospecimens and their mixtures such as tissue,
tissue lysate, cell lysate, serum, saliva and joint fluid,
monotypic molecule and their mixtures such as polynucleotides,
proteins, antibodies, glycoproteins, polysaccharides,
lipopolysaccharides, ligands, peptides, polypeptides, lipids,
drugs, drug candidates, antigens and the fragments, portions, and
components or any of above. The organic materials can also include
non-biological materials such as polymers, oligomers, dye
molecules, conducting polymers, responsive polymer, gas sensing
polymers, liquid crystals and metal organic frameworks (MOFs),
carbon nanotube, fullerene, grapheme, and their
nano/microstructures. In some embodiments, the patterned material
comprises capture agents. In some embodiments, the patterned
material comprises detectable targets. In other embodiments, the
patterned material comprises a material, such as cells or other
biological material to be assayed. In other embodiments, the
patterned material can comprise other organic or inorganic
substance for which the barcoded configuration is desired (e.g.
liquid crystal for LCD manufacturing, or gas selective polymers to
be used as gas sensors).
[0114] According to the patterning methods and systems herein
disclosed, a pattern and in particular a barcoded pattern or array
can be created on very small area and patterning of magnetic ID or
other material can therefore be performed onto small-sized
products.
[0115] In some embodiments, wherein the pattern is used for the
detection through capture agents, the capture agent is formed by a
polynucleotide and in particular a DNA polynucleotide, that bind
about 10 to 20 consecutive bases of a target RNA via complementary
hybridization. In some of those embodiments the arrays, substrates,
methods and systems herein disclosed can be used to detect
messenger RNA (mRNA) and in particular mRNA from a biospecimen
(e.g. tissue lysate). In some of those embodiments, another labeled
DNA stand (e.g. fluorescently labeled) is designed to bind to
.about.10-20 different bases of the captured mRNA for signal read
out. In some embodiments, a multiplexed measurement of a panel of
mRNA molecules can be performed on a barcode array patterned with
stripes of their capture agent DNA.
[0116] In some embodiments, wherein the pattern is used for the
detection, the target is a microRNA (miRNA) a type of short RNA
molecules (22 bases) that regulate gene expression at the
post-transcription level
[0117] In some embodiments, wherein the pattern is used for
detection, the target can be a transcription factor, and the
capture agent is a polynucleotide and in particular a DNA
polynucleotide having the same sequence of the binding site of the
transcription factor, or a portion thereof or an homologous
sequence thereof. In some embodiments, fluorescence-labeled or
biotin-labeled antibodies are then used for signal readout.
[0118] In some embodiments, the lines are formed by one or more
channels configured to host the material to be patterned. In
particular, in some embodiments the fluidic channel width can be
made ranging from 0.5 .mu.m to 1 cm. The height can be typically
> 1/10 of the channel width when a soft materials such as PDMS
is employed, and can be less if a harder material (e.g. glass,
silicon, polystyrene, PMMA, polycarbonate or epoxy) is used to make
the fluidic channels.
[0119] In embodiments when a two-layer device is used for
patterning arrays, the channel can be as short as 1 mm and up to
meters when the channel is shaped to cover the entire substrate
(e.g. a glass slide 1''.times.3'') for example by turning back and
forth on the substrate. In embodiments where a larger substrate is
used, the channel length can be longer since the length is defined
by the substrate and the application of interest.
[0120] The array can be in principle made into any custom-designed
shapes such as stripes, rings, concentric rings (see for example
the illustration of FIGS. 5 and 6), triangles, rectangles,
polyhedrons, stars, cross-bars, letters, pictures on flat, convex,
concaved or irregular substrates. In particular in FIG. 6 a
multiple ring pattern suitable to application such as a bio-assay
for detection of targets secreted by a sample such as a cell placed
in the middle, is shown. In particular the images of FIG. 6 show
the detection of proteins IL-2 and TNF-.alpha. visualized by Cy3
and Cy5 fluorescent probes.
[0121] In embodiments, wherein the channels are used to pattern
polynucleotides (e.g. DNA) or proteins (e.g. antibodies), the
channels width can be anywhere from 0.5 .mu.m to 1 cm and the
height can range from 1 .mu.m to 1 cm, and the length can any that
is allowed by the area of the given substrate. An exemplary 2-.mu.m
barcode array is shown in FIG. 7, wherein a barcoded array of
fluorescent DNA molecules manufactured according to the teaching of
the present disclosure, is illustrated. For optimum demonstrated
performance of polynucleotide detection using a complementary DNA
barcoded array, a channel width of 20 .mu.m and height of 20 .mu.m
are preferred when a 200-.mu.M capture DNA solution is used and the
developed array is visualized using fluorescence scanner. In
embodiments, wherein a DNA barcoded array is used to immobilize DNA
encoded antibodies and subsequent immuno-sandwich assay, the same
channel width and height are preferred (see below description of
DEAL technology).
[0122] In some embodiments, some or all of the substantially
parallel lines are connected to one another through at least one of
the ends. More particularly, in applications wherein the lines are
formed by channels the substantially parallel lines can be
connected to one another to form a single channels configured in a
serpentine-like shape. Serpentine-like channels allow the
fabrication of repeated barcode arrays over a large area, e.g. the
entire glass slide (1''.times.3''), in a single step of flowing
capture agents. It represents a significant advantage in
large-scale, low cost manufacture of barcoded arrays for detection
applications. In addition, it allows an assay to be executed in
multiple repeats at the same thus reduce the statistic errors. An
exemplary illustration of a serpentine-like channel is shown in
FIG. 8. Additional connections between the substantially parallel
lines of a pattern or multiple patterns (for example multiple
barcoded patterns connected to form a pyramid to increase DNA
loading in application wherein barcode is manufactured in
connection with DEAL technology).
[0123] The material to be patterned can be disposed along the
parallel lines according to a specific experimental design of
choice. For example, in embodiments where a plurality of capture
agents are patterned, the capture agents can be disposed with each
capture agent disposed along one line, or with two or more capture
agents located disposed along portions of a single channel. In
other embodiments, the material to be tested (and in particular
detected) can be patterned along one line or portion of a line of
the barcode. Exemplary illustrations of those embodiments are shown
in FIGS. 1 and 7.
[0124] In some embodiments, the patterned material can be used for
target detection. In those embodiments, typically capture agents
are patterned on the substrate, to form detectable capture agent
target complexes. In other embodiments, detectable targets are
patterned directly on the material. For example, a number of serum
samples from multiple patients can be patterned into a barcoded
array. In such array, each stripe contains the biomolecules in the
entire plasma proteome of that patient. This array can be exploited
to screen for antibodies, ligands, drug candidates, and comparison
of biological profiles among patients. Those embodiments are
exemplified for the barcoded arrays, substrates, methods and
systems of Examples 3-14 and illustrated in the related figures and
further described below.
[0125] In some embodiments, assays are performed in a
non-microfluidic environment. An exemplary illustration of those
embodiments is shown in FIG. 9, wherein execution of multiple
assays in twelve isolated wells using a barcoded array is
illustrated. In particular, the barcoded array illustrated in FIG.
9 is manufactured on a supporting glass slide including wells,
wherein. each well contains a different sample such as human serum.
In the experiments illustrated in FIG. 9, protein detection from
the different samples is visualized by fluorescence imaging.
[0126] In some embodiment, assays are performed in microfluidics
which allows handling particularly small amounts of biospecimens
(such as a finger prick of blood, tissue from skinny needle biopsy,
etc).
[0127] In some embodiments, the barcode array can be used to detect
multiple proteins and/or genes from a single cell via on-chip
single cell culture, lysis, mRNA and protein
isolation/purification, in particular using an integrated
microfluidic device such as the one described in the U.S.
Application entitled "Microfluidic Devices, Methods and Systems for
Detecting Target Molecules" Serial No. to be assigned filed on Jul.
16, 2008, Docket Number P235-US, incorporated herein by reference
in its entirety.
[0128] A further description of the arrays, substrates, devices
methods and systems of the present disclosure is provided with
reference to microfluidic applications wherein the sample is a
material of biological origin (bio sample) and the targets are
biomarkers. A person skilled in the art will appreciate the
applicability of the features described in detail for microfluidics
and biomarkers for non-microfluidic applications and/or for other
biologic, organic and inorganic samples and targets.
[0129] In some embodiments, the arrays, devices methods and systems
herein disclosed can be used to perform a surface bound bioassay
based on detection a biomolecule of interest in some biomaterial,
such as blood, serum, biological tissue, or as a component of a
cell culture (herein also indicated as bio-barcode assay).
[0130] The biological material can be pretreated so as to release
the biomolecules of interest, to remove biological material that
can interfere with binding of the biomolecules in the surface bound
bioassay. An exemplary pretreatment procedure includes separating
blood cells from blood plasma (or serum), and then measuring the
proteins from the plasma. In other procedures the separated cells
could be further separated into white and red blood cells, which
can be therefore subjected to further analysis. An exemplary
surface bound bioassay can be carried out as follows: The
biomolecule of interest is bound to a (primary or 1.degree.)
surface-bound capture agent molecule (e.g. an antibody or
complementary single-stranded DNA oligomer) that specifically
recognizes and binds to the biomolecule of interest. Typically, a
secondary (or 2.degree.) capture agent containing some label for
detection, such as a fluorescent molecule, is introduced to bind to
the surface-bound biomolecule.
