Synergistic Catalysis of Gold–Platinum Alloy Nanozymes: A Novel Colorimetric Sensor for ALP Detection in Complex Biological Matrices

<b>Background/Objectives</b>: Alkaline phosphatase (ALP) is a crucial enzyme in numerous pathological processes and a significant biomarker in clinical diagnostics. Conventional ALP detection methods are hampered by reliance on complex sample pretreatment, sophisticated instrumentation, time-consuming procedures, and high costs. This study aimed to develop a simple, rapid, and cost-effective colorimetric sensing method for ALP detection with enhanced resistance to matrix interference in biological samples. <b>Methods</b>: We designed a colorimetric assay based on bimetallic gold-platinum nanocatalysts (AuPt NPs) exhibiting peroxidase-like (POD-like) activity. The detection principle involves a dual-reaction cascade: (1) Alkaline phosphatase (ALP) catalyzes the conversion of trisodium L-ascorbic acid-2-phosphate (AA₂P) into ascorbic acid (AA), and (2) the generated AA reduces oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) produced by the catalytic activity of AuPt NPs. This method was evaluated for its detection performance in diluted human serum without complex sample pretreatment. <b>Results</b>: AuPt NPs exhibited resistance to biological matrix interference, enabling sensitive detection of ALP. The assay showed a linear ALP detection range of 0-90 mU·mL^-1 (R² = 0.994) and a limit of detection of 3.91 mU·mL^-1. In spiked human serum, recoveries were 95.45-111.97%, with negligible interference from ions and biomolecules. <b>Conclusions</b>: We developed a simple, rapid, and reliable colorimetric sensor for ALP detection based on AuPt NPs. It overcomes limitations of conventional methods, holding great potential for clinical diagnostics and point-of-care applications.