摘要
长期以来，由于电化学免疫传感器具有设计制造简单、高灵敏度、价格低廉而被广泛研究并已在生物检测中逐步得到了应用。然而，如何将生物活性组分有效的固定在电极表面的固定方法、降低甚至消除蛋白质在传感器上的非特异性吸附以及免疫传感器的重现性和重复使用性能等方面存在的问题阻碍了电化学免疫传感器的发展和应用。其中有效的生物活性组分的固定方法是构建性能优良的生物传感器的关键步骤。本论文研究的四个工作均采用静电吸附法固定生物分子，构建了四种生物化学传感器。
1，角叉胶富含磺酸基，是一种具有强负电性的天然聚电解质，通过静电吸附作用将抗体固定到电极表面制得了基于带强负电性物质作电极界面的电流型转铁蛋白免疫传感器。转铁蛋白抗原与响应电流在1.5-60μg mL~(-1)范围内呈准线性关系。线性方程为：
I=-0.5146c+59.06，相关系数为0.9800。
2，纳米金用于吸附固定抗体，已在免疫分析中得到广泛应用。近年来的研究发现，纳米金能降低一些物质在电极上发生反应的氧化还原的电位，并催化这些物质在电极上的电化学氧化还原反应。利用纳米金的上述特征，研制了基于壳聚糖-纳米金固定IgG抗体的荧光免疫传感器用于羊抗人IgG免疫球蛋白的测定。以对羟基苯丙酸为荧光底物，酶催化氧化对羟基苯丙酸生成二聚体，导致荧光增强，通过荧光增强与待测抗原含量呈反比而对抗原进行检测，荧光增强与待测抗原在1.12ng mL~(-1)-24.8μg mL~(-1)之间呈准线性关系，线性回归方程为：
I=-69.04c+133.5，相关系数为0.9860。
3，第四章，辣根过氧化物酶通过与纳米氧化锆之间的静电吸附作用而被固定在电极表面，以对苯二酚为电子媒介，通过测定酶催化双氧水产生的还原电流的大小来间接测定双氧水的浓度。响应电流与双氧水在3.6×10~(-6)-7.2×10~(-3)mol L~(-1)之间呈准线性关系。线性回归方程为：I=8.488×10~(-4)c+0.4892，相关系数为0.9960。
4，将含大量磺酸基的天然高分子角叉胶包埋于碳糊电极中制成表面带大量负电荷的电极界面固定辣根过氧化物酶，以对苯二酚为电子媒介，通过测定酶催化双氧水产生的还原电流的大小来间接测定双氧水的浓度。响应电流与双氧水在3.6×10~(-7)-7.2×10~(-3) mol L~(-1)之间呈准线性关系。线性回归方程为：I=2321c+11.85，相关系数为0.9990。
Electrochemical immunosensors are widely used for the assay of biological analytes. The advantages of this approach including their simple-design, high-sensitivity and low-cost attract substantial research efforts directed to the developments of some new electrochemical immunosensors in the last two decades. However, the method of immobilization, the prevention or elimination of nonspecific interactions, the reproducibility and the reusability still remain to be the key steps. The four parts of this thesis were all used electrostatic absorption to immobilizing biomolecules.1, Carrageenan which is the negatively charged natural polyelectrolyte, antibody is aggregated to the surface of the carrageenan-carbon paste electrode by electrostatic adsorption, such an immunosensor based on carrageenan-embedded in carbon paste electrode for transferrin assay has been constructed. The sensor exhibits a linear response to the transferrin in the concentration ranged from 1.5 μg mL-1 to 60 ug ml/-1, the regression equation can be expressed as follows:I = - 0.5146c + 59.06, correlation coefficient is 0.9960.2, It has been shown that gold nano-particles (colloid Au) can be used as a platform to immobilize antibodies by adsorption. Additionally, gold nano-particles can reduce the redox potential and catalyze the electrochemical redox reaction of some compounds on the electrode interface. Considering these characteristics of gold nano-particles, we designed a fluorescence immunosensor by immobilization of IgG antibody to a nano-Au monolayer modified chitosan-entrapped carbon paste. Using HPPA as substrate, HRP catalyze HPPA and form HPPA-dimer, which results in fluorescence increase of the substrate solution. The concentration of IgG antibody can be determined by fluorescence increase of HPPA-dimer. The sensor exhibits a linear response to IgG in the concentration ranged from 1.12 ng mL-1 to 24.8 μg mL-1, the regression equation can be expressed as: I = -69.04c + 133.5, correlation coefficient is 0.9800.3, In chapter IV, HRP was immobilized on electrode surface involving strong electrostatic absorption interaction between the enzyme and nano-zirconia dioxide. The concentration of H2O2 can be detected by determining the redox current of p-hydroquinone, which was oxidized by H2O2 in the presence of HRP. The linear response to hydrogen peroxide ranged from 3.6×10-6 to 7.2×10-3 mol L-1 was obtained.
The equation can be expressed as: I = 8.488 × 10-4c + 0.4892, the correlation coefficient is 0.9960.4, Carrageenan, which is the negatively charged natural polyelectrolyte and HRP enzyme were entrapped in the carbon paste electrode, thus a strong-negatively enzyme electrode has been constructed. The concentration of H2O2 can be detected by determining the redox current of p-hydroquinone, which was oxidized by H2O2 in the presence of HRP. The sensor exhibits a linear response in the concentration ranged from 3.6×10-7 to 7.2×10-3 mol L-1. The regression equation can be expressed as: I = 2321c + 11.85, correlation coefficient is 0.9990.
引文
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