唾液sIgA纳米免疫生物传感器的实验研究

发布时间：2018-01-07 19:34

本文关键词：唾液sIgA纳米免疫生物传感器的实验研究　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：唾液是人体重要体液之一,其成分相对稳定,人类唾液蛋白质组图发现唾液中有20%的蛋白质与血液中所含蛋白质相同。同时唾液也受全身各种因素和状态的影响,能够很好反映血液中各种蛋白质水平的变化,可作为疾病诊断、疗效判断、药物监测的参考指标,在疾病诊治中具有很好的应用前景。而且与血清标本相比,唾液采集无创伤,安全方便,无血源性疾病传染的危险,因此唾液诊断方法是当前检测方法的一个补充。但由于很多成分在唾液中的浓度远低于血液,以往的技术难以将其检测出来。随着生物传感技术迅速发展,电化学免疫传感技术因其方法简单、灵敏度高、选择性强等优点而被广泛应用于微量物质的测定。电化学免疫传感技术的基础是氧化还原活性中心与电极间的电子传递。它可以通过两种不同的途径实现,一方面活性中心与电极间的电子传递的介质非常重要;另一方面,生物活性分子的固定化对构筑生物传感器作用重大,这是影响生物传感器的稳定性、灵敏度和选择性的两个关键因素。本文选择唾液中高丰度蛋白质sIgA作为标志物,利用比表面积大、表面活性高的纳米材料和电子介体硫堇层层组装,通过静电吸附和金-氮(Au-N)、金-硫(Au-S)共价键作用实现了电子介体的有效固定,从而构建了性能优良的介体型唾液免疫传感器。同时,还对Nafion与纳米金、碳纳米管与纳米金结合作为固定基质在唾液免疫传感器领域的应用做了探索和研究。具体研究内容如下: 1.基于金纳米粒子/硫堇/Nafion膜修饰的可再生唾液sIgA免疫传感器的研制通过层层自组装将硫堇(ThI)和纳米金(GNPs)修饰到Nafion修饰的玻碳(GCE)电极表面,利用纳米金单层吸附唾液分泌性免疫球蛋白A(sIgA),最后用辣根过氧化物酶(HRP)封闭电极上的非特异性吸附位点,构建了一种检测唾液sIgA的新型电流型纳米免疫生物传感器。该生物传感器灵敏度高、特异性好、测试方便,检测线性范围为6.5-300 ng/mL,检出限为3.0 ng/mL;电流值达到95%稳态时间小于20 s。探讨了抗体及底物浓度、pH值和温度、孵育时间、干扰物对传感器的影响。该传感器与ELISA法相关性良好(r=0.98932,P0.001),可用于唾液sIgA的快速检测,从而判断人体局部免疫状况。 2.基于功能化碳纳米管/聚硫堇/金纳米粒子层层自组装修饰的可再生唾液sIgA免疫传感器的研制将壳聚糖(CHI)分散的羧基化多壁碳纳米管(MWNTs)吸附到GCE表面,形成负电荷界面,利用静电吸附和金-氮(Au-N)、金-硫(Au-S)共价键作用将阳离子电子媒介体THI和GNPs层层自组装到电极表面,通过GNPs单层吸附唾液sIgA,最后用HRP封闭电极上的非特异性吸附位点,同时放大电流信号,构建了一种检测唾液sIgA的新型纳米免疫生物传感器。实验结果表明,该传感器具有良好的准确性和稳定性;检测线性范围为3.0-350 ng/ml,r=0.98967(P0.001);检出限为1.5 ng/mL(S/N=3);电流值达到95%稳态时间小于20 s;可用0.1 mol/L的甘氨酸再生8次。探讨了底物浓度、抗体浓度、孵育时间、温度和pH值及干扰物质对该传感器的影响。该免疫传感器灵敏度高、特异性好、方便可再生,可用于唾液sIgA的快速检测,从而判断人体局部免疫状况。
[Abstract]:Saliva is one of the important components of the human body, relatively stable, human salivary proteins found in saliva protein and photos of 20% blood contained in the same protein. At the same time saliva is also affected by the various factors and systemic state, can well reflect the changes of protein levels in the blood, as disease diagnosis, curative effect evaluation, reference drug monitoring index, has a good application prospect in the diagnosis of disease. But compared with the serum samples of saliva collection without trauma, safe and convenient, without the risk of blood borne diseases infection, so saliva diagnosis method is a supplement to the current detection method. But because many of the components of concentration in saliva is far lower than that of blood in the past, the technology is difficult to be detected. With the rapid development of bio sensing technology, electrochemical immunosensor technology because of its simplicity, high sensitivity, adaptability and selection It is widely used for the determination of trace substances.
