Ultrasensitive multiplex digital elisa

Abstract

Described herein is a digital protein detection platform using an inexpensive, use and throw track-etched polycarbonate (PCTE) membrane. Wicking is used to fill the though holes of a piece of the membrane and conformally stick it to a sticky surface to form thousands of microwells without generating air bubbles. The digital biosensor achieved a dynamic range of pM–fM with a limit of detection of 100 aM. This digital "on" and "off" readout format circumvents any bias that usually occurs in other analog sensors based on absolute intensity, current and voltage measurements.

Description

[0001] ULTRASENSITIVE MULTIPLEX DIGITAL ELISA

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application Nos. 63 / 516,620, filed on July 31 , 2023, and 63 / 568,351, filed on March 21 , 2024, each of which is incorporated by reference herein in its entirety.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number CA241684 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Absolute and accurate quantification of several protein biomarkers present in a small volume of blood, cerebrospinal fluid, saliva, or urine can enable early disease diagnostics, prognosis, and monitoring of further treatment in patients. Current state of the art technique for protein detection is Enzyme-Linked Immunosorbent Assay (ELISA) which suffers from low limits of detection (~nM-pM) beyond which several clinically relevant protein biomarkers (~pM-fM) of cancer, neurodegenerative, cardiovascular, inflammatory, and autoimmune diseases remain undetected. To solve this issue, single-molecule arrays (Simoa) have been developed in which a protein molecule is sandwiched between an antibody-coated magnetic bead and an enzyme- conjugated detection antibody to form an immunocomplex. Subsequently, the immunocomplex is sampled into thousands of fL reaction chambers keeping the total protein concentration within the Poisson limit such that each bead contains either one or zero molecule. The fluorescence signal generated from the enzymatic reaction gives a digital readout providing an absolute quantification of the protein biomarker. However, Simoa are mostly performed in specially designed microwells which requires complex and expensive cleanroom-based microfabrication techniques, costly and bulky experimental setup, fluidics control setups such as syringe pump, centrifuge, vacuum, or other specialized equipment. Also, the total number of microwells present in the device which determines the sensor's dynamic range is limited by the master mold which is cumbersome to tune. Moreover, these complex workflows require a good amount of expertise and present a bottleneck for using them for point-of-care applications in resource-limited areas where a disposable sensor is preferable.

[0008] For digital quantification of proteins, the focus has been on the fabrication of small microwell-based picolitre reactors as ELISA has a linear amplification rate as opposed to PCR which is exponential. The ultrasmall volume of a reactor enhances the localized concentration of fluorescent products which can be subsequently detected by a regular fluorescence microscope. Several other methods have been reported in the literature apart from Simoa for digital assay. One common theme in all of them is the labor-intensive and complex microfabrication techniques used to form an array of thousands of microwells using very expensive and bulky instruments. Moreover, it is common to trap plenty of air bubbles into the microwells, thus impeding quantification. Hence, vacuum pumps and costly surface modifications / coatings are needed to alleviate them. Also, quite often the maximum number of microwells that can be obtained on a chip is fixed by the master mold which presents a limit for using these biosensors for massive multiplexing applications.

[0009] SUMMARY

[0010] One embodiment described herein is a method of detecting one or more analytes in a sample, the method comprising: (a) incubating a plurality of magnetic beads conjugated to one or more capture antibodies with a sample comprising a plurality of analytes, wherein the analytes are bound to a detection antibody and wherein at least one of the analytes bind to at least one of the one or more capture antibodies to form one or more immunocomplexes; (b) injecting the one or more immunocomplexes into a device comprising a fluid, a plurality of microwells affixed to a substrate, and a permanent magnet, wherein the plurality of microwells are formed by adhering a track-etched polycarbonate (PCTE) membrane on top of the substrate; (c) agitating the device for a period of time to position the one or more immunocomplexes into the microwells; (d) injecting one or more detection reagents into the device, wherein the detection reagents are converted into one or more signals after a period of incubation; (e) injecting an oil into the device, wherein the oil seals the microwells; and (f) measuring the at least one signal, wherein the at least one signal is indicative of the presence of the one or more immunocomplexes. In one aspect, the magnetic beads are conjugated to the one or more capture antibodies via a streptavidin-biotin interaction. In another aspect, the detection antibody is conjugated to an enzyme. In another aspect, the enzyme converts the one or more detection reagents into the one or more signals. In another aspect, the at least one signal is a fluorescence signal. In another aspect, the plurality of analytes is proteins. In another aspect, the track-etched polycarbonate (PCTE) membrane prevents bubble formation during immunocomplex injection. In another aspect, the substrate is polydimethylsiloxane (PDMS) coated on glass. In another aspect, the fluid is a droplet on top of the substrate. In another aspect, measuring the at least one signal comprises imaging the device with a fluorescent microscope. In another aspect, the method comprises simultaneously detecting at least two analytes in sample. In another aspect, the one or more capture antibodies comprises at least two different antibodies. In another aspect, different analytes are bound to different detection antibodies that are conjugated to different enzymes. In another aspect, the different enzymes convert the one or more detection reagents into the one or more different signals.

[0011] Another embodiment described herein is a device for detecting analytes in a sample, comprising: a glass substrate coated with a silicone polymer; a buffered liquid droplet, wherein the droplet is on top of the glass substrate; and a track-etched polycarbonate (PCTE) membrane that is adhered to the silicone polymer and forms microwells. In one aspect, the silicone polymer is polydimethylsiloxane (PDMS). In another aspect, the silicone polymer is mixed in a 10:1 weight ratio with a curing agent before coating the glass substrate. In another aspect, the silicone polymer coating is about 200 pm thick. In another aspect, the device comprises at least 1 ,000 microwells. In another aspect, the device comprises at least 1 ,000,000 microwells.

[0012] Another embodiment described herein is a method of making the device described herein, the method comprising immersing the PCTE membrane into the buffered liquid droplet until the PCTE membrane adheres to the silicone polymer coating. In one aspect, the method does not generate air bubbles.

[0013] DESCRIPTION OF THE DRAWINGS

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] FIG. 1 shows a schematic of digital ELISA platform

[0016] FIG. 2 shows duplex digital ELISA using B G and B-ALP and the colocalization of these targets in the assay.

[0017] FIG. 3A-I show schematics of the workflow of the digital ELISA platform using PCTE membrane. FIG. 3A shows a drop of PBS is put on top of a spin-coated glass slide containing a PDMS thin film. FIG. 3B shows the membrane is gently inserted into the solution to allow liquid loading into the pores without trapping bubbles. FIG. 3C shows the membrane is conformally fixed to the PDMS layer to form microwells. FIG. 3D shows an immunocomplex of the protein sandwiched between a detection and capture antibody. FIG. 3E shows suspended magnetic beads on top of membrane. FIG. 3F shows the increase in bead filling efficiency by using a permanent magnet and mechanical shaking. FIG. 3G-H show the injection of ELISA reagents (FIG. 3G) and oil (FIG. 3H) inside the microfluidics channel. FIG. 31 shows the imaging of the microwells.

[0018] FIG. 4A-E show images of the assay. FIG. 4A shows a bright field image of the PCTE membrane at 20* resolution. FIG. 4B shows distribution pore size of the previous image. FIG. 4C shows a bright field image of the microwells at 4* resolution showing ~20000 microwells in one frame. FIG. 4D shows a fluorescence image of the microwells when filled with 1 mM fluorescein. FIG. 4E shows an image of the device. FIG. 4F shows a fluorescence image at 10* resolution depicting all microwells filled with 1 mM fluorescein.

[0019] FIG. 5A-D shows single molecule enzymology studies using the digital platform. FIG. 5A shows a fluorescence image showing BpG and FDG reaction in 6 microwells at 50* resolution. FIG. 5B shows temporal intensity plots of 3 randomly selected microwells. FIG. 5C shows fluorescence image at 10* resolution and the inset shows zoomed in image. FIG. 5D shows a histogram of fluorescence intensities of microwells present in the inset showing bimodal distribution depicting the presence of a 1 and 2 enzymes trapped in a microwell.

[0020] FIG. 6A-D show images and data. FIG. 6A-B show fluorescence images at 10* resolution depicting the reaction of BpG and FDG (FIG. 6A) and B-ALP and 4-MUP (FIG. 6B) at various concentration of B G and B-ALP. FIG. 6C-D show semi-log graph showing the percentage of fluorescent microwells as a function of the concentration of BpG (FIG. 6C) and B- ALP (FIG. 6D) captured in the microwell array.

[0021] FIG. 7A-D show images and data. FIG. 7A shows images depicting the filling of microwells by using different concentration of magnetic beads. FIG. 7B shows bar graph showing number of beads captured per microwell using different concentration of magnetic beads. FIG. 7C shows image showing increase in bead filling using an external permanent magnet coupled with mechanical shaking. FIG. 7D shows a graph showing increase in single bead capture rate from -30% to 60%.

[0022] FIG. 8A-E shows digital ELISA assay between the BpG which was captured by streptavidin coated magnetic beads and FDG inside the microwells. FIG. 8A shows a brightfield image showing the empty as well as filled microwells using magnetic beads. FIG. 8B shows a fluorescence shows the reaction of BpG (2 pM concentration) and FDG. FIG. 80-D shows a 50* brightfield (FIG. 80) and its corresponding fluorescence image (FIG. 8D) confirming that only the magnetic bead containing microwells were in “on” state. FIG. 8E shows a composite image of both brightfield and fluorescence (Red) at 20 fM alongside with a zoomed in image. The zoomed in image shows the presence of empty microwells, microwells that contain a magnetic bead and are in “off’ state and microwells that contain a magnetic bead and are in “on” state (red color superimposed).

