【作者】 杨琴；

【导师】 赵元弟；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2011， 硕士

【摘要】 在植物体中存在着很多的信号分子如过氧化氢、一氧化氮等,它们之间能够相互协同、相互调节,对植物的生理活动进行调控。过氧化氢具有较强的氧化性以及细胞毒性,因此需要调节活性氧的量在一定的范围内;一氧化氮具有一定的还原作用,但是浓度太高时,同样对生物具有较大的毒害作用。当植株受到生物或者非生物的胁迫时,其体内的活性氧和一氧化氮的水平会发生变化,共同作用于植物的抗病防御体系。那么,探测植物体内的这些小的信号分子对研究植物的生命活动有着重要的意义。电化学生物传感器因检测方便迅速,无需外加示踪分子,电极探针易微型化,设备成本低等特点,不仅可以对生物体进行在体检测,还能连续监测这些小分子物质在植物体内浓度变化情况。本课题主要利用电化学修饰电极分别制备过氧化氢传感器和一氧化氮传感器,并应用于植物的在体监测。主要工作有以下内容:(1)采用循环伏安法在修饰有铂微颗粒的铂微柱电极上电沉积邻苯二胺,制得的过氧化氢传感器有着良好的电化学性能。测得过氧化氢在-0.1V电位下,得到的还原电流和其浓度之间呈良好的线性关系(R=0.995),检出限为25μmol/L。利用该过氧化氢传感器在体监测了油菜在受到草酸致病因子侵染时,不同部位产生的过氧化氢。实验结果表明,当油菜植株受到草酸胁迫时,会产生两次大量的活性氧爆发,第二次爆发的强度和持续时间都远大于第一次。第一次主要是感染细胞周围大量活性氧的产生,用于控制病原体的继续阔散;而第二次氧爆发主要是在植物受到胁迫后,自身防御体系调控所产生的强度更大并且持续时间更长的活性氧爆发。(2)利用聚邻苯二胺和Nafion膜包覆经过四磺化酞菁镍修饰的石墨电极(GE/NiTSPc/POPD/Nafion),制成一氧化氮传感器。在0.8V的工作电压下,一氧化氮在工作电极上具有较大的氧化电流,检测得到当一氧化氮的浓度在0.7μmol/L-8μmol/L时,其氧化电流与浓度之间具有良好的线性关系(R=0.995)。同时一氧化氮传感器对抗坏血酸(AA)、亚硝酸钠(NaNO2)和过氧化氢(H2O2)具有较好的抗干扰效果。当烟草受到紫外胁迫时,利用该一氧化氮传感器检测了其体内一氧化氮水平的变化。(3)将经过绝缘处理的铂丝和银丝利用环氧树脂封装在毛细管中,将银丝腐蚀形成一个孔洞,制成内缩式的Ag/AgCl参比电极。该集成式微电极具有良好的重现性,8次测量其式电位的标准偏差约为0.0032。之后我们在该集成式微电极的工作电极上修饰了分散有铂微粒的邻苯二胺,制得过氧化氢传感器,并成功的利用它在体监测了植物受侵染后所产生的活性氧爆发。该工作为利用电化学方法进行生物在体检测提供了一种新方法。更多还原

【Abstract】 There are many signal molecules in plant, which coolaborates and accommodates with each other for keeping natural vital movement of physiology and pathology in organism. Hydrogen peroxide owns strong oxidizability and toxicity in plants, it is necessary to keep Hydrogen peroxide in a normal range to prevent the healthy cell being killed. Similarly, as a signaling molecule, except for regulating the vital movement of plant, nitric oxide possesses the reducibility, but NO with high concentration in organism could poison the healthy cells. These signal molecules participate in the physiological process such as biotic and antibiotic stress, in that case, detecting these signal molecules in vivo is a great stimulative for researching the vital phenomena.It is a continuous process of signaling molecule change in plants. Because of convenient and simple operation, micromation of electrode, no requirement of tracer element and costing low, electrochemical biosensor is proper method to measure these small molecules continuously in vivo.In this paper, electrochemical modification of microsensor was employed to get the H2O2 sensor and NO sensor, which were applied to measured H2O2 and NO respectively in vivo. Main study content contains three parts as follows:(1)In order to gain H2O2 sensor, after Pt particulate scattered on the Pt working electrode, the polymer of o-phenylenedianime (o-PD) was electrodeposited on the Pt/Pt electrode. H2O2 sensor possesses good electrochemical capability, and cathodic current was measured at the potential of -0.1V, there is a good linear correlation between the cathodic current and concentration of H2O2 (R=0.995) with detection limit of 25μmol/L. H2O2 in oilseed rape induced by salicylic acid was detected on the Pt/Pt/POPD electrode in vivo successfully. The result of this study indicates that there are two oxide burst (OB) in oilseed rape when salicylic acid infect the plant. Besides, the intensification and duration of oxide burst of the second time is more than the first time. And generated H2O2 in different parts of plants were also tested, it is known that the OB for the first time is to stop the continuing spread of virus, and OB for the second time is the response of the defense of the whole plant. (2)?o-phenylenediamine (OPD) and Nafion is covered on the Graphite electrode after modified by electrodepositing a thin membrane of Nickel phthalocyanine (NiTSPc), which gains obvious anodic current when potential is +0.8V, and there is a good linear correlation between the anodic current and concentration of NO (R=0.995). Meanwhile GE/NiTSPc/POPD/Nafion electrode displays a good sensitivity and selectivity against the interferer of ascorbic acid (AA), nitrite (NaNO2) and peroxide (H2O2) from nitric oxide. Furthermore, NO induced by ultraviolet radiation in tobacco is detected successfully in vivo. As the result, lever of NO will increase twice when there is UV-C radiation stress.?(3) A new style integrated microelectrode was provided to application in vivo better. An insulating Pt wire (diameter 0.1mm) and a silver wire were inserted into a capillary and fixed with Epoxy, and silver wire was eroded with nitric acid to the cupped shape. This kind of integrated microelectrode possesses good electrochemical capability and repeatability with average potential 0.11V and standard deviation 0.0032. Then, as the method mentioned in chapter 2, the integrated microelectrode was decorated to H2O2 sensor, and it was employed to test the H2O2 in oilseed rape in vivo prosperously. From the result, it was seen there was only one time of OB. Because the area of working electrode is very tiny, and the current signal of first OB is weak in addition. The first current signal on the working electrode is covered by noise.更多还原