title: Unconventional application of conventional enzymatic substrate: first fluorogenic immunoassay based on enzymatic formation of quantum dots.
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 p-nitrophenyl phosphate (pNPP) leads to the formation of p-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd(2+) 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 λex = 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 p-nitrophenyl phosphate assay.
authors.affiliation: CICbiomaGUNE, Parque tecnológico de San Sebatián, Donostia-San Sebastián 20009, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Malashikhina
authors.forename: Gaizka
authors.initials: G
authors.lastname: Garai-Ibabe
authors.forename: Valeri
authors.initials: V
authors.lastname: Pavlov
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods; D045663:Quantum Dots / Q000737:chemistry*; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes; D000469:Alkaline Phosphatase