[0023] FIG. 9A-E show fluorescence images displaying the Digital ELISA assay between the BpG and FDG at various concentration of B[3G captured by streptavidin coated magnetic beads. FIG. 9F shows a log-log plot showing 4 logs of dynamic range and 200 aM limit of detection.

[0024] FIG. 10A-I show duplex digital ELISA using B G and B-ALP at different concentrations. FIG. 10A, FIG. 10D, and FIG. 10G show fluorescence illustrating B G and its reaction at a concentration of 300 pM, 3 pM, and 30 pM respectively. FIG. 10B, FIG. 10E, and FIG. 10H show fluorescence illustrating B-ALP and its reaction at a concentration of 700 pM, 70 pM, and 7 pM respectively. FIG. 10C, FIG. 10F, and FIG. 101 show composite images of each reaction sets showing both B-ALP (blue) and B[3G (green) fluorescence.

[0025] FIG. 11A-C show schematics of a comparison of two workflows for using the through holes of the PCTE membrane to obtain microwells for a digital assay. FIG. 11A is a diagram showing an example of a device where the membrane is first conformally sealed on the PDMS layer and then wetted with water. A PDMS micro-channel is applied to the top of the wetted membrane, and a fluorescein solution is inserted into the microchannel which is followed by oil sealing. The fluorescence image shows trapped bubbles inside the microwells. Scale bar is 20 pm. FIG. 11 B is a diagram showing an example of a device where the membrane is first wetted with water to remove all trapped air bubbles due to wicking. Afterwards, the membrane is conformally assembled over the PDMS layer and a PDMS micro-channel is applied to the top of the wetted membrane. Lastly, a fluorescein solution is inserted into the micro-channel which is followed by oil sealing. The fluorescence image shows that the microwells are filled without trapped bubbles. Scale bar is 20 pm. FIG. 11C is a diagram showing a Mem-dELISA device workflow for utilizing the through holes of the membrane for digital ELISA.

[0026] FIG. 12A-E show figures for the optimization of micro-channel height of a Mem-dELISA device for perfect sealing of microwells. FIG. 12A is a diagram showing schematics showing the height of the PDMS micro-channel used for injecting fluorescent liquid and oil for sealing of microwells. FIG. 12B is a graph showing the parametric variation of micro-channel height to completely remove fluorescent liquid by oil. The x-axis units are pm. The bars on the left side of the x-axis tick mark represent “Water Layer Removed,” while the bars on the right side represent “Water Island Removed.” The oil sealing method was very effective to remove the remnant water layer in hydrophobic membrane at a gap height of 300 pm and 100 pm. FIG. 12C and FIG. 12D show images depicting the sealing of microwells by oil after filling them with fluorescent solution for both hydrophilic (FIG. 12C) and hydrophobic (FIG. 12D) PCTE membranes. FIG. 12E shows a group of confocal z-stack images, of microwells filled with 1 mM fluorescein solution and sealed with oil depicting no crosstalk between individual microwells. Scale bar is 200 pm for FIG. 12C- D and 10 pm for FIG. 12E.

[0027] FIG. 13A-J show images and graphs of single molecule amplification results of B-ALP and B-pG in Mem-dELISA device. FIG. 13A are images that show sequential fluorescent images for digital enzymatic amplification for B-ALP at 10,15 and 25 mins. FIG. 13B is a graph that show the intensity distribution of each microwell extracted from the 25 mins image and fitted with sum of four Gaussians corresponding to microwells having 0,1 ,2 and 3+ B-ALP molecules. FIG. 13C is a graph that shows the comparison of the fraction of microwells having 0,1 ,2 and 3+ B-ALP molecules obtained experimentally by Gaussian fitting and obtained by Poisson’s distribution having A of 0.42. FIG. 13D are images of temporal fluorescence for digital enzymatic amplification for B-pG at 10,15 and 25 mins. FIG. 13E is a graph that shows the intensity distribution of each microwell extracted from the 25 mins B-pG image and fitted with sum of four Gaussians corresponding to microwells having 0,1,2 and 3 B-pG molecules. FIG. 13F shows the comparison of the fraction of microwells having 0,1 ,2 and 3 B-ALP molecules obtained experimentally by Gaussian fitting and obtained by Poisson’s distribution having A of 0.224. FIG. 13G are images that show the representative fluorescence of serial concentration dilution (100 pM - 0.1 pM) of B- PG. FIG. 13H is a graph showing the corresponding log-log plot of the % active wells as a function of B- G protein concentrations (n = 3). FIG. 131 are images that show the fluorescence of serial dilution in concentration (100 pM - 0.1 pM) of B-ALP molecules and FIG. 13J is a graph showing the corresponding plot of % active wells vs B-ALP concentration. Scale bar is 50 pm for FIG. 13A & FIG. 13D and 200 pm in FIG. 13G & FIG. 131.

[0028] FIG. 14A-G show graphs and images for the digital protein detection of B-ALP and B-pG protein molecules using magnetic beads in Mem-dELISA device. FIG. 14A is a surface plot that shows the Magnetic field from a permanent magnet obtained from COMSOL Multiphysics simulations. FIG. 14B is a graph that shows the variation of magnetic flux density (T) as a function of axial distance from the midpoint of the magnet obtained from FEM simulations. The experimentally obtained images of magnetic beads are divided in 3 zones. In I and II, magnetic beads chain formation was obtained which significantly reduces bead capture. Regime III is the working range whereas in regime IV the magnetic force from the magnet did not have any impact on the beads. FIG. 14C shows the loading efficiency of magnetic beads in microwells without using a magnet for three different magnetic bead densities: 2 * 107 / mL (left), 2.4 * 107 / mL (middle), and 1 x lO8 / mL (right). FIG. 14D shows a comparison of magnetic beads loading in microwells with gravity settling and using a magnet with mechanical shaking for 15 minutes. FIG. 14E is a log-log graph of the percent of fluorescent wells and variation with B- G concentration (n = 4). FIG. 14F is a log-log graph of the percent of fluorescent wells and B-ALP protein concentration (n = 4). FIG. 14G are images that show an overlay of bright field and red fluorescent image at 10 resolution. The zoomed image depicts the active wells (red) and inactive wells. Scale bar is 200 pm.

[0029] FIG. 15A-T show graphs and images of dual color protein detection using magnetic beads in Mem-dELISA device. FIG. 15A is a figure of dual color protein detection. FIG. 15B is an image showing the overlay image of the negative control (mixture of 4-MUP and RDG). *Note that even though we look at blue fluorescence to observe ALP amplification, the images are generated in green color for better visualization of the overlay image*. FIG. 15C and FIG. 15D are graphs showing the histogram of intensity distribution for negative control from both blue fluorescence (FIG. 15C) and red fluorescence (FIG. 15D) channels. The dotted line in both histograms represents the intensity value equal to mean + 5*standard deviation. FIG. 15E, FIG. 15H, FIG. 15K, FIG. 15N and FIG. 15Q are images showing the fluorescence from both filters along with the overlay image for the detection of B-ALP and B- G simultaneously in the Mem-dELISA device. The concentrations of both B-ALP and B-pG proteins were serially diluted while keeping their ratio constant. The B-ALP concentration varied from 1 pM to 100 aM while B-[3G concentration varied from 100 fM to 10 aM. FIG. 15F, FIG. 15G, FIG. 151, FIG. 15J, FIG. 15L, FIG. 15M, FIG. 150, FIG. 15P, FIG. 15R, and FIG. 15S are graphs showing the histograms of intensity distribution of microwells for both B-ALP (bins, FIG 15F, FIG. 151, FIG. 15L, FIG. 150, and FIG. 15R) and B- G (bins, FIG. 15G, FIG. 15J, FIG. 15M, FIG. 15P, and FIG. 15S) amplification in microwells with various concentration as mentioned in FIG. 15E, FIG. 15H, FIG. 15K, FIG. 15N, and FIG. 15Q. Note that FIG. 15E corresponds to FIG. 15F and FIG. 15G. FIG. 15H corresponds to FIG. 151 and FIG. 15J. FIG. 15K corresponds to FIG. 15L and FIG. 15M. FIG. 15N corresponds to FIG. 150 and FIG. 15P. The dotted line intensity was obtained from the negative control histogram. FIG. 15T is a graph showing B-ALP and B- G colocalization percentage and its comparison with double Poisson’s statistics prediction at various concentrations, mentioned in FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, and FIG. 15F. Scale bar is 200 pm for all images.

