Patent ID: 8486719

Claim:
An assay method, comprising: A) providing an assay mixture, comprising, in combination: a first interacting member, wherein the first interacting member is an enzyme, a second interacting member, wherein the second interacting member is a substrate for the enzyme, a first solid-phase support comprising a first specific binding agent selective for the first interacting member and a photosensitizer, wherein the photosensitizer is light excitable to activate oxygen to produce singlet oxygen, a second solid-phase support comprising a second specific binding agent selective for the second interacting member and a first emission system comprising a first energy acceptor activated by singlet oxygen, the first emission system activated by singlet oxygen to emit a first light signal characterized by a first emission spectrum, wherein specific interaction of the first interacting member and the second interacting member brings the photosensitizer and the first emission system into proximity, such that the irradiating generates the first light signal indicative of specific interaction of the first interacting member and the second interacting member, and a third solid-phase support comprising a third specific binding agent selective for the second interacting member, wherein the third specific binding agent selectively interacts with enzyme-modified substrate, and a second emission system comprising a second energy acceptor activated by singlet oxygen, the second emission system activated by singlet oxygen to emit a second light signal characterized by a second emission spectrum, wherein the first light signal and second light signal are distinguishable, wherein binding of the third specific binding agent with the second interacting member brings the photosensitizer and the second emission system into proximity, such that the irradiating generates the second light signal indicative of binding of the third specific binding agent and second interacting member, B) irradiating the assay mixture with a light source, thereby activating the photosensitizer to produce singlet oxygen; and C) detecting the first light signal and the second light signal, wherein detecting the first light signal is indicative of proximity of a first target bound to the first specific binding agent and a second target bound to the second specific binding agent and wherein detecting the second light signal is indicative of proximity of the first target bound to the first specific binding agent and third target bound to the third specific binding agent.