Granted Patent B2

US 12,460,249 · App. 15/914,729 · Granted Nov 4, 2025

Inventors: Alexander Edmund Johnson-Buck (Brighton, MA); Nils Walter (Ann Arbor, MI); Muneesh Tewari (Ann Arbor, MI)

Assignee: The Regents of the University of Michigan

C12Q1/6809C12N9/22C12P19/34C12Q1/6834C12Q1/6876C40B40/06G01N33/5308G01N33/542G01N33/54306

Abstract

Claims (20)

1. A system for detecting or quantifying a protein analyte in a sample, the system comprising:

(a) a protein analyte-specific capture probe that stably immobilizes a protein analyte to a surface;

(b) a query probe that binds to the protein analyte with a dissociation rate constant (k off ) for the analyte that is greater than 10 −2 s −1 ;

(c) a fluorescence microscope having single-molecule sensitivity and configured to provide a series of images of the surface; and

(d) a processor programmed to execute instructions to:

process each image of said series of images to measure signal intensities produced by a query probe binding to a protein analyte and/or by a query probe dissociating from a protein analyte;

record said signal intensities at the surface as a function of (x, y) position on the surface;

produce a differential signal intensity map comprising time-dependent signal intensity changes at the surface as a function of (x, y) position by subtracting an intensity value of each pixel P of each image N from a corresponding intensity value of the same pixel P of each subsequent image N+1, wherein the (x, y) position of each time-dependent signal intensity change is determined by processing the differential intensity map using centroid determination, least-squares fitting to a Gaussian function, least squares fitting to an Airy disk function, least squares fitting to a polynomial function, or maximum likelihood estimation;

cluster the time-dependent signal intensity changes at the surface using (x, y) position to produce event clusters; and

calculate a kinetic parameter for each event cluster to detect or quantify the protein analyte.

2. The system of claim 1 , wherein the query probe comprises an antibody, antibody fragment, aptamer, nanobody, kinase, phosphatase, acetylase, deacetylase, enzyme, polypeptide, or a small organic molecule.

3. The system of claim 1 , wherein the protein analyte-specific capture probe is a surface-bound capture probe.

4. The system of claim 3 wherein the protein analyte-specific capture probe is an antibody, antibody fragment, or nanobody.

5. The system of claim 1 , wherein the protein analyte is denatured.

6. The system of claim 1 wherein the surface is a solid support.

7. The system of claim 1 wherein a diffusible solid support comprises said surface.

8. The system of claim 1 wherein the protein analyte is a component of a macromolecular complex.

9. The system of claim 1 wherein the protein analyte comprises a post-translational modification.

10. The system of claim 9 , wherein a chemical affinity tag mediates a transient association between the protein analyte comprising a post-translational modification and the query probe.

11. The system of claim 1 further comprising a component configured to thermally denature the analyte prior to surface immobilization or further comprising a chemical denaturant to chemically denature the analyte prior to surface immobilization.

Assignments (1)

Continuity (2)

References Cited (400)