基于APTES和蛋白A联合固定化技术的乙肝病毒免疫生物传感器敏感膜的制作研究

发布时间：2018-01-05 14:32

本文关键词：基于APTES和蛋白A联合固定化技术的乙肝病毒免疫生物传感器敏感膜的制作研究　出处：《浙江大学》2012年硕士论文　论文类型：学位论文

【摘要】：稳定、高效的生物微纳免疫传感器在食品领域、环境监测领域及医疗诊断领域的广泛应用。生物传感器敏感膜固定化技术是生物传感器的关键核心技术。传统敏感膜固定方法(吸附法、共价结合法、交联法以及包埋法等)渐趋成熟但普遍存在固定生物分子失活,效价不高的问题。生物大分子定向固定可以提高生物材料固定的有效性,但是却存在牢固性和可重用性差等问题。针对上述问题,本文以乙肝免疫生物传感器敏感膜固定化技术研究为切入点,首先,提出了将传统化学共价结合和生物大分子定向固定化方法结合的生物传感器敏感膜固定化技术,它既利用了化学共价固定法的固定牢固性和特异性,又利用了蛋白八定向固定的抗空间位阻性和有效性高的特性。文中的化学共价修饰采用了能够有效控制修饰的APTES硅烷化固定方法,并实现了对于基底膜性质的有效控制。其次,为进一步提高传感器灵敏度等方面的性能,我们对固定化界面进行了纳米材料的修饰,通过控制材料表面纳米颗粒的大小、性质及反应条件,有效控制了纳米材料的纳米层面上的性质,进而有效利用了修饰材料的纳观效应。为验证上述固定化方法的有效性,我们通过接触角(WC八)傅里叶变换红外线光谱(FTIR)检测等实验方法定性验证了敏感膜的修饰结果,并用原子力显微镜对修饰过程进行了形貌学表征。首先,通过对比分析研究在经过或未经过APTES硅烷化修饰的硅片上固定蛋白八的效果,证明了APTES硅烷化修饰的优越性;其次,分析比较了通过APTES硅烷化纳米颗粒修饰后侨联蛋白A或不侨联蛋白A的两种方法,进一步证明蛋白八对于本固定方法中抗体定向固定,降低空间位阻,提高传感灵敏度的重要性。最后,通过双抗体夹心ELISA方法定性验证了上述联合固定化方法在微量固定抗体情况下的高活性。上述结果表明了通过将联合固定化技术与界面纳观效应有机结合可实现生物免疫传感器敏感膜的良好固定。
[Abstract]:Stable and efficient biosensors for biosensors in the field of food. Biosensor sensitive membrane immobilization technology is the key technology of biosensor. Traditional sensitive membrane fixation methods (adsorption method, covalent binding method) are widely used in the field of environmental monitoring and medical diagnosis. Crosslinking method and embedding method, etc.) but the problem of immobilization of biomolecules is common, such as inactivation and low titer of biomolecules. Directional fixation of biomolecules can improve the efficiency of biomaterials fixation. However, there are problems such as poor fastness and reusability. In order to solve the above problems, this paper focuses on the study of immobilization of sensitive membrane of hepatitis B immunosensor. The biosensor sensitive membrane immobilization technology which combines the traditional chemical covalent binding method and the biological macromolecule directional immobilization method is proposed. It not only makes use of the fixation fastness and specificity of the chemical covalent fixation method. The chemical covalent modification is based on the APTES silanization method which can effectively control the modification. And realized the effective control of the properties of the substrate membrane. Secondly, in order to further improve the sensor sensitivity and other aspects of the performance, we modified the immobilized interface nanomaterials. By controlling the size, properties and reaction conditions of nanocrystalline particles on the surface of the materials, the properties of the nanomaterials on the nanometer level are effectively controlled. Furthermore, the nano effect of the modified materials is effectively utilized to verify the effectiveness of the above immobilization methods. The modification results of the sensitive films were qualitatively verified by means of FTIR (contact angle WC8) Fourier transform infrared spectroscopy (FTIR). The modification process was characterized by atomic force microscope (AFM). Firstly, by comparing and analyzing the effect of immobilization of protein 8 on the silicon wafer modified with or without APTES silanization, the superiority of APTES silanization modification was proved. Secondly, two methods modified by APTES silanized nanoparticles were analyzed and compared. It was further proved that protein 8 was immobilized against antibodies in this method. Reduce the space steric resistance, improve the importance of sensing sensitivity. Finally. The double antibody sandwich ELISA method was used to qualitatively verify the high activity of the combined immobilization method in the case of microimmobilized antibodies. The above results show that the joint immobilization technique and the interface nano effect are organic junctions. It can achieve good fixation of biosensor sensitive membrane.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373;TP212.3

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