Simple and fast determination of biothiols using Fe3+-3, 3′, 5, 5′-tetramethylbenzidine as a colorimetric probe

Biothiols, generally including glutathione (GSH), cysteine (CySH), and homocysteine (HcySH), are important biomarkers and closely related to many diseases. In this work, a simple method for the spectrophotometric determination of the three biothiols was developed. The proposed method was based on the fact that Fe 3+ could oxidize 3, 3′, 5, 5′-tetramethylbenzidine (TMB) to oxidized TMB (ox-TMB) and induce a blue color solution corresponding to an absorption peak centered at 652 nm. However, the introduction of a biothiol compound could cause the reduction of ox-TMB which resulted in a blue color fading and a decrease of the absorbance. Based on this finding, we developed a method for the determination of biothiols with the help of UV–vis spectroscopy, and the influence of different factors including pH, temperature, and incubation time was studied when glutathione worked as an example. The detection limits for GSH, CySH, and HcySH were 1.04, 0.82, and 2.09 μM, respectively. More importantly, the suggested method was successfully applied for the determination of GSH in human serum samples with the recovery of 97%–112%. This method is simple, fast and without the use of complicated nanomaterials .