Target-responsive “signal-down” electrochemical biosensor based on an anti-biofouling hydrogel for glutathione detection

Glutathione (GSH), a ubiquitous biothiol, is critical in physiological processes, and abnormal fluctuations in its level are intimately associated with various diseases. However, real-time in vivo monitoring of GSH within tissues, such as the tumor microenvironment (TME), is fundamentally hindered by severely electrode biofouling and electrochemical interference from structural analogs. Herein, we report a novel target-responsive “Signal-Down” electrochemical biosensor based on an anti-biofouling hydrogel for GSH detection, which elegantly integrates robust anti-biofouling capabilities with highly specific target recognition. This sensing interface features a highly hydrated polyacrylamide (PAM) network, crosslinked by disulfide bonds and loaded with ferrocene/β-cyclodextrin (Fc@β-CD) electroactive probes. Upon exposure to GSH, the highly specific thiol-disulfide exchange reaction triggers the macroscopic degradation of the Fc-hydrogel. This spatial disruption drives the rapid outward diffusion of the redox probes, translating microscopic molecular recognition into a dramatic attenuation of the electrochemical signal. This biosensor not only successfully differentiated abnormal intracellular GSH levels in B16 melanoma cells from those in normal L929 fibroblasts in vitro, but also achieved in situ detection of pathological redox dysregulation in a melanoma-bearing mouse model. Ultimately, by uniquely coupling macroscopic material degradation with signal attenuation, this work establishes a reliable strategy for continuous, interference-free biosensing in highly complex and fouling-prone biological matrices.