| The Excellent physical and chemical properties make nano-Zn O be widely used in both traditional and emerging fields. At the same time, it is found that the cytotoxicity of Zn O can not be ignored when it was subdivided into nanoscale. As an important electrolyte, the function of Ca2+ is extensive and complex. Many important cellular physiological activities were closely related to the concentration changes of Calcium ions. So it is important to study the impact of Zn O nanoparticles on the Ca2+ concentration in organisms. Among various means that can detect the specific ion concentrations, ion-selective microelectrode technology can provide non-destructive response to specific ion activity, so that it is more suitable for the studies of ion transmembrane movement than other technique. In this thesis, we mainly focused on the preparation of the calcium ion selective microelectrode and the Ca2+ release of aloe protoplast caused by Zn O nanoparticles.We optimized the procedure to prepare calcium ion-selective microelectrodes and then tested their performances. The results indicated that the Nernst response range was of 10-1~10-5 mol/L with response slope of 26 m V/dec and R-Square of 0.99976, and response time was less than 1s.3~5 nm Zn O nanoparticles were prepared with sol-gel method. Aloe protoplasts with approximately spherical morphology and uniformly distributed chloroplasts were obtained by enzymolysis method. FDA testing results showed that the survival rate was better than 90%. The Zn O nanoparticles parepared were ultrasound dispersed in Ca2+-free culture medium to obtain 20, 30, 40 and 50 mg/L Zn O nanoparticle suspensions. Aloe protoplasts were washed Ca2+-free culture medium to reduce the background Ca2+ ions concentration(less than 10-5 mol/L), and then cultured in different concentrations of Zn O nanoparticle suspensions, respectively. Protoplasts cultured without Zn O nanoparticles were used as control. The concentrations of Ca2+ ions in the suspensions were measured with the above made Ca2+ ion-selective microelectrode. The results showed that in the early stages of the experiment, the concentration of calcium ions of the experimental group was distinctly higher than that of the control. And as time went on, the calcium ions concentrations of both experimental and control group increased gradually and eventually reached almost the same saturation level. But the time for the experimental group to reach this saturation value was less than that of the control group. These results indicated that Zn O nanoparticles could significantly accelerate the release of calcium from aloe cell protoplasts. The degree of the influence on the release speed of calcium ions of aloe cell protoplasts depends on the concentration of Zn O nanoparticles in the suspension. Within the range of 20~40 mg/L, the speed increased with increasing Zn O nanoparticle concentrations, but when the concentration of Zn O nanoparticles reached 50 mg/L, the acceleration effect decreased slightly. This may be related to the aggregation and cladding effect of Zn O nanoparticles. In addition, most of the protoplasts were still alive when the concentration of calcium ion reached to saturation value, this indicated that the speedy loss of calcium ions were not due to the death of protoplasts.The above results will contribute to in-depth understanding of the cytotoxicity mechanisms of Zn O nanoparticles from the perspective of calcium metabolism and may have important implications for the safe application of nano-Zn O in the future. |
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