DENSELY-PACKED ANALYTE LAYERS AND DETECTION METHODS
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These methods and systems may have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
1 . A method for determining a relative position of analytes deposited on a surface of a densely packed substrate, comprising:
(a) providing a substrate comprising a surface, wherein said surface is patterned or unpatterned and comprises a plurality of analytes deposited on said surface at discrete locations;
(b) performing a plurality of cycles of probe binding and signal detection on said surface, wherein a cycle of said plurality of cycles comprises:
(i) contacting said plurality of analytes with a plurality of probes from a probe set, wherein a probe of said plurality of probes comprises a detectable label, wherein said probe binds to an analyte of said plurality of analytes;
(ii) introducing said plurality of analytes to a solution comprising erythorbic acid; and
(iii) imaging said surface with an optical unit to detect one or more optical signals from said probe bound to said analyte.
2 . The method of claim 1 , wherein said solution further comprises glutathione.
3 . The method of claim 1 , further comprising determining an identity of one or more analytes of said plurality of analytes.
4 . The method of claim 1 , wherein said solution inhibits light-induced degradation of said plurality of analytes.
5 . The method of claim 1 , wherein said plurality of analytes are irradiated in said solution.
6 . The method of claim 3 , wherein said determining said identity of one or more analytes of said plurality of analytes comprises sequencing by synthesis.
7 . The method of claim 1 , wherein said solution further comprises tris(hydroxymethyl)aminomethane (Tris-HCl) or another pH buffering agent.
8 . The method of claim 1 , wherein said solution comprises about 1 mM to 10 mM erythorbic acid.
9 . The method of claim 2 , wherein said solution comprises about 5 mM to 20 mM glutathione.
10 . The method of claim 7 , wherein said solution comprises about 10 mM to 30 mM Tris-HCl.
11 .- 30 . (canceled)
31 . A method of controlling an average minimum center-to-center distance between one or more analytes, comprising:
exposing a plurality of analytes deposited on a surface to a gas-liquid interface to form a monolayer of analytes deposited across said surface,
wherein said plurality of analytes are treated with a repellant or attractive substance, and
wherein said plurality of analytes is deposited on said surface at an average center-center-to-center distance of less than about 500 nm.
32 . The method of claim 31 , wherein said surface is patterned or non-patterned.
33 . The method of claim 31 , wherein said gas-liquid interface comprises an air-water interface.
34 . The method of claim 31 , further comprising introducing said plurality of analytes to a solution comprising erythorbic acid.
35 . The method of claim 34 , wherein said solution comprises about 1 mM to 10 mM erythorbic acid.
36 . The method of claim 31 , wherein said plurality of analytes comprises a plurality of concatemers.
37 . The method of claim 31 , further comprising contacting said plurality of analytes with a plurality of probes, wherein a probe of said plurality of probes comprises a detectable label, wherein said probe binds to an analyte of said plurality of analytes.
38 . The method of claim 37 , further comprising imaging said surface with an optical unit to detect one or more optical signals from said probe bound to said analyte of said plurality of analytes.
39 . The method of claim 31 , wherein said plurality of analytes comprise one or more nucleic acid molecules.
40 . The method of claim 31 , wherein said repellant or attractive substance comprises zwitterionic features.