A label-free and modification-free ratiometric electrochemical strategy for enhanced natural enzyme detection using a bare electrode and nanozymes system.

Ratiometric electrochemical assays have been demonstrated to be more sensitive and selective in various sensing events, mainly due to their affordable built-in correction and good self-reference capability. But it is known that complicated modification and labeling operations usually are necessary for the construction of ratiometric electrochemical assays, therefore is a hot and important issue needing consideration carefully. We herein report a new yet simple bare electrode-based ratiometric electrochemical bioassay to achieve sensitive and selective analysis of alkaline phosphatase (ALP), using a liquid phase system that contains CoOOH nanozymes and commercially available indicator substrate. This proposed bioassay works based on the ratiometric change of dual electrochemical signals, arising from an exclusive target ALP-triggered hydrolysis of electrochemical substrate p-nitrophenyl phosphate (PNPP). In this design, the two hydrolyzed products of electrochemically active p-nitrophenol (PNP) and electrochemically inactive phosphate anion (PO43-) are responsible together for the ratiometric electrochemical analysis of ALP. PNP exhibits a straightforward current response toward ALP content; however, PO43- cannot show a direct electrochemical signal thus is rationally designed to offer an alternative response by linking it with the specific CoOOH nanozyme-catalyzed reaction of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, in which the nanozyme-catalyzed product oxTMB shows a direct reduction current at the GCE, and significantly decreases with increasing PO43- species due to the good inhibition of PO43- toward CoOOH nanozyme activity. As a result, a ratiometric electrochemical strategy for ALP analysis with a low limit of detection of 0.366 U/L (S/N = 3) was successfully achieved by integrating the above direct and indirect dual electrochemical responses. This developed bioassay can allow the quantitative diagnosis of ALP activity especially with a label-free and modification-free merit, therefore paving the way for simple, convenient, and portable electroanalytical tools in biosensing design and application.