Enzyme sensing based on a controllable oxidation reaction

We developed a new method for glutathione reductase (GR) enzyme sensing via a metal-controlled spontaneous oxidation reaction. A new complex HgL, composed of 3-benzothiazoliny-7-N,N-diethylaminocoumarin (L) and HgCl2, was used as an example for illustration. It was found that ligand L was released from complex HgL by a ligand exchange process in the presence of GSH, which was enzymatic reduced from GSSG in the presence of NADH. Subsequently, free ligand L was spontaneously oxidated to a laser dye (coumarin-6) with both fluorescence enhancement and color change. A good linear relationship between fluorescent intensity and GSH concentrations (in the range of 10–40 μM) elicits the potential use of HgL in GSH detection. Based on this result, complex HgL was applied to identify GR from other proteins/enzymes with good selectivity and sensitivity. HgL can also determine GR in the concentration range from 0.5 to 10 U/mL with a linear relationship. Furthermore, this novel sensing method was also expected to be a useful example for the design of other biosensing systems.