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US20090017455A1 - Methods and systems for detecting and/or sorting targets - Google Patents

Methods and systems for detecting and/or sorting targets Download PDF

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Publication number
US20090017455A1
US20090017455A1 US11/888,502 US88850207A US2009017455A1 US 20090017455 A1 US20090017455 A1 US 20090017455A1 US 88850207 A US88850207 A US 88850207A US 2009017455 A1 US2009017455 A1 US 2009017455A1
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Prior art keywords
polynucleotide
substrate
protein
target
encoded
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Inventor
Gabriel A. Kwong
Ryan C. Bailey
Rong Fan
James R. Heath
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California Institute of Technology
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Priority to US11/888,502 priority Critical patent/US20090017455A1/en
Assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY reassignment CALIFORNIA INSTITUTE OF TECHNOLOGY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FAN, RONG, HEATH, JAMES R, KWONG, GABRIEL A, BAILEY, RYAN C
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT EXECUTIVE ORDER 9424, CONFIRMATORY LICENSE Assignors: CALIFORNIA INSTITUTE OF TECHNOLOGY
Publication of US20090017455A1 publication Critical patent/US20090017455A1/en
Priority to US12/652,000 priority patent/US8354231B2/en
Priority to US12/901,151 priority patent/US8394590B2/en
Priority to US14/694,340 priority patent/US20160011189A1/en
Priority to US15/359,464 priority patent/US10928389B2/en
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present disclosure relates to detection of one or more targets, in particular biomarkers, in a sample such as a biological sample. More specifically, it relates to methods and systems for detecting and/or sorting targets.
  • Some of the techniques most commonly used in the laboratory for detection of single biological targets include gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), western blots, fluorescent in situ hybridization (FISH), Florescent activated cell sorting (FACS), Polymerase chain reaction (PCR), and enzyme linked immunosorbent assay (ELISA). These methods have provided the ability to detect one or more biomarkers in biological samples such as tissues and are also suitable for diagnostic purposes.
  • the collected data is integrated together within some model for the disease, such as a cancer pathway model (Weinberg, R. A., Cancer Biology . Garland Science: 2006), to generate a diagnosis.
  • a cancer pathway model Weinberg, R. A., Cancer Biology . Garland Science: 2006
  • performing these various measurements requires a surgically resected tissue sample.
  • the heterogeneity of such biopsies can lead to significant sampling errors since various measurements of cells, mRNAs, and proteins are each executed from different regions of the tissue.
  • the polynucleotide-encoded protein herein disclosed is comprised of a protein that specifically binds to a target and of an encoding-polynucleotide attached to the protein.
  • the encoding polynucleotide is comprised of a sequence that specifically binds to a substrate polynucleotide.
  • the substrate polynucleotide herein disclosed is attached to a substrate and is comprised of a sequence that specifically binds to the encoding polynucleotide.
  • assays including but not limited to assays for the detection and/or separation of targets, in particular biomarkers, such as cells, proteins and/or polynucleotides, can be performed according to the methods and systems herein disclosed.
  • the polynucleotide-encoded protein is used to specifically bind to a target in a polynucleotide-encoded protein-target complex
  • the substrate polynucleotide is used to bind the polynucleotide-encoded protein-target complex to the substrate for detection.
  • the methods and systems herein disclosed allow the advantageous performance of several assays in particular, in a microfluidic environment as it will be apparent to a skilled person upon reading of the present disclosure.
  • a method and a system to detect a target in a sample are disclosed, the method and system based on the combined use of a substrate polynucleotide attached to a substrate, and a polynucleotide-encoded protein comprised of a protein that specifically binds to the target and of an encoding polynucleotide that specifically binds to the substrate polynucleotide attached to the substrate.
  • the polynucleotide-encoded protein is contacted with the sample and the substrate polynucleotide for a time and under conditions to allow binding of the polynucleotide-encoded protein with the target in a polynucleotide-encoded protein-target complex, and binding of the encoding polynucleotide with the substrate polynucleotide thus providing a polynucleotide-encoded protein-target complex bound to the substrate polynucleotide.
  • the polynucleotide-encoded protein-target complex bound to the substrate polynucleotide is then detected by way of detecting techniques which will be identifiable by a skilled person upon reading of the present disclosure.
  • a substrate with a substrate polynucleotide attached to the substrate is provided, together with a polynucleotide-encoded protein comprising a protein that specifically binds to the target and an encoding-polynucleotide that specifically binds to the substrate polynucleotide.
  • a method and a system for detecting a plurality of targets in a sample are disclosed, the method and system based on the combined use of a plurality of substrate polynucleotides attached to a substrate and a plurality of polynucleotide-encoded antibodies.
  • each of the substrate polynucleotides is sequence specific and positionally distinguishable from another.
  • each of the polynucleotide-encoded proteins is comprised of a protein that specifically binds to a predetermined target of the plurality of targets and of an encoding polynucleotide that specifically binds to a sequence specific and positionally distinguishable substrate polynucleotide of the plurality of substrate polynucleotides.
  • each protein and encoding polynucleotide is bindingly distinguishable from another.
  • the plurality of polynucleotide-encoded antibodies is contacted with the sample and the plurality of substrate polynucleotides for a time and under conditions to allow binding of the antibodies with the targets in a plurality of polynucleotide-encoded protein-target complexes and binding of the encoding polynucleotides to the substrate polynucleotides.
  • the plurality of polynucleotide-encoded protein-target complexes bound to the plurality of substrate polynucleotides on the substrate is then detected by way of detecting techniques that will be identifiable by the skilled person upon reading of the present disclosure.
  • a substrate with the plurality substrate polynucleotides attached to the substrate is comprised, together with the plurality of polynucleotide-encoded antibodies.
  • a method and a system for detecting a plurality of targets in a sample are disclosed, wherein the targets comprise at least one target polynucleotide.
  • the method and system are based on the combined use of a plurality of substrate polynucleotides attached to a substrate, at least one polynucleotide-encoded protein and at least one labeled polynucleotide.
  • each substrate polynucleotide is sequence-specific and positionally distinguishable from another.
  • the at least one labeled polynucleotide specifically binds to the at least one target polynucleotide, with each labeled polynucleotide bindingly distinguishable from another.
  • the at least one polynucleotide-encoded protein is comprised of a protein that specifically binds to a predetermined target of the plurality of the targets and of an encoding polynucleotide that specifically binds to a sequence-specific and positionally distinguishable substrate polynucleotide of the plurality of substrate polynucleotides.
  • each protein and encoding polynucleotide is bindingly distinguishable from another, each protein is further bindingly distinguishable from each labeled polynucleotide, and each polynucleotide-encoded protein is bindingly distinguishable from each labeled target polynucleotide
  • the at least one labeled polynucleotide is contacted with the sample for a time and under conditions to allow binding of the labeled polynucleotide with the target polynucleotide to provide at least one labeled target polynucleotide, wherein the at least one labeled target polynucleotides is comprised of a sequence that specifically binds to a sequence-specific and positionally distinguishable substrate polynucleotide.
  • the at least one polynucleotide-encoded protein is contacted with the sample for a time and under conditions to allow binding of the protein with the target, in at least one polynucleotide-encoded protein-target complex.
  • the at least one labeled target polynucleotide and the at least one polynucleotide-encoded protein-target complex are contacted with the plurality of substrate polynucleotides for a time and under conditions to allow binding of the at least one labeled target polynucleotide with a corresponding substrate polynucleotide and binding of the at least one encoding polynucleotide with a corresponding substrate polynucleotide.
  • the labeled target polynucleotides and the polynucleotide-encoded protein-target complexes bound to the plurality of spatially located substrate polynucleotides on the substrate are then detected by use of detecting techniques that will be identifiable by the skilled person upon reading of the present disclosure.
  • a substrate with the plurality of substrate polynucleotides attached to the substrate is comprised together with, the at least one labeled polynucleotide and the at least one polynucleotide-encoded-protein.
  • the at least one labeled polynucleotide of the system is for the production of a labeled target polynucleotide that specifically binds to a sequence-specific and positionally distinguishable substrate polynucleotide.
  • a method and system for sorting targets of a plurality of targets is disclosed, the method and system based on the combined use of a plurality of substrate polynucleotides attached to a substrate and a plurality of polynucleotide-encoded antibodies.
  • the targets are cells and the method and systems are for sorting a plurality of cells.
  • each substrate polynucleotide is sequence-specific and positionally distinguishable from another.
  • each polynucleotide-encoded protein is comprised of a protein and of a 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 substrate polynucleotide of the plurality of substrate polynucleotides.
  • each protein and encoding polynucleotide is bindingly distinguishable from another.
  • the plurality of polynucleotide-encoded antibodies is contacted with the sample for a time and under conditions to allow binding of the antibodies with the targets, thus providing a plurality of polynucleotide-encoded protein-target complexes.
  • the plurality of polynucleotide-encoded protein-target complexes is then contacted with the plurality of substrate polynucleotides for a time and under conditions to allow binding of the encoding polynucleotides to the substrate polynucleotides attached to the substrate, thus sorting the plurality of targets in a plurality of polynucleotide-encoded protein-target complexes bound to the substrate.
  • a substrate with the plurality of substrate polynucleotides attached to the substrate is comprised together with the plurality of polynucleotide-encoded antibodies.
  • the substrate of each of the methods, systems and arrays disclosed herein is in operable association with a microfluidic component comprising a microfluidic feature for carrying a fluid. Accordingly, in the methods, at least contacting the encoding-polynucleotide and/or the labeled polynucleotide target with the substrate polynucleotide, can be performed in the fluid carried by the microfluidic feature. Additionally, each of the systems herein disclosed can further include the microfluidic component comprising the microfluidic feature.
  • a first advantage of the methods and systems disclosed herein is that, in each of the methods and systems herein disclosed, contacting the polynucleotide-encoded protein to the target can be performed before the protein is bound to the substrate.
  • targets such as cells
  • access of the target to the binding site of the protein cannot be impaired by the substrate and both the protein and the target molecule will have a complete orientational freedom in performing the contact, thus improving the sensitivity of any related assay performed with the disclosed methods and systems.
