A Universal Biosensing Platform Amplifying Signals Produced by NAD+/NADH-Dependent Enzymes

This project offers a novel approach to the analysis of different biomolecules with ultra-high sensitivity. The analytical goals are achieved by creating a universal biosensing platform that combines a biosensing part with a novel biochemical amplification part. The basic science interest of the proposed study is enhanced by the numerous applications of the novel biosensor and by simple, easily exchangeable, biosensor configuration for identifying specific biomolecules. The project opens novel possibilities in different areas of biosensing, including biomedical, environmental, forensic, and homeland security applications. The educational impacts include curricular developments as well as PhD student education and participation of undergraduate researchers. The research program addresses the challenge of enzyme-based biosensing with ultra-high sensitivity, particularly with the use of a universal biosensing platform adaptable to different analytes with minimum adjustments. A novel approach to signal amplification, providing the biosensing with a sub-nanomolar or even picomolar detection limit is achieved using artificial chimeric enzymes with allosteric properties. These enzymes are composed of a biocatalytic part of PQQ-dependent glucose dehydrogenase (GDH) and a biorecognition part selectively binding NADH or NAD+ cofactor that is combined with NADH/NAD+-dependent dehydrogenases. The universality of the offered approach is achieved because any NADH/NAD+-dependent dehydrogenase can be easily combined with the same standard artificial allosteric GDH enzyme selective to NADH/NAD+. The biocatalytic system including both enzyme types will be studied in an immobilized form with a fluorescent response, then on a nanomodified conductive electrode with an electrochemical (amperometric) response. The primary reaction biocatalyzed by an NADH/NAD+-dehydrogenase produces a stoichiometric response to the selected analyte in the form of the NADH or NAD+ cofactor. Then, the NADH or NAD+ molecules bind to a biorecognition part of the artificial GDH enzyme resulting in its conformational change. This change will be transduced to the biocatalytic part of the artificial GDH enzyme, switching it from the initial mute (OFF) state to the active (ON) state, thus triggering a biocatalytic cascade. Notably, the binding of a single NADH or NAD+ molecule activates the biocatalytic reaction providing a non-stoichiometric amplified response to the primary analyte. The study of amplified biosensing leads to novel biosensors with adaptivity to different analytes while using various target-selective NADH/NAD+-dehydrogenases in combination with the same artificial allosteric enzyme selective to NADH/NAD+. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.