Granted Patent B2

US 12,480,937 · App. 18/468,210 · Granted Nov 25, 2025

Inventors: Stuart Lindsay (Phoenix, AZ); Peiming Zhang (Phoenix, AZ)

Assignee: RECOGNITION ANALYTIX, INC.

G01N33/48721C12Q1/005G01N27/4146

Abstract

Claims (27)

1 . An array for direct measurement of protein activity comprising:

a plurality of devices, each device comprising a first electrode and a second electrode separated by a gap, and a protein attached to the first electrode via a first linker at a first attachment point and to the second electrode via a second linker at a second attachment point,

wherein the first linker and the second linker are distinct, and wherein each of the first and the second linkers is attached to a region of the protein that is inactive.

2 . The array of claim 1 , wherein the gap has a width of about 1.0 nm to about 20.0 nm.

3 . The array of claim 1 , wherein the first and second electrodes are separated by a dielectric layer, and wherein a passivation layer is disposed on the first and second electrodes.

4 . The array of claim 1 , wherein the protein is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase and an endonuclease.

5 . The array of claim 1 , wherein the protein is a polymerase.

6 . The array of claim 5 , wherein exonuclease activity of the polymerase is disabled.

7 . The array of claim 1 , wherein the protein is biotinylated.

8 . The array of claim 1 , wherein the first and/or the second linker comprises thiol-streptavidin.

9 . The array of claim 1 , wherein the protein and the first and/or second electrodes are biotinylated, and wherein the first and/or the second linker comprises a streptavidin molecule comprising at least two biotin binding sites.

10 . The array of claim 1 , wherein the first and/or second electrodes comprise a metal selected from the group consisting of gold, platinum, palladium, and ruthenium, or any combinations thereof.

11 . The array of claim 1 , wherein the first or the second linker is attached to the N-terminal region of the protein.

12 . The array of claim 11 , wherein the first or the second linker is attached to the exonuclease region of the polymerase.

13 . The array of claim 1 , wherein the array is configured to expose the plurality of devices to a solution comprising unincorporated dNTPs and/or a DNA template.

14 . The array of claim 1 , wherein the array is configured to apply a voltage bias to the first and second electrodes of each of the plurality of devices.

15 . The array of claim 1 , wherein the array is configured to detect fluctuations in current between the first and second electrodes.

16 . A method of sequencing a polynucleotide, said method comprising:

(a) introducing a primed polynucleotide template to the array of claim 1 , wherein the protein is a polymerase and wherein the polymerase is bound to the template;

(b) introducing a solution comprising dNTPs;

(c) applying a voltage bias between the first and second electrodes that is 100 mV or less;

(d) detecting fluctuations in current between the first and second electrodes that occur when each dNTP is incorporated as an additional nucleotide complementary to a corresponding nucleotide in the polynucleotide template; and

(e) determining the identity of each additional nucleotide being incorporated.

17 . The method of claim 16 , wherein each dNTP is present in the solution at about the same concentration.

18 . The method of claim 16 , wherein each dNTP is present at a concentration that is about equal to or above the saturation concentration of the template-bound polymerase.

19 . The method of claim 16 , wherein step (d) comprises detecting the presence of one or more current spike(s).

20 . The method of claim 19 , wherein step (e) comprises using the characteristics of each spike.

Assignments (3)

Continuity (5)

References Cited (331)