Engineering trienzyme cascade-triggered fluorescent immunosensor platform by sequentially integrating alkaline phosphatase, tyrosinase and horseradish peroxidase

Mulit-enzyme cascades are a major type of chemical transformations and play a crucial role in biolog-ical signal transduction and metabolism. Herein, a trienzyme cascade-triggered fluorescent immunosen-sor platform was constructed by sequentially integrating alkaline phosphatase (ALP), tyrosinase (TYR) and horseradish peroxidase (HRP). The proposed platform was based on HRP-induced a rapid in situ fluorogenic reaction between dopamine (DA) and 1,5-dihydroxynaphthalene (DHA) to produce a strong yellow azamonardine fluorescent compound (AFC). The obtained AFC was clearly characterized by high -resolution mass spectrum, 1 H NMR, 13 C NMR and theoretical calculations. The integration of the two -enzyme system (TYR and HRP) or three-enzyme system (ALP, TYR and HRP) led to a maximum of 400.0 -fold and 250.0-fold fluorescence enhancements, respectively. Using cardiac troponin I (cTnI) as the model antigen, a trienzyme cascade-triggered fluorescent immunosensor platform was developed for quantita-tive detecting cTnI in a wide linear range from 2 ng/mL to 150 ng/mL with a detection limit of 0.67 ng/mL. In addition, the proposed platform was successfully applied in detection of cTnI in serum of clin-ical patients. Overall, the developed fluorescent immunosensor performs powerful implications for re-searching enzyme cascade systems in the field of biomedicine.(c) 2023 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.