Assay methods

A method of detecting an analyte of interest in a sample comprising:

• a. contacting a complex on a surface comprising the analyte, a binding reagent for the analyte, a first detection reagent for the analyte comprising a first proximity probe, and a second detection reagent for the analyte comprising a second proximity probe, with one or more connector oligonucleotides comprising a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, wherein the contacting is performed under conditions sufficient for the first circularization probe and the second circularization probe to form a circular DNA template, and wherein the surface further comprises an anchoring reagent comprising an anchoring sequence;
• d. amplifying the circular DNA template to generate an amplicon comprising a plurality of detection sequences and an anchoring sequence complement;
• c. hybridizing the anchoring sequence to the anchoring sequence complement;
• d. hybridizing a plurality of detection probes to the plurality of detection sequences;
• and
• e. measuring the amount of analyte bound to the surface.

A method of detecting a plurality of analytes of interest in a sample comprising:

• a. forming a plurality of complexes on a surface comprising a plurality of discrete binding domains, each complex comprising an analyte of the plurality of analytes, a binding reagent for the analyte, a first detection for the analyte comprising a first proximity probe, and a second detection reagent for the analyte comprising a second proximity probe, wherein each complex is in a discrete binding domain of the surface, and wherein each binding domain comprises an anchoring reagent comprising an anchoring sequence;
• b. contacting each complex of the complex of the plurality of complexes with one or more connector oligonucleotides comprising:
• a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, wherein the contacting is performed under conditions sufficient for the first circularization probe and the second circularization probe to form a circular DNA template, thereby forming a plurality of circular DNA templates;
• c. amplifying the circular DNA template of each complex to generate a plurality of amplicons each comprising a plurality of detection sequences and an anchoring sequence complement;
• d. hybridizing the anchoring sequence to the anchoring sequence complement in each binding domain;
• e. hybridizing a plurality of detection probes to the plurality of detection sequences in each binding domain;
• and
• f. measuring the amount of the plurality of analytes bound to the surface.

A kit for the measurement of an analyte of interest in a sample, the kit comprising:

• a. a surface comprising a binding reagent for the analyte and an anchoring reagent comprising an anchoring sequence;
• and
• b. in one or more containers, compartments, or vessels:
• i. a first detection reagent for the analyte and a second detection reagent for the analyte, wherein the first and second detection reagents comprise a first proximity probe and a second proximity probe, respectively;
• ii. one or more connector oligonucleotides comprising a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, wherein the one or more connector oligonucleotides further comprises a detection sequence complement;
• and
• iii. one or more detection probes complementary to the detection sequence complement.