  • a third advantage of the methods and systems disclosed herein is that in each of the methods and systems herein disclosed, biofouling, i.e. non-specific binding of non-encoded protein to the substrate, is greatly reduced when compared to the protein-based methods and systems of the art, therefore allowing a more efficient binding and, when detection is desired, a more accurate quantitative detection of the target molecule in the sample when compared with antibodies based methods and system of the art.
  • a fourth advantage of the methods and systems disclosed herein is that the multiplexed detection and/or separation of a higher number of targets can be performed when compared to the protein-based methods and systems of the art. This is due to several factors.
  • a first factor is that the reduced biofouling associated with the use of a polynucleotide-encoded protein in combination with a substrate polynucleotide attached to a substrate allows a more efficient binding and detection of the polynucleotide-encoded protein-target complexes to the substrate.
  • a second factor is that the size of the substrate polynucleotide in the method system herein disclosed is much smaller, than the corresponding anchoring molecules used in the protein-based methods and systems of the art. As a consequence, a higher density of proteins can be assembled on the substrate in comparison with the prior art techniques (e.g., about 5,000 spots per square inch versus 96 well plates of techniques like ELISA).
  • a fifth advantage of the methods and systems disclosed herein is that in each of the methods and systems herein disclosed it is possible to detect and separate in a single substrate chemically different targets, including biomarkers such as polynucleotides, proteins, and cells that have a different surface marker. Accordingly, the methods and systems herein disclosed allow the multiplexed detection and/or separation of genes, proteins and cells within the same environment.
  • a further advantage of the methods and systems for sorting targets herein disclosed is that the methods and systems herein disclosed make the sorted cells immediately available for post-sorting analysis, which is particularly relevant in the embodiments wherein the targets are cells that are made available for post-sorting analysis of gene and protein expression in the cells.
  • An additional advantage of the methods and systems herein disclosed when used to perform diagnostic assays is that multiplexed detection of multiple biomarkers from a same region of tissue can be performed on a single substrate.
  • a further advantage of the methods and systems used to perform diagnostic assays is that the biomarkers can be chemically distinct biomarkers such as cells, mRNAs and proteins and that the detection can be a quantitative detection and/or a qualitative.
  • a still further advantage of the methods and systems herein disclosed when used to perform a diagnostic assay is that they allow detection of complex genomic and/or proteomic profiles that, when compared with pre-determined profiles provide diagnostic indications for diseases characterized by perturbed regulatory networks, such as cancer.
  • Another advantage of the methods and systems herein disclosed when used to perform a diagnostic assay is the possibility to analyze a small amount of biological sample in a multiparameter fashion, and be able to bridge the three relevant areas of biological information, that of the genes (represented by DNA), proteins, and cells.
  • the multiplexed multiparameter microfluidic methods and systems herein disclosed are particularly advantageous when the targets are biomarkers from a tissue in view of the reduced amount of sample required to perform the analysis which minimizes the need to euthanize mice.
  • the methods and systems performed in a microfluidic environment herein disclosed allow a detection of a target that is included in a sample in a small quantities allowing detection of molecules present in the sample at a concentration down to about a 10 femtoMolar.
  • Still further advantages of the methods and systems herein disclosed, when the substrate is in operable association with a microfluidic component when used to perform a diagnostic assay, are to allow the multiplexed detection of biomarkers, including chemically distinct biomarkers such as polynucleotides, proteins and cells.
  • a further additional advantage of the diagnostic methods and systems herein disclosed, in embodiments wherein the substrate is in operable association with a microfluidic component, is to allow performance of multiplexed multiparameter assays on a single sample from the same microscopic region of an heterogeneous tissue.
  • the methods and systems herein disclosed also minimize the sampling errors associated with heterogeneous biopsies required to perform the various measurements of the diagnostic method and systems for the detection of multiple chemically distinct biomarkers of the art.
  • FIG. 1 is a schematic illustration of a coupling strategy utilized to prepare polynucleotide-encoded-protein herein disclosed.
  • Panel a is a schematic illustration of a reaction for the preparation of an antibody;
  • Panel b is a schematic illustration of a reaction the preparation of a polynucleotide;
  • Panel c is an illustration of the polynucleotide-encoded antibody resulting from the conjugation of the antibody shown in Panel a and the polynucleotide shown in Panel b;
  • Panel d shows a gel mobility shift assay showing that the number of polynucleotide strand A1′ attached to the antibody can be controlled by adjusting the amount of coupling molecule to antibody as shown in Panel a.
  • lanes I-IV corresponds to stoichiometric ratios of 300:1, 100:1, 50:1, 25:1 of the coupling molecule to antibody respectively;
  • FIG. 2 is a schematic illustration of the conjugation chemistry of a polynucleotide-encoded protein disclosed herein.
  • Panel a shows a schematic illustration of the conjugation chemistry between a polynucleotide and the protein streptavidin;
  • Panel b shows the assembly of the polynucleotide-encoded streptavidin with a protein containing biotin, which is the ligand of streptavidin;
  • SA indicates the streptavidin protein, Biotin-Protein: indicates a protein containing the ligand biotin;
  • FIG. 3 shows diagrams illustrating the optimization of polynucleotide loading of polynucleotide-encoded antibodies for cell surface marker recognition herein disclosed.
  • Panel a shows FACS plots comparing ⁇ -CD90.2/FITC-polynucleotide conjugates (FITC-DNA-labeled ⁇ -CD90.2) with FITC ⁇ -CD90.2 antibody having no polynucleotide attached to antibody (FITC ⁇ -CD90.2) along with a negative control with no antibody and no polynucleotide encoded antibody (unlabeled).
  • the florescent intensity corresponding to the FITC channel is given on the x axis, the y axis corresponding to a null florescent channel;
  • Panel b shows histograms of the mean fluorescent intensities for different numbers of FITC-polynucleotide attached to the antibody; on the x axis the number of polynucleotides attached to the antibody are reported, on the y axis the mean fluorescence intensity is reported;
  • FIG. 4 is a schematic illustration of a combined use of polynucleotide-encoded antibodies and substrate polynucleotides herein disclosed;
  • FIG. 5 illustrates an embodiment of the methods and systems wherein the polynucleotide-encoded protein is based on the streptavidin biotin system and the targets are cells.
  • Panel a shows assembly of the polynucleotide-encoded streptavidin according to FIG. 2 , wherein the biotin containing protein is the Major histocompatibility complex (MHC) and preassembly of the polynucleotide-encoded straptavidin onto the substrate before the cells of interest are exposed to the glass substrate.
  • Panel b shows exposure of the microarray following binding of the polynucleotide-encoded MHC to the cells in solution;
  • MHC Major histocompatibility complex
  • FIG. 6 illustrates a method of detecting a plurality of targets using polynucleotide-encoded antibodies and substrate polynucleotide herein disclosed.
  • Panel a shows a schematic illustration of a combined used of a plurality of polynucleotide-encoded antibodies herein disclosed in combination with substrate polynucleotides
  • Panel b shows a related immunoassay performed using polynucleotide-encoded antibodies and substrate polynucleotide herein disclosed;
  • FIG. 7 shows a spatially encoded protein array using encoded polynucleotide-encoded antibodies and substrate polynucleotides herein disclosed.
  • Panel a shows an immunoassay performed with three identical goat ⁇ -human IgG (labeled with Alexa488, Alexa594, or Alexa 647 dyes) and tagged with polynucleotides A1′, B1′ and C1′ respectively; shows a schematic representation of the results of the immunoassays from the portion of the array of Panel a indicated by a white bar; the scale bar shown in the Figure corresponding to 1 mm;
  • FIG. 8 shows the results of an immunoassay showing minimization of non specific protein absorption resulting from the combined used of polynucleotide-encoded antibodies and substrate polynucleotide herein disclosed.
  • Panel a shows a microarray simultaneously exposed to goat ⁇ -human IgG-Alexa488/A1′, goat ⁇ -human IgG-Alexa647/C1′ each conjugated with a specific polynucleotide and goat ⁇ -human IgG-Alexa594 with no pendant DNA
  • Panel b shows a schematic representation of the results of the immunoassays from the portion of the array of Panel a indicated by a white bar; the scale bar shown in the Figure corresponding to 1 mm;
  • FIG. 9 illustrates the results of the in silico orthogonalization of substrate polynucleotides wherein each substrate polynucleotide is orthogonal to the others and bind to their corresponding antibody specific polynucleotides.
  • Panel a shows a glass slide printed with three substrate polynucleotides exposed to two polynucleotide-encoded antibodies complementary to two out of the three substrate polynucleotides;
  • Panel b shows the secondary structure formed from the hybridization of A1 in silico hybridization in silico of the two substrate polynucleotides complementary to the antibody specific polynucleotide;
  • Panel c shows generation in silico of additional substrate polynucleotide with the constraints that each strand be orthogonal with each other and with their corresponding complements;
  • Panel d shows a set of 6 orthogonal sequences, listed 5′ to 3′ end;
  • FIG. 10 illustrates a method for performing multiplexed cell sorting using the polynucleotide-encoded antibody and the substrate polynucleotide herein disclosed.
