Profiling of Biofluid Metabolites with a Kinetically Differentiated Binary Biosensing Platform

Biofluid metabolites have a crucial linkage with the health of the human body, and developing a universal method for metabolite monitoring is imperative for disease diagnosis and health management. Herein, we report a kinetically differentiated binary biosensing platform that is specifically responsive to NAD(P)H for profiling diverse biofluid metabolites. The kinetically differentiated binary biosensing platform comprises a cyanine derivative dye with fast reaction kinetics and a quinolinium derivative dye with slow reaction kinetics. Compared to the traditional unitary strategy for NAD(P)H detection, the linear range of the binary biosensing platform is widened by up to 20 times. NAD(P)H are ubiquitous cofactors in living systems, and metabolite production generally involves the consumption or generation of NAD(P)H. Thus, biofluid metabolites can be easily quantified by measuring the variation of NAD(P)H concentration during biochemical reactions with the binary biosensing platform. In this study, serum sorbitol, 2-hydroxybutyric acid (2HB), and α-ketoglutarate (AKG) were all quantified by the binary biosensing platform with accuracies higher than 93%. The kinetically differentiated binary biosensing platform can be extended to the analysis of any molecule that can react directly or indirectly with NAD(P)H. In addition, we constructed a paper-based assay with the binary biosensing platform, and the test papers showed good promise in the point-of-care (POC) profiling of biofluid metabolites. This study proposes a simple strategy to expand the calibration range of traditional unitary detection systems and further provides a universal paradigm for high throughput profiling of disease-associated biomolecules, which offers good promise in disease diagnosis and health management.