[0131] The bio-barcode can be manufactured patterning the capture
agents of choice on a substrate along substantially parallel lines.
In certain microfluidic applications the substantially parallel
lines can be formed by channels or channel portions. Exemplary
illustration of different embodiments wherein capture agents are
attached to a surface in a bio-barcode are shown in FIG. 10
(capture agents DNA molecules for detection of polynucleotide (e.g.
mRNA and microRNA) to be configured in a barcoded array), FIG. 11
(DNA-encoding antibodies to enable immuno-sandwich assay on barcode
array allowing detection of proteins, cell surface markers,
glycoproteins, virus and bacteria in multiplex) and FIG. 12
(schematic illustration showing how increased DNA loading helps to
enhance detection sensitivity in application wherein the
bio-barcode is coupled with DEAL technology see below).
[0132] Patterning of capture agents, for example, antibody arrays
for detecting proteins or complementary DNA arrays for detecting
polynucleotides, results in an increased sensitivity of molecules
such as polynucleotide, nucleic acid (mRNA, miRNA, DNA etc), An
increased sensitivity could be in particular associated with two
factors: (1) the increased loading of capture DNA using poly-amine
to coat substrate surface (for embodiments wherein the capture
agent is a polynucleotide and in particular DNA) and (2) the
reduced feature size with respect to conventional pin spotted
arrays (e.g. 20 .mu.m in barcoded array vs. 200 .mu.m in
conventional pin-spotted array) lowers the diffusion barrier and
leads to high binding efficiency.
[0133] In some embodiments the capture agents include one or more
component. In particular, in some embodiments the capture agents
can be formed by a substrate polynucleotide and a polynucleotide
encoded-protein in application of the technology (herein also
identified as DEAL) described in U.S. patent application Ser. No.
11/888,502 herein incorporated by reference in its entirety.
[0134] Accordingly, the wording "substrate polynucleotide" as used
herein refers to a polynucleotide that is attached to a substrate
so to maintain the ability to bind to its complementary
polynucleotide. A substrate polynucleotide can be in particular
comprised of a sequence that specifically binds and is thereby
defined as complementary with an encoding-polynucleotide of a
polynucleotide encoded protein.
[0135] The wording "polynucleotide-encoded protein" refers to a
polynucleotide-protein complex comprising a protein component that
specifically binds to, and is thereby defined as complementary to,
a target and an encoding polynucleotide attached to the protein
component. In some embodiments, the encoding polynucleotide
attached to the protein is protein-specific. Those embodiments can
be used to perform assays that exploit the protein-specific
interaction to detect other proteins, cytokines, chemokines, small
molecules, DNA, RNA, lipids, etc., whenever a target is known, and
sensitive detection of that target is required. The term
"polynucleotide-encoded antibody" as used herein refers to a
polynucleotide-encoded protein wherein the protein component is an
antibody.
[0136] In the polynucleotide-encoded proteins herein disclosed each
protein specifically binds to, and is thereby defined as
complementary to, a pre-determined target, and each encoding
polynucleotide-specifically binds to, and is thereby defined as
complementary to, a pre-determined substrate polynucleotide.
[0137] In embodiments wherein the protein is an antibody, the
protein-target interaction is an antibody-antigen interaction. In
embodiments wherein the protein is other than an antibody, the
interaction can be receptor-ligand, enzyme-substrate and additional
protein-protein interactions identifiable by a skilled person upon
reading of the present disclosure. For example, in embodiments
where the protein is streptavidin, the protein-target interaction
is a receptor-ligand interaction, where the receptor is
streptavidin and the ligand is biotin, free or attached to any
biomolecules. An exemplary schematic illustration is shown in FIG.
12.
[0138] When coupled with the DEAL technique, the amount of
polynucleotides that is deposited onto a given spatial location
within the bio-barcode array can be controlled in view of the
desired sensitivity and concentration range over which the
biomolecule of interest can be detected. By using two or more
stripes within the same bio-barcode array, each optimized to detect
the same biomolecule but over different concentration ranges, the
concentration range over which that protein can be detected, as
compared to a conventional assay, can be dramatically
increased.
[0139] The concentration range of DNA detectable with a Bio-Barcode
array coupled with DEAL can be as low as 1 pM to 100 nM using 200
.mu.M loading of capture DNA on 20 .mu.m barcode stripes. Target
molecules suitable for this technique include messenger RNAs, micro
RNAs, the fragments of genomic DNAs, viral DNA, bacterial DNA, and
synthesized polynucleotides.
[0140] Some embodiments wherein the Bio-Barcode is coupled with
DEAL shows an increased sensitivity if compared with embodiments
wherein protein capture agents are patterned directly on a
substrate. In particular, in some embodiments wherein antibodies
are patterned directly into barcoded array with fabrication methods
that require application of high temperatures when the antibodies
are attached to the substrate, all the target molecules that can be
detected by DEAL are in principle detectable, but a lower
sensitivity might be seen due to the poor stability of the antibody
in a dry state.
[0141] When coupled with the DEAL technique, the bio-barcode array
withstands the processing conditions associated with micro fluidics
chip fabrication. As a consequence, the Bio Bar .Bar Code array can
be advantageously manufactured as illustrated in the exemplary
procedure outlined below with reference to an exemplary array
including 10 antibodies used as capture agents (10 CAs) labeled
with single stranded DNA used as encoding polynucleotide.
[0142] The 10 antibodies against the biomarker of interest are
chemically labeled with single-stranded DNA (ssDNA) oligomers. The
complementary ssDNA' oligomers can be deposited onto regions of a
surface. DNA hybridization assembles the 10 CAs onto those
particular regions.
[0143] The 10 CAs are patterned using microfluidics channels. The
channel widths and densities are limited by what can be
patterned--smaller channels and higher densities than are practical
using other methods are readily achieved. Typically channels of
widths of at least 10 micrometers, spaced by distances of at least
50 micrometers, are most practical for typical bioassays, such as
analyzing multiple proteins from serum. This allows for large
numbers of measurements to be carried out in a relatively small
microfluidics channel.
[0144] Spot sizes significantly smaller than 10 micrometers are
also possible with this technique, as are significantly higher spot
densities. These may be useful for more specialized applications,
such as would be required for measuring a panel of protein
biomarkers and other biomolecules from circulating tumor cells,
cancer stem cells, and other extremely rare cell types.
[0145] The bio-barcode patterned microfluidics channels are readily
aligned with other microfluidics channels, such as are used for the
handling of the biological specimen from which the assays are
performed. For example, alignment markers that are utilized to
align the bio-barcode micro fluidics channels can also be utilized
to assemble the microfluidics channels for handling the biological
sample. This is standard fabrication practice.
[0146] The density of 1.degree. CAs that can be deposited onto such
a small spot can be significantly higher than what can be achieved
using spotting methods. Repeated depositions of 10 CAs through the
same microfluidics channels can achieve a very high surface loading
of the 10 CAs. Conversely, the DEAL technique utilizes
single-stranded DNA (ssDNA) oligomers as capture agents for the 10
CA antibodies that are, in turn, utilized to detect proteins. The
DNA can be loaded at very high levels using the bio-barcode Array
because of the high solubility of DNA in water. This, in turn, can
lead to very high coverage of the 1.degree. antibody CAs.
[0147] Multiple numbers and classes of capture agents can be placed
on specific, microscopic locations on a surface using microfluidic
patterning of the 10 capture agents. In this way, the panel of
biomolecules is detected by detecting labeling signals (for
example, fluorescence) from the region of the surface where the
pattern of 10 capture agents was placed.
[0148] In some embodiments, wherein the arrays, substrates methods
and systems herein disclosed are performed in microfluidics, the
capture agents can be attached on the location with a method to
attach molecule along a predetermined pattern herein disclosed. In
those embodiments, using a microchannel-guided flow-patterning
approach, a barcode arrays can be manufactured that are at least an
order of magnitude denser than conventional microarrays. In some
embodiments, this result can be accomplished by creating a mold,
e.g. a polydimethylsiloxane (PDMS) mold containing the desired
number of microfluidic channels, e.g. 13-20 parallel microfluidic
channels, with each channel conveying a different biomolecule
capture agent. A skilled person will understand that the number of
channels can readily be expanded to include 100 or more different
capture agents; whereas in microcontact printing, the patterning
difficulty increases exponentially as the number of proteins
printed is increased, due to the challenges of aligning multiple
stamps to print multiple proteins.
[0149] In some embodiments, the barcoded array is a DEAL barcoded
array. In some of those embodiments poly-amine coated glass
surfaces can be use to allow significantly higher DNA loading than
do more traditional aminated surfaces. DNA "bars" of 2 micrometers
in width could be successfully patterned. In some exemplary
embodiments, described herein an about 20-micrometer (.mu.m)
channel width was chosen because the fluorescence microarray
scanner has a resolution of 5 .mu.m.
[0150] In those embodiments a 10-fold increase in array density is
achieved as compared to a typical pin-spotted DNA array (i.e. 150
.mu.m spot diameters at 300 .mu.m pitch), and greatly expands the
numbers of proteins that can be measured within a microfluidic chip
disclosed herein for a given sample size. In particular, in some
embodiments, simultaneous detection of 12 to 20, up to 50 or even
more than 50 proteins. This feature can be used in applications
where detection of multiple targets is desired, for example
detection of a biological profiles but also a variety of waste
gases (e.g. from car engine exhaustion) or pollutes in a
sample.
