Peroksidaz Aktivitesine Sahip Nanopartikül/Mikroküre Kompozitlerinin Sentezi ve Glutatyon Tayininde Kullanılması
Özet
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.
