2.3.5. Determination of Thiol Content by Ellman’s Reagent Method

The standard linearity graph of the thiourea was plotted against the concentration on the x-axis and the corresponding absorbance on the y-axis. For this purpose, 2 mg of thiourea were accurately weighed and added in a test tube to prepare a 1 mL solution using a 0.5 M phosphate buffer of pH 8.0 as the solvent. Furthermore, the serial dilutions were prepared from this solution in the concentration range of 10–100 μg/mL. The absorbance of the prepared dilutions was determined by using a UV–visible spectrophotometer (PG Instruments T80, Leicestershire, United Kingdom) at 280 nm.

The thiol contents of TPLX were determined by using Ellman’s reagent technique [13]. In the 1st step, a 2% w/v solution of the polymer conjugate was prepared by dissolving it in a sufficient volume of deionized water. After that, 1350 μL of 0.1 M PBS at pH 7.4 were prepared. Then 150 μL of Ellman’s reagent were prepared by dissolving 3.96 mg of reagent in 10 μL of PBS, and 150 μL of polymer conjugate were added to 1350 μL of deionized water. The mixture was incubated at room temperature for 90 min, and then its absorbance was measured at a wavelength of 412 nm with a UV spectrophotometer [14]. The amount of the thiol group attached to the polymer was determined by a thiourea calibration curve.