Methods and systems for integrated on-chip single-molecule detection

Patent metadata

Field	Value
Publication number	US-12306093-B2
Application number	US-202117513877-A
Country	US
Kind code	B2
Filing date	Oct 28, 2021
Priority date	Apr 29, 2019
Publication date	May 20, 2025
Grant date	May 20, 2025

Abstract

The present disclosure provides methods and systems for performing single-molecule detection using fabricated integrated on-chip devices. Provided herein is a composition, comprising: (a) a substrate; (b) a structured nucleic acid particle coupled to the substrate, wherein a biological entity is coupled to the structured nucleic acid particle; and (c) a fluorescent detection agent, wherein the fluorescent detection agent comprises an affinity reagent attached to another structured nucleic acid particle, wherein the affinity reagent is bound to the biological entity.

First claim

What is claimed is:

1. A composition, comprising:
a. a substrate;
b. a first structured nucleic acid particle coupled to the substrate, wherein a biological entity is coupled to the first structured nucleic acid particle; and
c. a fluorescent detection agent, wherein the fluorescent detection agent comprises an affinity reagent attached to a second structured nucleic acid particle, wherein the affinity reagent is bound to the biological entity, wherein the first structured nucleic acid particle is coupled to the substrate by binding between a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is attached to the substrate, and wherein the second oligonucleotide comprises a strand of the structured nucleic acid particle.

2. The composition of claim 1 , wherein the biological entity comprises a protein.

3. The composition of claim 1 , wherein the biological entity comprises a small molecule, a DNA, an RNA, a glycoprotein, a metabolite, a carbohydrate, an enzyme, or an antibody.

4. The composition of claim 1 , wherein the biological entity consists essentially of a single protein.

5. The composition of claim 1 , wherein the first structured nucleic acid particle comprises a nucleic acid origami.

6. The composition of claim 5 , wherein the nucleic acid origami comprises a long nucleic acid strand and one or more short nucleic acid strands.

7. The composition of claim 6 , wherein the nucleic acid origami comprises at least 100 short nucleic acid strands.

8. The composition of claim 7 , wherein the nucleic acid origami comprises a landing surface that preferentially contacts the substrate.

9. The composition of claim 1 , wherein the second structured nucleic acid particle comprises a nucleic acid origami.

10. The composition of claim 1 , wherein the first structured nucleic acid particle comprises a short nucleic acid strand hybridized to a long nucleic acid strand.

11. The composition of claim 1 , wherein the fluorescent detection agent comprises two or more affinity reagents coupled to the second structure nucleic acid particle.

12. The composition of claim 1 , wherein the affinity reagent comprises an antibody or an antibody fragment.

13. The composition of claim 1 , wherein the affinity reagent comprises an aptamer.

14. The composition of claim 1 , wherein the affinity reagent comprises a mini-peptide binder or a peptimer.

15. The composition of claim 1 , wherein the affinity reagent comprises a known degree of nonspecificity.

16. The composition of claim 1 , wherein the substrate comprises silicon, silica, fused silica, quartz, or glass.

17. The composition of claim 1 , wherein the substrate comprises a microstructure that is coupled to the first structured nucleic acid particle.

18. The composition of claim 1 , wherein the substrate further comprises a passivating layer.

19. The composition of claim 1 , wherein the substrate further comprises a functionalized region.

20. The composition of claim 1 , wherein the functionalized region comprises a filamentous molecule.

21. The composition of claim 20 , wherein the filamentous molecule comprises an oligonucleotide or a nanoparticle.

22. The composition of claim 20 , wherein the first structured nucleic acid particle comprises a moiety that is bound to the filamentous molecule.

23. The composition of claim 1 , wherein the substrate comprises a light-sensing device.

24. The composition of claim 23 , wherein the light-sensing device comprises a charge-coupled device, a complementary metal-oxide semiconductor sensor, a charge injection device, or a JOT image sensor.

Assignees

• Nautilus Subsidiary Inc

Inventors

• Indermuhle Pierre
• Sorelle Elliott
• Stern David
• Mallick Parag
• Patel Sujal M

Classifications

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• G01N2021/7793

  Sensor comprising plural indicators · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N33/54366

  Apparatus specially adapted for solid-phase testing · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title