Signal encoding and decoding in multiplexed biochemical assays

A method of unambiguously detecting any unique combination of presence or absence of at least five polynucleotide analytes in a plurality of droplets, the method comprising:

• (a) providing a sample comprising, or potentially comprising, at least one of said at least five polynucleotide analytes;
• (b) forming a mixture of said sample and at least five hybridization probes, wherein each of said at least five hybridization probes further comprises at least one fluorophore and at most four fluorophores;
• (c) partitioning said mixture into said plurality of droplets;
• (d) exciting said at least one fluorophore to generate one or more signals if one or more of said at least five polynucleotide analytes is present in said plurality of droplets, wherein said one or more signals comprise at least one signal generated by excitement of said at least one fluorophore;
• (e) measuring said one or more signals to generate a cumulative intensity measurement, wherein said cumulative intensity measurement corresponds to the presence of a unique combination of presence or absence of said at least five polynucleotide analytes in said sample;
• and
• (f) determining whether each of said least five polynucleotide analytes is present, in any unique combination of presence or absence, based on said cumulative intensity measurement,
• wherein the method does not require any step of immobilization of said at least five polynucleotide analytes or mass spectrometry.