Synthesis of nanoparticle/ microsphere composites with peroxidase activity and their use in the determination of glutathione

Silica and magnetic microspheres in the monodisperse porous form used as a supporting material are synthesized using the multi-step hydrolysis-condensation method. The surface of magnetic silica microspheres was coated with silica shell. The gold nanoparticles synthesized by the Turkevich method were immobilized onto the silica-coated magnetic silica and bare silica microspheres. Surface morphology and size distribution of the microspheres were analyzed by scanning electron microscopy (SEM). The surface atomic composition and the chemical structure were investigated by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction method (XRD), respectively. The surface area and the pore size distribution were determined by using the nitrogen adsorption and desorption method (BET / Brunauer-Emmett-Teller). The magnetization properties of magnetic microsphere were investigated using vibrating sample magnetometry (VSM). The peroxidase-like activity of the synthesized microspheres was investigated by varying pH, substrate concentration, and the nanozyme concentration. OPD was used as substrate in the peroxidase-like activity measurements. Measurements were made with UV-visible spectrophotometry. Lineweaver-Burk graphs were plotted using OPD utilization rate and substrate concentration values and Michaelis Menten constant (Km) demostrating the affinity between enzyme and substrate was calculated for nanozym structure. Km values of gold nanoparticle immobilized silica coated magnetic silica microspheres and gold nanoparticle immobilized silica microsphere were calculated as 522,47 µM and 816,48 µM. A biosensor was developed for glutathione determination based on the variation of peroxidase-like activity of microsphere depends on glutathione concentation by adsorption glutathione to the surface of the microsphere. Adsorption of glutathione onto the microsphere surface was analyzed using High Performance Liquid Chromatography (HPLC). Glutathione in buffer solution and in human serum were determined using the synthesized nanozymes under appropriate conditions. The linear change of adsorbance values corresponding to glutathione concentration was shown, and the determination of glutathione between 325 - 3253 µM concentration in buffer medium was performed with gold nanoparticle immobilized silica and silica coated magnetic silica microspheres. It has been shown that glutathione can be determined in human serum at concentrations between 2.5-50 µM.