[0030] FIG. 16A-H show graphs and images of Mem-dELISA device utilization for performing single EV assay to study the effect of Paclitaxel drug treatment on breast cancer cell lines. FIG. 16A is a figure showing a schematic of the immunocomplex of EV sandwiched between CD63 coated magnetic beads and two reporter antibodies (EpCAM and GPC-1) conjugated with ALP and beta galactosidase enzyme, respectively. FIG. 16B are images showing the western blots of CD63, GPC-1 and EpCAM proteins from cell lysate as function of drug concentration (10 nM, 100 nM, and 500 nM) for MDA-MB-231 and MDA-MB-468 cell lines. FIG. 16C and FIG. 16D show the overlayed fluorescent images (red: GPC-1 and green: EpCAM) obtained from duplex digital ELISA of EVs as a function of drug dosage for both MDA-MB-231 and MDA-MB-468 cell lines, respectively. Left to right images represent untreated control, 10 nM,100 nM and 500 nM Paclitaxel drug treatment. FIG. 16E and FIG. 16G are graphs showing the plot of % of EpCAM and GPC-1 protein content on EVs as function of drug concentration for MDA-MB-231 and MDA- MB-468 cell lines, respectively. FIG. 16F and FIG. 16H are graphs showing the % of colocalized EVs (both EpCAM and GPC-1) as a function of drug concentration for MDA-MB-231 and MDA- MB-468 cell lines, respectively. Scale bar is 200 pm.

[0031] FIG. 17 is a diagram and image of the final assembled Mem-dELISA device.

[0032] FIG. 18 is a diagram showing that image processing involves an initial step aimed at noise reduction and the preliminary determination of the approximate coordinates of the microwell center. Following the identification of these coordinates, a 4 * 4 pixel square is generated as illustrated in the leftmost image. This square undergoes a translational shift of up to 2 pixels in both the x and / directions to obtain the maximum microwell intensity, thereby facilitating accurate determination of the microwell coordinates. A square, characterized by the maximum intensity, is subsequently selected as the focal region for acquiring the precise (x, y) coordinates of the microwell center.

[0033] FIG. 19 is an image and graph showing a bright field image of a PCTE membrane (top) and a graph showing the distribution of membrane pore sizes (bottom).

[0034] FIG. 20A-B show graphs of ALP and beta galactosidase amplification at various concentrations. FIG 20A is a graph showing bulk results of ALP amplification as a function of concentration using 2x diluted 4-MUP. FIG. 20B is a graph showing bulk amplification of beta galactosidase at various concentration while keeping the ALP concentration fixed at 70 pM. For this reaction 4-MUP and RDG were mixed together.

[0035] FIG. 21 A-F show schematics for the workflow of random encapsulation of B-[3G or B-ALP proteins followed by injection and oil sealing. FIG. 21A is a figure showing a drop of aqueous solution on top of a spin-coated glass slide containing a PDMS thin film. FIG. 21 B is a figure showing the membrane is conformally fixed to the PDMS layer. FIG. 21 C and FIG. 21 D are figures showing the loading of the microwells. FIG. 21 E is a figure showing the oil sealing of microwells. FIG. 21 F is a figure showing the imaging of the microwells.

[0036] FIG. 22 shows images of the random encapsulation of B-[3G or B-ALP molecules inside the microwells using 30 pM B- G and 100 pM B-ALP molecules. FIG. 23 shows a plot of the domain geometry for simulating a permanent magnet with magnetization of 750 kA / m.

[0037] FIG. 24 shows images of the bright field (left) and its corresponding fluorescence image at 50x resolution (right) confirming that only the magnetic bead containing microwells were in ‘on’ state.

[0038] FIG. 25A-B show images representative of a digital assay with a serial dilution using 1 pM - 10 aM of (FIG. 25A) B- G and (FIG. 25B) B-ALP. FIG. 25A are images representative of a digital assay with a serial dilution using 1 pM - 10 aM of B-[3G. FIG. 25B are images representative of a digital assay with a serial dilution using 1 pM - 10 aM of B-ALP.

[0039] FIG. 26A-H show plots representative scatter plots of fluorescence intensity (B-ALP and B-pG) of the microwell vs microwell ID for digital assay. The sample concentration was serially diluted while keeping the B-ALP to B- G ratio constant at 10. FIG. 26A is a scatter plot where the B-ALP concentration varied from 1 pM-100 fM. FIG. 26B is a scatter plot where the B- G concentration varied from 1 pM-100 fM. FIG. 26C is a scatter plot where the B-ALP concentration varied from 100fM-10fM. FIG. 26D is a scatter plot where the B-[3G concentration varied from 100fM-10fM. FIG. 26E is a scatter plot where the B-ALP concentration varied from 10 fM-1 fM. FIG. 26F is a scatter plot where the B-[3G concentration varied from 10 fM-1 fM. FIG. 26G is a scatter plot where the B-ALP concentration varied from 1fM-100 aM. FIG. 26H is a scatter plot where the B-[3G concentration varied from 1fM-100 aM.

[0040] FIG. 27 shows a schematic of the normalization scheme for protein co-localization assay for GPC-1 and Epcam proteins present on EVs using set theory.

[0041] FIG. 28A-D show images depicting the filling of microwells with beads. FIG. 28A is an image showing the filling by using 2 x 107beads / mL. FIG. 28B is an image showing the filling by using 2.4 x 107beads / mL. FIG. 28C is an image showing the filling by using 1 x 108beads / mL. FIG. 28D is an image showing an increase in bead filling at 2.4 x 107beads / mL concentration using an external permanent magnet coupled with mechanical shaking.

[0042] DETAILED DESCRIPTION

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0044] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0045] As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not.

[0046] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0047] As used herein, the term “or” can be conjunctive or disjunctive.

[0048] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0049] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0050] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about’ refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0051] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.

[0052] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0053] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0054] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.

[0055] As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0056] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0057] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0058] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments. As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0059] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0060] Described herein is a novel user friendly, power, and clean room lithography free digital protein detection platform using an inexpensive, use and throw track-etched polycarbonate (PCTE) membrane. As used herein, “track-etched” refers to a polycarbonate (PCTE) membrane formed by bombarding the polymer thin film with high energy particles. This results in “tracks” that are “etched” into the membrane, forming pores. Wicking is used to fill the though holes of a piece of the membrane and conformally stick it to a sticky surface to form thousands of microwells without generating air bubbles. The microwells were used to perform duplex digital enzymatic reaction of free-floating beta galactosidase and alkaline phosphatase enzymes. Subsequently, the bead filling inside the microwells was optimized using a permanent magnet and mechanical shaking. In the end, streptavidin functionalized magnetic beads were used to capture biotin-|3- galactosidase and digital ELISA was performed using fluorescein di p-D-galactopyranoside substrate. The digital biosensor achieved a dynamic range of pM-fM with a limit of detection of 100 aM. This digital “on” and “off” readout format circumvents any bias that usually occurs in other analog sensors based on absolute intensity, current and voltage measurements.

[0061] One embodiment described herein is a method of detecting one or more analytes in a sample, the method comprising: (a) incubating a plurality of magnetic beads conjugated to one or more capture antibodies with a sample comprising a plurality of analytes, wherein the analytes are bound to a detection antibody and wherein at least one of the analytes bind to at least one of the one or more capture antibodies to form one or more immunocomplexes; (b) injecting the one or more immunocomplexes into a device comprising a fluid, a plurality of microwells affixed to a substrate, and a permanent magnet, wherein the plurality of microwells are formed by adhering a track-etched polycarbonate (PCTE) membrane on top of the substrate; (c) agitating the device for a period of time to position the one or more immunocomplexes into the microwells; (d) injecting one or more detection reagents into the device, wherein the detection reagents are converted into one or more signals after a period of incubation; (e) injecting an oil into the device, wherein the oil seals the microwells; and (f) measuring the at least one signal, wherein the at least one signal is indicative of the presence of the one or more immunocomplexes. In one aspect, the magnetic beads are conjugated to the one or more capture antibodies via a streptavidin-biotin interaction. In another aspect, the detection antibody is conjugated to an enzyme. In another aspect, the enzyme converts the one or more detection reagents into the one or more signals. In another aspect, the at least one signal is a fluorescence signal. In another aspect, the plurality of analytes is proteins. In another aspect, the track-etched polycarbonate (PCTE) membrane prevents bubble formation during immunocomplex injection. In another aspect, the substrate is polydimethylsiloxane (PDMS) coated on glass. In another aspect, the fluid is a droplet on top of the substrate. In another aspect, measuring the at least one signal comprises imaging the device with a fluorescent microscope. In another aspect, the method comprises simultaneously detecting at least two analytes in sample. In another aspect, the one or more capture antibodies comprises at least two different antibodies. In another aspect, different analytes are bound to different detection antibodies that are conjugated to different enzymes. In another aspect, the different enzymes convert the one or more detection reagents into the one or more different signals.

[0062] Another embodiment described herein is a device for detecting analytes in a sample, comprising: a glass substrate coated with a silicone polymer; a buffered liquid droplet, wherein the droplet is on top of the glass substrate; and a track-etched polycarbonate (PCTE) membrane that is adhered to the silicone polymer and forms microwells. In one aspect, the silicone polymer is polydimethylsiloxane (PDMS). In another aspect, the silicone polymer is mixed in a 10:1 weight ratio with a curing agent before coating the glass substrate. In another aspect, the silicone polymer coating is about 200 pm thick. In another aspect, the device comprises at least 1 ,000 microwells. In another aspect, the device comprises at least 1 ,000,000 microwells.

