A miniaturized heterogeneous fluorescence immunoassay utilizing radiationless energy transfer to gold surfaces for fluorescence quenching is described. The phase-separation fluorescence immunoassay (PSFIA), a competitive heterogeneous assay, is carried out in the nanowells of a gold-coated nano-titer plate (NTP). Small analytes such as atrazine and histamine can be detected in nanoliter volumes with low limits of detection, maintaining a high degree of parallelization and miniaturization. The assay is characterized and optimized with respect to gold-layer thickness, surface concentration of immobilized analyte derivative, and antibody concentration. Various immobilization strategies are discussed.
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