Unconventional
Application of Conventional Enzymatic
Substrate: First Fluorogenic Immunoassay Based on Enzymatic Formation
of Quantum Dots
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Abstract
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 Cd<sup>2+</sup> with S<sup>2–</sup> 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<sub>ex</sub> = 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<sup>–1</sup> of analyte antibody with a linear range up to 10 ng mL<sup>–1</sup>. 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