[0063] Another embodiment described herein is a method of making the device described herein, the method comprising immersing the PCTE membrane into the buffered liquid droplet until the PCTE membrane adheres to the silicone polymer coating. In one aspect, the method does not generate air bubbles. It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0064] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0065] Clause 1 . A method of detecting one or more analytes in a sample, the method comprising:

[0066] (a) incubating a plurality of magnetic beads conjugated to one or more capture antibodies with a sample comprising a plurality of analytes, wherein the analytes are bound to a detection antibody and wherein at least one of the analytes bind to at least one of the one or more capture antibodies to form one or more immunocomplexes;

[0067] (b) injecting the one or more immunocomplexes into a device comprising a fluid, a plurality of microwells affixed to a substrate, and a permanent magnet, wherein the plurality of microwells are formed by adhering a track-etched polycarbonate (PCTE) membrane on top of the substrate;

[0068] (c) agitating the device for a period of time to position the one or more immunocomplexes into the microwells;

[0069] (d) injecting one or more detection reagents into the device, wherein the detection reagents are converted into one or more signals after a period of incubation; (e) injecting an oil into the device, wherein the oil seals the microwells; and

[0070] (f) measuring the at least one signal, wherein the at least one signal is indicative of the presence of the one or more immunocomplexes.

[0071] Clause 2. The method of clause 1 , wherein the magnetic beads are conjugated to the one or more capture antibodies via a streptavidin-biotin interaction.

[0072] Clause 3. The method of clause 1 or 2, wherein the detection antibody is conjugated to an enzyme.

[0073] Clause 4. The method of clause 3, wherein the enzyme converts the one or more detection reagents into the one or more signals.

[0074] Clause 5. The method of any one of clauses 1-4, wherein the at least one signal is a fluorescence signal.

[0075] Clause 6. The method of any one of clauses 1-5, wherein the plurality of analytes is proteins.

[0076] Clause 7. The method of any one of clauses 1-6, wherein the track-etched polycarbonate

[0077] (PCTE) membrane prevents bubble formation during immunocomplex injection.

[0078] Clause 8. The method of any one of clauses 1-7, wherein the substrate is polydimethylsiloxane (PDMS) coated on glass.

[0079] Clause 9. The method of any one of clauses 1-8, wherein the fluid is a droplet on top of the substrate.

[0080] Clause 10. The method of any one of clauses 1-9, wherein measuring the at least one signal comprises imaging the device with a fluorescent microscope.

[0081] Clause H . The method of any one of clauses 1-10, wherein the method comprises simultaneously detecting at least two analytes in sample.

[0082] Clause 12. The method of clause 11, wherein the one or more capture antibodies comprises at least two different antibodies.

[0083] Clause 13. The method of clause 11 or 12, wherein different analytes are bound to different detection antibodies that are conjugated to different enzymes.

[0084] Clause 14. The method of clause 13, wherein the different enzymes convert the one or more detection reagents into the one or more different signals.

[0085] Clause 15. A device for detecting analytes in a sample, comprising: a glass substrate coated with a silicone polymer; a buffered liquid droplet, wherein the droplet is on top of the glass substrate; and a track-etched polycarbonate (PCTE) membrane that is adhered to the silicone polymer and forms microwells. Clause 16. The device of clause 15, wherein the silicone polymer is polydimethylsiloxane (PDMS).

[0086] Clause 17. The device of clause 15 or 16, wherein the silicone polymer is mixed in a 10:1 weight ratio with a curing agent before coating the glass substrate.

[0087] Clause 18. The device of any one of clauses 15-17, wherein the silicone polymer coating is about 200 pm thick.

[0088] Clause 19. The device of any one of clauses 15-18, wherein the device comprises at least 1 ,000 microwells.

[0089] Clause 20. The device of any one of clauses 15-18, wherein the device comprises at least 1 ,000,000 microwells.

[0090] Clause 21. A method of making the device of any one of clauses 15-20, the method comprising immersing the PCTE membrane into the buffered liquid droplet until the PCTE membrane adheres to the silicone polymer coating.

[0091] Clause 22. The method of clause 20, wherein the method does not generate air bubbles.

[0092] EXAMPLES

[0093] Reagents and Materials

[0094] Biotinylated p-galactosidase (B- G) and Biotin Alkaline Phosphatase Conjugated (B-ALP) were purchased from Rockland Immunochemicals (PA, USA). Dynabeads M-280 Streptavidin, Dynabeads™, Biotin conjugated CD326 (EpCAM) monoclonal antibody, RIPA buffer and Eppendorf™ LoBind microcentrifuge tubes were purchased from Thermo Fisher Scientific (MA, USA). Track-etched polycarbonate membranes were purchased from Sigma Aldrich (St. Louis, MO, USA) and Sterlitech Corporation (WA, USA). Resorufin [3-D-Galactopyranoside (RDG), Fluorescein di(P-D-galactopyranoside) (FDG), streptavidin-p-galactosidase (S- G), Biotin Alkaline Phosphatase Conjugated (S-ALP), Tween-20, Bovine Serum Albumin (BSA) and silicone oil were purchased from Sigma Aldrich (St. Louis, MO, USA). The polydimethylsiloxane and curing agent (Sylgard 184 silicon elastomer kit) was purchased from Dow Corning (Ml, USA). 4- Methylumbelliferyl phosphate (4-MUP) liquid substrate was purchased from Millipore sigma (St. Louis, USA). Human Glypican 1 (GPC-1) biotinylated antibody was purchased from R&D systems (MN, USA). Biotinylated anti-CD63, anti-vinculin and anti-GPC-1 antibodies were purchased from Abeam (MA, USA). Anti-EpCAM antibody was purchased from Invitrogen (MA, USA) and anti- CD63 was purchased from BD Biosciences (NJ, USA). Anti-rabbit and anti-mouse HRP- conjugated IgG secondary antibodies were purchased from Cell Signaling Technology (MA, USA). Laemmli sample buffer, nonfat dry milk (NFDM) and ECL substrate kit were purchased from Bio-rad (CA, USA). Dulbecco’s Modified Eagle Medium (DMEM) and penicillin-streptomycin were purchased from Corning (NY, USA), and fetal bovine serum (FBS) was purchased from Gibco (NY, USA). Paclitaxel was purchased from Selleck Chemicals (TX, USA).

[0095] Device Fabrication

[0096] A mixture of the curing agent and polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) base, with a weight ratio of 1 :10, was prepared and degassed for one hour to eliminate air bubbles. Subsequently, the PDMS was spin-coated onto a 50 * 75 mm glass slide and left overnight for curing, forming a thin film of approximately 200 pm. For the creation of the negative mold for the top channel, a 0.3 mm thick piece of KAPTON® Tape (McMaster-Carr) was cut into the shape of a converging-diverging microchannel using a Graphtec Cutting Pro FC7000MK2-60 cutting machine. The tape was then affixed to a petri dish. Then the PDMS was poured into the mold and degassed again to remove any trapped air bubbles, followed by overnight curing at 60 °C. After curing, a 1 mm biopsy punch was used to punch holes for the inlet and an outlet.

[0097] Device Assembly

[0098] The PDMS thin film was washed with isopropyl alcohol and then dried with nitrogen gas. A 500 pL drop of 1 x PBS was put in the center of the glass slide having a thin film of the PDMS. Subsequently, a piece of PCTE membrane measuring ~ 1 cm x 0.5 cm was cut and slowly immersed into the droplet using forceps. The membrane was gently pushed until it made contact with the PDMS surface. Finally, the flat surface of the forceps was utilized to ensure the conformal binding of the membrane with the PDMS surface. The membrane demonstrates adhesive characteristics to PDMS, leading to a conformal coating on the elastomeric substrate. The previously fabricated PDMS-based microchannel was placed on top of the membrane and was pressed until it established contact with the PDMS thin film. The excessive buffer solution at the edges of the PDMS channel was then slowly removed using Kimwipes™. The assembled device is shown in FIG. 17.

[0099] Microwell Filling Experiment using Fluorescein

[0100] Following the assembly of the device, a 200 pL solution of fluorescein (1 mM) was introduced through a pipette into the inlet of the upper PDMS channel. Subsequently, 200 pL of silicone oil was pipetted until it entirely displaced the fluorescein solution in the microchannel. For visualization of the microwell filling performance, MetaMorph v7.7.9 software was used to do z- stack imaging using a Nikon Eclipse Ti confocal microscope attached to an iXonEM+ cooled CCD camera attached to it.

[0101] Beta Galactosidase and B-ALP Assay on Bulk Solution

[0102] For measuring the BfJG fluorescence signal in bulk solution, 96-well black plates were used to measure the fluorescence signal. 100 pL of different concentration BALP were added to each blank well. The stock solution of 4-MUP was diluted 4 times and 100 pL was injected into the same wells. After 1 hour, the contents were measured using a microplate reader (Tecan Infinite M200 Pro) at 360 nm. Like B[3G study, 96-well black plates were used to measure the fluorescence signal for B-ALP. 100 pL of different concentration BALP were added to each blank well. 100 pL of 500 pM of FDG was injected into the same wells. After 1 hour, the contents were measured using a microplate reader (Tecan Infinite M200 Pro) at 488 nm.

[0103] Digital Enzyme Assays without Magnetic Beads

[0104] After the completion of device assembly steps, a solution comprising 100 pL of B-ALP or / and B-[3G (spiked in incubation buffer) each at different concentration were mixed and injected into the microchannel. The solution was incubated for 5 minutes to enable stochastic encapsulation of enzyme molecules inside the microwell. Afterwards, a solution containing 100 pL of 200 pM Fluorescein di(P-D-galactopyranoside) (FDG) and 100 pL of undiluted 4- Methylumbelliferyl phosphate (4-MUP) substrates was prepared and injected into the microchannel. Immediately afterwards, 200 pL of silicone oil was pipetted into the channel to seal all the microwells. The microwells were then imaged after 30 minutes following incubation of the device in a dark chamber. The DAPI filter was used to measure alkaline phosphate amplification at 1s exposure while amplification of beta galactosidase was observed using Rhodamine filter at an exposure of 500 ms for all imaging steps in digital assay.