[0151] The protein assay can be carried out on the 10 CAs array as
described above. Use of DNA hybridization as an assembly strategy
allows for multiple proteins to be detected within the same
microenvironment, since the various 10 CA antibodies for the
various proteins to be detected can be each labeled with a
different ssDNA oligomer. Also use of DNA hybridization as an
assembly strategy allows preparation of the substrate including
ssDNA in early in the fabrication process so that a substrate
including the ssDNA can be treated, dried out, heated, shipped and
provided to the final user in a ready to use systems that also
include complementary capture agents. Exemplary applications are
described in Examples 1 to 7 and in the related figures describe
the bar-code array patterning technique and DEAL bar-code chips for
protein detection.
[0152] A person skilled in the art would understand that the array
herein disclosed can include patterning a variety of biological
materials, e.g. DNA, proteins, sera and tissue lysates, using micro
fluidic channels. The Bio Bar-code Array method can be applied to
the fabrication of bio-chips and integrated biosensing devices for
high-density, multiplexed and sensitive detection of DNA and
proteins in clinic diagnostics of human diseases like cancers, and
for high-throughput drug screening. In some embodiments the
patterning is based upon a new, yet simple and reliable
approach--micro channel guided surface patterning of a large number
of different biological species to fabricate a small-size,
high-density array.
[0153] The systems herein disclosed can be provided in the form of
arrays or kits of parts. An array sometimes referred to as a
"microarray" includes any one, two or three dimensional arrangement
of addressable regions bearing a particular molecule associated to
that region. Usually the characteristic feature size for
microarrays is micrometers.
[0154] In a kit of parts, various components can be comprised in
the kit independently. In some embodiments, a patterned substrate
can be provided together with a label and/or other reagents
suitable to perform detection. In some embodiments, a device
suitable for detecting the pattern can also be included.
[0155] In embodiments, wherein the patterned substrate is
integrated with deal technology a system can include
polynucleotide-encoded proteins and a patterned substrate comprised
in the kit independently. Molecules comprised in the kit (e.g. the
polynucleotide-encoded protein) can in particular be included in
one or more compositions, with each molecule in a composition
together with a suitable vehicle carrier or auxiliary agent.
[0156] The substrate provided in the system can have substrate
polynucleotides attached thereto or other molecule attached
according to the desired pattern. In some embodiments, the
substrate polynucleotides, or the material to be patterned can be
further provided as an additional component of the kit. Additional
components can include labeled polynucleotides, labeled antibodies,
labels, microfluidic chip, reference standards, and additional
components identifiable by a skilled person upon reading of the
present disclosure. In particular, the components of the kit can be
provided, with suitable instructions and other necessary reagents,
in order to perform the methods here disclosed. The kit will
normally contain the compositions in separate containers.
Instructions, for example written or audio instructions, on paper
or electronic support such as tapes or CD-ROMs, for carrying out
the assay, will usually be included in the kit. The kit can also
contain, depending on the particular method used, other packaged
reagents and materials (i.e. wash buffers and the like).
[0157] Additional applications in which the patterned material is
not limited to a biological sample will be identifiable by the
person skilled in the art. In particular in some embodiments, the
patterned material can be used for magnetic identity (ID) of
small-sized products, which can include but are not limited to
products carrying a biological material. For example, a magnetic ID
bar has been widely used in tracking a product. But conventional
magnetic ID pad is too large to be used for a small-sized subject
such as a small camera CMOS chip, a fine jewel and a tiny artifact.
Those embodiments are exemplified for the barcoded arrays,
substrates, methods and systems in Example 15.
[0158] Further details concerning the identification of the
suitable carrier agent or auxiliary agent of the compositions, and
generally manufacturing and packaging of the kit, can be identified
by the person skilled in the art upon reading of the present
disclosure.
EXAMPLES
[0159] The methods and system herein disclosed are further
illustrated in the following examples, which are provided by way of
illustration and are not intended to be limiting the scope of the
present disclosure.
Example 1
Fabrication and Use of a Barcoded Chip with Integrated DEAL
Technology
[0160] A Barcoded chip was fabricated according to the procedure
schematically illustrated in FIG. 13 Panel A.
[0161] A silicon elastomer (PDMS) stamp was molded from a
lithographically patterned silicon master. Then it was thermally
bonded onto a poly-amine coated glass slide on which different
biomolecule solutions are flowed into the parallel microchannels.
Once the solutions evaporate completely, the PDMS stamp is peeled
off and the glass side will be baked to create a robust
Bio-Bar-code array. The bar-code stripes can be made 2-20 .mu.m in
width and spacing, leading to increased array density compare to
conventional microarrays. In principle, there is no limit for the
number of primary molecules like DNA that can be patterned using
this technique. It indeed enables the fabrication of a large-scale,
high-density biomolecule array for systems biology and disease
diagnostics.
[0162] More particularly, a polydimethylsiloxane (PDMS) mold
containing 13-20 parallel microfluidic channels, with each channel
conveying a different DNA oligomer as DEAL code, was fabricated by
soft lithography. The PDMS mold was bonded to a polylysine-coated
glass slide via thermal treatment at 80.degree. C. for 2 hours. The
polyamine surfaces permit significantly higher DNA loading than do
more traditional aminated surfaces. DNA "bars" of 2 micrometers in
width have been successfully patterned using this technique. In the
present study, a 20-micrometer (.mu.m) channel width was chosen
because the fluorescence microarray scanner used by applications
has a resolution of 5 .mu.m. Nevertheless, the current design
already resulted in a DNA barcode array an order of magnitude
denser than conventional microarrays fabricated by pin-spotting.
The coding DNA solutions (A-M for the cancer serum test and AA-HH
for the finger-prick blood test) prepared in 1.times.PBS were
flowed into individual channels, and then allowed to evaporate
completely. Finally, the PDMS was peeled off and the substrate with
DNA barcode arrays was baked at 80.degree. C. for 2-4 hours. The
DNA solution concentration was .about.100 .mu.M in all experiments
except in the hCG test, leading to a high loading of
.about.6.times.10.sup.13 molecules/cm.sup.2 (assuming 50% was
collected onto substrate).
[0163] The array so created was used in a bio assay as illustrated
in FIG. 13 Panel B. An integrated microfluidic device was placed
onto the bio-bar-code chip microfluidic channels. There was no need
of fine alignment to integrate the bio-bar-code pattern with the
microfluidic systems. Different samples such as patient sera,
tissue lysates can be assayed in each microfluidic channels,
respectively. The array depicted in FIG. 13 panel B enables
high-through biodetection with minimum sample consumption.
[0164] The experiments described above can be modified to modulate
sensitivity and detectable range of targets according to the
experimental design of choice. A possible modification is
illustrated in FIG. 8 which shows a schematic illustration of a
mask design of a 13-channel patterning chip, wherein the letter A-M
indicate the channels for flowing different DNA molecules.
Additional modifications include subjecting the array to poly-amine
surface modification, e.g. with the procedure exemplified in
Example 2 below, to allow increased DNA loading. This modification
leads to higher sensitivity and broader dynamic range as illustrate
in the exemplary procedure of Example 3 below.
Example 2
Fabrication of a DEAL Barcoded Chip with an Increased DNA
Loading
[0165] During microchannel-guided flow-patterning of the DEAL
barcode arrays, the glass surface was modified by treatment with
poly-L-lysine (a poly-amine), yielding a three-dimensional matrix
for DNA adsorption and markedly increasing the amount of DNA
loading
[0166] The results are illustrated in FIG. 14, which shows the
effects of poly-lysine coating on an assay performed with DEAL
technology. More particularly, FIG. 14 shows detection of protein
targets using the barcoded array manufactured with low and high
loading of primary DNA molecules and the resulting difference in
the protein detection. As shown in the schematic illustration of
panel (a) polylysine coating of the PDMS support results in an
increased loading of DNA oligomer codes.
[0167] In particular, the DNA-loading density is estimated to be
6.times.10.sup.13 molecules/cm.sup.2 in our experiments, an order
of magnitude higher than typical loading densities on amino-silane
coated glass slides. As a result, the protein detection sensitivity
was improved by an order of magnitude, and the dynamic range was
increased to 4 orders of magnitude, as compared with 2-3 orders of
magnitude for the small-molecule amine (i.e.
amino-propyl-triethoxyl silane, APTES) functionalized glass
surface. Exemplary results of this comparative analysis is
illustrated in FIG. 14 Panel (b) detection of three human cytokines
(IFN-.gamma., TNF-.alpha., and IL-2) using substrates coated with
amino-silane and polylysine, respectively is shown.
Example 3
Barcoded Chip with ELISA-Like Sensitivity
[0168] A series of experiments performed by the applicants showed
that a barcode chip integrated with DEAL technology renders a high
density array for multiplexed protein measurements. Moreover, the
DEAL barcoded chip also demonstrates a marked improvement in
sensitivity as compared to conventional pin-spotted
microarrays.
