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

ABSTRACT 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 (H 2 O 2 )-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 H 2 O 2 , which triggers the expression of the lux CDABE reporter gene cluster and produces bioluminescence in the H 2 O 2 -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. IMPORTANCE The increasing public focus on health management has fueled the development of decentralized diagnostic testing. While biosensors are ideally suited to this application area, their dependence on molecular specificity mandates individualized transducers for each target. Consequently, multiplied engineering efforts and resource expenditure substantially restrict practical deployment. This study addresses this challenge through the integration of a universal signal transduction pathway with multiple substrate-specific oxidases, enabling simultaneous quantification of diverse molecular targets within a single biosensing platform. Clinical validation with 17 urine specimens confirmed the system’s robust analytical performance. Our findings establish a technological foundation for cost-effective, rapid, and multiplexed home-testing devices, showing substantial promise for advancing disease surveillance and personalized healthcare management.