The basis of electrochemical immunosensors is redox active center and electron transfer between the electrode. It can be achieved through two different pathways, one electron transfer between electrode and the active center of the medium is very important; on the other hand, the immobilization of bioactive molecules on the construction of major biological sensor, which is affecting the stability biosensor, two key factors of sensitivity and selectivity.
The high abundance of protein sIgA in saliva as a marker, with large surface area, nano materials and electron mediator thionine high surface activity layer by layer, through electrostatic adsorption and gold - nitrogen (Au-N), gold sulfur (Au-S) covalent interaction to achieve the electronic mediator effectively fixed, so as to construct the excellent performance of the medium saliva immune sensor. At the same time, the Nafion and gold nanoparticles, carbon nanotubes and nano gold as a combination of application in the field of saliva immune sensor immobilization matrix makes the exploration and research. The main research contents are as follows:
1. development of a renewable saliva sIgA immunosensor based on gold nanoparticles / thio /Nafion membrane
Through the layers of self-assembly of Thionine (ThI) and gold nanoparticles (GNPs) modified glassy carbon electrode modified with Nafion (GCE) surface by using gold nanoparticle monolayer adsorption of salivary secretory immunoglobulin A (sIgA), finally using horseradish peroxidase (HRP) blocking non-specific adsorption sites on the electrode, construct a new current type nano immunosensor for detection of salivary sIgA. The biosensor has high sensitivity, good specificity, convenient testing, detection of the linear range of 6.5-300 ng/mL, the detection limit was 3 ng/mL; the current value reached 95% steady state time is less than 20 s. of antibody and substrate concentration, pH value and temperature, incubation time effect, disruptors on the sensor. The sensor and ELISA good correlation (r=0.98932, P0.001), can be used for rapid detection of sIgA in saliva, so as to determine the human body local immune status.
2. development of sIgA immunosensor based on functional carbon nanotube / polythio / gold nanoparticle self redecorating and decorating regenerative saliva
The chitosan (CHI) dispersed carboxylated multiwalled carbon nanotubes (MWNTs) adsorbed on the surface of GCE, forming a negative charge interface, using electrostatic adsorption and gold - nitrogen (Au-N), gold sulfur (Au-S) covalent interaction of cationic mediator THI and GNPs self-assembly on the electrode surface by GNPs monolayer adsorbed salivary sIgA, finally used HRP to block the non-specific adsorption site on the electrode, and the amplified current signals, constructed a sialic sIgA nano immunosensor. The experimental results show that the sensor has good accuracy and stability; the linear range of detection for 3.0-350 ng/ml r=0.98967 (P0.001); detection the limit is 1.5 ng/mL (S/N=3); current value reached 95% steady state time is less than 20 s; available 0.1 mol/L glycine 8 times of regeneration were discussed. The concentration of substrate, antibody concentration, incubation time, temperature and pH value of the sensor and interfering substances on the. The immunosensor has high sensitivity, good specificity, convenient and renewable, and can be used for rapid detection of saliva sIgA, so as to judge the local immune status of the human body.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【参考文献】