  • Panel a shows a homogeneous assay in which polynucleotide-encoded antibodies are combined with the cells, and then the mixture is introduced onto the spotted DNA array microchip;
  • Panel b shows polynucleotide-encoded antibodies assembled onto a spotted DNA array, followed by introduction of the cells;
  • Panel c shows brightfield and fluorescence microscopy images of multiplexed cell sorting experiments where a 1:1 mixture of mRFP-expressing T cells (red channel) and EGFP-expressing B cells (green channel) is spatially stratified onto spots A1 and C1, corresponding to the encoding of ⁇ -CD90.2 and ⁇ -B220 antibodies with A1′ and C1′, respectively;
  • Panel d is a fluorescence micrograph of multiplexed sorting of primary cells harvested from mice
  • a 1:1 mixture of CD4+ cells from EGFP transgenic mice and CD8+ cells from dsRed transgenic mice are separated to spots A1 and C1 by utilizing polynucleotide-encoded conjugates ⁇ -CD4-A1′ and ⁇ -CD8-C1′, respectively;
  • FIG. 11 is a schematic illustration of a combined use of polynucleotide-encoded antibodies and substrate polynucleotides herein disclosed for cell sorting and/or co-detection of chemically distinct molecules;
  • FIG. 12 illustrates the ability of a polynucleotide-encoded protein to detect a plurality of targets according to an embodiments of the methods and systems herein disclosed;
  • Panel a shows a microarrays exposed to an antibody specific for antigen IL4 encoded with polynucleotide C1 and a polynucleotide complementary to polynucleotide B1 labeled with a fluorophore;
  • Panel b shows a schematic representation of the embodiment of the methods and systems herein disclosed used to perform the assay;
  • Panel c shows a schematic representation of the results of the assay illustrated in the portion of panel A identified by a white bar;
  • FIG. 13 shows microscopy images demonstrating simultaneous cell capture and multiparameter detection of genes and proteins, the scale bar shown in the Figure corresponding to 300 ⁇ m;
  • FIG. 14 shows a protein array used in an embodiment of the method for detecting targets herein disclosed assembled in microfluidics
  • FIG. 15 shows fluorescence and brightfield images of DNA-templated protein immunoassays executed within microfluidic channels, the 600 ⁇ m micrometer wide channels being delineated with white dashed lines.
  • Panel a shows a two-parameter immunoassay performed using polynucleotide-encoded antibodies in combination with substrate polynucleotides herein disclosed;
  • Panel b shows detection of a target concentration series in an embodiment of the method and system herein disclosed wherein the detection is performed using fluorescence based techniques;
  • Panel c shows detection of a target concentration series in an embodiment of the method and system herein disclosed wherein the detection is performed using Au electroless deposition as a visualization and amplification strategy;
  • FIG. 16 is a schematic illustration of a combined use of polynucleotide-encoded antibodies and substrate polynucleotides wherein the polynucleotide-encoded antibodies are labeled with metal nanoparticles according to an embodiment of the methods and systems herein disclosed;
  • FIG. 17 is an additional schematic illustration of the combined use of FIG. 16 , showing the polynucleotide-encoded antibody target complex bound to the substrate and labeled with metal nanoparticles according to an embodiment of the methods and systems herein disclosed;
  • FIG. 18 is a schematic illustration of a device and related method to detect a signal from polynucleotide-encoded antibodies labeled with metal nanoparticles according to an embodiment of the methods and systems herein disclosed;
  • FIG. 19 shows detection of a proteomic with a method and system herein disclosed wherein the detection is performed using Au electroless deposition as a visualization and amplification strategy.
  • Panel a shows detection at concentration of about 100 pM;
  • Panel b shows detection at concentration of about 100 femtoM;
  • Panel c shows detection at concentration of about 100 attoM;
  • FIG. 20 shows detection of a proteomic with a method and system herein disclosed wherein the detection is performed using Au electroless deposition as a visualization and amplification strategy.
  • Panel a shows detection at concentration of about 100 pM;
  • Panel b shows detection at concentration of about 1 pM;
  • Panel c shows detection at concentration of about 10 fM;
  • Panel d shows detection at concentration of about 100 aM;
  • Panel e shows an histogram correlating the numbers of proteins counted (y axis) versus their concentration in solution (x-axis);
  • FIG. 21 shows detection of a proteomic of 3 proteins (IFN- ⁇ , TNF- ⁇ and IL-2) from tissue culture media spiked with the three proteins with a method and system herein disclosed wherein the detection is performed using Au electroless deposition as a visualization and amplification strategy.
  • Panel a shows detection of IFN- ⁇ ;
  • Panel b shows detection of TNF- ⁇ ;
  • Panel c shows detection of IL-2;
  • FIG. 22 shows detection of a proteomic of 3 proteins (IFN- ⁇ , TNF- ⁇ and IL-2) from a serum sample spiked with the three proteins (Panel a) and from the serum of a healthy human (Panel b) with a method and system herein disclosed wherein the detection is performed using Au electroless deposition as a visualization and amplification strategy;
  • FIG. 23 is a diagram illustrating the calibration and quantification of the protein marker, Pten, with an embodiment of the methods and systems herein disclosed;
  • Panel a shows a diagram wherein the average fluorescent intensity of the signal detected from the microfluidic experiments illustrated in Panels b and c, is illustrated;
  • Panel b shows the raw data from the calibration lanes for recombinant pten;
  • Panel c shows the raw fluorescent data from the samples from two cell lines, one is the null U87, expressing basal levels of pten, and the other is the U-87-pten overexpressing cell samples; and
  • FIG. 24 illustrates the pathway from serum biomarker discovery via tandem mass spectrometry (Panel a or 1) to antibody validation and selection (Panel c or 3) via large scale SPR (Panel b or 2) to validating clinical pathways with an embodiment of the methods and systems herein disclosed.
  • polynucleotide-encoded proteins are used in combination with substrate polynucleotides to detect one or more targets in a sample.
  • detect 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.
  • target indicates an analyte of interest.
  • 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.
  • biomolecule indicates a substance compound or component associated to a biological environment including but not limited to sugars, aminoacids, peptides proteins, oligonucleotides, polynucleotides, polypeptides, organic molecules, haptens, epitopes, biological cells, parts of biological cells, vitamins, hormones and the like.
  • 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.
  • sample 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.
  • nucleotide indicates an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof.
  • 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.
  • 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.
  • 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.
  • polypeptide indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof.
  • 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.
  • amino acid refers to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D an L optical isomers.
  • 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.
  • protein 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.
  • antibody 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).
  • 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.
  • 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.
  • 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.
  • substrate polynucleotide 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.
  • substrate 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.
  • 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.
  • the protein-target interaction is an antibody-antigen interaction.
  • the interaction can be receptor-ligand, enzyme-substrate and additional protein-protein interactions identifiable by a skilled person upon reading of the present disclosure.
  • 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.
  • each substrate polynucleotide and encoding polynucleotide is bindingly distinguishable from another.
  • each substrate polynucleotide of a substrate is sequence specific and positionally distinguishable from another.
  • bindingly distinguishable 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.
  • positionally distinguishable refers to with reference to molecules, indicates molecules that are distinguishable based on the point or area occupied by the molecules. Accordingly, positionally distinguishable substrate polynucleotides are substrate polynucleotide that occupy different points or areas on the substrate and are thereby positionally distinguishable.
  • polynucleotide-encoded protein herein disclosed can be produced with common bioconjugation methods, such as chemical cross-linking which include techniques relying on the presence of primary amines in the protein to be bound (usually found on Lysine residues).
  • polynucleotide-encoded-protein can be produced by the covalent conjugation strategy shown in FIGS. 1 and 2 for polynucleotide-encoded antibodies ( FIG. 1 ) and a polynucleotide-encoded streptavidin ( FIG. 2 ).
  • Identical bioconjugation chemistry can be used for the production of any polynucleotide-encoded-protein such as polynucleotide-encoded streptavidin, as exemplified in Example 2 and illustrated in FIG. 2 .
  • the number of encoding polynucleotides to be conjugated with a particular polynucleotide-encoded protein can be varied.
  • the number of polynucleotides attached to the protein component can be modulated to minimize the size and therefore the steric hindrance of the pending moieties while still maintaining binding specificity.
  • the optimization can be performed by way of procedures exemplified in Example 3 and illustrated in the related in FIG. 3 .
  • Example 3 and FIG. 3 different batches of polynucleotide-encoded antibodies were made, in which the total number of polynucleotides linked to each antibody were varied. Because the encoding polynucleotides of FIG.
  • Example 3 contained a fluorophore, the binding efficiency of each variant for cell surface markers could be tested out using FACS. It should be noted that there are other analogous techniques to measure and optimize antibody binding affinity as a function of polynucleotide loading, including techniques which directly measure the binding kinetics of antibodies such as surface plasmon resonance (SPR) and isothermal titration calorimetery (ITC).
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetery
  • the number of encoding polynucleotides to be attached to each protein can be any from 1 to 6.
  • attaching 3 encoding polynucleotides per protein provides the further advantage of minimizing the steric effects of labeling and therefore allowing a labeling of a polynucleotide-encoded protein with a plurality of encoding polynucleotides for high affinity hybridization with the complementary substrate polynucleotide.
  • the length of the polynucleotide forming the pending moieties can also be controlled to optimize binding of the polynucleotide-encoded protein to the substrate.
  • the length of the encoding polynucleotides can be optimized for orthogonalization purposes as illustrated in Example 8 and FIG. 9 and further discussed below.
  • polynucleotide-encoded protein is a polynucleotide-encoded antibody.
  • a skilled person will be able to adapt the teaching provided for the polynucleotide-encoded antibodies to other polynucleotide-encoded proteins upon reading of the present disclosure.
  • the substrate polynucleotides can be produced by normal techniques in the field. For example, first the polynucleotides can be chemically synthesized. The polynucleotides can then be pin spotted according the paradigm outlined by Pat Brown at Stanford (Schena M, Shalon D, Davis R W, Brown P O. Science. 1995 Oct. 20; 270(5235): 467-70). The substrate polynucleotides so produced can be then attached to a substrate according to techniques identifiable by a skilled person upon reading of the present disclosure. Particularly, suitable polynucleotides for the production of substrate polynucleotides include at least 75 mers long on polylysine substrates.
  • the encoding polynucleotides and/or the substrate polynucleotides are orthogonalized to minimize the non-specific binding between encoding-polynucleotide and substrate polynucleotide.
  • orthogonalized polynucleotides include polynucleotides whose sequence is computationally generated to minimize incomplete base pairing, metastable states and/or other secondary structures to minimize non specific interactions between polynucleotides and non linear secondary interactions in the polynucleotide usually associated with random generation of the relevant sequences.
  • orthogonalization refers to the process by which a set of polynucleotides are generated computationally, in which incomplete base pairing, metastable states and other secondary structures are minimized, such that a polynucleotide only binds to its complementary strand and none other.
