An enzyme-gated precipitation-polarity reversal strategy for versatile bioassay

Unidirectional "signal-on/off" modalities remain a fundamental constraint in photoelectrochemical (PEC) biosensing, not only constricting dynamic range but also elevating susceptibility to false-positive or false-negative outcomes. To circumvent this limitation, we introduce an innovative "enzyme-gated precipitation-polarity reversal" (EGPPR) mechanism that inherently suppresses spurious signals while substantially amplifying detection sensitivity. This strategy facilitates a simplified, label-free detection of acetylcholinesterase (AChE) and glutathione reductase (GR) within homogeneous solutions. Upon target enzyme catalysis, enzyme-generated thiol moieties chelate free metal ions, thereby precluding the formation of insoluble metal ferrocyanide. The unbound [Fe(CN)₆]^4- ions in solution subsequently undergo surface coordination with Bi₄Ti₃O₁₂ photoanodes, instigating an instantaneous photocurrent reversal from anodic to cathodic polarity. The magnitude of this directional transition exhibits a logarithmic correlation with enzyme concentrations, achieving linear detection ranges of 0.05-50 U/L for AChE and 0.1-50 U/L for GR, with detection limits of 0.013 U/L and 0.024 U/L for AChE and GR, respectively. Through facile substitution of substrate-metal ion pairs, this strategy was conceptually demonstrated with AChE and GR models, and could in principle be extended to other thiol-generating enzymes, pending further experimental validation. This offers new insights for PEC sensing methodologies with directional signal outputs.