Distance-RegulatedElectron Transfer of Nanozyme Hybridsfor Selective Glutathione Detection

Nanozymes offer a cost-effective and stable alternative to natural enzymes but often suffer from limited selectivity, requiring further modifications for targeted applications. This study introduces a distance-regulated electron-transfer (DRET) probe based on nanozymes, designed to enable selective enzymatic responses to specific targets. The hybrid DRET system comprises carbon dot (CD)-conjugated iron oxide nanoclusters (IONs), with interparticle distances controlled by linkers. The system catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), producing a blue color as a readout of enzymatic activity. Compared to its individual components, the DRET system exhibits significantly enhanced enzymatic efficiency, likely due to improved electron transfer facilitated by the CDs surrounding the IONs. By introducing linkers of varying lengths, the relationship between interparticle distance and enzymatic activity was systematically explored. To demonstrate its utility, a glutathione (GSH)-responsive DRET probe was engineered using cystamine as the linker, which is cleaved in the presence of GSH. This cleavage reduces the synergistic enzymatic effect, resulting in a proportional decrease in TMB oxidation and color intensity. The GSH-DRET system showed high specificity for GSH, outperforming responses to ions and other metabolites. Moreover, it enabled accurate detection of GSH concentration in human plasma solution (imitating human samples) via simple absorbance measurements. These findings highlight the potential of the DRET nanozyme system as a versatile field detection tool, with possibilities for expanded applications through diverse linker strategies.