  • orthogonalization techniques used in this disclosure include orthogonalization performed according to the paradigm outlined by Dirks et al. (Dirks, R. M.; Lin, M.; Winfree, E.; Pierce, N. A. Nucleic Acids Research 2004, 32, (4), 1392-1403)
  • the encoding-polynucleotides and the corresponding complementary substrate polynucleotides are orthogonalized polynucleotides having the sequences from SEQ ID NO: 7 to SEQ ID NO 18 (see Example 8 and related Table 1)
  • Additional orthogonalized polynucleotides can be further identified by way of methods and procedures, such as in silico orthogonalization (i.e. computerized orthogonalization) of polynucleotides exemplified in Example 8 and illustrated in FIG. 9 , and additional procedures that would be apparent to a skilled person upon reading of the present disclosure.
  • silico orthogonalization i.e. computerized orthogonalization
  • the methods and systems herein disclosed can be used for performing assays for the detection of targets, including mono-parameter assays, and multiparameter assays, all of which can be performed as multiplex assays.
  • the term “monoparameter assay” as used herein refers to an analysis performed to determine the presence, absence, or quantity of one target.
  • the term “multiparameter assay” refers to an analysis performed to determine the presence, absence, or quantity of a plurality of targets.
  • the term “multiplex” or “multiplexed” assays refers to an assay in which multiple assays reactions, e.g., simultaneous assays of multiple analytes, are carried out in a single reaction chamber and/or analyzed in a single separation and detection format.
  • the methods and systems herein disclosed can advantageously used to perform diagnostic assays, wherein the target(s) to be detected are predetermined biomarkers associated with a predetermined disease.
  • the target(s) to be detected are predetermined biomarkers associated with a predetermined disease.
  • Those embodiments are particularly advantageous in a diagnostic approach where different classes of biomaterials and biomolecules are each measured from a different region of a typically heterogeneous tissue sample, thus introducing unavoidable sources of noise that are hard to quantitate.
  • the polynucleotide-encoded protein and substrate polynucleotide are used in combination as schematically illustrated in FIG. 4 wherein the polynucleotide-encoded proteins are polynucleotide-encoded antibodies.
  • a polynucleotide-encoded antibody ( 10 ) is provided in combination with a substrate ( 100 ).
  • the polynucleotide-encoded antibody ( 10 ) is comprised of an antibody ( 11 ) and an encoding-polynucleotide ( 12 ).
  • the substrate ( 100 ) has a substrate polynucleotide ( 120 ) bound to a substrate surface.
  • the encoding polynucleotide ( 12 ) is complementary to the substrate polynucleotide ( 120 ) so that when contacted the substrate polynucleotide ( 120 ) and the encoding polynucleotide ( 12 ) hybridize.
  • the polynucleotide-encoded antibodies herein disclosed form a protein array that can be contacted with a sample to detect a target in the sample.
  • the embodiment of FIG. 4 is particularly advantageous for detecting and/or sorting protein-targets.
  • some or all of the polynucleotide-encoded antibodies are contacted with the sample before contacting the polynucleotide-encoded-antibodies with the complementary substrate polynucleotide.
  • the antibodies and the one or more corresponding targets can bind in absence of the substrate, e.g., in a solution phase, where both molecules have a complete orientational freedom and the access of the target to the binding pocket of the antibody is not impaired by the substrate.
  • surface-induced protein denaturation does not occur because the polynucleotide-encoded antibodies remain in solution preserving the tertiary fold of the protein.
  • FIGS. 5 , 7 , 8 11 and 13 Exemplary embodiments showing some of the above advantages are illustrated in FIGS. 5 , 7 , 8 11 and 13 .
  • the antibody-target complex bound to the substrate is eventually detected from the substrate.
  • detection of the complex is performed by providing a labeled molecule, which includes any molecule that can specifically bind a polynucleotide-encoded-protein 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 polynucleotide-encoded protein-target complex and the labeling signal from the label compound bound to the polynucleotide-encoded protein-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.
  • 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.
  • detectably distinguishable 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, chemiluminescent 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.
  • the plurality of labeled molecules is contacted with the plurality of polynucleotide-encoded protein-target complexes for a time and under condition to allow binding of the plurality of polynucleotide-encoded protein-target complexes with the plurality of labeled molecules.
  • the labeling signal is then detected from the plurality of labeled molecules bound to the plurality of polynucleotide-encoded protein-target complexes on the substrate.
  • the detection method can be carried either via fluorescent based readouts, in which the labeled antibody is labeled with fluorophore which includes but not exhaustively small molecular dyes, protein chromophores, quantum dots, and gold nanoparticles.
  • fluorophore which includes but not exhaustively small molecular dyes, protein chromophores, quantum dots, and gold nanoparticles.
  • 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.
  • the readout comes from dark field scattering of gold particles, single molecule digital proteomics is enabled. Additional techniques are identifiable by a skilled person upon reading of the present disclosure and will not be further discussed in details.
  • 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, chemiluminescent 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.
  • fluorophore refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image.
  • labeling signal indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemoluminescence, production of a compound in outcome of an enzymatic reaction and the likes.
  • one specific target is detected.
  • contacting the polynucleotide-encoded antibodies with the target can be performed before or after contacting the polynucleotide-encoded antibody with the substrate.
  • the embodiments wherein contacting the polynucleotide antibodies with the target is performed before contacting the polynucleotide-encoded antibody with the substrate are particularly suitable to sort or detect cells.
  • the efficiency and specificity of the binding between antibody and target is maximized even for a detection of a single target.
  • the target capture is not driven by antibody to cell surface marker interactions, but rather by the increased avidity of antibody specific polynucleotide for the corresponding strands on the microarray through cooperative binding, greatly increasing capture efficiency.
  • This advantage is particularly relevant for target cells that can be efficiently captured so that with this process it is typical to see a DNA spot entirely occupied by a confluent layer of cells. (see Example 5 and FIG. 5 ).
  • contacting the polynucleotide-encoded antibodies with the target is performed after contacting the polynucleotide-encoded antibody with the substrate are particularly suitable to sort or detect proteins with high sensitivity.
  • Exemplary embodiments of methods and systems herein disclosed wherein contacting the polynucleotide-encoded antibodies with the target is performed after contacting the polynucleotide-encoded antibody with the substrate are exemplified in Examples 12, and 13 and illustrated in FIGS. 15 , 19 , 20 , 21 , 22 , 23 , 24 ( c ).
  • competition for the same specific substrate polynucleotide between a polynucleotide-encoded-proteins bound to the target and polynucleotide-encoded-proteins not bound to the target can be eliminated and the sensitivity of the assay consequently increased.
  • concentration of polynucleotides on the substrate can be optimized so that higher concentration of polynucleotide-encoded proteins can be bound to the substrate, which will in turn result in higher concentrations of correctly assembled complex, which in turn increase the overall detection sensitivity, by virtue of equilibrium thermodynamics law that govern each binding.
  • Monoparameter assays that can be performed with the methods and systems exemplified in FIGS. 4 and 5 and in Example 5, include but are not limited to, any assays for the detection of single markers in serum, single protein detection in biological samples, cell sorting according to one surface marker and further assays identifiable by a skilled person upon reading of the present disclosure.
  • detection of a plurality of targets is performed, according to a strategy schematically illustrated in FIG. 6 .
  • a plurality of polynucleotide-encoded antibodies ( 10 , 20 and 30 ) is produced, each polynucleotide-encoded antibody able to specifically bind to a predetermined target with the antibody component ( 11 , 21 and 31 ) and to bind to a complementary substrate polynucleotide with the encoding-polynucleotide component. ( 12 , 22 and 32 ).
  • a substrate is generated with sequence specific positionally distinguishable substrate polynucleotides ( 12 , 122 , and 132 ).
  • polynucleotide-encoded antibodies ( 10 ), ( 20 ) and ( 30 ) are then contacted with the substrate polynucleotide ( 112 ), ( 122 ) and ( 132 ) and upon binding of the antibody specific polynucleotide with the corresponding substrate polynucleotide, polynucleotide-encoded antibody complexes self assemble on the substrate.
  • a protein array composed of a plurality of bindingly distinguishable and positionally distinguishable antibodies is produced.
  • Those embodiments are particularly advantageous for sorting and/or detecting different protein-targets with a high sensitivity. Exemplary illustrations of those embodiments are shown in Examples 9, 10 and 12 and in FIGS. 10 , 12 , 13 and 15 a.
  • the plurality of polynucleotide-encoded antibodies is contacted with a sample for detection of the related target before contacting the substrate polynucleotides.
  • the methods and systems herein disclosed can be used to perform multiplexed multiparameter assays wherein due to the improved sensitivity and selectivity associated with binding of antibody and target in absence of a substrate and in view of the reduced biofouling and protein denaturation, a large number of biomarkers can be efficiently detected in a quantitative and/or qualitative fashion. Exemplary illustrations of those embodiments are shown in Examples 9, 10 and 12 and in FIGS. 10 , 12 , 13 and 15 .
  • Multiparameter assays that can be performed with the methods and systems exemplified in Examples 9, 10 and 12 and illustrated in FIGS. 10 , 12 , 13 and 15 include but are not limited to any proteomic analysis, tissue analysis, serum diagnostics, biomarker, serum profiling, multiparameter cell sorting, single cell studies, and additional assays identifiable by a person skilled in the art upon reading of the present disclosure.
  • the combined use schematically illustrated in FIG. 6 can be applied in a method for sorting a plurality of targets which is particularly advantageous when the plurality of targets is composed of different types of cells, and in particular primary cells.
  • the polynucleotide-encoded antibody is preferably contacted with the sample including the cells before contacting the substrate according to procedure exemplified in Example 9 and illustrated in FIG. 10 .
  • Embodiments of the methods and systems wherein the plurality of targets is composed of different types of cells are particularly advantageous over corresponding methods and systems of the art such as panning in which cells interact with surface marker-specific antibodies printed onto an underlying substrate (Cardoso, A. A.; Watt, S. M.; Batard, P.; Li, M. L.; Hatzfeld, A.; Genevier, H.; Hatzfeld, J. Exp. Hematol. 1995, 23, 407-412).