[0169] In particular, a side-by-side comparison study was performed
by running DEAL assays on three cytokines under identical
conditions. Using the microchannel-guided flow patterning method, a
glass slide was patterned with DNA oligomers A, B, C and a blank
control O. Each bar was 20 .mu.m in width. The DNA solutions were
all 50-100 .mu.M. The pin-spotted array was printed at the
Institute for Systems Biology at 100 .mu.M concentration. The
typical spot size was 150-200 .mu.m. Six sets of spots were printed
corresponding to oligomers A, B, C, D, E, and F. Poly-l-lysine
coated slides were used for both types of arrays.
[0170] Before the DEAL assay, the capture antibodies were
conjugated to DNA oligomer codes as follows: A' to IFN-.gamma., B'
to TNF- and C' to IL-2. Protein standards were diluted in 1%
BSA/PBS solution at concentrations ranging from 1 fM to 1 nM. The
incubation time for each step (blocking, conjugate hybridization,
sample binding, detection-antibody binding, and
fluorescent-molecule binding) was 30 min. The bar width was 20
.mu.m.
[0171] The results are illustrated in FIG. 15 wherein immunoassays
run on DEAL barcode arrays is shown. In particular, as illustrated
in Panel (a) detection of three human cytokines (A: IFN-.gamma., B:
TNF-.alpha., C: IL-2, O: negative control) was proven to be
concentration dependent. In the illustration of Panel (a) the
bar-code array has a sequence of ABCOABCOABCOA (herein, "0" denotes
that no 1.degree. DNA was flowed in such microchannel). This data
show proteins can be detected at concentration as low as 1 pM.
Concentration dependence is indicated by the diagram of Panel (b)
where quantitation of fluorescence intensity is plotted versus
TNF-.alpha. concentration. The line profile for the results
obtained with 1-pM protein sample as indicated in Panel (a), is
shown in the diagrams of Panel (c).
[0172] As a further comparison, the sensitivity obtained in ELISA
assays (using antibody pairs and protein standards from
eBioscience) is projected to be .about.10 pg/mL (0.8 pM) for
TNF-.alpha.. Therefore, those experiments show that the DEAL
barcode array combines ELISA-like sensitivity with a high degree of
multiplexing for protein measurements.
[0173] In addition, the TNF-.alpha. detection sensitivity of the
DEAL barcode arrays was higher and the projected sensitivity limit
was better than 1 pM, as compared to 10-100 pM for conventional
microarrays as illustrated in the comparative assay performed under
the same condition using a conventional pin-spotting method of
Panel (d) further illustrated in the comparative Example 6 below.
These results confirmed that the barcoded chip has much higher
sensitivity and increased linear range for protein measurements, as
compared with a conventional assay.
Example 4
Use of a Barcoded Array for DNA Detection
[0174] A barcoded array was used in a bio assay for detection of
DNA. In particular, a polynucleotide (DNA) was patterned on a
substrate and used to detect a complementary polynucleotide in a
sample. The results illustrated in FIG. 16 show that the patterned
DNA oligomers exhibit a high affinity for binding their
complementary strands.
[0175] In particular, in FIG. 16 panel A, fluorescence images are
reported taken before and after hybridization of an A' strand to
its Alexa 532 labeled complementary stand. Three different strands
of DNA oligomers, nonfluorescent A, Alexa 532 labeled B (red) and
Alexa 635 labeled (dark green) were flow-patterned on a
polyL-lysine slide to form this bar-code chip. "0" denotes a
non-patterned channel for bland control. After applying the
Alexa-532 labeled A' molecule s (its concentration is 1 nanomolar,
these DNA molecules are complementary to the surface bound A
stands), a clear and strong green fluorescence band emerges,
indicating highly effective and specific sensing of A' DNA
molecules.
[0176] The line profile of fluorescence intensity across the whole
set of bar-code array is shown in FIG. 16 Panel B. In the
illustration of FIG. 16, A' is the target polynucleotide that was
added into sample b and detected by fluorescence change in the
location indicated by an asterisk.
Example 5
Use of Barcoded Array for Protein Detection
[0177] A barcoded array assembled as disclosed herein was used for
protein detection according to an experimental approach developed
by the applicants.
[0178] In particular, applicants developed a multiplexed assay of
12 plasma proteins using DEAL barcode arrays. In a first test, the
level of cross-reactivity of each antigen with DEAL stripes that
are not specific to that antigen was assessed. DNA-encoding capture
antibodies and biotinylated detection antibodies for all 12
antigens were used as usual, but a distinct antigen (10 nM) was
added to each assay lane. Cy5-Streptavidin (red-fluorescence tag)
was run as usual to visualize the extent of analyte capture.
[0179] The reference marks (DNA strand M) were visualized in all
lanes with fluorescent green Cy3-M' DNA molecules. The 12 proteins
showed a negligible extent of cross-talk. In a second test, assays
were performed on serial dilutions of all 12 proteins on the DEAL
barcode chip in view of the limitation imposed by the particular
devices used, each allowing a maximum of 12 parallel assays to be
executed. In the specific experimental approach of choice for this
setting 6 lanes were used for cross-talk validation and 6 lanes
were used for dynamic range studies.
[0180] The results are illustrated in FIG. 17 which shows
cross-reactivity check and dilution curves for all 12 proteins. In
particular, the DEAL barcode images and line profiles from a single
device of panel (a) show minimal cross-talk and a series of
standard antigens ranging from 1 nM to 1 pM for all 12 proteins. In
the experiments shown in panel (a), 2 proteins were combined in
each assay lane (FIG. 17 panel (a)).
[0181] All proteins were assayed on the same chip over the
concentration range of 1 nM down to 1 pM (except PSA and TGF-b: 5
nM to 5 pM), and quantified the fluorescence signal vs.
concentration for all 12 antigens as illustrated in FIG. 17 panel
(b), where dilution curves for all 12 proteins are shown.
[0182] In this experiment, all the concentrations were imaged using
the Genepix scanner at the same laser power (55 for 635 nm, 15 for
532 nm), optical gain (500 for 635 nm and 400 for 532 nm), and
brightness/contrast (92/90) in order for quantitative comparison.
Apparently, the estimated sensitivity varies a lot from .about.0.3
pM (e.g. IL-1.beta. and IL-12) to 30 pM (TGF-.beta.) largely
depending on the antibodies being used. For example, the TGF-b
antibody pair has a relatively lower binding affinity and a poorer
detection limit in ELISA (according to the spec sheet, it is
.about.70 pg/mL compared to 5-10 pg/mL for most other cytokines).
Predictably, this gave rise to a poorer performance in the DEAL
assay. Although these curves clearly show a dynamic response of
DEAL signals with respect to antigen concentrations, the variation
remains pretty large as compared to bulk-scale immuno-assay such as
ELISA.
[0183] Detection probes are not limited to fluorescent dyes, but
can be any others that are capable to transduce signal from
captured targets to optical, magnetic or electrical read out.
[0184] In particular, an alternative method of detection is
provided by use of gold nanoparticles as probes. An exemplary
illustration of detection performed using gold nanoparticles is
shown in FIG. 18, wherein detection of target protein IL-1.beta.
using gold nanoparticles as the probe is shown.
[0185] In particular, in the example of FIG. 18, 40-nm gold
nanoparticles were used to visualize the captured protein (e.g.
IL-1.beta.) of interest from human serum).
[0186] Additional examples of labels and method of detections are
illustrated the U.S. Application entitled "Methods and Systems for
Detecting and/or Sorting Targets" Ser. No. 11/888,502 filed on Aug.
1, 2007, incorporated herein by reference in its entirety.
Example 6
Comparative Example Related to Use of a Barcoded Array and a
Conventional Microarray for Protein Detection
[0187] Comparative experiments were performed on the barcode array
of example 3 and a conventional microarray printed using
pin-spotting technique. The results illustrated in FIG. 15 panel d,
show how apparently, the conventional microarray only achieved
sensitivity 1-2 orders of magnitude worse than the DEAL barcoded
chips.
[0188] A side-by-side comparison study was performed by running
DEAL assays on three cytokines under identical conditions on a
barcoded and a pin spotted microarrays under the experimental
conditions illustrated in Example 3. The pin-spotted array was
printed at the Institute for Systems Biology at 100 .mu.M
concentration. The typical spot size was 150-200 .mu.m. Six sets of
spots were printed corresponding to oligomers A, B, C, D, E, and F.
Poly-l-lysine coated slides were used for both types of arrays.
Further details are illustrated in Example 3.
[0189] The results illustrated in FIG. 15 panel e show that
barcoded array exhibits greater performance with higher sensitivity
than does the conventional array.
[0190] In particular, these results demonstrate that the detection
sensitivity of the DEAL barcode arrays was higher and the projected
sensitivity limit was better than 1 pM, as compared to 10-100 pM
for conventional microarrays (FIG. 15 panel e).
[0191] The only difference between the barcoded and conventional
pin-spotted platforms used in the experiment shown in FIG. 15 is
the feature size. The barcode array has a line-width of 20 .mu.m,
whereas the spot size in conventional arrays is more than 150
.mu.m. The mechanism for improved sensitivity in the DEAL barcode
assay is not completely understood. A possible explanation which is
not intended to be limited is that the improved sensitivity could
be attributed to a reduced kinetic barrier and decreased diffusion
time. These results are consistent with a recent report which
demonstrated that DNA microarrays with smaller spot sizes could
detect DNA with increased sensitivity.
