Detection of Glutathione in Vitro and in Cells by the Controlled Self-Assembly of Nanorings

Taking advantage of a reduction-controlled biocompatible condensation reaction and self-assembly, we have developed a new method for the determination of glutathione (GSH) concentration in vitro and in HepG(2) human liver cancer cells. Upon reduction by GSH under physiological conditions (pH 7.4 in buffer), the small molecule CBT-Cys(SEt) condenses and self-assembles into nanorings, increasing the UV absorbance at 380 nm (with significant linear correlation in the 0-87 mu M GSH range and a limit of detection of 1 mu M). This method is also selective to GSH rather than cysteine in biological samples. Through the use of added internal standards, we successfully determined the concentration of GSH in HepG2 cells to be 14.96 mu M (2.99 fmol/cell). To better understand the mechanism of nanoring self-assembly, the condensation product of CBT-Cys(SEt) formed using different concentrations of GSH and different reaction times were characterized by transmission electron microscopy (TEM).