  • the efficiency of cell capture on the substrate is improved with respect to prior art methods and systems, due to the use of polynucleotide to bind the antibody to the substrate (see FIG. 5 and FIG. 10 ).
  • those preferred embodiments do not have the same limitations as conventional spotted protein microarrays, such as antibodies that are not always oriented appropriately on a surface, and/or antibodies that can dry out and lose functionality.
  • any of the embodiments to sort cells has several advantages over methods and systems to sort cells known in the art such as FACS, since the cells sorted by the methods and systems herein disclosed are immediately available for post-sorting analysis of gene and/or protein expression.
  • the methods and systems herein disclosed perform a spatially multiplexed sorting of multiple cells that is particularly effective in sorting cells according to multiple cells surface markers and is not limited by the number of spectrally distinct fluorophores that can be utilized to label the cell surface markers used for the sorting, as exemplified in Example 9 and related FIG. 10 .
  • the combined use depicted in FIG. 6 can be applied to detection of a plurality of chemically distinct targets according to the approach schematically illustrated in FIG. 11 .
  • the approach is illustrated for separation of a plurality of distinct biomarkers such as DNA cells and proteins.
  • the methods and systems herein disclosed are performed to separate cells ( 1 ) (see FIG. 11 , arrow A1) and analyze the relevant genomic and proteomic signature (see FIG. 11 , arrow A2) using a substrate ( 2 ) with a plurality of substrate polynucleotides ( 3 ) attached thereto in a multiparameter assay for the analysis of cells, genes and proteins.
  • the sample is contacted with a plurality of polynucleotide-encoded antibodies to allow formation of a plurality of polynucleotide-encoded biomarker complexes that are then contacted to a substrate such as a DNA array wherein the antibody specific polynucleotides specifically bind the corresponding DNA strands.
  • a labeled polynucleotide that specifically bind to the target polynucleotide can further be contacted with the sample for the production of a labeled target polynucleotide that specifically binds a predetermined DNA strands on the substrate.
  • the labeled target polynucleotide is eventually contacted with the substrate polynucleotide and detected.
  • the cells, protein and DNA biomarkers are sorted and then detected in a single substrate, thus allowing advantageous performance of multiplexed multiparameter assays.
  • polynucleotides as a common assembly strategy for cells, cDNAs, and proteins, it is possible to optimize the substrate conditions for high DNA loading onto the spotted substrates, and for complementary DNA loading on the antibodies.
  • This and the reduced biofouling associated with polynucleotide based binding of antibodies on the substrate allows performance of highly sensitive sandwich assays for protein detection, as well as high efficiency cell sorting (compared with traditional panning).
  • An exemplary method and system to perform detection of chemically different biomarkers is described in Example 10 and illustrated in FIG. 13 .
  • Assays to sort targets performable with the methods and systems exemplified in Examples 9, 10, 12 and 13 and illustrated in FIGS. 13 , 10 c , 10 d 15 a , 22 , 23 , 24 , include any assay that requires detection of a particular target (including but not limited to cell targets, protein-target or gene targets) in a mixture, which will be identifiable by a skilled person upon reading of the present disclosure.
  • high sensitivity detection of single or multiple targets can be performed by using antibodies labeled with metal nanoparticles for the detection, followed by electroless metal deposition.
  • any of the methods and systems herein disclosed can be performed by using a metal nanoparticle (in particular Au nanoparticles) as a labeling molecule to detect the encoded-polynucleotide protein-target complex bound to the substrate.
  • a metal nanoparticle such as a gold nanoparticle
  • the labeled molecule e.g., a second antibody
  • Metal particles, such as Au nanoparticles have unique optical properties in that a particle that is much smaller than the wavelength of visual light can still be readily imaged using light scattering.
  • FIGS. 16 and 17 show schematically an exemplary embodiment of the methods and systems herein disclosed, wherein the labeling molecule includes a metal nanoparticle such as a gold nanoparticle.
  • a gold nanoparticle 210
  • a linker molecule 211
  • a 2° antibody 212
  • On the 1 AB 213
  • one or more ssDNA oligomers 214
  • the target to be detected 217
  • the assay itself will be measured on a surface ( 216 ) that has been coated with ssDNA′ ( 215 ).
  • Exemplary embodiments are further illustrated in FIGS. 18 to 22 and exemplified in Example 13.
  • An advantage of some embodiments of the methods and systems herein disclosed when metal nanoparticles are used for labeling is that there is no need to calibrate the immunoassay each time a protein measurement is done, since amount of protein counted represents an absolute measurement. Fluorescence or absorbance assays, by comparison, represent relative measurements, since they are dependent upon background fluorescence (absorbance) levels, light amplification electronics, photobleaching effects (for fluorescence), etc.
  • the nanoparticle-based digital methods and systems herein disclosed can be advantageously used for: (1) the ultrasensitive detection of proteins at high attoMolar levels (10 3 -10 6 fold improvement over conventional ELISA immunoassays) and over a broad concentration range; (2) the multiplexed detection of several proteins on the same chip; and (3) the detection of extracellular signaling molecules, cytokines, in human patient sera.
  • Some embodiments of the methods and systems herein disclosed wherein labeling and detection is performed by using metal nanoparticles is based on a detection system, such as a Raleigh scattering mechanism that allows for the indirect visualization of individual plasmonic nanoparticles, in this case 40 nm Au nanoparticles, that are conjugated to detection antibodies to realize single protein counting.
  • a graphical software interface can be utilized to digitally count the absolute number of particles and to thus quantitate the amount of proteins.
  • Those embodiments are in sharp contrast to conventional quantitation methods using averaged signal readout after amplification.
  • the methods and systems herein disclosed that use metal nanoparticles as label compounds are able to multiplex the detection by simultaneously counting different kinds of proteins from the same biological sample.
  • a further advantage of the methods and systems herein disclosed wherein metal nanoparticles are used as label compounds over highly sensitive protein detection techniques of the art that are based upon variants of the ELISA scheme are the possibility to eliminate an amplification of the signal and associated additional noise and time required for performance.
  • the prior art methods all require some sort of amplification step, and each method requires some level of calibration that must be carried out for every assay performed. For example, methods in which the 2° AB is labeled with DNA, and that DNA is amplified using the polymerase chain reaction (PCR) have been reported. It is this amplified DNA that is detected and then correlated to the measured protein concentration.
  • PCR polymerase chain reaction
  • the 2° AB is labeled with a gold nanoparticle, and then silver metal is deposited (via electroless deposition) onto that gold nanoparticle in order to generate an amplified absorbance signal.
  • the amplification step itself introduces noise into the assay, and requires an additional amount of time—often a significant amount of time.
  • This application would be particularly advantageous for detection the field of proteomics ( FIGS. 21 an 22 ), and/or detection of biomarkers present at a very low concentration in a small volume sample, e.g., a drop of blood ( FIGS. 19 and 20 ).
  • the substrate of any of the methods and systems herein disclosed can be associated with a microfluidic component so to allow performance of microfluidic based assays.
  • Microfluidic-based assays offer advantages such as reduced sample and reagent volumes, and shortened assay times (Breslauer, D. N.; Lee, P. J.; Lee, L. P. Mol. BioSyst. 2006, 2, 97-112).
  • the surface binding assay kinetics are primarily determined by the analyte (protein) concentration and the analyte/antigen binding affinity, rather than by diffusion (Zimmermann, M.; Delamarche, E.; Wolf, M.; Hunziker, P. Biomedical Microdevices 2005, 7, (2), 99-110).
  • microfluidic refers to a component or system that has microfluidic features e.g. channels and/or chambers that are generally fabricated on the micron or sub-micron scale.
  • the typical channels or chambers have at least one cross-sectional dimension in the range of about 0.1 microns to about 1500 microns, more typically in the range of about 0.2 microns to about 1000 microns, still more typically in the range of about 0.4 microns to about 500 microns.
  • Individual microfluidic features typically hold very small quantities of fluid, e.g from about 10 nanoliters to about 5 milliliters, more typically from about 100 nanoliters to about 2 milliliters, still more typically from about 200 nanoliters to about 500 microliters, or yet more typically from about 500 nanoliters to about 200 microliters.
  • microfluidic components can be included in an integrated device.
  • integrated device refers to a device having two (or more) components physically and operably joined together. The components may be (fully or partially) fabricated separate from each other and joined after their (full or partial) fabrication, or the integrated device may be fabricated including the distinct components in the integrated device.
  • An integrated microfluidic array device includes an array component joined to a microfluidic component, wherein the microfluidic component and the array component are in operable association with each other such that an array substrate of the array component is in fluid communication with a microfluidic feature of the microfluidic component.
  • a microfluidic component is a component that includes a microfluidic feature and is adapted to being in operable association with an array component.
  • An array component is a component that includes a substrate and is adapted to being in operable association with a microfluidic component.
  • microfluidic systems can also be provided in a modular form.
  • Module describes a system or device having multiple standardized components for use together, wherein one of multiple different examples of a type of component may be substituted for another of the same type of component to alter the function or capabilities of the system or device; in such a system or device, each of the standardized components being a “module”.
  • the sensitivity of the assay can also be increased to detect targets at a concentration as low as 10 fM, including biomarkers (e.g. proteins in human sera) previously considered below detectable levels by any other techniques.
  • biomarkers e.g. proteins in human sera
  • microfluidic methods and systems herein disclosed accordingly allow optical read out of assays that are 100-1000 fold more sensitive than corresponding methods and system of the art (see FIG. 14 ). Accordingly, a further advantage of the microfluidic methods and systems herein disclosed is the possibility of using said methods and systems as a digital technique—i.e. a technique for the quantitative detection of protein via single molecule counting. This application would be particularly advantageous for detection in the field of proteomics ( FIG. 14 ), and/or detection of biomarkers present at a very low concentration in a small volume sample (e.g., a drop of blood)
  • microfluidic methods and systems herein disclosed allow performance of both (i) mono step assays (wherein the polynucleotide-encoded antibodies the target(s) and labeled antibodies are contacted in a single step) and (ii) multi-steps assays (wherein the substrate is sequentially exposed to polynucleotide-encoded antibodies, target(s), and then secondary antibody) in a reduced amount of time, with samples reduced in size and with a higher sensitivity when compared with corresponding microfluidic methods and system of the art and with other non-microfluidic methods and systems for molecule detection (see Examples 11 and 12).