Example 7
Use of a Barcoded Array for Detection of Multiple Different
Targets
[0192] A barcoded array integrated with DEAL technology was used to
detect multiple proteins as illustrated in FIG. 19. In particular
FIG. 19 shows the use of DEAL bar-code immunoassay for the
detection of five different proteins. The proteins are detected
within an area that is less than would be required for the
detection of a single protein using a conventional spotted
microarray.
[0193] The results illustrated in FIG. 19 show in particular
multiple proteins simultaneously detected using a DEAL bio-barcode.
Panel A shows a schematic illustration of DEAL bar-code array for
co-detection of a variety of proteins at the same time, including
cytokines, chemokines, growth factors, intracellular signaling
molecules and cancer markers. Panel B shows a multiparameter DEAL
Bar-code immunoassays of 5 proteins at the same time, detected from
human reference serum that was spiked with the five proteins: hCG,
TNF-.alpha., IL-2, IL-a, and IL-1.beta.. In principle, bar-code
array can provide high density assay of a much greater number of
protein s simply by increasing the number of microchannel s used in
flow patterning.
[0194] The detection of multiple targets was performed according to
the schematic representation of FIG. 20 that shows the microfluidic
device used in patient serum measurement In particular, FIG. 20
panel A shows. the schematic of the operation of a microfluidic
device that is bonded onto a barcode array glass slide.
[0195] FIG. 20 Panel B shows a schematic illustrating the method to
introduce fluid into microfluidic devices for molecular detection
and in particular interfacing the outside sample loading/injection
systems to the microfluidic device using plastic tubing and metal
pins.)
Example 8
Use of a Barcoded Array to Detect Proteins Over a Broad Dynamic
Concentration Range
[0196] A bio-barcode integrated with DEAL technology was used to
detect biomarkers as illustrated in FIG. 21. In particular FIG. 21
illustrates the increased dynamic range of a barcoded array when it
is utilized with DEAL technology. The data show measurements of
hCG, a pregnancy test marker, in human serum using the DEAL
bar-code immunoassay that can cover the huge dynamic range >4
orders of magnitude.
[0197] In particular, the results illustrated in FIG. 21, show that
an expanded range of concentrations that can be detected from a
single DEAL-based bio-barcode, demonstrated here for the detection
of hCG. hCG is a pregnancy test marker, as well as a serum cancer
marker. By varying the primary DNA oligomer concentration that
binds the 1.degree. antibody capture agent during the initial flow
patterning step, a single set of bar-code can distinguish the hCG
concentration spanning from 25000 mIU/mL to O.25 mIU/mL (not shown)
in a single step.
Example 9
Barcoded Array for Detecting a Biological Profile: Detection of
Human Chorionic Gonadotropin (hCG) Over a Period of Time
[0198] Applicants performed a test on a series of standard human
chorionic gonadotropin (hCG) spiked human serum samples provided by
the National Cancer Institute (NCI). hCG is widely used for
pregnancy testing, and also serves as a biomarker for gestational
trophoblastic tumors and germ cell cancers of the ovaries and
testes.
[0199] The results from these hCG assays are shown in FIG. 22,
which illustrate measurement of human chorionic gonadotropin (hCG)
spiked in sera using a microfluidic DEAL barcode chip on an
integrated platform including a barcoded array manufactured as
described in U.S. Application entitled "Microfluidic Devices,
Methods and Systems for Detecting Target Molecules" Serial No. to
be assigned filed on Jul. 16, 2008, Docket Number P235-US herein
incorporated by reference in its entirety.
[0200] In Panel a of FIG. 22, fluorescence images of DEAL barcodes
used in measuring standard hCG samples and two unknowns, are shown.
The bars used to measure hCG were patterned with DNA strand A at
different concentrations. TNF-.alpha. encoded by strand B was
employed as a negative control. The lane indicated with REF
represents the reference marker, while the other lanes indicate hCG
test results in which the DNA was patterned from solutions at
concentrations that varied from 2 .mu.M-200 .mu.M. A negative
control using TNF-.alpha. was also included.
[0201] ELISA-like sensitivity (.about.1 mIU/mL), but with a broader
detectable concentration range (.about.10.sup.5), was demonstrated
by quantitating fluorescence intensity. Moreover, even without
photon integration, the analyte concentrations over a large range
can be readily estimated by eye through pattern-recognition of the
full barcode (See also indication in Example 5).
[0202] Quantitation of fluorescence signals obtained at different
DNA loading was also performed as indicated in panel (b) of FIG.
22. In such a barcoded array, the bar with high DNA-loading
rendered great sensitivity at low analyte concentrations, whereas
the bar with low DNA-loading was used to readily discriminate
samples with high analyte concentrations. The two unknowns were
also assayed and the results are in good agreement with ELISA tests
run at NCI Laboratories.
[0203] Applicants noted that the hCG level is tracked during
pregnancy, with concentrations in the blood increasing from
.about.5 mIU/mL in the first week of pregnancy to
.about.2.times.10.sup.5 mIU/mL in ten weeks. The microfluidic
barcoded arrays used in the experiments herein described can
accurately cover such a broad physiological hCG range.
Example 10
Barcoded Array for Detecting a Biological Profile: Protein
Profiling in Cancer Patients
[0204] A barcoded array was used to detect a biological profile as
illustrated in FIG. 23. In particular, FIG. 23 shows the use of an
integrated microfluidic DEAL barcoded device for human serum
protein profiling. The serum samples from 12 cancer patients were
measured in such prototype clinic test platform.
[0205] The protein profile obtained from this experiment exhibits
unique patterns for individual patients, suggesting the efficacy of
DEAL bar-code assay for serum-based cancer diagnosis and
personalized medicine. This result displays a great indication for
using a barcoded device and in particular an integrated DEAL
barcode device for diagnostics and in particular human disease
diagnostics.
[0206] In particular, the results of FIG. 23 show that the
integrated DEAL Bio bar-code device can be used for rapid,
sensitive and high-throughput protein measurements out of cancer
patient sera. Panel A illustrates the design of the integrated
microfluidic device that can conduct a dozen of serum assays at the
same time in a highly automated fashion. Blue denotes the
microfluidic channels for delivery of all reagents and samples.
Magenta shows the control channel for pressure-actuated valves
where they intersect the microfluidic channels. Overlay is a
representative image of DEAL bar-code chip visualized by Cy5
fluorescence probes.
[0207] Measurement of 12 proteins out of 11 cancer patient serum
samples and reference serum is illustrated in Panel B. The number
denotes each individual lanes used for protein detection out of a
patient sample.
[0208] Statistics of 12 protein level present in the serum samples
from 12 different patients (SI-S 12), among which S1-5 are breast
cancer patients while S6-S11 are prostate cancer patients, is shown
in Panel C. Each patient displays a unique pattern of serum
proteins that are thought to be associated with their unique
molecular origin of cancer.
[0209] A chart listing the specification and medical history of
cancer patients is shown in panel D. Several unique signatures can
be seen by comparing the medical record and the serum protein
profile measured from DEAL bar-code assay.
Example 11
Barcoded Array for Detecting a Biological Profile: Additional
Protein Profiling in Cancer Patients
[0210] To further assess the utility and reproducibility of
barcoded array for clinical blood samples, applicants measured a
panel of 12 proteins from small amounts of serum from 24 cancer
patients in a DEAL barcoded microfluidic device. The proteins in
this panel included prostate specific antigen (PSA), as well as
eleven proteins secreted by various white blood cells. Each barcode
was measured many times for each serum sample.
[0211] The stored serum samples from 11 breast cancer patients (all
female) and 11 prostate cancer patients (all male) were acquired
from Asterand. Two unknowns were acquired from Sigma-Aldrich.
Nineteen out of 22 patients were Caucasian and the remaining three
were Asian, Hispanic and African-American. The medical history is
summarized in the supplementary materials.
[0212] Finger pricks were performed using BD Microtainer
Contact-Activated Lancets. Blood was collected with SAFE-T-FILL
capillary blood collection tubes (RAM Scientific), which we
pre-filled with 80 .mu.L of 25 mM EDTA solution. A 10 .mu.L volume
of fresh human blood from a healthy volunteer was collected in this
EDTA-coated capillary, dispensed into the tube, and rapidly mixed
by inverting a few times. The spiked blood sample was prepared in a
similar means except that 40 .mu.L of 25 mM EDTA solution and 40
.mu.L of recombinant solution were mixed and pre-added in the
collection tube. Then 2 .mu.L of 0.5 M EDTA was added to bring the
total EDTA concentration up to 25 mM.
[0213] Execution of blood separation and plasma protein measurement
was performed using an integrated platform extensively described in
U.S. entitled "Microfluidic Devices, Methods and Systems for
Detecting Target Molecules" Serial No. to be assigned filed on Jul.
16, 2008, Docket Number P235-US herein incorporated by reference in
its entirety.
[0214] The integrated platforms were first blocked with the buffer
solution for 30-60 minutes. The buffer solution prepared was 1% w/v
Bovine Serum Albumin Fraction V (Sigma) in 150 mM 1.times.PBS
without calcium/magnesium salts (Irvine Scientific). Then
DNA-antibody conjugates (.about.50-100 nM) were flowed through the
plasma assay channels for .about.30-45 min. This step transformed
the DNA arrays into capture-antibody arrays. Unbound conjugates
were washed off by flowing buffer solution through the channels. At
this step, the integrated platform was ready for the blood test.
