Integration of substrate-specific enzymes and a peroxide biosensor for detection of glucose, uric acid, and cholesterol

Conventional whole-cell biosensors function as “one analyte, one sensor” systems to ensure high specificity relying on transcriptional regulatory protein cascades to induce reporter gene expression, leading to the development of distinct sensor systems to monitor multiple target substances. To address this constraint and broaden analyte detection capabilities, we developed a modular biosensing platform that couples a key metabolite-responsive biosensor with various substrate-specific enzymes to detect a range of target molecules. This study demonstrated that the integration of a general hydrogen peroxide (H2O2)-responsive biosensor with glucose oxidase, uricase, and cholesterol oxidase enabled the detection of three clinically relevant biomarkers: glucose, uric acid, and cholesterol. Upon substrate-specific enzyme conversion, these three biomarkers generate H2O2, which triggers the expression of the luxCDABE reporter gene cluster and produces bioluminescence in the H2O2-responsive biosensor Pseudomonas putida KT2440[pPahpc]. The system has demonstrated sensitive detection of glucose (10–200 µM), uric acid (5–125 µM), and cholesterol (1.25–100 µM). Validation with 17 clinical urine specimens confirmed the reliability of our system for quantifying glucose and uric acid, thereby establishing its potential diagnostic utility.