  • An additional advantage associated with microfluidic methods and systems herein disclosed includes the possibility of performing in a microfluidic environment any assay that involves substrate-supported antibodies, which would not have survived microfluidic chip assembly with the use of previous techniques.
  • the methods and systems herein disclosed allow the multiplexed multiparameter detection, sorting and of biomarkers of interest and related diagnostic analysis. Exemplary illustration of applications of the methods and systems herein disclosed for diagnostic analysis are described in Example 14 and shown in FIGS. 23 and 24 , and any additional assay identifiable by a skilled person upon reading of the present disclosure.
  • 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 is micrometers.
  • FIGS. 4 , 5 , 6 , 7 , 8 , 9 and 10 provide exemplary microarrays.
  • the polynucleotide-encoded proteins and a substrate are comprised in the kit independently.
  • the polynucleotide-encoded protein is included in one or more compositions, and each polynucleotide-encoded protein is in a composition together with a suitable vehicle carrier or auxiliary agent.
  • the substrate provided in the system can have substrate polynucleotide attached thereto.
  • the substrate polynucleotides 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.
  • 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).
  • DNA encoded antibodies were generated according to the two step strategy illustrated in FIG. 1 .
  • an aldehyde functionality was introduced to the 5′-aminated oligonucleotide via succinimide chemistry, using commercially available reagents ( FIG. 1 Panel a).
  • a hydrazide moiety was introduced via reaction with the lysine side chains of the respective antibody ( FIG. 1 Panel a).
  • DNA-antibody conjugate formation was then facilitated via stoichiometric hydrazone bond formation between the aldehyde and hydrazide functionalities. Conjugate formation and control over DNA-loading was verified by PAGE electrophoresis ( FIG. 1 Panel b).
  • AlexaFluor 488, 594, and 647-labeled polyclonal Goat anti-Human IgGs were purchased from Invitrogen.
  • Monoclonal Rabbit anti-Human Interleukin-4 (clone: 8D4-8), non-fluorescent and APC-labeled Rabbit anti-Human Tumor Necrosis Factor- ⁇ (clones: MAb1 and MAb11, respectively), and non-fluorescent and PE-labeled Rabbit anti-Human Interferon- ⁇ (clones: NIB42 and 4S.B3, respectively) were all purchased from eBioscience.
  • Non-fluorescent and biotin-labeled mouse anti-Human Interleukin-2 (clones: 5344.111 and B33-2, respectively) were purchased from BD Biosciences. All DNA strands were purchased with a 5′-amino modification from the Midland Certified Reagent company. Sequences for all six 26-mers and their respective designations are given in Table I below together with the respective name/identifier by which the sequences are listed in the enclosed Sequence Listing
  • succinimidyl 4-hydrazinonicotinate acetone hydrazone in DMF (SANH, SolulinkTM) was added to the antibodies at variable molar excess of (1000:1 to 5:1) of SANH to antibody. In this way the number of hydrazide groups introduced to the antibodies was varied.
  • succinimidyl 4-formylbenzoate in DMF (SFB, SolulinkTM) was added at a 20-fold molar excess to 5′ aminated 26mer oligomers in PBS. This ratio of SFB to DNA ensured complete reaction of the 5′ amine groups to yield 5′ aldehydes.
  • Varied oligomer (strand A1′) loading unto ⁇ -human IL-4 was measured by gel mobility shift assay (see FIG. 1 Panel b).
  • the average number of attached oligonucleotides can be controlled.
  • FACS fluorescence activated cell sorting
  • VL3 and A-20 cells were incubated for 20 min. on ice with 0.5 ⁇ g of FITC-conjugated Rat Anti-Mouse CD90.2 (Thy1.2, BD Pharmingen, clone 30-H12, catalog #553012) in 100 ⁇ L PBS-3% FCS. Cells were also incubated with equimolar amounts of ⁇ -CD90.2/FITC-DNA conjugates characterized by various FITC-DNA loadings. Cells were washed once with PBS-3% FCS and then were analyzed by flow cytometry on a BD FACSCantoTM instrument running the BD FACSDivaTM software.
  • FIG. 3 FACS plot (Panel a) and histograms (Panel b) comparing ⁇ -CD90.2/FITC-DNA conjugates with the commercially-available FITC ⁇ -CD90.2 antibody (no DNA) are shown.
  • the conjugates bind to VL3 cells (100%) with minimal non-specific interactions with A20 (1.3%).
  • the overall fluorescent intensities are lower by a factor of 10, with slightly higher non-specific binding to A20.
  • the histogram of the mean fluorescent intensities for various FITC-DNA loadings illustrated in Panel b shows that the fluorescence increases are roughly linear when the number of DNA strands is increased from 1 to 2 to 3, corresponding to the 1, 2 and 3 chromophores (1 per strand). For higher loadings, the fluorescence plateaus and then decreases.
  • each strand of DNA is attached to one fluorophore only (i.e. conjugates with one DNA strand has a fluorophore to antibody ratio of 1:1) whereas the commercial antibodies generally have more than one fluorophore per antibody (i.e. fluorescent antibodies have a fluorophore to antibody ratio>1).
  • the factor of 10 less fluorescence should not be strictly interpreted as a 10 ⁇ reduction in the binding affinity of the DNA antibody conjugates, although it is possible that the oligomer steric effects discussed earlier do account for some reduction in relative fluorescence intensity.
  • Direct measurement of the affinity of the DNA antibody conjugate compared with the corresponding unmodified antibody using methods like Surface Plasmon Resonance (SPR) can provide more conclusive information.
  • a further optimization of polynucleotides loading of the polynucleotide-encoded-antibodies was performed as follows. Two different lengths of complementary polynucleotides were invested. One set had an overlap of 16 bases, the other an overlap of 20 bases. Orthogonal DNA sequences for set of 16 or 20 were designed according to procedures exemplified in Example 8 below, and it was discovered empirically that 16 bases did not have the variability in the total number of sequences possible to generate large numbers of orthogonal sequences. In moving to 20 bases, the initial pool of possible sequences dramatically increased and computing orthogonal sequences seemed to be much easier. It should be noted that the total number of possible sequences is exponential, (4 n , where n is the length of the complementary region).
  • DNA microarrays were printed via standard methods by the microarray facility at the Institute for Systems Biology (ISB—Seattle, Wash.) onto amine-coated glass slides.
  • the DNA microarrays were printed with various combination of oligomers having sequences SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, and SEQ ID NO 18,
  • Typical spot size and spacing were 150 and 500 ⁇ m, respectively.
  • Poly-lysine slides were made in house. Blank glass slides were cleaned with IPA and water in a sonication bath for 10 minutes each. They were then treated with oxygen plasma at 150 W for 60 sec., and then quickly dipped into D 1 water to produce a silanol terminated, highly hydrophilic surface. After drying them with a nitrogen gun, poly-L-lysine solution (Sigma P8920, 0.1% w/v without dilution) was applied to the plasma treated surfaces for 15 minutes, and then rinsed off with D 1 water for several seconds. Finally, these treated slides were baked at 60° C. for 1 hr. These slides were then sent to ISB and printed as described above.
  • FIG. 5 is an example of using DNA-encoded streptavidin to perform cell sorting experiments.
  • the DNA-encoded streptavidin is first exposed to its ligand, biotin labeled protein at a ratio of 4:1 biotin-MHC: DNA-encoded streptavidin.
  • the protein is the major histocompatiblity complex (MHC).
  • MHC major histocompatiblity complex
  • the tetramer is allowed to bind to the substrate for 30 minutes and rinsed in PBS before subsequence exposure of 2 ⁇ 10 6 cells onto the array.
  • DNA-encoded MHC is first allowed to bind to the same number of cells on ice for 20 min. before subsequent exposure to the underlying DNA array.
  • the cell capture efficiencies between the two panels are apparent. Solution phase capture for pMHC complexes is much higher than the panning analog. Of notice is the enhanced cell capture efficiency of the latter series of events.
  • the polynucleotide-encoded protein approach for spatially localizing antibodies was demonstrated using three identical goat anti-human IgGs, each bearing a different molecular fluorophore and each encoded with a unique DNA strand. A solution containing all three antibodies was then introduced onto a microarray spotted with complementary oligonucleotides. After a two-hour hybridization period and substrate rinse, the antibodies self-assembled according to Watson-Crick base-pairing.
  • antibody microarrays were generated by first blocking the DNA slide with 0.1% BSA in 3 ⁇ SSC for 30 minutes at 37° C. The slides were washed with dH 2 O and blown dry. A 30 ⁇ l solution containing DNA-antibody conjugates (3 ⁇ SSC, 0.1% SDS, 0.1% BSA, 15 ng/ ⁇ l of each conjugate) was sandwiched to the array with a microscope slide, and incubated at 37° C. for 4 hours. Arrays were then washed first in 1 ⁇ SSC, 0.05% SDS at 37° C. with gentle agitation, then at 0.2 ⁇ SSC, then finally at 0.05 ⁇ SSC. The slides were blown dry and scanned with a Gene Pix 4200 A two-color array scanner (Axon InstrumentsTM).
  • the DNA-encoded 1° antibody (15 ng/ ⁇ l), antigen (3 ng/ ⁇ l) and fluorescently-labeled 2° antibody (0.5 ng/ ⁇ l) were combined in a single tube. After 2 hour incubation at 37° C., the formed antibody-antigen-antibody complexes were introduced to the microarrays as described above in Example 3. Subsequent wash steps and visualization were identical
  • FIGS. 6 and 7 The results are shown in FIGS. 6 and 7 , wherein a spatially encoded-protein array with a scale bar that corresponds to 1 mm is shown.
