基于pH指示剂显色效应的改进型酶联免疫检测方法研究

发布时间：2019-04-04 11:20

【摘要】：传统酶联免疫吸附剂测定(ELISA)易受干扰,灵敏度低等一些缺点限制了其广泛应用。近些年对传统ELISA的改进已经做了许多努力,使其在生物传感检测中得到了很好的应用。纳米材料问世之后由于其一系列优点,如比表面积大,吸附能力强,催化效率高,生物相容性好等一直备受关注。非金属的纳米材料具有纳米材料的优点,不含有贵金属,绿色前驱体,成本低、无污染,因此受到更多的青睐。廉价的pH指示剂代替酶的使用,降低了实验成本,化学性质稳定,反应后颜色变化显著,实验结果可视化。本文中,利用非金属纳米材料作为信号载体,pH指示剂作为信号直接放大平台通过π-π共轭负载到纳米材料上,疾病标记物与纳米材料通过化学键牢固结合,在96-微孔板中构建成完整的免疫传感器。向微孔板的孔中加入定量的释放剂,释放剂与疏水性的指示剂分子反应,转化为亲水性的离子从纳米材料上释放到溶液中,引起溶液颜色发生变化。该方法实现了对疾病标记物的可视化超灵敏检测。具体检测内容如下:(1)设计了一种基于酚酞(PP)指示剂变色效应的改进传统ELISA,实现对癌胚抗原(CEA)的高灵敏可视化检测。类石墨烯氮化碳(g-C_3N_4)纳米材料由于具有较大的比表面积,容易制备,化学稳定性高,成本低,被羧基化后的g-C_3N_4(cC_3N_4)被用来作为该体系中信号分子的载体。免疫传感器制备成功后,加入碱性释放剂(AS),PP分子遇碱反应生成离子,释放到溶液中,引起溶液颜色发生变化。优化实验条件后,实现了对CEA的高灵敏可视化检测。检测范围是0.5 pg/mL到100 ng/mL,最低检测限(LOD)为0.34 pg/m L。这种方法是一种无酶的改进型ELISA,可以命名为PILISA。PILISA遵循酶联免疫吸附测定形式,同时避免了传统ELISA的缺点,为实际临床检测提供了一种很有发展前景的方法。(2)心血管疾病是引起全球死亡率高的主要原因之一,而心脏病标志物肌钙蛋白I(cTnI)和C-反应蛋白(CRP)的测定分别用于检测和诊断急性心肌梗死和冠状动脉综合症。使用聚多巴胺(PDA)包裹的g-C_3N_4(PDA-C_3N_4)作为信号载体,是由于PDA表面具有丰富的官能团,既可以与生物分子结合,又可以吸附大量的指示剂分子百里香酚酞(TP)和溴酚红(BR),设计的新型免疫传感器实现了对心脏病标记物cTnI和CRP的可视化双检测。灵敏度高,检测范围宽(cTnI:0.5 pg/m L-8 ng/mL;CRP:0.5 pg/m L-10 ng/m L),其中LOD分别为0.32 pg/m L和0.28 pg/mL。根据检测结果可以看出,该免疫传感器的稳定性、选择性和重现性都是令人满意的。(3)介孔二氧化硅(MSN)由于其特殊的孔状结构,比表面积大和易功能化等优点得到了广泛的应用。将MSN功能化,使其孔内带有芳香结构,能与疏水性的pH指示剂分子通过π-π共轭结合。MSN与聚乙烯亚胺(PEI)作用,使其表面显正电性,与表面带有负电的抗体分子作用连接。因此功能化的MSN作为信号载体参与构建免疫传感器,实现了对前列腺抗原(PSA)的可视化高灵敏检测。检测浓度范围宽(0.5 pg/m L-8 ng/m L),检测限低(0.21 pg/m L),并且该新型的免疫传感器具有良好的稳定性,选择性和重现性。此外,蛋白质生物标志物的比色检测由于其简单、快速、经济和稳定的信号输出特征在临床医学治疗中具有潜在的应用。
[Abstract]:The traditional enzyme-linked immunosorbent assay (ELISA) is susceptible to interference and low sensitivity, and has limited its wide application. In recent years, the improvement of the traditional ELISA has done a lot of efforts to make it a good application in the biological sensing detection. Because of its series of advantages, such as large specific surface area, strong adsorption capacity, high catalytic efficiency and good biocompatibility, the nano-material has been of great concern. The non-metal nanometer material has the advantages of nano material, no noble metal, green precursor, low cost and no pollution, thus being more favored. The cheap pH indicator replaces the use of the enzyme, the experimental cost is reduced, the chemical property is stable, the color change after the reaction is remarkable, and the experimental result is visualized. In this paper, a non-metallic nano-material is used as the signal carrier, and the pH indicator is loaded on the nano-material by the carbon-carbon coanda by using a pH indicator as a signal direct amplification platform, and the disease marker and the nano material are firmly combined with the nano material through a chemical bond, and a complete immune sensor is constructed in the 96-well plate. A quantitative release agent is added into the pores of the microporous plate, the release agent reacts with the hydrophobic indicator molecules, and the ions which are converted into the hydrophilic are released from the nano material to the solution to cause the color of the solution to change. The method realizes the visual and ultra-sensitive detection of the disease marker. The specific detection content is as follows: (1) a modified traditional ELISA based on the color change effect of the phenolphthalein (PP) indicator is designed to realize the high-sensitivity visual detection of the carcinoembryonic antigen (CEA). The graphene nitride (g-C _ 3N _ 4) nano-material has a large specific surface area, is easy to prepare, has high chemical stability and low cost, and the g-C _ 3N _ 4 (cC _ 3N _ 4) after being modified is used as the carrier of the signal molecule in the system. After the preparation of the immune sensor is successful, the alkaline releasing agent (AS) is added, the PP molecule is reacted with alkali to generate ions, and the solution is released to the solution to cause the color of the solution to change. And the high-sensitivity visual detection of the CEA is realized after the experimental conditions are optimized. The detection range is 0.5 pg/ mL to 100 ng/ mL and the minimum detection limit (LOD) is 0.34 pg/ mL. This method is an enzyme-free modified ELISA, which can be named as PILISA. PILISA follows the enzyme-linked immunosorbent assay format, while avoiding the disadvantages of conventional ELISA, And provides a promising method for the actual clinical detection. (2) Cardiovascular disease is one of the main causes of high global mortality, and the determination of cardiac troponin I (cTnI) and C-reactive protein (CRP) is used to detect and diagnose acute myocardial infarction and coronary syndrome, respectively. The g-C _ 3N _ 4 (PDA-C _ 3N _ 4) wrapped with the polydopamine (PDA) is used as the signal carrier, because the PDA surface has rich functional groups, can be combined with the biological molecule, and can adsorb a large number of indicator molecules thymol phthalide (TP) and bromophenol red (BR), The design of new type of immunosensor realizes the visual double-detection of the cardiac markers cTnI and CRP. High sensitivity, wide detection range (cTnI: 0.5 pg/ m L-8 ng/ mL; CRP: 0.5 pg/ m L-10 ng/ m L), where LOD is 0.32 pg/ m L and 0.28 pg/ mL, respectively. It can be seen from the results of the test that the stability, selectivity and reproducibility of the immunosensor are satisfactory. (3) Mesoporous silica (MSN) has been widely used because of its special pore-like structure, large specific surface area and easy functionalization. The msn is functionalized so that its pores are provided with an aromatic structure in which the hydrophobic pH indicator molecules can be co-linked with the hydrophobic pH indicator molecules. The effect of MSN and polyethyleneimine (PEI) on the surface is positively and electrically connected to the negatively charged antibody molecules on the surface. Therefore, the functionalized MSN is used as the signal carrier to participate in the construction of the immune sensor, and the visualization and high-sensitivity detection of the prostate antigen (PSA) is realized. The detection concentration range is wide (0.5 pg/ m L-8 ng/ m L), the detection limit is low (0.21 pg/ m L), and the novel immunosensor has good stability, selectivity and reproducibility. In addition, the colorimetric detection of protein biomarkers has potential application in clinical medicine therapy due to its simple, rapid, economical and stable signal output characteristics.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212.3

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