Nucleic acid linked immune-sandwich assay (NULISA)

1 . An assay method for detecting one or multiple different analytes in a sample, comprising:

(1) mixing one or more first binding moieties, wherein each of the one or more first binding moieties comprises a first binder and a first presenting group, one or more second binding moieties, wherein each of the one or more second binding moieties comprises a second binder and a second presenting group, and the sample in a solution, wherein for each pair of a first binding moiety and a second binding moiety:

(i) the first binder and the second binder bind to an analyte of the one or multiple different analytes and form an immunocomplex,

(ii) the immunocomplex is captured on a first solid surface in contact with the solution via binding between the first presenting group and a first receiving group coupled to the first solid surface, and

(iii) the first binding moiety further comprises a first target label and the second binding moiety further comprises a second target label;

(2) washing the first solid surface to remove unbound molecules;

(2a) releasing the immunocomplex from the first solid surface;

(2b) introducing a second solid surface and capturing the immunocomplex on the second solid surface via binding between the second presenting group and a second receiving group coupled to the second solid surface;

(3) generating a reporter from the immunocomplex based on proximity between the first target label and the second target label; and

(4) detecting the reporter, thereby detecting the analyte.

2 . The assay method of claim 1 , wherein (i) the first target label comprises a first identity barcode (“ID”) that is analyte-specific (“target ID”); (ii) the second target label comprises a second target ID; or (iii) both (i) and (ii).

3 . The assay method of claim 2 , wherein (i) the reporter comprises the first target ID; (ii) the reporter comprises the second target ID; or (iii) both (i) and (ii).

4 . The assay method of claim 3 , wherein the reporter is a nucleic acid reporter, wherein:

(i) the first target ID in the reporter is a complementary sequence of the first target ID in the first binding moiety;

(ii) the second target ID in the reporter is a complementary sequence of the second target ID in the first binding moiety; or

(iii) both (i) and (ii).

5 . The assay method of claim 1 , further comprising:

a step 2(c) between step 2(b) and step (3):

washing the second solid surface to remove unbound molecules.

6 . The assay method of claim 5 , further comprising a step (2d): releasing the immunocomplex from the second solid surface by disrupting the binding between the second presenting group and the second receiving group.

7 . The assay method of claim 6 , further comprising a step (2e): incorporating a sample label comprising an ID that is sample-specific (“sample ID”) (i) to the first target label, (ii) to the second target label, or (iii) to both the first target label and the second target label.

8 . The assay method of claim 1 , wherein step (1) comprises forming the immunocomplex in the solution before capturing the immunocomplex on the first solid surface.

9 . The assay method of claim 8 , wherein

(i) the first presenting group is a first nucleic acid tag (a “first tag”) and the first receiving group is a first nucleic acid capture probe (a “first probe”); or

(ii) the second presenting group is a second nucleic acid tag (a “second tag”) and the second receiving group is a second nucleic acid capture probe (a “second probe”).

10 . The assay method of claim 9 , wherein (i) the first probe is conjugated with biotin, which binds streptavidin or avidin that is directly coupled to the first solid surface; or (ii) the second probe is conjugated with biotin, which binds streptavidin or avidin that is directly coupled to the second solid surface.

11 . The assay method of claim 5 , wherein:

(i) the first presenting group is conjugated to the first binder;

(ii) the first presenting group is non-covalently bound to the first binder;

(iii) the first presenting group is conjugated to the first target label;

(iv) the first presenting group is non-covalently bound to the first target label; or

(v) the first presenting group is part of the first target label, and

wherein:

(i) the second presenting group is conjugated to the second binder;

(ii) the second presenting group is non-covalently bound to the second binder;

(iii) the second presenting group is conjugated to the second target label;

(iv) the second presenting group is non-covalently bound to the second target label; or

(v) the second presenting group is part of the second target label.

12 . The assay method of claim 7 , wherein the sample label is:

(i) a double-stranded nucleic acid molecule comprising two 5′ overhangs;

(ii) a double-stranded nucleic acid molecule comprising two 3′ overhangs;

(iii) a double-stranded nucleic acid molecule comprising a 5′ overhang and a 3′ overhang;

(iv) a double-stranded nucleic acid molecule comprising a 5′ overhang and a blunt end; or

(v) a double-stranded nucleic acid molecule comprising a 3′ overhang and a blunt end, and

wherein the sample label:

(i) hybridizes with the first target label via an overhang of the sample label;

(ii) hybridizes with the second target label via an overhang of the sample label; or

(iii) both (i) and (ii).

13 . The assay method of claim 1 , further comprising, in step (1), mixing a reference analyte.

14 . The assay method of claim 1 , wherein one or more nucleic acid-based internal reference is added in one or more steps.

15 . The assay method of claim 14 , wherein the one or more nucleic acid-based internal reference are physically restrained.

16 . The assay method of claim 14 , wherein the one or more nucleic acid-based internal reference comprises binding moieties to be captured by one or more solid surface, and/or comprises ligation sites that are compatible with ligation reagents.

17 . The assay method of claim 5 , wherein the immunocomplex is released from the first and/or the second solid surface using an elution buffer with a pH greater than about 10.0.

18 . The assay method of claim 5 , wherein the immunocomplex is released from the first and/or the second solid surface using an elution buffer with a pH greater than about 11.0.

19 . The method of claim 17 , wherein the elution buffer is a solution of NaOH.

20 . The method of claim 19 , wherein the NaOH solution has a concentration of about 10 mM or a pH of about 12.0.

21 . The method of claim 1 , wherein the assay method detects multiple different analytes in the same sample in parallel.

22 . The method of claim 21 , wherein the assay method detects at least twenty different analytes in the same sample in parallel.