Enzyme-nanozyme cascade-amplified ratiometric fluorescence immunoassay for the ultrasensitive detection of saxitoxin

Herein, a ratiometric fluorescence (RF) immunoassay platform coupling with enzyme-nanozyme cascade amplification strategy was fabricated for precise and ultrasensitive quantification of saxitoxin (STX). In this strategy, oxidase-like cobalt oxyhydroxide nanoflakes (CoOOH NFs) catalyzed the oxidation of non-fluorescent o-phenylenediamine (OPD) into fluorescent 2,3-diaminophenazine with the maximum emission at 568 nm. This nanozyme-triggered fluorogenic reaction was cascaded with the alkaline phosphatase (ALP)-catalyzed hydrolysis of ascorbic acid 2-phosphate into ascorbic acid (AA) through the redox reaction between AA and CoOOH NFs. During the redox reaction, CoOOH NFs were reduced into Co2+ without oxidase-like activity, whereas AA was converted into dehydroascorbic acid (DHAA). Consequently, an ALP activity-dependent RF response was achieved by coupling with the condensation reaction between OPD and the DHAA, which yields fluorescent 3-(1,2-dihydroxy ethyl) furo[3,4-b] quinoxalin-1(3 H)-on with the maximum emission at 430 nm. Using the anti-STX polyclonal antibody and the commercial ALP-labelled secondary antibody as the recognition unit and signal transducing module, an ultrasensitive and accurate RF-ELISA for STX was developed with a detection limit of 3.2 pg/mL, which was 12-fold more sensitive than that of classic colorimetric ELISA. This work opened up new ways for ultrasensitive and accurate detection of STX and other analytes in food safety supervision.