  • the antibodies assemble with the DNA on the substrate thus converting the >900 spot complementary DNA chip into a multi-element antibody microarray (see FIG. 7 ). This observation implied that quite large antibody arrays can be assembled in similar fashion.
  • any protein array is likely be limited by interference from non-specific binding of proteins.
  • three antibodies were similarly introduced onto a microarray: two antibodies having complementary DNA-labeling spotted oligonucleotides and a third unmodified antibody.
  • a microarray was simultaneously exposed to goat ⁇ -human IgG-Alexa488/A1′, goat ⁇ -human IgG-Alexa647/C1′ polynucleotide-encoded conjugates and goat ⁇ -human IgG-Alexa594 with no pendant DNA.
  • the slide was not thoroughly rinsed following hybridization and accordingly a high background signal due to non-specific adsorption of non-encoded fluorescently-labeled antibody was observed.
  • FIG. 8 is an illustration of the resistance of the polynucleotide encoded-protein approach towards non-specific protein absorption.
  • spot B1 did not have fluorescence from non-complementary IgG conjugates nor did it exhibit fluorescence from proteins not encoded with DNA (goat ⁇ -human IgG-Alexa594).
  • DNA sequences were designed with the objective of minimizing any intra- and intermolecular interactions between the sequences and the complementary targets, at 37° C.
  • the computational design was performed using the paradigm outlined by Dirks et al. (Dirks, R. M.; Lin, M.; Winfree, E.; Pierce, N. A. Nucleic Acids Research 2004, 32, (4), 1392-1403).
  • six orthogonal sequences have been designed and empirically verified and are reported in Table 2.
  • VL-3 T cells thymic lymphoma line (Groves, T.; Katis, P.; Madden, Z.; Manickam, K.; Ramsden, D.; Wu, G.; Guidos, C. J. J. Immunol. 1995, 154, 5011-5022)
  • A20 B cells mouse B cell lymphoma (Kim, K. J.; Langevin, C. K.; Merwin, R. M.; Sachs, D. H.; Asfsky, R. J. Immunol. 1979, 122, 549-554), purchased from ATCC) were engineered to express mRFP and EGFP, respectively, using standard retroviral transduction protocols.
  • Antibodies against surface markers for each of these cell lines, ⁇ -CD90.2 for VL-3 and ⁇ -B220 for A20 (eBioscience), were encoded as described above with DNA strands A1′ and B1′, respectively.
  • cells were passaged to fresh culture media [RPMI 1640 (ATCC) supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and 0.05 mM ⁇ -mercaptoethanol] at a concentration of 10 6 cells/100 ⁇ l media and incubated with DNA-antibody conjugate (0.5 ⁇ g/100 ⁇ l) for 30 minutes on ice. Excess conjugate was removed from the supernatant after centrifugation, after which cells were resuspended in fresh media.
  • RPMI 1640 ATCC
  • DNA-antibody conjugate 0.5 ⁇ g/100 ⁇ l
  • CD4+ and CD8+ T cells were purified from EGFP and dsRed transgenic mice (obtained from Jackson Laboratories), respectively, using standard magnetic bead negative selection protocols and the BD IMagTM cell separation system. Prior to polynucleotide-encoded based fractionation, the purity of these populations was analyzed by FACS and found to be greater than 80%.
  • GFP-expressing B cells (106/100 ⁇ l) were located on B1 spots after labeling with ⁇ -B220-B1′ (0.5 ⁇ g/100 ⁇ L).
  • ⁇ -B220-B1′ 0.5 ⁇ g/100 ⁇ L.
  • a TNF- ⁇ ELISA pair with C1′-encoded 10 and APC-labeled 2° antibodies were introduced along with 0.5 ng/ ⁇ l FITC-labeled A1′ and allowed to hybridize for a period of 30 minutes at room temperature.
  • the slide was then rinsed with TBS+MgCl 2 and visualized via brightfield and fluorescence microscopy.
  • the cell panning experiments were performed in parallel; 5 ⁇ g of a-CD3/C3′ conjugate in 1 ml RPMI media was incubated on a microarray for 1 hour on ice before rinsing in 0.5 ⁇ PBS, then deionized water. The slide was not blown dry, but gently tapped on the side to remove the majority of the excess solution, keeping the array hydrated. Jurkats (5 ⁇ 10 6 /200 ⁇ L) were immediately placed on the array for one hour on ice. Subsequent wash and visualization steps are identical.
  • Panels a and b show brightfield images showing the efficiency of the homogeneous cell capture process according to an embodiment of the methods and systems herein disclosed.
  • Panel a a homogeneous assay is described in which DNA labeled antibodies are combined with the cells, and then the mixture is introduced onto the spotted DNA array microchip.
  • Panel b DNA labeled antibodies are first assembled onto a spotted DNA array, followed by introduction of the cells. This heterogeneous process is similar to the traditional panning method of using surface bound antibodies to trap specific cells.
  • the polynucleotide-encoded protein based cell sorting was compared with panning by evaluating homogeneous cell capture (solution phase cell capture) and heterogeneous capture of cells (surface confined cell capture).
  • the homogeneous DNA-encoded protein method exhibited a higher cell capture efficiency.
  • the increase in capture efficiency can be attributed to several factors.
  • the DNA-antibody conjugates are allowed to properly orient and bind to the cell surface markers in solution.
  • Cell capture is not driven by antibody to cell surface marker interactions, but rather by the increased avidity of the multivalent DNA-antibody conjugates for the complementary DNA strands on the microarray through cooperative binding, greatly increasing capture efficiency.
  • Similar trends have been reported for nanoparticle, DNA hybridization schemes (Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760).
  • panning methods which are analogous to a heterogeneous DNA-antibody defined arrays herein disclosed, the capture agents are restricted to adopt a random orientation on the surface. The activity of the antibodies is reduced, simply because of improper orientation for interaction with the cell surface markers, decreasing maximum avidity and cooperation with neighboring antibodies.
  • Panel c brightfield and fluorescence microscopy images of multiplexed cell sorting experiments are shown, where a 1:1 mixture of mRFP-expressing T cells (red channel) and EGFP-expressing B cells (green channel) is spatially stratified onto spots A1 and C1, corresponding to the encoding of ⁇ -CD90.2 and ⁇ -B220 antibodies with A1′ and C1′, respectively.
  • two unique DNA strands were conjugated to antibodies raised against the T cell marker CD90.2 (Thy1.2) and the B cell marker CD45R (B220), respectively.
  • Multiplexed DNA-antibody-based cell sorting was demonstrated by spatially separating a 1:1 mixture of monomeric Red fluorescent protein (Campbell, R.
  • mRFP mRFP-expressing T cells
  • VL-3 murine thymic lymphoma
  • EGFP-expressing B cells mouse B cell lymphoma
  • Panel d a fluorescence micrograph of multiplexed sorting of primary cells harvested from mice.
  • a 1:1 mixture of CD4+ cells from EGFP transgenic mice and CD8+ cells from dsRed transgenic mice is separated to spots A1 and C1 by utilizing polynucleotide-encoded conjugates ⁇ -CD4-A1′ and ⁇ -CD8-C1′, respectively.
  • Primary cells are usually more fragile than established cell lines. This is due to the fact that they have to be extracted (usually by enzymatic digestions) from the surrounding tissues, a process that can lead to decreased viability. Moreover, the culture process often selects for clones characterized by greatly increased viability as well as proliferation potential.
  • a generalized cell sorting technology must therefore also work on primary cells with minimal sample manipulation.
  • a synthetic mixture of CD4+ and CD8+ T cells was isolated via magnetic negative depletion from EGFP- and dsRED-transgenic mice, respectively. The mixture was stratified using ⁇ -CD4 and ⁇ -CD8 DNA-antibody conjugates. As shown in FIG. 10 d , the two cell types were separated to different spatial locations according to the pendant DNA encoding.
  • a multiparameter analysis (cells, mRNAs and proteins) was performed according to the strategy schematically described in FIG. 12 .
  • FIG. 11 is an illustration of the polynucleotide-encoded protein method for cell sorting and co-detection of proteins and cDNAs (mRNAs).
  • Antibodies against proteins (for cell sorting) or other proteins (including cell surface markers) are labeled with distinct DNA oligomers. These conjugates may then be combined with the biological sample (cells, tissue, etc.) where they bind to their cognate antigens.
  • parallel self assembly according to Watson-Crick base pairing, localizes the bound species to a specific spatial location allowing for multiplexed, multiparameter analysis.
  • An immunoassay was performed to illustrate the ability of polynucleotide-encoded protein herein disclosed to detect a plurality of targets, including chemically different targets.
  • the assay was performed for the detection of protein target IL4 and a polynucleotide B1.
  • an antibody specific to the protein target IL4 was encoded with polynucleotide C1 and a polynucleotide complementary to polynucleotide B1 was prepared.
  • the polynucleotide complementary to polynucleotide B1 was incubated together with the C1′ encoded anti-IL4 as described above.
  • a fluorophore secondary antibody to IL4 was introduced, and the simultaneous detection of the protein target IL4, and the oligonucleotide B1 performed as illustrated in FIG. 12 .
  • GFP-expressing B cells were tagged with B1′ DNA-encoded antibody conjugates and spatially located onto spots (B1) encoded with the complementary oligonucleotide.
  • the resulting brightfield and fluorescence microscopy images, shown in FIG. 13 demonstrate the validity of a platform according to an embodiment of the methods and systems herein disclosed, for simultaneously extending across different levels of biological complexity.
  • FIG. 13 shows microscopy images demonstrating simultaneous cell capture at spot B1 and multiparameter detection of genes and proteins, at spots A1 and C1, respectively.
  • the brightfield image shows EGFP-expressing B cells (green channel) located to spots B1, FITC-labeled (green) cDNA at A1, and an APC-labeled TNF- ⁇ sandwich immunoassay (blue) encoded to C1.
  • the scale bar corresponds to 300 ⁇ m.
  • a compatible surface may be an activated ester glass slide to which amine-DNA and proteins can both covalently attach.
