| Zinc Oxide nanoparticles (ZnO NPs) is widely used in the field of biomedicine, daily cosmetics, electronics, environment, etc., due to its excellent physical and chemical properties. However, recent studies confirmed that direct or indirect contact with ZnO NPs will have a negative impact living things. Ca2+ ions is important to maintain the normal function and regulate the physiological activities of cells. The study of effect of ZnO NPs on the intracellular metabolism of Ca2+ can help to evaluate the biological safety of ZnO NPs.In this thesis, calcium ion selective microelectrode was used to study the Ca2+ release of aloe vera cell protoplast in low calcium and low permeability test solution after ZnO NPs treatment.The protoplast was prepared by enzymatic hydrolysis of fresh aloe leaves. The preparation process was optimized to increase the yield and productivity. ZnO nanoparticles were purchased from Sigma Aldrich with diameters less than 50 nm. ZnO nanoparticles were ultrasonically dispersed into calcium free culture medium to obtain 120 mg/L suspension. Borosilicate glass tube (diameter 0.1 mm) was drawn into microelectrode with tip diameter of 2-3 ?m. After silanization, electrolyte filling, calcium ion exchanger (LIX) inhalation, internal reference electrode insertion, calcium ion selective microelectrode was prepared. The conversion efficiency of the electrode is higher than 90%. The detection limit is 10-6 mol/L. Response time t95% < 1 s and the stability is good.Aloe protoplasts with good activity was divided equally into control group and experimental group. The experimental group was then transferred into culture medium dispersed 120 mg/L ZnO NPs suspensions. The control group was transferred into the same kind of culture medium, excepted that no ZnO NPs was added. The two groups of protoplasts were cultured for 10 hours in the same condition. After cultivation, the protoplasts were transferred into the low osmotic solution without calcium, respectively. Real time measurement of electronic potential was carried out by calcium ion selective microelectrode near the cell. The relationship between the potential value and the concentration of calcium ion was given by Nernst's equation.The experimental results showed that the extracellular Ca2+ concentration was relatively stable before the death and rupture of the protoplast. When the protoplast ruptured, the Ca2+concentration exhibited a sharp rise and a slow descending, and finally tended to the background value. The time dependence curve of the calcium ion concentration was an inverted bell pulse.The pulse was studied and found that the distribution of the leading-edge time of the pulse of the control group satisfied the normal distribution, the mean value was 9.74 s. While for the experimental group, the distribution of the leading-edge time changed dramatically and did not satisfy the normal distribution. Most of the time lied in about 11.97 s, which is longer than that of the control. The reason may be that treatment of ZnO NPs changed the characteristics of the cell membrane.Analysis of the peak value of the Ca2+ concentration pulse signal showed that the peak values of both groups were in accord with the normal distribution and the significant level was 0.413. The peak potential of the experimental group was -6.59 mV, and the peak potential of the control group was -6.30 mV, both corresponding to a calcium ion concentration of about 10-2mol/L level, which was much higher than the normal calcium concentration in the cytoplasm of Aloe Vera, about 10-6 mol/L. Because the protoplast was cultured in low calcium environment, there is less possibility for cells to uptake a large amount of calcium ions from the surrounding environment.To sum up, the effect of zinc oxide nanoparticles on plant cells was studied by ion selective microelectrode technique, which provided a new basis for the further study of the mechanism of the biological toxicity of NPs ZnO. |
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