Two blood samples prepared as mentioned above were flowed into the
integrated platforms within 1 minute of collection. The integrated
platform quickly separated plasma from whole blood, and the plasma
proteins of interest were captured in the assay zone where DEAL
barcode arrays were placed. This whole process from finger-prick to
plasma protein capture took <10 minutes. In the cancer-patient
serum experiment, the as-received serum samples were flowed into
the integrated platforms without any pre-treatment (i.e. no
purification or dilution). Afterwards, a mixture of biotin-labeled
detection antibodies (.about.50-100 nM) for the entire protein
panel and the fluorescence Cy5-straptavidin conjugates (.about.100
nM) were flowed sequentially into the integrated platforms to
complete the DEAL immunoassay. The unbound fluorescence probes were
rinsed off by flowing the buffer solution for 10 minutes. At last,
the PDMS chip was removed from the glass slide. The slide was
immediately rinsed in 1/2.times.PBS solution and deionized water,
and then dried with a nitrogen gun. Finally, the DEAL barcode slide
was scanned by an Axon Instruments Genepix Scanner.
[0215] The serum samples from 24 cancer patients were assayed using
two chips, each containing 12 separate assay units that were
operated in parallel. In every assay unit, 50 sets of DEAL barcodes
were placed in the detection channel for statistical sampling of
the serum. In all experiments, 25 .mu.L of patient serum, or 500
nanoliters per barcode, was used for each assay. The white-blood
cell secreted proteins included inflammatory molecules and
cytokines. These proteins are employed by immune cells for
intracellular communication, and have significant implications in
tumor microenvironment formation, and in tumor progression and
metastasis. Thus, this panel provides information on both cancer
and the immune system.
[0216] Experiments were repeated at least 2-3 times. In every
integrated platform, multiple sets of barcode arrays were patterned
in a single assay channel to allow simultaneous parallel
measurements. For example, 50 sets of barcode were used in assaying
a cancer patient serum sample, with each barcode detecting the full
panel of proteins. Quantitation of fluorescence signal was
performed using either the Genepix software or imageJ (NIH). In
processing the cancer-patient data, the background intensity for
each channel was individually identified, and then re-assigned to a
common background level of 20 arbitrary units. The intensities of
all "bars" in a given channel are normalized to that channel's
background. Therefore, the data in FIG. 10 corresponds to the bar's
fluorescence intensity differences relative to its own channel's
background, plus the common background level of 20. This treatment
minimizes interference from non-specific background signal, but
could make it indistinguishable between the positive results with
high background (e.g. B10) and the true negative results (e.g. B9
and B11).
[0217] The results are illustrated in FIGS. 24 and 25, which show
the related profile of cancer patients (FIG. 24) together with
their medical history (FIG. 25).
[0218] In particular, fluorescence images each showing four sets of
representative barcodes obtained from the 24 patient samples are
shown in FIG. 24. The proteins measured include cancer marker PSA
and eleven cytokines also indicated in details in FIG. 25. In the
barcode image panel, the left two columns were performed on the
same chip while the right two were from the other. The samples were
randomly picked in the assay to minimize arbitrary bias. B01-B11
denote 11 samples from breast cancer patients, P01-P11 denote those
from prostate cancer patients, whereas the S01 and S02 are unknown
samples from a different supplier.
[0219] The medical records for all patients are summarized in FIG.
24 which shows a brief summary of cancer patient medical records.
The two unknowns are not included in the table.
[0220] A more detailed medical history of the patients is included
in the following table 1.
TABLE-US-00001 TABLE 1 Medical Record of cancer patients. GLEASONS
PATIENT CANCER GENDER/AGE RACE UICC STAGE SCORE OTHERS B01 Breast
Female/62 Caucasian T2N0M0 wine 200 mL/day B02 Breast Female/79
Caucasian T4N2M0 B03 Breast Female/71 Caucasian T1cNXM0 1-2
drinks/day B04 Breast Female/72 Caucasian T2NXM0 hypertension B05
Breast Female/89 Caucasian T3N0MX arthritis B06 Breast Female/56
Asian T1NXM0 B07 Breast Female/54 Caucasian T2N2M0 hypertension,
obesity B08 Breast Female/55 Caucasian T2NxM0 1-5 cigs/day, wine
200 mL/day B09 Breast Female/83 Caucasian T4N0M0 Coronary artery
disease, cerebral atherosclerosis B10 Breast Female/63 Hispanic
T3N2MX 6-10 cigs/day, hyperthyroid, hypertension, osteoarthritis
B11 Breast Female/63 Caucasian T1NXM0 arterial hypertension P01
Prostate Male/51 Caucasian T2cNXM0 4 + 3 = 7 P02 Prostate Male/64
Caucasian T3bN0MX 3 + 4 = 7 P03 Prostate Male/47 Caucasian T2cN0M0
3 + 3 = 6 hypertension P04 Prostate Male/55 Caucasian T2bN0M0 3 + 3
= 6 11-20 cigs/day P05 Prostate Male/73 Caucasian T3aNXMX 4 + 4 = 8
hypertension, 11-20 cigs/day P06 Prostate Male/64 Caucasian T3N0M0
chronic bronchitis, 11-20 cigs/day P07 Prostate Male/60 Caucasian
T3aN0M0 3 + 4 = 7 gastroesophageal reflux P08 Prostate Male/72
African Am. T2aNXMX 3 + 3 = 6 1-5 cigs/day P09 Prostate Male/78
Caucasian T3aN1MX 4 + 3 = 7 hypertension, atrial fibrillation P10
Prostate Male/66 Caucasian T2aN0MX 3 + 3 = 6 hypertension, 11-20
cigs/day P11 Prostate Male/47 Caucasian T2cN0M0 3 + 3 = 6
hypertension S01 Unknown S02 Unknown
[0221] Many proteins were successfully detected with high
signal-to-noise, and the barcode signatures are distinctive among
patients. Most assays show a relatively low fluorescence
background. However, the assays on P05, P04, P10 and B10 were
characterized by a high, interfering background. These high
background assays all correlate with patients that were heavy
smokers (.about.11-20 cigs/day); only one serum sample from a heavy
smoker did not exhibit a high background (P06). The reason for this
high background fluorescence remains unclear. A possible cause is
the elevated blood content of the fluorescent carboxyhemoglobin
formed in lung. While this identification of smokers constitutes
unexpected information from the IBBCs, it also means that, for
these patients, some pre-purification of the plasma or serum will
be required in order to assay serum protein levels.
[0222] The protein panels used in the cancer-patient serum
experiment (panel 1) and finger-prick blood test (panel 2), the
corresponding DNA codes, and their sequences are summarized in
Tables 2 and 3. These DNA oligomers were synthesized by Integrated
DNA Technologies (IDT), and purified by high pressure liquid
chromatography (HPLC). The quality was confirmed by mass
spectrometry.
