Single-Molecule Oxidoreductase Activity Analysis for Activity-Based Diagnosis Based on Proteoform Alterations

We developed a single-molecule enzyme activity assay platform for NAD(P)⁺-dependent oxidoreductases, leveraging a new NAD(P)H-responsive fluorogenic probe optimized for microdevice-based fluorometric detection. This platform enabled the detection of enzyme activities in blood and cerebrospinal fluid (CSF), including lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinases. We demonstrate its potential for activity-based diagnosis by detecting altered populations of enzyme activity species in blood and CSF from liver damage in brain tumor patients.