[0105] Magnetic Bead Seeding in Microwells and Digital Assay

[0106] 50 pL of magnetic beads from the commercial vial (10 mg / mL) was taken and diluted to 25 times to make a stock solution. Then 50 pl of the stock solution was taken in microcentrifuge tubes, washed 3 times with 1 x PBS and incubated with PBST (1 x PBS, 1 % BSA, and 0.1 % Tween 20) for 2 hours. A NdFeB cylinder magnet (DCC-N52, K&J Magnetics) was used to concentrate the beads and facilitate washing steps. 100 pL of different B[3G or B-ALP was injected into each tube after washing 3 times with 1x PBS and were incubated overnight on with gentle shaking. Afterwards, the beads were washed 3 times and 50 pL of 1 x PBS was injected into each tube. After filling the microwells by 1x PBS, the solution containing magnetic beads was poured on top of the wetted membrane. The permanent magnet was kept at 5 mm below the PDMS coated glass slide. The slide was manually shaked for 15 minutes for increasing bead filling inside the microwells. Later, the PDMS based microchannel was gently put of the top and was sealed manually. After that the subsequent substrate solution was injected into the chip and incubated for 10 minutes. The microwells were then sealed with silicone oil and were later imaged using a CCD camera (Retiga EXi, Qlmaging) connected to an Olympus 1X71 microscope. The complete workflow has been described in FIG. 3.

[0107] Digital Enzyme Assays with Magnetic Beads

[0108] 10 mL stock solutions of washing buffer (5x PBS + 0.1% Tween 20) and incubation buffer (1 x PBS + 2% BSA + 0.05% Tween 20) were prepared and stored at 4 °C for subsequent use. 50 pL of magnetic beads (Dynabeads M-280 Streptavidin, Dynabeads™) from the commercial vial (10 mg / mL) was taken and diluted 25 times to make a stock solution of magnetic beads. Subsequently, a 50 pL of this stock solution was taken in low protein binding microcentrifuge tubes, washed 3 times with washing buffer and incubated in the incubation buffer for a duration of 2 hours. Following the incubation, the beads were washed 3 times and were subsequently incubated with 100 pL of incubation buffer spiked with B-[3G and / or B-ALP into the tube. This mixture underwent a 2-hour incubation period while being rotated. Afterwards, the tubes were washed 3 times in the washing buffer to remove nonspecifically bound molecules and incubated in the incubation buffer to a final volume of 100 pL. After wetting the membrane as outlined in “Device assembly” section, the solution containing magnetic beads was pipetted on top of the wetted membrane. The NdFeB cylinder magnet (DCC-N52, K&J Magnetics) was kept at an optimized distance below the PDMS coated glass slide. The glass slide was manually shaked in the x-y plane for 15 minutes to enhance bead filling within the microwells. Later, the PDMS based microchannel was gently put on the top to complete the device assembly as described earlier. Subsequently, a solution consisting of 100 pL of 200 pM FDG and 100 pL of undiluted 4-MUP substrates were mixed in PBS and introduced into the microchannel. Immediately thereafter, 200 pL of silicone oil was pipetted into the channel to achieve the sealing of all microwells. The device was then incubated in a dark chamber and was subsequently imaged after half an hour.

[0109] Image Processing and Data Analysis All fluorescence images were processed using a custom developed code. More details can be found in FIG. 18. GraphPad Prism has been used for graphical representation along with statistical analysis. Unless otherwise specified, all data used in this study are shown as mean ± standard deviation.

[0110] Membrane as Microwells Chamber

[0111] PCTE membranes, commercially produced through ion irradiation and subsequent track etching, exhibit a high density of uniformly sized cylindrical through-holes. These holes are available in a range of desired diameters (from 10 nm to 30 pm) and possess low protein binding properties. In the Mem-dELISA technology, we ingeniously utilize the through holes of the PCTE membranes to repurpose them as microwells by strategically blocking one end as described in FIG. 1. To accomplish this, a drop of 1xPBS was first put on top of a glass slide spin-coated (1000 rpm, 2 minutes) with a thin film of PDMS (FIG. 1B). The membrane was fully immersed into the PBS solution using forceps to remove all the air bubbles via capillary wicking of water inside the through holes. After successfully eliminated all trapped air bubbles, we capitalize on the adhesive nature of the PCTE membrane to a PDMS surface. The forceps were gently used to place the membrane on the PDMS surface, where it adhered to form microwells. Afterwards, a PDMS based microchannel was put on top to develop the integrated Mem-dELISA platform. The PDMS based microchannel permits the flow of different reagents on top of membrane, diffusion of reagents to the microwells and ensures complete sample partition within each microwell after sealing with oil. Injection of a fluorescein solution followed by oil sealing showed complete segregation of microwells by oil as shown in the fluorescent image obtained in FIG. 1B.

[0112] The initial wicking action of the membrane is crucial to the removal of trapped bubbles. If the membrane is first attached to the PDMS surface followed by the placement of the PBS droplet above, trapped air bubbles were consistently observed in the majority of microwells (FIG. 1A). Using commercially available micron-sized (~2.8-pm) magnetic beads for protein capture, we opted to develop our Mem-dELISA technology by utilizing a 5-pm pore size membrane. The selected membrane exhibits uniformly sized microwells with a low population of overlapping pores as evident from the bright field image (see FIG. 19).

[0113] With the initial workflow finalized, we optimized the height of the PDMS based microchannel to enable seamless sealing of microwells by oil (FIG. 12A). After the device assembly, a 1 mM solution of fluorescein was injected into the microchannel followed by oil injection to partition individual microwells. Two distinct types of hydrophobic and hydrophilic commercial membranes were also utilized in the experiment to understand the effect of the surface property of membrane. The height of the microchannel was systematically varied for each type of membrane from 100-1000 pm. Since the surface of the PCTE membrane is uneven, a high shear is necessary to remove the remnant water layer present on the top of the membrane. For hydrophilic membrane, the layer of water could not be removed until the microchannel height was reduced to 100 pm. Even with this lowered channel height, many water islands were observed thus corrupting sample partitioning (FIG. 12C). In case of hydrophobic membranes, excellent sample partition was observed when microchannel height was reduced to 300 pm and less as shown in FIG. 12B and 12D. The images show a clear separation of individual picolitre reactors containing a fluorescein solution. From these findings, we selected the microchannel height of 300 pm for our subsequent experiments. Z-stack confocal imaging was performed to orthogonally confirm the removal of all air bubbles within the microwells in the hydrophobic membrane as shown in FIG. 12E. As we move in z-direction, the microwells transition into focus and recede out of focus at 25 pm. The results indicate that the height of the cylindrical pores is around 20 pm which aligns closely to the manufacturers specifications of 21 pm. Using these results, we estimate that the volume of one microwell is ~0.4 pL while the membrane has a well density of 4 x 105wells / cm2.

[0114] Since we are utilizing the wicking to remove all trapped bubbles inside the membrane, our method provides an extremely low-cost solution for microwell fabrication and operation as compared to other methods that utilize complex lithography techniques. Additionally, it eliminates the need for expensive vacuum pumps and surface modification of microwells to render them super-hydrophilic. Furthermore, the quantity of microwells is contingent upon the size of the membrane and can be readily scaled to ~106wells with a ~2.5 cm2section of 5 pm pore size membrane for highly multiplex assays. This distinguishes our technique from other fabricationbased methods where the microwell count is constrained by the dimensions of the master mold.

[0115] Single-molecule Assay for Free Floating B- G and B-ALP

[0116] Since the commercialization of Quanterix’s Simoa® technology for digital ELISA, great progress has been made in utilizing the power of digital counting of protein molecules using femtoliter-sized chambers. Notably, the enzymatic amplification in their device is executed exclusively through the utilization of beta-galactosidase enzymatic reaction with its substrate. It is imperative to recognize that a solitary enzymatic amplification within a singular microwell is insufficient for elucidating the colocalization of multiple proteins within nanocarriers, such as extracellular vesicles which are extensively studied nowadays. In this study, we have selected beta galactosidase and alkaline phosphatase as our two candidate enzymes for performing duplex enzymatic reaction within the same microwell. To perform duplex assay, it’s important to test the enzyme cross reactivity with their substrates. Various enzyme-substrate combinations were systematically tested to ascertain the combination exhibiting minimal fluorescence crosstalk and inhibitory behavior as summarized in Table 1. From our study, beta galactosidase-RDG and alkaline phosphatase-4 MUP substrate combination showed the best results. The B-[3G reacts with RDG to give red fluorescence signal (resorufin: Emission 584 nm) signal and B-ALP reacts with 4-MUP to give a blue fluorescence signal (methylumbelliferone: Emission 445 nm) that can be separated by using Rhodamine and DAPI filters respectively. We also performed a bulk cross reactivity study in which the concentration of B-pG varied from 0 to 20 nM while keeping the B-ALP concentration fixed at 0.7 nM (FIG. 20). The blue fluorescence remained roughly constant while the green fluorescence increased with the concentration of B-[3G confirming minimum cross reactivity between the two enzymes.

[0117] An analysis of various enzyme-substrate combinations was used to ascertain the combination exhibiting minimal fluorescence crosstalk and inhibitory behavior, Table 1.

