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

Abstract

The present disclosure provides methods and systems for performing single-molecule detection using fabricated integrated on-chip devices. In an aspect, the present disclosure provides a method for on-chip detection of an array of biological, chemical, or physical entities, comprising: (a) providing an array of light sensing devices; (b) immobilizing the array of biological, chemical, or physical entities on a substrate of the array of light sensing devices; (c) exposing the array of biological, chemical, or physical entities to electromagnetic radiation sufficient to excite the array of biological, chemical, or physical entities, thereby producing an emission signal of the array of biological, chemical, or physical entities; (d) using the array of light sensing devices, acquiring pixel information of the emission signal of the array of biological, chemical, or physical entities without scanning the array of light sensing devices across the array of biological, chemical, or physical entities; and (d) detecting the array of biological, chemical, or physical entities based at least in part on the acquired pixel information.

1 .- 27 . (canceled) 
     
     
         28 . A method, comprising:
 (a) providing an array of biological entities on a substrate;   (b) contacting the array of biological entities with a fluorescent detection reagent;   
       wherein the fluorescent detection reagent comprises an affinity reagent attached to a first structured nucleic acid particle, and wherein the first structured nucleic acid particle comprises a nucleic acid origami; and
 (c) detecting presence or absence of a fluorescent signal from the fluorescent detection reagent at each biological entity of the array of biological entities. 
 
     
     
         29 . The method of  claim 28 , wherein a biological entity of the array of biological entities is immobilized to the substrate by a second structured nucleic acid particle. 
     
     
         30 . The method of  claim 29 , wherein providing the array of biological entities on the substrate comprises immobilizing the biological entities on the substrate. 
     
     
         31 . The method of  claim 30 , wherein immobilizing the biological entity to the substrate comprises attaching the second structured nucleic acid particle to the substrate. 
     
     
         32 . The method of  claim 30 , further comprising, before immobilizing the array of biological entities on the substrate, attaching the biological entity of the array of biological entities to the second structured nucleic acid particle. 
     
     
         33 . The method of  claim 29 , wherein the second structured nucleic acid particle comprises a short nucleic acid strand hybridized to a long nucleic acid strand. 
     
     
         34 . The method of  claim 33 , wherein the second structured nucleic acid particle comprises a nucleic acid origami. 
     
     
         35 . The method of  claim 30 , wherein immobilizing the array of biological entities on the substrate comprises non-covalently attaching the array of biological entities on the substrate. 
     
     
         36 . The method of  claim 30 , wherein immobilizing the array of biological entities on the substrate comprises covalently attaching the array of biological entities on the substrate. 
     
     
         37 . The method of  claim 29 , wherein the second structured nucleic acid particle is coupled to the substrate by a functional group. 
     
     
         38 . The method of  claim 37 , wherein the second structured nucleic acid particle is coupled to the substrate by a binding between a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is attached to the substrate and the second oligonucleotide comprises a strand of the structured nucleic acid particle. 
     
     
         39 . The method of  claim 28 , further comprising, before providing the array of biological entities on the substrate, modifying the substrate with functional groups that enhance attachment of the biological entities to the substrate. 
     
     
         40 . The method of  claim 28 , wherein the substrate comprises a passivating layer. 
     
     
         41 . The method of  claim 40 , wherein the functional groups and the passivating layer are patterned on the substrate. 
     
     
         42 . The method of  claim 28 , wherein a biological entity of the array of biological entities comprises a protein. 
     
     
         43 . The method of  claim 28 , wherein a biological entity of the array of biological entities comprises a small molecule, a DNA, an RNA, a glycoprotein, a metabolite, a carbohydrate, an enzyme, or an antibody. 
     
     
         44 . The method of  claim 28 , wherein a biological entity of the array of biological entities comprises a single protein bound to a single second structured nucleic acid particle. 
     
     
         45 . The method of  claim 28 , wherein the fluorescent detection reagent comprises two or more affinity reagents attached to the first structured nucleic acid particle.