Unconventional Application of Conventional Enzymatic Substrate: First Fluorogenic Immunoassay Based on Enzymatic Formation of Quantum Dots

In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of <i>p</i>-nitrophenyl phosphate (pNPP) leads to the formation of <i>p</i>-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd²⁺ with S^2– ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λ_ex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL^–1 of analyte antibody with a linear range up to 10 ng mL^–1. The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric <i>p</i>-nitrophenyl phosphate assay