[0118] After successful demonstration of bubble-free partitioning of liquid into microwells, the Mem-dELISA platform was tested to study single molecule amplification of B- G and B-ALP enzymes. The complete workflow is described in FIG. 21. Following the device assembly and filling each microwell with PBS, a solution of B-ALP (300 pM) was quickly injected into the chip. The chip was then incubated for a minute, allowing for the stochastic encapsulation of B-ALP molecules within the microwells through diffusion from the microchannel to the microwells. Afterwards, 2x diluted 4-MUP substrate was injected into the microchannel followed by sealing of the microwells with oil. The time-elapsed images of these microwells are shown in FIG. 13A where the fluorescence intensity increases with time depicting enzymatic reaction between B- ALP and 4-MUP substrate. The intensity of each microwell at 25 minutes image was extracted using a custom developed code and plotted as a histogram, fitted with the sum of four Gaussian functions depicting the presence of 0, 1 , 2 and 3+ enzymes in the microwells (FIG. 13B). Theoretically, the encapsulation of enzyme molecules in the empty microwells follows a Poisson

[0119] ^e~Akdistribution: P( / c: A) = — - — . Here P( / c: A) denotes the random probability of encapsulating k molecules in a microwell and A is the average number of molecules per microwell. At the calculated lambda of 0.42, the expected probabilities of 0, 1 , 2 and 3+ enzymes per microwells are 65.71 %, 27.60%, 5.80% and 0.01% respectively. The experimentally observed values obtained from Gaussian fitting matched well with the theoretical estimates from Poisson distribution as shown in FIG. 13C. The presence of discrete peaks is consistent with earlier reports. Similarly, we did the same experiment to study the enzymatic amplification of beta galactosidase enzyme with its substrate (100 pM RDG) by keeping B-[3G enzyme concentration at 30 pM. The sequential images capturing the temporal evolution of fluorescence intensity increase in microwells are presented in FIG. 13D. As described earlier, the fluorescence intensity associated with each microwell was extracted from the image captured at the 25 minutes timestamp. Subsequently, a histogram was generated and superimposed with the composite fit derived from the summation of four Gaussian functions (FIG. 13E). The obtained experimental values matched well with the theoretical estimates from Poisson distribution with A = 0.224 (FIG. 13F). The discrete fluorescence peaks observed with B-[3G enzyme and RDG substrate reaction in microwells are also consistent with the previous literature reports.

[0120] The analysis shown above confirms the encapsulation and amplification of single molecules of B- G and B-ALP within individual microwells in separate experiments at a particular concentration. We now proceeded to vary the concentrations of both B-[3G and B-ALP in separate experiments within the range of 100-0.1 pM. With the sequential reduction in enzyme concentrations, the total number of wells lighting up decreased exponentially based on Poisson statistics as shown in the series of fluorescent images in FIG. 13G and FIG. 3I. A log-log plot revealing the percentage of fluorescent microwells as a function of the concentration is depicted in FIG. 13H and 131. The biosensor had a working concentration range 10-0.1 pM for both B- G and B-ALP. This analysis shows that the Mem-dELISA methods and protocols have no effect in the biochemical reaction between different enzymes and their respective substrates.

[0121] After optimizing the individual reactions of B- G and B-ALP separately on the microwells, duplex assay was performed. The B-[3G (30 pM) and B-ALP (100 pM) were mixed and incubated in the microchannel for a minute. The RDG and 4-MUP substrates were mixed and immediately injected into the microchannel followed by immediate sealing with oil. It is important to note that the final temperature of the mixture influenced the reaction kinetics (data not shown). In our experience, both the substrates are warmed up to 37 °C before mixing them together for optimal results. FIG. 22 displays a superimposed fluorescence image derived from the blue and red fluorescence channels. It is important to note that for better visualization of overlayed images, the blue channel images have been colored with green hues. Therefore, the green and red hues correspond to the fluorescence signature of 4-MU and resorufin, respectively. Consistent with our expectations, the green and red fluorescent channels exhibited a random distribution, with certain microwells displaying both green and red fluorescence concurrently (yellow / orange microwells) which indicates co encapsulation of both B- G and B-ALP in the same microwell. Hence, successful demonstration of the feasibility of dual-color digital enzyme assays is presented in this section and will be explored further in the upcoming sections.

[0122] Magnetic Beads Seeding Optimization

[0123] In the earlier section we demonstrated the capture of free-floating beta galactosidase and alkaline phosphate enzyme molecules in microwells for performing digital assays. However, relying only on diffusion to capture free-floating protein particles leads to detection of a very small fraction of protein molecules as more than 99.9% of the sample is lost. Hence, to increase the sensitivity of the digital sensor, magnetic beads are used to concentrate the free-floating proteins into the microwells. Moreover, to increase the percentage of magnetic bead inside the microwells, permanent magnets have been used although it can cause adverse effects such as bead chain formation that significantly reduces the bead capture efficiency. To develop more mechanistic insights of the system, COMSOL Multiphysics simulations of a permanent bar magnet was performed using the ‘Magnetic fields, no current’ module to understand the variation of magnetic flux density as a function of distance from the permanent magnet as shown in FIG. 14A. The simulation geometry and details are presented in FIG. 23. The simulations show that the magnetic flux density varies as ~1 / r3with r is the radial distance from the magnet FIG. 14B. Moreover, the magnetic force exerted on the magnetic bead by the permanent magnet is directly proportional to the square of the gradient of the magnetic field. This relationship also underscores the sensitivity of the force acting on the magnetic bead to variations in radial distance from the permanent magnet. Unfavorably, the strong field from the magnet often causes undesirable chain formation between magnetic beads which negatively impacts the digital assay. Hence, the distance of the membrane surface from the permanent magnet needs to be optimized so to concentrate the beads on the membrane surface without causing undesirable chain formation.

[0124] The experimental results indicate the occurrence of large undesired magnetic bead chains when the beads were directly positioned atop the magnet, as illustrated in zone I of FIG. 14B. Note that the origin is taken on the plane of the membrane which is -1 .2 mm distant from the magnet’s surface. With an increasing separation between the magnet and the membrane, the prominent large chunks of bead aggregates progressively diminish as indicated by zone II image. Moreover, the large bead chain formation was greatly reduced when the magnet was positioned approximately 4 mm away from the glass slide. The operational effectiveness of the magnetic force was found to be optimal within the range of 4 mm to 1.6 cm from the glass slide, where no significant formation of magnetic bead chains was observed. Beyond 2 cm, the magnetic force was not sufficient to pull all the free-floating beads in the solution phase on top of the membrane. For all the experiments, the magnet was kept at 5 mm from the glass slide.

[0125] Initial experiments were performed with magnetic beads without using a permanent magnet. The magnetic beads themselves are expected to be captured into the microwell following Poisson distribution, imposing a theoretical limit of the maximum percentage (< 37%) of single bead occupancy in the microwell chamber. We observed the same behavior when we seeded the magnetic beads at three different bead densities on top of the membrane piece and utilized gravity settling of beads to fill the microwells (FIG. 14C). The majority of the microwells remained empty in these cases with less than 40% of the microwells are filled with magnetic beads by using gravity settling (FIG. 28). As the bead density was increased to (108 / m0, single bead occupancy decreased, and double bead occupancy increased drastically. Hence, (2.4 x 107 / ml) bead density was selected for further optimization using a magnet. After the beads settled on the membrane surface, the Mem-dELISA chip was mechanically shaken for 15 minutes under the influence of the magnetic field from a permanent magnet. This technique improved the single bead capture from -34% to >60% (FIG. 4D). In total, the final bead capture efficiency was increased to > 80% using a permanent magnet and mechanical shaking.

[0126] We offer a simple mechanistic explanation for this improved result. During mechanical shaking, an impulse is given to the particle in the horizontal direction while the magnet is kept in the vertical direction. A simple force balance on a magnetic bead reveals that the drag acts in the horizontal direction whereas the magnetic force along with gravity acts in the vertical direction. The net resultant vector will be inclined at an angle to the vertical direction. Hence the bead will bounce on the membrane, thus substantially elevating the probability of locating a nearby microwell. Once the bead is captured into the microwell, the high surface tension prevents the bead from exiting the microwells.

[0127] Another notable point is that the microwells in our membranes are more separated and deeper as compared to other devices. During processes such as dendritic growth or chain formation of magnetic beads, the trapped particles amplify the magnetic field, thereby capturing additional particles atop them. Importantly, this enhanced field is limited to only a few bead diameters. Hence, we hypothesize that well-separated wells (beyond one particle diameter) and large number of microwells is the key to prevent chain formation and improve sensitivity. The randomly distributed and well-separated deep microwells of the PCTE membrane facilitate the convenient utilization of a permanent magnet to increase the bead loading efficiency.

