Investigation of Intracellular Reactions Using Ultramicroelectrodes

1.通过电化学刻蚀铂丝、用软玻璃封接铂细丝、抛光针尖等工艺，制备了针尖半径小于5微米的超微铂圆盘电极。对所制备的微电极的电化学性质进行了研究，发现利用微电极进行氧还原的恒电位安培法和差示安培法可以提供局部区域的氧浓度。用恒电流安培法测定了羽毛苔藓原生质体的胞内氧浓度。光照射后细胞内氧还原电流增大，表明用本方法可以实时示踪细胞内氧。从位于细胞附近的微电极进行的差示安培测量的详细分析中，可以在单个细胞水平上确定氧气的流入或流出。用靠近膜的微电极测量了Ru(NH_3)_(63+)和Fe(CN)_(63-)通过平面类脂膜离子通道的通透性。并用安培法测定了活性原生质体的膜渗透选择性。将镍卟啉和超氧化物歧化酶/过氧化氢酶分别固定在微电极上，制成了微型NO传感器和微型O2传感器。我们将这些微型传感器植入大鼠脑内，并在体内测量了NO和O2的浓度

1. Ultramicro Pt disk electrodes with tip radii less than 5mum were fabricated via the processes ; electrochemical etching of Pt wire, sealing the Pt fine wire with a soft glass, and polishing the tip. The electrochemical characteristics of the resultant microelectrodes was investigated and found that constant potential amperometry and differential amperometry for oxygen reduction using the microelectrodes afford the oxygen concentration in the local region.2. Intracellular oxygen concentration in a protoplast from Bryopsis plumosa was determined by constant current amperometry. The intracellular oxygen reduction current increased upon light irradiation, indicating that the real-time tracing of the intracellular oxygen is possible by the present method. From the detailcd analyzes of diferential amperometric measurements using the microelectrode located near the cell, the oxygen influx or efflux was determined at a single cell level.3. The permeability of Ru (NH3) 63+ and Fe (CN) 63- through an ion channel in a planar lipid membrane was evaluated by amperometric measurements using microelectrode located near the membrane. The membrane permselectivity of the viable protoplast was also carried out by amperometry.4. Micro NO sensor and micro O_2 sensor were fabricated by immobilizing respectively Ni porphyrin and superoxide dismutase/catalase onto microelectrodes. We implanted these microsensors into rat brain and measured in vivo concentrations of NO and O_2