Multifunctional biosensors for analysis of brain functions with measurements of bioactive substances in the brain.

本项目的目的是开发多功能生物传感器，可以检测影响中枢神经系统神经元活动的内源性物质的局部动力学。研制了一种新型生物胺传感器。我们在体外测试了传感器的特性，并尝试在体内条件下应用。采用碳纤维电极作为体内伏安法的工作电极。这些电极事先通过在0.1 M H SO溶液中施加三角形电流脉冲（0-2V, 70 Hz， 60秒）进行电化学激活。使用这些电极的DOPAC（多巴胺代谢物）和5-HIAA（5-羟色胺代谢物）的校准曲线（氧化电流与浓度）在1muM和10muM浓度之间证实是线性的。为了评价探针的功能，我们连续测量了自由运动大鼠下丘脑外侧区（LHA; feeding center）血清素的释放量。碳纤维电极与聚四氟乙烯涂层铂铱导线相结合，可以长时间记录神经元的活动。结果表明，血清素在1800小时左右开始升高，2100小时左右达到最大值，随后逐渐降低，3 ~ 4小时内恢复到1800小时左右的水平。30个神经元与血清素同时释放，其中12个神经元活性增加（40%），7个神经元活性降低（23%），11个神经元活性无变化（37%）。结果表明，这种探针可用于分析脑功能和测量生物活性物质。

The purpose of this project was to develop multifunctional biosensors which can detect local kinetics of endogenous substances influencing neuronal activity in the central nervous system. New type of biogenic amine sensors using voltammetry system was developed. We examined characteristics of the sensors in vitro and tried to apply in vivo conditions.As a working electrode of in vivo voltammetry, carbon fiber electrodes were used. These electrodes were electrochemically activated beforehand by applying a trinagular current pulses (0-2V, 70 Hz, 60 sec)in 0.1 M H SO sulution. Calibration curves (oxidation current vs. concentration) of DOPAC (metabolite of dopamine) and 5-HIAA (metabolite of serotonin) using these electodes were confirmed to be linear between 1muM and 10muM concentration.To evaluate the function of probe, serotonin release in the lateral hypothalamic area (LHA; feeding center) was measured continuously in freely moving rats. The carbon fiber electrode was combined with teflon-coated platinum-iridium wires which enabled to record the neuronal activity for long period. The results indicated that the serotonin release in the LHA began to increase at around 1800 hr and reached to the maximum level at around 2100 hr, then gradually decreased and returned to the level at around 1800 hr within 3 or 4 hours. Thirty neurons recorded simultaneously with serotonin release, 12 neurons (40%) increased their activity, 7 (23%) decreased and 11 (37%) showed no change. The results indicate that this kind of probe is useful for analysis of the brain functions with measurements of bioactive substances.