SPR生物传感器用于检测吲哚乙酸和脱落酸

发布时间：2018-06-13 19:20

本文选题：SPR生物传感器 + IAA　；参考：《山东农业大学》2017年硕士论文

【摘要】：3-吲哚乙酸(IAA)和脱落酸(ABA)是普遍存在的植物激素,在植物生长发育过程中起着重要的作用。IAA最基本的功能不但可以加速和调控植物的生长发育,而且还可以阻碍植物的生长和根、茎、组织建成的作用。ABA的基本作用是促进和抑制植物生长,促进叶子与果实的脱落,影响花期等作用。目前这两种植物激素的检测方法大都存在样品前处理复杂,费时,成本高,重现性和选择性差等缺点。因此,本研究采用了具有实时监测、样品无需预处理、快速灵敏检测等优点的表面等离子体共振(SPR)生物传感器检测IAA和ABA这两种植物激素。为了提高SPR传感器的灵敏度,本文采用两种方法:一种是将金/银合金纳米粒子作为敏化材料引入SPR生物传感器中,另一种是通过加长检测链的方式来提高SPR生物传感器检测的灵敏度。本文包括以下两部分实验内容:(1)两种SPR生物传感器的构建以及对IAA的检测:一种是传统模式SPR生物传感器(传感器1),另一种是引入Au/Ag合金纳米粒子作为增敏材料的SPR生物传感器(传感器2)。首先用不同的物质修饰SPR的芯片。传感器1:先在裸金片上修饰MPA,再修饰蛋白A,蛋白A能与MPA末端的被EDC/NHS活化后的羧基结合使蛋白A修饰到芯片上,用盐酸乙醇胺对芯片进行灭活,IAA抗体的非抗原结合位点能与蛋白A结合使IAA抗体修饰到芯片上,最后进行对IAA的检测。传感器2:先在裸金片上修饰HDT,Au/Ag合金纳米粒子可与金硫键、金银键结合从而使其修饰到芯片上,接下来在传感器2上依次修饰MPA、蛋白A、IAA抗体的方法与传感器1相同。结果两种传感器均能成功的检测IAA,传感器1对IAA浓度为175~350μg/L所对应的波数变化进行线性回归分析,得到线性方程为y=-6.58+0.103 x,R2=0.994,检测限为25μg/L(S/N=3);传感器2对IAA浓度为17.5~250μg/L所对应的波数变化进行线性回归分析,得到线性方程为y=4.27+0.104 x,R2=0.999,检测限为2.2μg/L(S/N=3)。说明传感器2中引入Au/Ag合金纳米粒子确实可以有效的提高检测的灵敏度,此外本实验所构建的SPR生物传感器具有良好的稳定性,特异性及重现性。(2)构建两种SPR生物传感器并成功检测ABA:一种是MPA构建的SPR生物传感器(传感器1),另一种是巯基己酸构建的SPR生物传感器(传感器2)。通过分子自组装技术对SPR生物传感器的芯片进行修饰。传感器1依次修饰HDT、Au/Ag合金纳米粒子、MPA、蛋白A、IAA抗体,最后对ABA进行检测。传感器2与传感器1的不同之处在于将传感器1的MPA换成了巯基己酸,增长了3个碳链,通过加长检测链的方法来提高SPR生物传感器的灵敏度。结果两种SPR生物传感器均能成功的检测ABA,传感器1对ABA浓度为13.1~23.8μg/L所对应的波数变化进行线性回归分析,得线性方程为y=1.29+0.074 x,R2=0.995,检测限为2.3μg/L(S/N=3);传感器2对ABA浓度为3.3~40μg/L所对应的波数变化进行线性回归分析,得线性方程为y=1.12+0.044 x,R2=0.992,检测限为0.56μg/L(S/N=3)。说明传感器2通过加长检测链确实可以有效提高检测的灵敏度,此外本实验所构建的SPR生物传感器具有良好的精密度,稳定性,特异性及重现性。
[Abstract]:3- indoloacetic acid (IAA) and abscisic acid (ABA) are common plant hormones and play an important role in plant growth and development. The basic function of.IAA not only accelerates and regulates the growth and development of plants, but also hinders plant growth and root, stem and group formation, the basic role of.ABA is to promote and inhibit planting. At present, most of these two kinds of plant hormone detection methods are complicated, time-consuming, expensive, high cost, and poor selectivity. Therefore, this study uses the advantages of real-time monitoring, samples without preprocessing, rapid and sensitive detection and so on. In order to improve the sensitivity of IAA and ABA, two kinds of plant hormones are detected by the SPR biosensor. In order to improve the sensitivity of SPR sensors, this paper adopts two methods: one is to introduce gold / silver alloy nanoparticles into SPR biosensors as sensitized materials. The other is to improve the sensitivity of SPR biosensor detection by the way of lengthening the detection chain. This article includes two parts of the experiment: (1) the construction of two kinds of SPR biosensors and the detection of IAA: one is the traditional mode SPR biosensor (sensor 1), the other is the SPR biosensor (sensor 2) which introduces the Au/Ag alloy nanoparticles as the sensitizing material (the sensor 2). First, the SPR chip is modified with different substances. 