Embodiments of this disclosure encompass methods, systems and probes for the detection of a target analyte in a sample. The method uses of the detection of at least two distinct sites on an analyte molecule, or the pairing of two distinct sites on two adjacent and contacting molecules, the sites being integral to the structure of a single molecule or positioned near one another due to the three-dimensional structure of the polypeptide. At least one of the detectable sites may be formed by a modification of a larger molecule. The methods of detecting a target analyte comprise contacting a sample with a pair of probes, each probe comprising a binding moiety capable of specifically binding to a target analyte o a tag thereon, and an oligonucleotide tail that comprises a PCR initiator region proximal to the target analyte binding moiety, a barcoding region uniquely associated with the target analyte binding moiety, and a connector-hybridizing region complementary to a region of a connector oligonucleotide; hybridizing a connector oligonucleotide to the connector- hybridizing regions of the probes and ligating the connector-hybridizing regions of the probes; PCR amplifying the ligated oligonucleotide tails; hybridizing the amplification product with a substrate-immobilized oligonucleotide that as regions complementary to the barcoding regions of the probes; digesting any single-strand DNA molecule; hybridizing a signaling oligonucleotide to the product of the previous step; and detecting the signal, thereby detecting the presence of the analyte in the sample.
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