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EP-4735888-A1 - ULTRASENSITIVE MULTIPLEX DIGITAL ELISA

EP4735888A1EP 4735888 A1EP4735888 A1EP 4735888A1EP-4735888-A1

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.

Inventors

  • SHARMA, Himani
  • YADAV, VIVEK
  • SENAPATI, SATYAJYOTI
  • CHANG, HSUEH-CHIA

Assignees

  • University of Notre Dame du Lac

Dates

Publication Date
20260506
Application Date
20240730

Claims (20)

  1. 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. 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. 3. The method of claim 1 or 2, wherein the detection antibody is conjugated to an enzyme.
  4. 4. The method of claim 3, wherein the enzyme converts the one or more detection reagents into the one or more signals.
  5. 5. The method of any one of claims 1—4, wherein the at least one signal is a fluorescence signal.
  6. 6. The method of any one of claims 1-5, wherein the plurality of analytes is proteins.
  7. 7. The method of any one of claims 1-6, wherein the track-etched polycarbonate (PCTE) membrane prevents bubble formation during immunocomplex injection.
  8. 8. The method of any one of claims 1-7, wherein the substrate is polydimethylsiloxane (PDMS) coated on glass.
  9. 9. The method of any one of claims 1-8, wherein the fluid is a droplet on top of the substrate.
  10. 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. 11. The method of any one of claims 1-10, wherein the method comprises simultaneously detecting at least two analytes in sample.
  12. 12. The method of claim 11 , wherein the one or more capture antibodies comprises at least two different antibodies.
  13. 13. The method of claim 11 or 12, wherein different analytes are bound to different detection antibodies that are conjugated to different enzymes.
  14. 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. 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. 16. The device of claim 15, wherein the silicone polymer is polydimethylsiloxane (PDMS).
  17. 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. 18. The device of any one of claims 15-17, wherein the silicone polymer coating is about 200 pm thick.
  19. 19. The device of any one of claims 15-18, wherein the device comprises at least 1 ,000 microwells.
  20. 20. The device of any one of claims 15-18, wherein the device comprises at least 1 ,000,000 microwells.

Description

ULTRASENSITIVE MULTIPLEX DIGITAL ELISA CROSS-REFERENCE TO RELATED APPLICATIONS 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. FEDERALLY SPONSORED RESEARCH 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. BACKGROUND 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. 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. SUMMARY 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