PROXIMITY ASSAY WITH DETECTION BASED ON HYBRIDISATION CHAIN REACTION (HCR)

The present invention provides a method for detecting an analyte in a sample, said method comprising a) contacting said sample with a set of proximity probes comprising at least first and second proximity probes, which probes each comprise an analyte-binding domain capable of binding directly or indirectly to said analyte and a nucleic acid domain, such that the proximity probes can simultaneously bind, directly or indirectly, to the analyte, wherein i) the nucleic acid domains of said first and second proximity probes comprise regions capable of mediating an interaction involving said domains when under permissive conditions; and ii) the nucleic acid domain of one of said first and second probes comprises an HCR initiator region comprised within a metastable secondary structure such that it is unable to initiate an HCR reaction until released from said metastable secondary structure; b) introducing permissive conditions to allow the nucleic acid domains of said first and second probes to interact with each other when said probes have both bound directly or indirectly to the analyte, wherein said interaction results in unfolding of the metastable secondary structure of the nucleic acid domain of the first or second probe to release a single-stranded HCR initiator region; c) performing an HCR reaction using at least two HCR monomers, wherein the first HCR monomer comprises a region of complementarity to the HCR initiator region and hybridisation of the HCR initiator region to the first HCR polymer begins the HCR reaction to form a polymer; and d) detecting the polymer thereby to detect the analyte.