TABLE-US-00002 TABLE 2 List of protein panels and corresponding DNA
codes. DNA-code Human Plasma Protein Abbreviation Panel (1) A/A'
Interferon-gamma IFN-.gamma. B/B' Tumor necrosis factor-alpha
TNF-.alpha. C/C' Interleukin-2 IL-2 D/D' Interleukin-1 alpha
IL-1.alpha. E/E' Interleukin-1beta IL-1.beta. F/F' Transforming
growth factor beta TGF-.beta. G/G' Prostate specific antigen
(total) PSA H/H' Interleukin-6 IL-6 I/I' Interleukin-10 IL-10 J/J'
Interleukin-12 IL-12 K/K' Granulocyte-macrophage colony GM-CSF
stimulating factor L/L' Monocyte chemoattractant protein-1 MCP-1
M/M' Blank control/reference Panel (2) AA/AA' Interleukin-1 beta
IL-1.beta. BB/BB' Interleukin-6 IL-6 CC/CC' Interleukin-10 IL-10
DD/DD' Tumor necrosis factor-alpha TNF-.alpha. EE/EE' Complement
Component 3 C3 FF/FF' C-reactive protein CRP GG/GG' Plasminogen
Plasminogen HH/HH' Prostate specific antigen (total) PSA
TABLE-US-00003 TABLE 3 List of DNA sequences used for spatial
encoding of antibodies Sequence SEQ ID Tm (50 mM Name Sequence) NO
NaCl) .degree. C. A 5'-AAA AAA AAA AAA AAT CCT GGA GCT AAG TCC
GTA-3' 1 57.9 A' 5' NH3- AAA AAA AAA ATA CGG ACT TAG CTC CAG GAT-3'
2 57.2 B 5'-AAA AAA AAA AAA AGC CTC ATT GAA TCA TGC CTA-3' 3 57.4
B' 5' NH3AAA AAA AAA ATA GGC ATG ATT CAA TGA GGC -3' 4 55.9 C
5'-AAA AAA AAA AAA AGC ACT CGT CTA CTA TCG CTA-3' 5 57.6 C' 5'
NH3-AAA AAA AAA ATA GCG ATA GTA GAC GAG TGC -3' 6 56.2 D 5'-AAA AAA
AAA AAA AAT GGT CGA GAT GTC AGA GTA-3' 7 56.5 D' 5' NH3-AAA AAA AAA
ATA CTC TGA CAT CTC GAC CAT -3' 8 55.7 E 5'-AAA AAA AAA AAA AAT GTG
AAG TGG CAG TAT CTA-3' 9 55.7 E' 5' NH3-AAA AAA AAA ATA GAT ACT GCC
ACT TCA CAT -3' 10 54.7 F 5'-AAA AAA AAA AAA AAT CAG GTA AGG TTC
ACG GTA-3' 11 56.9 F' 5' NH3-AAA AAA AAA ATA CCG TGA ACC TTA CCT
GAT -3' 12 56.1 G 5'- AAA AAA AAA AGA GTA GCC TTC CCG AGC ATT-3' 13
59.3 G' 5' NH3-AAA AAA AAA AAA TGC TCG GGA AGG CTA CTC-3' 14 58.6 H
5'- AAA AAA AAA AAT TGA CCA AAC TGC GGT GCG-3' 15 59.9 H' 5'
NH3-AAA AAA AAA ACG CAC CGC AGT TTG GTC AAT-3' 16 60.8 I 5'- AAA
AAA AAA ATG CCC TAT TGT TGC GTC GGA-3' 17 60.1 I' 5' NH3-AAA AAA
AAA ATC CGA CGC AAC AAT AGG GCA-3' 18 60.1 J 5'- AAA AAA AAA ATC
TTC TAG TTG TCG AGC AGG-3' 19 56.5 J' 5' NH3-AAA AAA AAA ACC TGC
TCG ACA ACT AGA AGA-3' 20 57.5 K 5'- AAA AAA AAA ATA ATC TAA TTC
TGG TCG CGG-3' 21 55.4 K' 5' NH3-AAA AAA AAA ACC GCG ACC AGA ATT
AGA TTA-3' 22 56.3 L 5'- AAA AAA AAA AGT GAT TAA GTC TGC TTC GGC-3'
23 57.2 L' 5' NH3-AAA AAA AAA AGC CGA AGC AGA CTT AAT CAC-3' 24
57.2 M 5'- Cy3-AAA AAA AAA AGT CGA GGA TTC TGA ACC TGT-3' 25 57.6
M' 5' NH3-AAA AAA AAA AAC AGG TTC AGA ATC CTC GAC-3' 26 56.9 AA' 5'
NH3-AAAAAAAAAAGTCACAGACTAGCCACGAAG-3' 27 58 BB 5'-
AAAAAAAAAAGCGTGTGTGGACTCTCTCTA-3' 28 58.7 BB' 5'
NH3-AAAAAAAAAATAGAGAGAGTCCACACACGC-3' 29 57.9 CC 5'-
AAAAAAAAAATCTTCTAGTTGTCGAGCAGG-3' 30 56.5 CC' 5'
NH3-AAAAAAAAAACCTGCTCGACAACTAGAAGA-3' 31 57.5 DD 5'-
AAAAAAAAAAGATCGTATGGTCCGCTCTCA-3' 32 58.8 DD' 5'
NH3-AAAAAAAAAATGAGAGCGGACCATACGATC-3' 33 58 EE 5'-
AAAAAAAAAAGCACTAACTGGTCTGGGTCA-3' 34 59.2 EE' 5'
NH3-AAAAAAAAAATGACCCAGACCAGTTAGTGC-3' 35 58.4 FF 5'-
AAAAAAAAAATGCCCTATTGTTGCGTCGGA-3' 36 60.1 FF' 5'
NH3-AAAAAAAAAATCCGACGCAACAATAGGGCA-3' 37 60.1 GG 5'-
AAAAAAAAAACTCTGTGAACTGTCATCGGT-3' 38 57.8 GG' 5'
NH3-AAAAAAAAAAACCGATGACAGTTCACAGAG-3' 39 57 HH 5'-
AAAAAAAAAAGAGTAGCCTTCCCGAGCATT-3' 40 59.3 HH' 5'
NH3-AAAAAAAAAAAATGCTCGGGAAGGCTACTC-3' 41 58.6 *All amine-terminated
strands were linked to antibodies to form DNA-antibody conjugates
using SFB/SANH coupling chemistry as described by R. Bailey et
al..sup.1 Codes AA-HH were used in the experiment which examined
fresh whole blood from a heathy volunteer. Codes A-M were used for
the molecular analyses of cancer patient serum samples.
Example 12
Barcoded Array for Detecting a Biological Profile: Quantitative
Protein Profiling in Cancer Patients
[0223] The blood barcodes measured throughout the experiments
illustrated in Example 10 were unique for each patient.
[0224] FIGS. 26 to 28 show quantitation and clustering of cancer
patient barcode data obtained using a barcode array designed as
exemplified in Example 8. In particular, FIG. 26 shows layout of
the barcode array used in this study. Strand M denotes the
reference (control). FIG. 27 illustrates a plot showing
quantitation of fluorescence signals of all proteins (left axis)
detected as shown in FIG. 21A for all cancer patients (from left:
B01-B11, P01-P11, S01 and S02). S01 and S02 are two unknown serum
samples. FIG. 28 shows an exemplary manual clustering of cancer
patients derived on the basis of protein patterns. First, all
prostate cancer patients are clearly identified by PSA. Second,
both breast and prostate cancer patients exhibit possible
subpopulations with distinct cytokine profiles.
[0225] The fluorescence signals intensity for all the patient
samples are plotted in FIG. 26. The cancer marker, PSA, clearly
distinguishes between the breast and prostate cancer patients, and
allowed for the unknown samples, S01 and S02, to be assigned to
prostate cancer patients. Applicants then performed a manual
clustering of patients on the basis of protein signals and
generated the map schematically illustrated in FIG. 27 to assess
the potential of this technology for patient stratification. This
approach is only going to be as good as the biomarker panel itself,
and the number of serum samples profiled is small. Nevertheless,
the results are encouraging. For example, the measured profiles of
breast cancer patients can be classified into three
subsets--non-inflammatory, IL-1.beta. positive, and TNF-.alpha.
positive. The prostate cancer patient data exhibits a generally
higher level of inflammation, and those inflammation-positive
samples can also be classified as shown in FIG. 27. An interesting
observation is the lack of IL-10 signal for most patients. IL-10 is
a cytokine production suppressor that functions as an
anti-inflammatory mediator, and its absence may reflect deviation
from normal immune homeostasis in local tumor sites. Applicants
have initiated studies involving a larger number of proteins and a
much larger number of blood samples. Researches have been focused
on developing technologies for multiplexed measurement of
cytokines, and serum cytokine profiling has shown relevance in
cancer diagnostics and prognostics. The results described above
have clearly demonstrated that integrated platforms can be applied
to the multiparameter analysis of human health-relevant
proteins.
[0226] The principal goal behind developing the integrated platform
was to be able to measure the levels of a large number of proteins
in human blood within a few minutes of sampling that blood, so as
to avoid protein degradation that can occur when plasma is stored.
In a typical 96 well plate immunoassay, the biological sample of
interest is added, and the protein diffuses to the surface-bound
antibody. Under sufficient flow conditions, diffusion is no longer
important, and the only parameter that limits the speed of the
assay is the protein/antibody binding kinetics (the Langmuir
isotherm), thus allowing the immunoassay to be completed in just a
few minutes.
Example 13
Barcoded Array for Detecting a Biological Profile: Human Plasma
Proteome
[0227] Use of a barcoded array was tested to verify improved
sensitivity for plasma protein assays.
[0228] The human plasma proteome is comprised of three major
classes of proteins--classical plasma proteins, tissue leakage
proteins, and cell-cell signaling molecules (cytokines and
chemokines). Cell-cell signaling molecules are biologically
informative in a variety of physiological and pathological
processes, i.e. tumor host immunity and inflammation.
[0229] The results of a first series of experiments performed by
the Applicants are illustrated in FIG. 29, wherein a detection of
target protein other than cytokines TNF-.alpha., and Interleukins
such as IL-6, IL-10 is shown. In particular, FIG. 29, shows
detection of molecules such as CRP, C3 and plasminogen associated
with biological profile such inflammation response (CRP),
complement system (C3) and liver toxicity response (CRP and
plasminogen).
[0230] The results of a second series of experiments performed by
the Applicants is summarized in the diagram of FIG. 30, showing a
schematic of human plasma proteome (refer to N. L. Anderson and N.
G. Anderson, Molecular & Cellular Proteomics 11, 845,
2001).
[0231] As shown in FIG. 30, the concentration range of plasma
proteins spans 12 orders of magnitude and the lowest end is
approximately at the detection limit of mass spectrometry--a
high-throughput protein profiling technique. The state-of-the-art
for clinical protein measurements is still the ELISA assay. Yet
ELISA is a low-throughput process, requiring a large amount of
sample and long duration to complete a multiparameter plasma
protein measurement. The high performance of the DEAL barcode chip,
especially its increased sensitivity, is a key to realizing highly
multiplexed measurements of a panel of proteins, including the low
abundance cytokines, from small quantities of clinical blood
samples.
[0232] Applicants therefore concluded that the DEAL barcode assay
has a markedly high sensitivity, comparable to ELISA, leading to
the feasibility of multiplexed detection of plasma proteins
including low-abundance cell-cell signaling molecules, e.g.
cytokines and chemokines, from a small quantity of sample.
