Nano-hydroxyapatite For Determination Of Hydrogen Peroxide In The Cells

Reactive oxygen species (ROS), including hydroxyl radicals (·OH), superoxide anions (O2-), peroxynitrite (ONOO-), and hydrogen peroxide (H2O2), etc., are ubiquitous in life and death processes of cells. They are considered as the mediators of the biochemistry of cellular pathology and may be involved in a number of pathological events connected with lipid peroxidation, organ injury, DNA damage, tumor promotion, etc. Among the ROS, H2O2 is the most stable species, and has captured the interests of many researchers since it can exert diverse pathological and/or physiological effects in living systems and participate in the physiological processes in a concentration-dependent manner. Therefore, the quantitatively determination of the intracellular and extracellular H2O2 could lead to a better understanding of the clinical consequence of the enhancement in H2O2 concentration as well assisting in studies designed to elucidate the biological effect of H2O2 in cells. This thesis studies a HRP-based H2O2 biosensor based on the reduction of H2O2 with the use of hydroxyapatite (HAP) nanostructures as the enzyme immobilization matrix. Moreover, the biosensor can be used for the determination of the extracellular H2O2 released by RAW 264.7 murine macrophage cells. The main results of this thesis are expressed as following:1. The preparation and characterization of HAP. HAP nanostructures were synthesized by a chemical precipitation method with use of Ca(OH)2 and H3PO4. The prepared HAP were characterizaed by XRD, SEM, TEM, FTIR et al, XRD indicated that the as-synthesized HAP nanostructures had a highly crystalline hexagonal phase, and no impurity phases could be found. TEM image indicated that the HAP nanostructures had a needle-like morphology with an average size of 20～25 nm in width and 160～180 nm in length. SEM images could provide additional information on the assembly of the HRP on the surface of HAP nanostructures. The image indicated that the needle-like nanostructures distributed almost uniformly on the entire surface of the GC electrode, exhibiting a special three-dimensional porous structure. The results of FTIR spectra also demonstrated the successful preparation of HAP nanostructures.2. The assembly and direct electron transfer characteristics of HRP on the surface of HAP, and electrocatalytic activity of the HRP-HAP toward the reduction of H2O2. HRP was assembled on the surface of negatively charged HAP via the electrostatic interaction to form a HRP-HAP hybrid. The HRP-HAP hybrid was characterized by several techniques, such as UV-vis, FTIR and CV et al, and the results showed that HRP could rapidly and effectively be adsorbed on the surface of HAP with high stability without changing the native structure of HRP and the structure properties of HAP. HRP-HAP/GC electrode was fabraicated by modifying HRP-HAP hybrid on the GC electrode surface. The cyclic voltammetric showed that HAP could facilitate the effective DET between HRP and the electrode surface. The HRP-HAP/GC electrode exhibited a pair of well-defined and nearly symmetrical redox peaks with the cathodic (Epc) and anodic (Epa) peak potential of ca.-401 mV and -338 mV(vs. SCE) and the formal potential of (-370 mV), which was almost independent on the scan rates. The surface electron transfer rate constant ks was 3.77 s-1. Besides, the response displayed a good linear range from 5μM to 0.82 mM at a detection potential of-400 mV (with a correlation coefficient of 0.998). The detection limit was estimated to be 0.1μM. These results indicated the suitability of the proposed biosensor to practical applications.3. The measurement of the concentration of H2O2 in RAW 264.7 murine macrophage cells by the HRP-HAP/GC electrode. The voltammetric responses of HRP-HAP/GC electrode were attributed to the releasing of H2O2 from RAW 264.7 murine macrophage cell. According to the relationship between the current and the concentration of H2O2, the H2O2 concentration in the cell was ca.8.0 nM. The developed method was also used to measure H2O2 in RAW 264.7 murine macrophage cell in response to fMLP (N-formyl-methiony-L-leucy-L-phenylalanine) treatment since fMLP was known to induce the generation of H2O2 in RAW 264.7 murine macrophage cell. The amount of H2O2 in RAW 264.7 murine macrophage cell was significantly increased with the enhancement of the fMLP concentration, and it reached the highest value (ca.17 nM) at fMLP concentration of 0.3μM at the time of 30 min. Afterwards the concentration decreases gradually and reached the relatively stable value. Therefore, the proposed method could be used for studying the intracellular kinetics of H2O2 generation.