Fluorescent enzyme-linked immunosorbent assay based on alkaline phosphatase-responsive coordination polymer composite.

The fabrication of alkaline phosphatase (ALP)-responsive coordination polymer (CP) composite is demonstrated for establishing a fluorescent immunoassay. The CP composite (ThT@GMP/Eu) was synthesized by encapsulating thioflavin T (ThT) into the CP host that was composed of europium ion (Eu3+) and guanine monophosphate (GMP). The ThT@GMP/Eu composite shows a strong fluorescence in aqueous solution due to the confinement effect of GMP/Eu CPs, which restricts the conformational rotation of ThT. However, upon the addition of ALP, the structure of GMP/Eu CPs was disrupted to release ThT into solution. This results in the quenching of the fluorescence of ThT@GMP/Eu. The fluorescence of ThT@GMP/Eu has a linear response that covers 0.8 to 120 mU/mL ALP with a detection limit of 0.26 mU/mL and exhibits excellent specificity towards ALP against other enzymes. On this basis, inspired by the wide application of ALP as an enzyme label in enzyme-linked immunosorbent assay (ELISA), an ALP-based fluorescent immunoassay was further developed for the detection of mouse immunoglobulin G (mIgG). The developed immunoassay displays a linear fluorescent response towards mIgG from 0.8 to 100 ng/mL, and the detection limit is 0.16 ng/mL. The fluorescent immunoassay was successfully applied to the determination of mIgG in serum samples. Schematic of the responsivity of ThT@GMP/Eu to ALP that hydrolyzes GMP to release ThT, which leads to fluorescent quenching, and its application in the construction of a fluorescent immunoassay for mIgG determination.