Optically decodable microcarries, arrays and methods

A hybridization assay for at least one of a multiplicity of nucleic acid sequences in an analyte comprising the steps of:

• (a) contacting said analyte with a mixture of encoded microcarriers having immobilized on their surfaces
• (i) a hybridization probe for one of said multiplicity of sequences, whose hybridization to said at least one sequence can be detected, and
• (ii) a coding scheme comprising a plurality of signaling hairpins that are not hybridization probes for said multiplicity of sequences, including said at least one sequence, comprising quenched, fluorophore-labeled hairpin molecules each comprising an interacting affinity pair separated by a linking moiety, one member of said affinity pair having bound thereto at least one quenched fluorophore, wherein interaction of the affinity pair is disruptable to unquench said at least one fluorophore by a physical or chemical change in a condition of its environment, wherein the disruption of the interaction of at least one affinity pair occurs at a first level of said condition and the disruption of the interaction of at least another affinity pair occurs at a second level of said condition, and wherein said disruptions are optically differentiable, and wherein the coding scheme for identifying individual microcarriers in said mixture comprises a combination of multiple spectrally differentiable fluorophores and multiple affinity pairs disruptable at detectably different levels of said condition;
• (b) forming a distributed array of said microcarriers wherein location of said microcarriers in said distributed array is not used to identify said at least one nucleic acid sequence;
• (c) determining which microcarriers have hybridization probes hybridized to said at least one nucleic acid sequence of said analyte;
• and
• (d) optically decoding the microcarriers having said at least one nucleic acid sequence hybridized to its hybridization probes to identify said at least one nucleic and sequence by changing said condition to said detectably different levels to disrupt quenching, and detecting changes in fluorescence from the signaling hairpins.