[0128] Magnetic Bead-Based Multiplex Assay

[0129] Following the optimization of the magnetic bead loading step, digital protein assays were conducted on the Mem-dELISA device to improve the limit of detection from Pico (10“12) to 10 Atto (10-18) molar range for B-[3G molecules captured by streptavidin-coated magnetic beads. For an initial test, 1 pM concentration of B- G was incubated with streptavidin-coated magnetic beads. At this concentration, it is expected that each magnetic bead should capture at least one copy of B-pG , if not more. To confirm this hypothesis, both brightfield and its corresponding fluorescence image were taken at 50* resolution for comparison (FIG. 24). The microwells that contain no magnetic bead are in ‘off’ state, while microwells that contain a magnetic bead are in ‘on’ state (drawn circles). The marked circles on the fluorescence image are the empty microwells that did not show any signal. This confirms that only the magnetic beads present in the microwells were in ‘on’ state. Next, the concentration of B- G was reduced to 20 fM and a similar analysis was performed. At this concentration, only a fraction of magnetic beads should capture B-pG molecule, and a composite image is made using both brightfield and fluorescence (Red) as shown in FIG. 14G. The zoomed-in image shows the presence of empty microwells, microwells that contain a magnetic bead and are in ‘off’ state and microwells that contain a magnetic bead and are in ‘on’ state (drawn circles). These initial tests validated the capture of B-[3G by streptavidin- coated magnetic beads.

[0130] Hence, the concentration of B-[3G was systematically reduced by an order of magnitude from 1 pM to 10 aM as shown by the series of images in FIG. 25A. FIG. 14E shows a log-log graph depicting the variation of % of fluorescent wells as a function of B-PG protein concentration. The dotted line is obtained from the negative controls, showing that 0.03% of microwells will be fluorescent. The calibration curve showed 5 logs of dynamic range (100 fM-10 aM) and limit of detection of -10 aM. It is important to note that the utilization of magnetic beads for capturing free-floating protein molecules has resulted in an improved limit of detection by 5 orders of magnitude, shifting from 100 fM to 10 aM compared to cases without magnetic beads (FIG. 13H). Similarly, the concentration of B-ALP was also varied from 1 pM-10 aM as shown in FIG. 25B and a similar calibration plot was made as discussed above in FIG. 13F.

[0131] Next, we tested the Mem-dELISA device to perform duplex digital protein assay by detecting both B-pG and B-ALP proteins simultaneously in a single experiment (FIG. 15A). Since the base substrates used in these reactions (RDG and 4-MUP) also produce a fluorescent signal, it was necessary to perform a negative control to obtain the baseline intensity. The intensities of all microwells were extracted from images (FIG. 15B) captured from both fluorescence filters (Blue and Red) and plotted as histograms as shown in FIG. 15C and FIG. 15D. As observed in negative controls, <0.03% of the microwells light up at an intensity threshold of five standard deviations from the mean value. Consequently, a microwell is designated as being in an 'on' state if its intensity exceeds this specified threshold. To perform the duplex digital assay, for the first experiment, 1 pM of B-ALP and 100 fM of B-pG were mixed and incubated with magnetic beads. As expected, at these concentrations, the majority of microwells were in ‘on’ state as shown in FIG. 15E. The concentration of both protein molecules was serially decreased by an order of magnitude in subsequent experiments and the results were shown as composite images in FIG. 15 (H, K, N, and Q). Since the blue fluorescence from 4-MUP substrate was consistently present in all microwells, its fluorescence was used for accurate position identification for all microwells using an in-house developed MATLAB script. As the concentration decreases by an order of magnitude, the number of microwells crossing the threshold intensity decreases as well for both B-pG and B-ALP. For efficient data visualization, the histogram of the intensity of microwells was plotted for all concentrations of B-pG and B-ALP in FIG. 15 (F, G, I, J, L, M, O, P, R & S). The threshold intensity in these histograms is obtained from the negative controls described earlier. As the concentration of the protein molecules decreased, the histogram peaks shifted towards the left indicating that the number of microwells lighting up also decreased significantly. Additionally, the microwell intensity from each microwell was also extracted from a representative image and plotted as a function of the microwell ID for various concentrations in FIG. 26 for better visualization of this trend. Furthermore, the capture of protein molecules by magnetic beads shall also obey the Poisson’s distribution as described in previous reports. For a duplex assay, since a single bead is present to capture two free floating protein molecules, the combined probability of capturing both protein molecules on the same magnetic bead shall be the multiplication of two independent Poisson distributions. In FIG. 15T, the percentage of microwells having both B- G and B-ALP proteins is plotted as a function of their concentrations which matches well with the theoretical predictions of double Poisson’s distribution.

[0132] Simultaneous Detection of Free Floating B$G and B-ALP

[0133] To perform the duplex assay, it was important to test the enzyme cross reactivity with their substrate. First, we performed bulk cross reactivity study in which the concentration of B[3G was varied from 0 to 20 nM while keeping the B-ALP concentration fixed at 0.7 nM. The blue fluorescence remained roughly constant while the green fluorescence increased with the concentration of B G confirming minimum cross reactivity between the enzymes. Finally, after optimizing the individual reactions of B G and B- ALP in both bulk as well on the chip, duplex assay was performed. The B$G reacts with FDG to give green fluorescence and B-ALP reacts with 4-MUP to give a blue, fluorescent reaction product (methylumbelliferone). To show the preliminary experiments, different concentration of B G and B-ALP were first injected into the microchannel. Initially, 300 pM of BfJG and 700 pM B-ALP were injected into the chip as before shown in FIG. 5. At this high concentration, all microwells contained the fluorescence products from both reactions as shown in FIG. 10A-C. In the second case 3pM of B$G and 70 pM B-ALP were taken. As depicted in FIG. 10D-F, the number of green fluorescence wells is less as compared to blue fluorescence wells. Finally, in the third case 30pM of B G and 7 pM B-ALP were injected into the chip and the number of green fluorescence wells outnumber the blue ones FIG. 10G-I.

[0134] A novel low cost, user-friendly, power and lithography free digital biosensor is described using a commercial PCTE membrane achieving a dynamic range of pM-fM with a limit of detection of 100 aM. Single bead filling inside the microwells was increased from nominal sub 30% to 62% using a permanent magnet kept at an optimized distance and through mechanical shaking. The deep and well separated microwells also reduce chain / dendrite formation of magnetic beads. Finally, a duplex digital ELISA platform was developed for simultaneous detection of B G and 70 pM B-ALP in the same experiment. The developed method can be massively multiplexed when coupled with bar coded beads. Moreover, the technology can also be utilized to digitally detect specific extracellular vesicles (EVs) containing cancerous proteins such as GPC-1 and mutated EGFR. Collectively, the inexpensive (< $0.1 per membrane) digital ELISA platform can be utilized for duplex digital quantification of protein biomarkers thus enabling point of care applications in laboratory resource constraints areas.

[0135] Magnetic Bead Functionalization with Antibodies

[0136] The 50 pL stock solution of streptavidin conjugated magnetic beads was aliquoted in low protein binding microcentrifuge tubes, washed 3 times with washing buffer and incubated in the incubation buffer for a duration of 2 hours. 50 pL of 100* (diluted in PBS + 0.05% Tween 20) diluted Biotin-conjugated CD63 antibody were incubated with the magnetic beads in a shaker for 3 hours. Following this incubation, the beads underwent three successive washes in the washing buffer and were subsequently stored in the incubation buffer at 4 °C until further use.

[0137] Extracellular Vesicles (EVs) Isolation

[0138] MDA-MB-231 and MDA-MB-468 cells were cultured in DMEM supplemented with 10% FBS and 1 % penicillin-streptomycin at 37°C with 5% CO2. EV-depleted media was made with FBS that had been centrifuged at 100,000 x g for 9 hours to remove bovine EVs. EV-depleted media was added to confluent flasks and incubated for 48 hours. For paclitaxel treatment conditions, paclitaxel was added to the EV-depleted media. The media was then collected and spun at 400 x g for 5 min to remove cell debris, and the supernatant was centrifuged at 100,000 x g for 1 h to pellet EVs. The EV pellet was washed once with PBS, then resuspended in PBS and stored at -80 °C for downstream analysis.

[0139] Multiplex Digital Protein Assay of EVs

[0140] The complete workflow of digital ELISA is described in FIG. 11C. The 50 pL of CD 63 antibody functionalized magnetic beads described earlier were incubated with 100 pL of EVs (100x diluted in incubation buffer from ultracentrifugation) for 2 hours under constant rotation. The beads were washed 3 times in the washing buffer and were later incubated with the incubation buffer. Parallelly, biotinylated GPC-1 detection antibody was incubated with 20 pM of S-ALP and 1 nM biotinylated EpCAM was incubated with 20 pM of S-pG respectively for 2 hours to attach enzymes on antibodies. The stored magnetic beads were then incubated with 100 pL of GPC-1 & S-ALP enzyme complex for 30 minutes under rotation. The immunocomplex was then washed 8 times with the washing buffer to remove non-specifically bound molecules. Similarly, 100 pL of biotinylated EpCAM & S-[3G complex was then incubated with the beads for half an hour under constant rotation and was washed 8 times to form the immunocomplex. This magnetic bead based immunocomplex was used to perform digital protein detection using the steps described in earlier sections.

[0141] Western Blot

[0142] Cells were lysed in RIPA buffer (Thermo Scientific) and spun at 16,000 g for 20 min to obtain the protein supernatant. Samples were prepared for SDS-PAGE in Laemmli sample buffer (BIO-RAD) and heated to 70 °C for 10 minutes. SDS-PAGE was performed with 20 pg of protein per sample in a polyacrylamide gel, and the protein was electrophoretically transferred to a nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk (NFDM, BIORAD) TBS with Tween 20 (TBST, abeam), then incubated overnight with a primary antibody mixture containing either anti-vinculin (1 :5000, abeam), anti-GPC1 (1 :1000, abeam) and anti- EpCAM (1:50, Invitrogen) primary antibodies or anti-vinculin and anti-CD63 (1 :500, BD Biosciences) antibodies in TBST with 5% NFDM. After washing with TBST, the membranes were incubated for 1 hour in TBST containing anti-rabbit (1 :1000, Cell Signaling Technology) and antimouse (1 :1000, Cell Signaling Technology) HRP-conjugated IgG secondary antibodies. After washing with TBST, the protein was visualized using an ECL substrate kit (BIO-RAD) and imaged with a ChemiDoc-lt2 system.