Example 14
Assay Performed in a Barcoded Array
[0233] For the assays shown in the Examples 3-13 illustrated in the
related figures, a DEAL immunoassay was used. To detect each
protein, a pair of antibodies was chosen. One is conjugated to the
secondary DNA strands that are complementary to the primary DNA
strands flow-patterned on glass slides. This antibody also serves
to capture proteins being detected, and then the biotin-labeled
detection came in to bind to the same protein creating
immunosandwich structure. Finally, Cy-3 or Cy5labled fluorescent
streptavidin was used to visualize the results of bar-code through
streptavidin-biotin binding.
[0234] Detection of human cytokine proteins prepared at different
concentrations was first tested (FIG. 15). The results show the
detection is highly specific, and exhibits increased sensitivity
comparable to ELISA. Then, a multiparameter (up to 5 proteins)
detection was demonstrated as in FIG. 16. TNF-a exhibits the best
signal intensity due to the high affinity of the 10 anti-TNF-a AB.
Having the high loading of primary DNA oligomers and by varying DNA
concentrations in flow-pattering step, it is shown the a single
bar-code can detect protein like hCG across a huge dynamic range,
several orders of magnitube better than any conventional protein
detection methods (FIG. 21). Finally, an integrated microfluidic
device was fabricated, which comprises of a two-layer PDMS
microfluidic chip bonded on to a bar-DEAL barcode glass chip, that
allows rapid, sensitive detection of 13 different proteins at the
same time out of 12 different human serum samples. The DEAL
bar-code devices for the first time provide a highly multiplexed
(as in protein microarray and mass spectrometry) method for protein
detection at an ultra-high sensitivity as good as the state-of-art
ELISA assay.
[0235] Barcoded array patterning is a generic technique that can be
exploited to pattern DNA, protein, or even sera and tissue lysates.
The inverse-phase bar-code array (serum or lysate array) can be
used for high throughput drug screening and biomarker
discovering.
Example 15
Manufacturing a Barcoded Array for Magnetic ID
[0236] A schematic representation of a method to manufacture a
magnetic ID barcode on a small object such as a ring is shown in
FIG. 31.
[0237] A PDMS microfluidic channels with a small exposed contact
area can be manufactured using two-layer lithography (it means
there are two layers of fluidic channels. The bottom layer can be
contacted with the substrate e.g. the small-sized product and the
fluid can be introduced from the upper layer that contains embedded
fluidic channels to join the bottom layer channels at the small
contact area to the large inlets at the sides of the PDMS
device.
[0238] Once this PDMS device is attached onto the small subject, a
number of distinct different molecules were flowed to the contact
area to create a DNA barcoded array. Next, a library of
complementary DNA-magnetic nanoparticle conjugates can be
synthesized.
[0239] Therefore, the fabrication of magnetic barcode can be
realized by simply immersing the small-sized subject patterned with
DNA barcodes into a solution that contains several complementary
DNA-magnetic nanoparticle conjugates. The different combination of
complementary DNA-magnetic nanoparticle conjugates gives rise to a
distinct magnetic ID barcode that can be readily read with a
magnetoresistive scan head.
[0240] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the devices, systems and
methods of the disclosure, and are not intended to limit the scope
of what the inventors regard as their disclosure. Modifications of
the above-described modes for carrying out the disclosure that are
obvious to persons of skill in the art are intended to be within
the scope of the following claims. All patents and publications
mentioned in the specification are indicative of the levels of
skill of those skilled in the art to which the disclosure pertains.
All references cited in this disclosure are incorporated by
reference to the same extent as if each reference had been
incorporated by reference in its entirety individually.
[0241] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background,
Detailed Description, and Examples is hereby incorporated herein by
reference. Further, the hard copy of the sequence listing submitted
herewith and the corresponding computer readable form are both
incorporated herein by reference in their entireties.
[0242] It is to be understood that the disclosures are not limited
to particular compositions or biological systems, which can, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting. As used in this
specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the content clearly
dictates otherwise. The term "plurality" includes two or more
referents unless the content clearly dictates otherwise. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the disclosure pertains. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the specific
examples of appropriate materials and methods are described
herein.
[0243] A number of embodiments of the disclosure have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the present disclosure. Accordingly, other embodiments are
within the scope of the following claims.
REFERENCES
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Sequence CWU 1
1
41133DNAartificial sequenceSynthetic Polynucleotide 1aaaaaaaaaa
aaaatcctgg agctaagtcc gta 33230DNAartificial sequenceSynthetic
Polynucleotide 2aaaaaaaaaa tacggactta gctccaggat 30333DNAartificial
sequenceSynthetic Polynucleotide 3aaaaaaaaaa aaagcctcat tgaatcatgc
cta 33430DNAartificial sequenceSynthetic Polynucleotide 4aaaaaaaaaa
taggcatgat tcaatgaggc 30533DNAartificial sequenceSynthetic
Polynucleotide 5aaaaaaaaaa aaagcactcg tctactatcg cta
33630DNAartificial sequenceSynthetic Polynucleotide 6aaaaaaaaaa
tagcgatagt agacgagtgc 30733DNAartificial sequenceSynthetic
Polynucleotide 7aaaaaaaaaa aaaatggtcg agatgtcaga gta
33830DNAartificial sequenceSynthetic Polynucleotide 8aaaaaaaaaa
tactctgaca tctcgaccat 30933DNAartificial sequenceSynthetic
Polynucleotide 9aaaaaaaaaa aaaatgtgaa gtggcagtat cta
331030DNAartificial sequenceSynthetic Polynucleotide 10aaaaaaaaaa
tagatactgc cacttcacat 301133DNAartificial sequenceSynthetic
Polynucleotide 11aaaaaaaaaa aaaatcaggt aaggttcacg gta
331230DNAartificial sequenceSynthetic Polynucleotide 12aaaaaaaaaa
taccgtgaac cttacctgat 301330DNAartificial sequenceSynthetic
Polynucleotide 13aaaaaaaaaa gagtagcctt cccgagcatt
301430DNAartificial sequenceSynthetic Polynucleotide 14aaaaaaaaaa
aatgctcggg aaggctactc 301530DNAartificial sequenceSynthetic
Polynucleotide 15aaaaaaaaaa attgaccaaa ctgcggtgcg
301630DNAartificial sequenceSynthetic Polynucleotide 16aaaaaaaaaa
cgcaccgcag tttggtcaat 301730DNAartificial sequenceSynthetic
Polynucleotide 17aaaaaaaaaa tgccctattg ttgcgtcgga
301830DNAartificial sequenceSynthetic Polynucleotide 18aaaaaaaaaa
tccgacgcaa caatagggca 301929DNAartificial sequenceSynthetic
Polynucleotide 19aaaaaaaaat cttctagttg tcgagcagg
292030DNAartificial sequenceSynthetic Polynucleotide 20aaaaaaaaaa
cctgctcgac aactagaaga 302130DNAartificial sequenceSynthetic
Polynucleotide 21aaaaaaaaaa taatctaatt ctggtcgcgg
302230DNAartificial sequenceSynthetic Polynucleotide 22aaaaaaaaaa
ccgcgaccag aattagatta 302330DNAartificial sequenceSynthetic
Polynucleotide 23aaaaaaaaaa gtgattaagt ctgcttcggc
302430DNAartificial sequenceSynthetic Polynucleotide 24aaaaaaaaaa
gccgaagcag acttaatcac 302530DNAartificial sequenceSynthetic
Polynucleotide 25aaaaaaaaaa gtcgaggatt ctgaacctgt
302630DNAartificial sequenceSynthetic Polynucleotide 26aaaaaaaaaa
acaggttcag aatcctcgac 302730DNAartificial sequenceSynthetic
Polynucleotide 27aaaaaaaaaa gtcacagact agccacgaag
302830DNAartificial sequenceSynthetic Polynucleotide 28aaaaaaaaaa
gcgtgtgtgg actctctcta 302930DNAartificial sequenceSynthetic
Polynucleotide 29aaaaaaaaaa tagagagagt ccacacacgc
303030DNAartificial sequenceSynthetic Polynucleotide 30aaaaaaaaaa
tcttctagtt gtcgagcagg 303130DNAartificial sequenceSynthetic
Polynucleotide 31aaaaaaaaaa cctgctcgac aactagaaga
303230DNAartificial sequenceSynthetic Polynucleotide 32aaaaaaaaaa
gatcgtatgg tccgctctca 303330DNAartificial sequenceSynthetic
Polynucleotide 33aaaaaaaaaa tgagagcgga ccatacgatc
303430DNAartificial sequenceSynthetic Polynucleotide 34aaaaaaaaaa
gcactaactg gtctgggtca 303530DNAartificial sequenceSynthetic
Polynucleotide 35aaaaaaaaaa tgacccagac cagttagtgc
303630DNAartificial sequenceSynthetic Polynucleotide 36aaaaaaaaaa
tgccctattg ttgcgtcgga 303730DNAartificial sequenceSynthetic
Polynucleotide 37aaaaaaaaaa tccgacgcaa caatagggca
303830DNAartificial sequenceSynthetic Polynucleotide 38aaaaaaaaaa
ctctgtgaac tgtcatcggt 303930DNAartificial sequenceSynthetic
Polynucleotide 39aaaaaaaaaa accgatgaca gttcacagag
304030DNAartificial sequenceSynthetic Polynucleotide 40aaaaaaaaaa
gagtagcctt cccgagcatt 304130DNAartificial sequenceSynthetic
Polynucleotide 41aaaaaaaaaa aatgctcggg aaggctactc 30
* * * * *