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Fluorescence Immunoassay Based on the Alkaline Phosphatase Triggered in Situ Fluorogenic Reaction of o-Phenylenediamine and Ascorbic Acid

Zhao, Dan, Li, Juan, Peng, Chuanyun, Zhu, Shuyun, Sun, Jian, Yang, Xiurong
Analytical chemistry 2019 v.91 no.4 pp. 2978-2984
alkaline phosphatase, antigens, ascorbic acid, biomarkers, enzyme-linked immunosorbent assay, fluorescence, fluorescent antibody technique, fluorescent dyes, hepatoma, models
Inspired by the special reducing capability of ascorbic acid (AA), ascorbic acid 2-phosphate (AA2P) has been extensively utilized as a substrate in current alkaline phosphatase (ALP) activity assays owing to the ALP-triggered transformation of AA2P into AA. However, such assays usually require AA-related complicated and laborious synthesis and/or signal generation procedures. Herein, we report an interesting in situ fluorogenic interaction between o-phenylenediamine (OPD) and AA, which inspires us to put forward a novel and simple AA2P/OPD-participated fluorescence turn-on ALP activity assay for the first time, and then the corresponding ALP-based fluorescence enzyme-linked immunosorbent assay (ELISA) has also been developed by means of the conventional ELISA platforms. According to the convenient and facile detection process with clear response mechanism, our fluorogenic reaction-based assay exhibits good sensitivity, selectivity, and excellent sensing performance, which ensures fluorescence ELISA to potentially be applied in clinical diagnosis by employing a well-studied biomarker of hepatocellular carcinoma, α-fetoprotein (AFP) as the model analyte. Such original ELISA via in situ formation of fluorophore from scratch gives a new sight to develop other potential immunoassay platforms in early clinical diagnosis by controlling the target antigens in the near future.