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基于海藻胶与聚丙烯腈丙烯酸生物传感器的构筑及性能研究
【作者】 朱道彬；
【导师】 单丹；
【作者基本信息】 扬州大学 ， 物理化学， 2008， 硕士
【摘要】 1.基于以海藻胶为固定载体的安培型葡萄糖生物传感器由于海藻胶具有的独特的亲水性和溶胀性、多孔性,具有较好得生物兼容性,可以用于固定生物大分子。而海藻胶特有的成膜性可有效地固定生物大分子,加入DMF,可以大大改善其凝胶性能,从而制备新型的生物传感器。用N,N—二甲基甲酰胺（DMF）/海藻胶（ALG）固定葡萄糖氧化酶（GOD）,制备了DMF/ALG/GOD/Pt电极.本文讨论了海藻胶浓度,膜的厚度以及酶的量对电极的影响。该酶电极在工作电位0.6V、测量溶液pH为6.5,传感器对葡萄糖表现出快速的电化学响应特性,宽广的线性检测范围(5×10-6 1×10-2 M)以及良好的操作稳定性。实验结果表明固定化葡萄糖氧化酶的表观米氏常数为6.37mM,酶催化反应的表观活化能为24.8 kJ mol-1。此外,本文还系统地研究了该传感器构建的最佳参数以及溶液pH值、操作电位和系统温度等使用条件对酶传感器响应电流的影响。2.基于海藻胶/类水滑石杂化材料的葡萄糖生物传感器研究了采用一种新颖的载体材料阴离子粘土（类水滑石:LDHs）与海藻胶（ALG）杂化材料固定葡萄糖氧化酶（GOD）制备的安培型生物传感器。该杂化材料结合了有机生物聚合物（海藻胶）与无机粘土类水滑石二者的优点。将此材料用与固定葡萄糖氧化酶可以不用交联,这样可以保持酶的活性。并通过红外对该材料进行了表征。结果表明葡萄糖氧化酶（GOD）在复合膜中保持了原有的结构特征。LDHs/ALG/GOD酶电极传感器在热稳定性方面具有明显的改进,该生物传感器表现出一些优异的分析性能:宽广的线性检测范围(1×10-5 2×10-3 M)、高的灵敏度（70 mA cm-2 M-1）、低的检测下限（1μM,信号噪声比为3）。此外,该电极具有较好的稳定性与令人满意的重现性。3. Hb/PAA-co-PAN仿生膜的直接电化学和电催化将血红蛋白（Hb）固载在多孔丙烯腈与丙烯酸共聚物膜（PAA-co-PAN）中,形成了一种新型的仿生膜。在pH 7.0的PBS缓冲溶液中,丙烯腈与丙烯酸共聚物-血红蛋白仿生膜的的循环伏安扫描范围内有一对较好的氧化还原峰,这对峰产生于血红蛋白辅基血红素Fe（III）/Fe（II）电对的氧化还原。血红蛋白中Fe（III）/Fe（II）的标准峰电位EΘ随pH的增加而呈线性下降,直线斜率为-53.5 mV·pH-1,这说明电化学反应是电子传递伴随质子转移的过程。在丙烯腈与丙烯酸共聚物膜中血红蛋白的吸收峰位置跟它原来的位置相似,这说明在仿生膜中血红蛋白保持了它原有的高级结构。丙烯腈与丙烯酸共聚物固定化血红蛋白具有生物催化功能,可以实现过氧化氢的电催化;线性范围是5×10-5～2.1×10-3M。当信噪比（S/N）为3时,检测下限是5×10-5M。
【Abstract】 1. Amperometric Detection of Glucose with Glucose Oxidase Immobilized in alginateAlginate （ALG）, have been demonstrated as attractive material for the immobilization of biomolecules onto physical transducers, due to their hydrophilic, swelling, and porosity properties. A biosensor has been prepared with N ,N—Dimethylformamide（DMF） and alginate（ALG） as immobilization matrix. The influence of concentration of ALG and the thickness of membrane and quantity of the enzyme to the biosensor response have been discussed in detail. Amperometric Detection of Glucose with an unmediated sensor at 0.6V （vs.SCE） results in a rapid response, a wide linear range of 5×10-6 1×10-2 M, as well as good operational stability. The results indicated that the apparent Michaelis-Menten constant （KMapp） for the sensor was found to be 6.37 mM and the activation energy for enzymatic reaction is calculated to be 24.8 kJ mol-1.In addition, effects of pH value, applied potential, temperature and electrode construction were investigated and discussed.2. Electrochemical study of alginate modified layered double hydroxides composite matrix: Potential application in the development of amperometric biosensorsA new type of amperometric glucose biosensor based on alginate/layered double hydroxides organic-inorganic composite film was described. This hybrid material combined the advantages of organic biopolymer, alginate, and inorganic layered double hydroxides. Glucose oxidase （GOD） immobilized in the material maintained its activity well as the usage of glutaraldehyde was avoided. The composite films have been characterized by Fourier transform infrared （FT-IR）. The results indicated that GOD retained the essential feature of its native structure in the composite film. The enzyme electrode provided a linear response to glucose over a concentration range of 1×10-5 - 2×10-3 M with a sensitivity of 70 mA M-1 cm-2 and a detection limit of 1μM based on S/N=3. Furthermore, the biosensor exhibited excellent long-term stability, and satisfactory reproducibility.3. Studies on direct electron transfer and biocatalytic properties of hemoglobin in poly（acrylonitrile-co-acrylic acid） matrixHemoglobin （Hb） immobilized in poly（acrylonitrile-co-acrylic acid）, a new type of biologic film was achieved. The Hb/PAA-co-PAN film exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks for Hb Fe（III）/Fe（II） redox couple in a pH 7.0 phosphate buffer. The formal potential of Hb heme Fe（III）/Fe（II） couple varied linearly with the increase of pH in the range of 4.0-8.0 with a slope of -53.5 mV pH-1, which implied that a proton transfer is accompanied with each electron transfer in the electrochemical reaction. Position of Soret absorption band of Hb/PAA-co-PAN film suggested that the Hb kept its secondary structure similar to its native state in the PAA-co-PAN matrix. The Hb in PAA-co-PAN matrix was act as a biologic catalyst to catalyze reduction of hydrogen peroxide. The electrocatalytic response showed a linear dependence on the H2O2 concentration ranging from 5×10-5～2.1×10-3M with a detection limit of 5×10-5M at 3 .