1: first modified MPA on bare gold slices and then modified protein A. Protein A can be combined with the carboxyl of EDC/NHS at the end of MPA to modify the protein A on the chip and inactivate the chip with ethanolamine hydrochloride. The non antigen binding site of IAA antibody can be combined with protein A to modify the IAA antibody to the chip. Finally, IAA is detected. Sensor 2:. First, the HDT is modified on the bare gold plate. The Au/Ag alloy nanoparticles can be combined with gold and silver bonds with gold and silver bonds to make them modified to the chip. Then, the MPA, protein A, IAA antibody are the same as the sensor 1. The results of the two sensors are all successful in detecting IAA, and sensor 1 to the IAA concentration corresponding to 175~350 u g/L. Linear regression analysis is carried out, the linear equation is y=-6.58+0.103 x, R2=0.994, the detection limit is 25 g/L (S/N=3); sensor 2 is linear regression analysis on the wave number of IAA concentration corresponding to 17.5~250 u g/L, and the linear equation is y=4.27+0.104 x, R2=0.999, and the detection limit is 2.2 micron g/L. The alloy nanoparticles can effectively improve the sensitivity of the detection. In addition, the SPR biosensor constructed by this experiment has good stability, specificity and reproducibility. (2) two kinds of SPR biosensors are constructed and ABA: is a SPR biosensor (sensor 1) constructed by MPA, and the other is the SPR generation of mercapto hexanoacid. The sensor (sensor 2). Modifies the SPR biosensor chip by molecular self-assembly technology. Sensor 1 modifies HDT, Au/Ag alloy nanoparticles, MPA, protein A, IAA antibody in turn, and finally detects ABA. The difference between sensor 2 and sensor 1 is that the MPA of the sensor 1 is replaced by mercapto hexanoprol acid and 3 carbon chains are increased. The sensitivity of the SPR biosensor was enhanced by the method of lengthening the detection chain. Results two kinds of SPR biosensors were able to detect ABA successfully. Sensor 1 had linear regression analysis on the wave number changes corresponding to the ABA concentration of 13.1~23.8 mu g/L. The linear equation was y=1.29+0.074 x, R2= 0.995, and the detection limit was 2.3 mu g/L (S/N=3); sensor 2 pairs of ABA. The linear regression analysis of the wave number variation corresponding to 3.3~40 mu g/L is obtained. The linear equation is y=1.12+0.044 x, R2=0.992, and the detection limit is 0.56 mu g/L (S/N=3). It shows that the sensor 2 can effectively improve the sensitivity of the detection by adding a long detection chain. Furthermore, the SPR biosensor built in this experiment has good precision and stability. Specificity and reproducibility.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212.3

【参考文献】