Hydrogel-Embedded Platinum–Ruthenium Alloy Peroxidase Mimic as a Visual Sensor for “Signal-on” Alkaline Phosphatase Detection

Nanozyme-assisted alkaline phosphatase (ALP) assay is a commonly used detection technique. However, this type of sensor faces several challenges, including a "signal-off" detection model, dependence on oxidase mimics, and the need for spectrometers. In this study, by optimizing stabilizers, we obtain chitosan-coated platinum–ruthenium nanoparticles (CS-Pt/Ru NPs) for the first time, which have superior peroxidase (POD) mimic activity. CS-Pt/Ru nanozymes can catalyze phenol oxidation in the presence of 4-aminoantipyrine and hydrogen peroxide to produce an enhanced red color, while phenol can be obtained by ALP-mediated hydrolysis. Thus, ALP activity is quantified by absorbance in a "signal-on" model. This approach demonstrates a remarkably low limit of detection (0.01 U·L–1) and a wide linear range (0.03–10 U·L–1). In addition, we combine CS-Pt/Ru nanozymes with a hydrogel to form a CS-Pt/Ru@Gel sensor, which enables a visual ALP assay using a smartphone. It can detect ALP activity as low as 0.1 U·L–1, with a linear range of 0.15–10 U·L–1. Finally, both Pt/Ru NPs and Pt/Ru@Gel are successfully employed to detect ALP activity in clinical blood samples. Our study develops an excellent hydrogel-supported POD nanozyme system and paves the way for an ALP assay using a "signal-on" and visual model.