  • the inventors have found that the loading capacity of these slides for DNA is diminished, resulting in poor signal intensity when compared with DNA printed on conventionally prepared amine slides.
  • unreacted esters are hydrolyzed back to carboxylic acids, which are negatively charged at normal hybridization buffers (pH 7), electrostatically reducing the DNA interaction.
  • the substrate conditions for high DNA loading onto the spotted substrates, and for complementary DNA loading on the antibodies can be optimized. This leads to highly sensitive sandwich assays for protein detection, as well as high efficiency cell sorting (compared with traditional panning).
  • Microfluidic-based assays offer advantages such as reduced sample and reagent volumes, and shortened assay times (Breslauer, D. N.; Lee, P. J.; Lee, L. P. Mol. BioSyst. 2006, 2, 97-112).
  • the surface binding assay kinetics are primarily determined by the analyte (protein) concentration and the analyte/antigen binding affinity, rather than by diffusion (Zimmermann, M.; Delamarche, E.; Wolf, M.; Hunziker, P. Biomedical Microdevices 2005, 7, (2), 99-110).
  • a microfluidics-based polynucleotide-encoded-protein approach was evaluated by bonding a polydimethylsiloxane (PDMS)-based microfluidic channel on top of a DNA microarray.
  • PDMS polydimethylsiloxane
  • microfluidic channels were fabricated from polydimethylsiloxane (PDMS) using conventional soft lithographic techniques.
  • the goal was to fabricate robust microfluidics channels that could be disassembled after the surface assays were complete for optical analysis.
  • Master molds were made photolithographically from a high resolution transparency mask (CadArt) so that the resulting fluidic network consisted of 20 parallel channels each having a cross-sectional profile of 10 ⁇ 600 ⁇ m and were 2 cm long. This corresponds to channel volumes of 120 nl.
  • a silicone elastomer (Dow Corning Sylgard 184TM) was mixed and poured on top of the mold.
  • the PDMS was removed from the mold and sample inlet and outlet ports punched with a 20 gauge steel pin (Technical InnovationsTM).
  • the microfluidic channels were then aligned on top of the microarray and bonded to the substrate in an 80° C. oven overnight.
  • Microfluidic devices were interfaced with 23 gauge steel pins and TygonTM tubing to allow pneumatically controlled flow rates of ⁇ 0.5 ⁇ l/min.
  • TBS Tris Buffered Saline
  • Each channel was blocked with 1.0% BSA in TBS prior to exposure to DNA-antibody conjugates or immunoassay pairs for 10 minutes under flowing conditions.
  • After a 10 minute exposure to conjugates or antigens under flowing conditions channels were washed with buffer for 2 minutes and the microfluidics disassembled from the glass slide in order to be scanned. Immediately prior to imaging, the entire slide was briefly rinsed in TBS, blown dry and imaged on an array scanner as described above.
  • a non-fluorescent, DNA-encoded 1° antibody was combined with antigen and a fluorescently labeled (no DNA) 2° antibody.
  • a fluorescent signal will be spatially encoded only if an antibody-antigen-antibody sandwich is successfully formed in homogeneous solution and localized onto the microarray.
  • DNA-encoded antibody sandwich assays self-assembled to their specific spatial locations where they were detected, as shown in FIG. 15 a .
  • This multi-protein immunoassay also took 10 minutes to complete.
  • FIG. 15 b and FIG. 15 c The results are shown in FIG. 15 b and FIG. 15 c wherein visualization was performed using a fluorescent 2° antibody (panel b) and Au electroless deposition as a visualization and amplification strategy (panel c), respectively.
  • the assay peaked with a sensitivity limit of around 1 nM on slides printed at saturating concentrations of 5 ⁇ M of complementary DNA (data not shown).
  • a sensitivity limit of around 1 nM on slides printed at saturating concentrations of 5 ⁇ M of complementary DNA (data not shown).
  • complementary DNA data not shown.
  • primary DNA-antibody conjugates were laid down first on the surface, before exposure to antigen and secondary antibody. This is because at lower concentrations of antigen, the signals decrease, due to the high ratio of antigen-unbound primary antibody competing with antigen-bound primary for hybridization to the DNA array.
  • excesses were washed away before subsequent exposure to antigen and secondary antibody, increasing signal.
  • microfluidics-based Au amplification experiments were performed in a manner similar to the one disclosed above, with the notable exception that a biotin-secondary antibody was used instead of a fluorescently labeled antibody.
  • Au-streptavidin Naprobes
  • the entire slide was then amplified with gold enhancer kit (Nanoprobes) according to manufacturer's protocol.
  • IL-2 interleukin Adopting these improvements, the presence of IL-2 interleukin can be readily detected at a concentration limit less than 10 fM ( FIG. 15 c ), representing at least a 1000-fold sensitivity increase over the fluorescence based microfluidics immunoassay.
  • Digital proteomics were detected using DNA encoded antibody in combination with DNA arrays according to the strategy described in FIGS. 16 and 17 .
  • assays have been performed to detect certain cytokines (IL2, TNF- ⁇ and IFN- ⁇ ). All experiments were performed in a manner analogous to the 3-step immunoassays described above with the notable exception that a 40 nm Au particle is used and the detection scheme is a dark field light scattering microscope.
  • the 2° antibodies were labeled with 40 nm Au nanoparticles, which are readily detected by dark-field light scattering microscopy. More specifically a 40 nanometer Au nanoparticle-Streptavidin conjugate was used as the detection probe for the digital assay.
  • Detection of the relevant digital immunoassays was performed with the method illustrated in FIG. 18 .
  • scattered light is measured using a dark-field microscope objective.
  • the plasmonic response of even very small Au particles is readily picked detected.
  • the individual particles are counted either manually or using an automated software package for particle counting.
  • the scattering color of all of the particles is very similar—yellow-to-green. This is because the Au nanoparticles (10) are of a fairly narrow size range ( ⁇ 60 nanometers diameter).
  • An optical filter can be utilized in the light scattering microscope to eliminate all other scattered colors and thus reduce background.
  • FIGS. 19 and 20 show the sensitivity of the digital assay performed according to an embodiment of the methods and systems herein disclosed.
  • the signal from this protein can be easily identified at concentrations as low as 100 attoMolar.
  • FIGS. 19 and 20 show the representative dark field images of TNF- ⁇ Digital immunoassays performed at different concentrations with a method and system herein disclosed. ImageTM, a scientific graph processing software provided by NIH, was used automatically count the particle numbers. The number of gold nanoparticles vs TNF- ⁇ concentration is plotted in the histogram of FIG. 20 .
  • cytokine proteins IL2, TNF- ⁇ and IFN- ⁇
  • human serum purchased from Sigma-Aldrich
  • AuNP based assay performed above.
  • FIG. 21 the images of Panel a were collected from a serum sample that was spiked with the three proteins: IFN- ⁇ ; TNF- ⁇ , and IL-2.
  • the images of Panel b are from a digital immunoassay that was measured from the serum of a healthy human according to an embodiment of the methods and systems herein disclosed. All three of these proteins are typically present at below-detectable concentrations in human serum.
  • TNF- ⁇ is below the detectable limit, but IFN- ⁇ and IL-2 are present at the few femtoMolar (10 ⁇ 15 M) concentration levels. This amount of protein is well below the detection limit of a conventional absorbance or fluorescent ELISA or even immunoassay performed with another embodiment of the methods and systems herein disclosed.
  • TNF- ⁇ exhibits the best signal intensity due to the high affinity of the 1° anti-TNF- ⁇ AB.
  • the background is near zero, and that the dynamic range of detected proteins is at least 10 6 .
  • These types of assays have been utilized to detect certain cytokines (IL2, TNF- ⁇ and IFN- ⁇ ) out of healthy human serum. This has not been previously possible, as those proteins are present (by our measurements) at a level of only 1-5 femtoM. It is to be noted that once the antibody/protein affinities have been characterized, these types of assays are absolute and quantitative—meaning that they do not require calibration.
  • FIG. 23 an embodiment is illustrated wherein the technology is applied to the detection of the biomarker pten, which is an important marker in glioblastoma (brain cancer).
  • Methods and systems herein disclosed have been used in a fluorescent based assay first to calibrate a device by using recombinant pten as the standard (FIG. 23 , Panels a and b).
  • the calibration of the protein pten is shown with the left 7 bins, ranging from 25 nM to 375 pM.
  • the right 3 bins represent pten-positive and pten-null samples.
  • the inventors then proceeded to quantitate pten expression levels in the glioblastoma cell line U87 (Panels a and c). It is apparent that reasonable levels of pten (1 nM) are detectable using methods and systems herein disclosed as illustrated in FIG. 23 .
  • liver toxicity studies can be performed using the methods and systems herein disclosed.
  • the results in liver are particularly interesting because the liver is the second largest organ in the human body (the first is the skin) and is in constant contact with the blood. Thus it is highly likely that perturbations at this organ will result in a notable change in the amount of protein biomakers found in serum that are liver specific.
  • FIG. 24 An exemplary pathway from serum biomarker discovery to clinical validation is illustrated in FIG. 24 .
  • a first step in serum biomarker discovery involves the proteomic analysis of the proteins in the blood via current state of the art in tandem mass spectrometry. Accordingly an initial protein list of about 25 proteins was discovered to be upregulated or downregulated following administration of high levels of acetomaniphen to murine model using tandem mass spectrometry ( FIG. 24 Panel a ( 1 ). In particular, the peptides that are detected are mapped back to generate a list of candidate protein biomarkers. These biomarkers and their associated capture agents (antibodies) are screened and verified using the state of the art in surface plasmon resonance. In particular, a particularly effective antibody pairs was validated using SPR ( FIG. 24 Panel b ( 2 ).
  • these verified protein capture agents are translated into a microfluidic system according to an embodiment herein disclosed, allowing the monitoring of serum biomarkers in blood.
  • a chip was designed and tested to detect 4 liver specific serum proteins and 3 immune specific proteins from whole serum ( FIG. 24 Panel c ( 3 ). The results shown in FIG. 24 indicate that all targets were detected without difficulty from serum.

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