Enzyme-Linked Immunoassays Using Nanosecond Fluorometric Detection

Abstract

Nanosecond temporal resolution is combined with an enzyme-linked immunosorbent assay (ELISA) to improve the lower limit of detection for a plant virus, brome mosaic virus. The method uses alkaline phosphatase as the enzyme link and β-naphthyl phosphate as the substrate. Enzymatic activity produces the highly fluorescent tag β-naphthol. The 8.9-ns lifetime of the tag allows temporal discrimination against the assay blank, providing a 64× improvement in the detection limit as compared to a steady-state measurement, and a ∼4100× improvement over a standard ELISA method incorporating the chromogenic substrate p-nitrophenyl phosphate.

Optical demodulation system for digitally encoded suspension array in fluoroimmunoassay

“…7 By measuring the amount of captured biomolecules, the concentration of target biomolecules in samples could be deduced proportionally. In fluoroimmunoassay analysis, the amounts of captured molecules are calculated by collecting the fluorescence emission from the label, 8,9 which indicates that the quantitative analysis of analyte can be achieved by the fluorescence measurement of a single microbead. 10,11 Once various types of microbeads can specifically capture different analytes in the same sample and can be recognized with each other, the microbead-based detection mode can endow suspension array the potential of multiplexing.…”