[0143] EVs based dELISA

[0144] EVs are an emerging class of promising highly heterogeneous circulating biomarkers that play important roles in shuttling molecular cargo from host cell to recipient cell, thereby facilitating intercellular communication, modulating drug resistances and immune response. Therefore, by first principles, alterations in the protein expression of EVs derived from tumors are expected to exhibit a strong correlation with the protein expression in the host tumor cells. In the case of breast cancer, the molecular cargo of EVs has been associated with prediction of therapy outcome and drug resistance. Though the EVs heterogeneity in size and molecular cargo has been well documented, bulk EV analysis methods (Dynamic Light Scattering, Nanoparticle Tracking Analysis, ELISA, and Western blots) have been predominantly used while advances have been made to perform single EV analysis using single-particle interferometric reflectance imaging with fluorescence, nanoparticle tracking analysis, microfluidic resistive pulse sensing, and nanoflow cytometry. Nonetheless, most single EV analysis platforms that analyze the molecular biomarkers suffer from low dynamic range (~2 logs), interference from non-targets and inherent issues with fluorescent probes such as protein autofluorescence and photobleaching. The developed dual color Mem-dELISA biosensor with 5 logs of dynamic range utilizes enzymatic amplification instead of fluorescent labelling and can easily overcome these challenges. Moreover, the platform utilizes efficient wash protocols using high ionic strength buffer (5x PBS) to minimize electrostatic interactions between non targets and beads. The high throughput analysis of different surface proteins simultaneously on the surface of a single EV without EV lysis provides a holistic approach to capture the heterogeneity with improved reproducibility to allow accurate diagnostic and therapeutic predictions. Multiple protein analysis on the same EVs improves normalization of data with a reference protein to minimize experimental bias caused by upstream EV isolation steps.

[0145] We utilized the multiplex Mem-dELISA platform to detect the two proteins colocalized on the surface of a single EV derived from cell culture media of breast cancer cell lines. As a proof- of-concept study, we studied the effect of chemotherapy (paclitaxel) treatment on two triplenegative breast cancer cell lines: MDA-MB-231 and MDA-MB-468 by quantifying the colocalization of GPC-1 and EpCAM proteins on the surface of EVs. Several reports suggest that both EpCAM and GPC-1 are biomarkers of breast cancer and are present in both MDA-MB-231 and MDA-MB-468 cell lines. EpCAM has been attributed to increased drug resistance and poor prognosis. We hypothesized that the normalized ratio of colocalized EpCAM-GPC-1 on EVs would provide insights on the effect of paclitaxel drug treatment on chemo-resistance for use in drug screening and therapy management.

[0146] Magnetic beads coated with CD63, a known tetraspanin marker, were selected to capture EVs isolated by ultracentrifugation from cell culture. An immunocomplex was formed where an EV is sandwiched between a CD63 capture antibody and enzyme coated GPC-1 and EpCAM detection antibody FIG. 16A. While forming this immunocomplex, it is crucial to exercise caution during the incubation of reporter antibodies mixture to minimize the antibody crosstalk. In our case, the steps of conjugating an enzyme (beta galactosidase / alkaline phosphate) to a detection antibody involves the incubation of biotinylated detection antibodies with streptavidin-conjugated enzymes to create a detection antibody-enzyme adduct. However, biotin and streptavidin have strong affinity for each other and can conjugate if both are incubated together. We hence incubated biotinylated detection antibodies and streptavidin conjugated enzymes sequentially during the reporter incubation step with the magnetic beads and employed three washes of magnetic beads after each incubation to minimize the cross-conjugation of enzymes and antibodies.

[0147] The presence of GPC-1 and EpCAM in the cell lysate was confirmed by western blot (FIG. 16B). For characterization of the effect of Paclitaxel drug treatment on the EVs derived from breast cancer cells, first the cell lines were treated with different concentrations of paclitaxel (10 nM, 100 nM, and 500 nM). Following the drug treatment, the EVs were isolated from the cell culture media by ultracentrifugation. FIG. 11 C shows the complete workflow of the Mem-dELISA platform for the duplex detection of GPC-1 and EpCAM proteins after immunocomplex formation. FIG. 16C and 16D shows the series of representative fluorescence images from both breast cancer cell lines to elucidate the effect of drug treatment on EVs for both MDA-MB-231 and MDA- MB-468 cell lines. Images from the left to right images represent the digital assay performed on EVs which were obtained from untreated control, 10 nM, 100 nM, and 500 nM Paclitaxel drug treated cell lines. The red color corresponds to GPC-1 positive EVs while the green color corresponds to EpCAM positive EVs. The fraction of the EVs belonging to GPC-1 only, EPCAM only and colocalized (both GPC-1 and EpCAM) is calculated based on set theory as shown in FIG. 27. As shown in FIG. 16F and 16H, for both the breast cancer cell lines, as the concentration of paclitaxel was increased, the colocalized fraction of GPC-1 and EpCAM monotonically decreased. In contrast, the fraction of GPC-1 increases with increase in drug treatment concentration (FIG. 16E and 16G). However, in the case of EpCAM alone, the fraction decreases monotonically with an increase of paclitaxel. These results suggest that the drug treatment suppresses the expression of EpCAM but not GPC-1. It also indicates that cancerous cells still exist and may require higher doses of chemotherapy to suppress the expression GPC-1. Interestingly, this selective suppression cannot be identified by Western blot analysis of cell lysate as evident in FIG. 16B. This is likely due to the presence of dispersed soluble proteins in the cell culture media that generate false positive signals. This result suggests that our mem-dELISA EV colocalization assay provides more tumor state-relevant information than Western blot analysis of cell lysate.

Claims

CLAIMSWhat is claimed:

1. A method of detecting one or more analytes in a sample, the method comprising:(a) incubating a plurality of magnetic beads conjugated to one or more capture antibodies with a sample comprising a plurality of analytes, wherein the analytes are bound to a detection antibody and wherein at least one of the analytes bind to at least one of the one or more capture antibodies to form one or more immunocomplexes;(b) injecting the one or more immunocomplexes into a device comprising a fluid, a plurality of microwells affixed to a substrate, and a permanent magnet, wherein the plurality of microwells are formed by adhering a track-etched polycarbonate (PCTE) membrane on top of the substrate;(c) agitating the device for a period of time to position the one or more immunocomplexes into the microwells;(d) injecting one or more detection reagents into the device, wherein the detection reagents are converted into one or more signals after a period of incubation;(e) injecting an oil into the device, wherein the oil seals the microwells; and(f) measuring the at least one signal, wherein the at least one signal is indicative of the presence of the one or more immunocomplexes.

2. The method of claim 1 , wherein the magnetic beads are conjugated to the one or more capture antibodies via a streptavidin-biotin interaction.

3. The method of claim 1 or 2, wherein the detection antibody is conjugated to an enzyme.

4. The method of claim 3, wherein the enzyme converts the one or more detection reagents into the one or more signals.

5. The method of any one of claims 1—4, wherein the at least one signal is a fluorescence signal.

6. The method of any one of claims 1-5, wherein the plurality of analytes is proteins.

7. The method of any one of claims 1-6, wherein the track-etched polycarbonate (PCTE) membrane prevents bubble formation during immunocomplex injection.

8. The method of any one of claims 1-7, wherein the substrate is polydimethylsiloxane (PDMS) coated on glass.

9. The method of any one of claims 1-8, wherein the fluid is a droplet on top of the substrate.

10. The method of any one of claims 1-9, wherein measuring the at least one signal comprises imaging the device with a fluorescent microscope.

11. The method of any one of claims 1-10, wherein the method comprises simultaneously detecting at least two analytes in sample.

12. The method of claim 11 , wherein the one or more capture antibodies comprises at least two different antibodies.

13. The method of claim 11 or 12, wherein different analytes are bound to different detection antibodies that are conjugated to different enzymes.

14. The method of claim 13, wherein the different enzymes convert the one or more detection reagents into the one or more different signals.

15. A device for detecting analytes in a sample, comprising: a glass substrate coated with a silicone polymer; a buffered liquid droplet, wherein the droplet is on top of the glass substrate; and a track-etched polycarbonate (PCTE) membrane that is adhered to the silicone polymer and forms microwells.

16. The device of claim 15, wherein the silicone polymer is polydimethylsiloxane (PDMS).

17. The device of claim 15 or 16, wherein the silicone polymer is mixed in a 10:1 weight ratio with a curing agent before coating the glass substrate.

18. The device of any one of claims 15-17, wherein the silicone polymer coating is about 200 pm thick.

19. The device of any one of claims 15-18, wherein the device comprises at least 1 ,000 microwells.

20. The device of any one of claims 15-18, wherein the device comprises at least 1 ,000,000 microwells.

21. A method of making the device of any one of claims 15-20, the method comprising immersing the PCTE membrane into the buffered liquid droplet until the PCTE membrane adheres to the silicone polymer coating.

22. The method of claim 20, wherein the method does not generate air bubbles.

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