Biosensor-enabled single-molecule enzyme activity profiling for function-based molecular counting
Year: 2026
Type: preprint
Abstract: ABSTRACT Single-molecule enzyme activity analysis enables proteoform-resolved quantification of catalytically active enzyme molecules but remains limited by the need for enzyme-specific fluorogenic substrates, particularly for enzymes involved in central metabolism. Here we establish a biosensor-based framework that integrates genetically encoded fluorescent metabolite biosensors with single-molecule enzyme activity profiling (SEAP), enabling native enzymatic reactions to be monitored without enzyme-specific fluorogenic substrates. By employing different biosensors, the framework was applied to distinct metabolic enzymes, including lactate dehydrogenase and pyruvate kinase. Biosensor-based SEAP enables absolute quantification of active enzyme molecules, transforming conventional activity measurements (U/mL) into molecular abundance (molecules/mL). Applying this approach to single-cell functional proteoform analysis revealed functional heterogeneity in lactate dehydrogenase activity during T-cell activation. In addition, analysis of circulating enzymes showed that much of the plasma lactate dehydrogenase pool could be quantitatively accounted for by physiological erythrocyte turnover. These results establish genetically encoded fluorescent protein-based metabolite biosensors as modular optical transducers for single-molecule functional enzyme analysis across cellular and circulating samples.
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
Funders: Naito Foundation, Japan Agency for Medical Research and Development, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Chugai Pharmaceutical, Japan Society for the Promotion of Science, Core Research for Evolutional Science and Technology, Precursory Research for Embryonic Science and Technology, ACT-X
Awards: G1352590317, Spatiotemporal Effect of Metabolic Imprinting on Immune System, Massive Expansion of the Palette of Fluorescent Protein-based Sensors for Multiplexed Imaging of Signaling and Metabolism, G2142007452, G3763141689, 膵がん・大腸がんの発症リスク予測血液バイオマーカーの開発, G4197712963, G4245581419, Development of bioimaging sensors based on next-generation fluorescent proteins, Advance of enzymomics study for establishment of disease diagnosis platform, G4768537516, Ultra-sensitive detection of disease-related proteins by cascade activation of fluorescent signals, Development of a series of chemigenetic fluorescence biosensors for multicolor and multiplexing bioimaging, Advance of enzymomics study by fully automated synthesis of chemical probes, G6880519004, G8178606013
OpenAlex ID: W7214364987