基于新型多功能磁性纳米材料平台高效检测C反应蛋白

THE EFFICIENT DETECTION OF C REACTIVE PROTEIN BASED ON THE NEW MULTIFUNCTIONAL MAGNETIC NANOMATERIAL PLATFORM

汪珍玉

11849113

硕士

生物工程领域工程

葛东亮

2020-06-02

2020-07-20

哈尔滨工业大学

深圳

C反应蛋白是炎症的客观制造者之一，血清中CRP的浓度在感染、组织损伤或其他炎症条件下会增加上千倍，而且CRP是心血管疾病的重要危险因素。因此，检测和控制CRP水平对临床感染性疾病和心血管疾病的治疗至关重要。目前，用于CRP测定的方法有乳胶凝集法、单项免疫扩散法、免疫比浊法、电化学发光免疫分析法、胶体金免疫层析法、酶联免疫吸附试验和时间分辨荧光免疫层析法等。这些方法存在一些缺点如耗时耗力、灵敏度低等，所以探索一种快速、简便、可靠的CRP检测方法具有重要的预防和治疗意义。本研究以生物化学、材料化学、纳米科学、有机化学、分子生物学、免疫学等知识为理论指导，基于新型多功能磁性纳米材料平台，建立了一种新型的检测方法，用于在液体中无标记特异性检测和收集急性炎症与心血管疾病标记物C反应蛋白。免疫磁珠是由磁珠表面结合免疫配基制成的，用磁性纳米材料作为固相，在其表面包裹高分子层，最后通过修饰，共价交联免疫配基形成的。本研究用活化剂EDC/NHS活化羧基磁珠表面的羧基基团，在羧基磁珠表面固定CRP抗体，制备免疫磁珠，搭建新型多功能磁性纳米材料平台，检测CRP蛋白。本研究首先研究偶联液MES的pH值、活化剂、活化反应时间、抗体加入量、偶联反应时间等条件对新型多功能磁性纳米材料平台富集CRP效率的影响，确定免疫磁珠制备的最优实验条件：偶联液为pH=6.5的MES、活化剂为EDC/NHS、抗体加入量为70 μg、偶联反应时间为2小时。实验表明采用EDC/NHS活化羧基磁珠，使纳米粒子进行表面改性，表面改性可有效提高纳米粒子和CRP抗体的偶联能力。在与CRP抗体接触后，活化后的羧基磁珠能够和CRP抗体结合。通过对磁珠进行表征，制备的表面偶联CRP抗体的磁珠粒径增加。偶联抗体前后，纳米颗粒大小的变化与CRP抗体浓度有很好的相关性。同时制备好的免疫磁珠能够特异性地识别CRP，在液体中与CRP进行抗原抗体反应，从而达到富集检测CRP的效果，并且富集率能够达到99%以上。本研究建立了一种基于多功能磁性纳米材料的平台，分离检测CRP的方法，为后续开发新型的CRP检测试剂盒以及自动化检测CRP的仪器提供了理论依据。

C reactive protein is one of the objective producers of inflammation, because serum levels of CRP can increase thousands of times in response to infection, tissue damage, or other inflammatory conditions, and CRP is an important risk factor for cardiovascular disease. Therefore, detection and control of CRP levels are essential for the treatment of clinical infectious diseases and cardiovascular diseases. At present, the methods for the determination of CRP include latex agglutination test, single radil immunodiffusion, turbidimetric inhibition immunoassay, electrochemiluminescence immunoassay, gold immunochromatographic assay, enzyme linked immunosorbent assay and time resolved fluoroimmunoassay. These methods have some disadvantages such as time extensive and low sensitivity. Therefore, it is important to explore a fast, simple and reliable method for detecting CRP for infectious and cardiovascular diseases prevention and treatment.This research uses the knowledge of biochemistry, material chemistry, nanoscience, organic chemistry, molecular biology, immunology as the theoretical guidance. Based on the new multifunctional magnetic nanomaterial platform, this study established a new detection method for detecting and collecting the markers of acute inflammation and cardiovascular disease, C reactive protein (CRP), in liquid without the use of labeling markers.Immunomagnetic beads are made by combining immune ligands on the surface of magnetic beads. Using magnetic nanomaterials as the solid phase, the polymer layer is wrapped on the surface of the immunomagnetic beads. In this study, the carboxyl group on the surface of carboxyl beads was activated by the activator EDC/NHS. Immunomagnetic beads were prepared by immobilizing CRP antibody on the surface of carboxyl beads, then the immunomagnetic beads were used to enrich and detect CRP in the liquid.In this study, the effects of buffer pH value, activator, activation reaction time, antibody addition amount, coupling reaction time and other conditions of coupling on the CRP enrichment efficiency of the new multifunctional magnetic nanomaterial platform were studied. After that, the optimal experimental conditions for the preparation of immunomagnetic beads were determined: the coupling solution was MES with pH=6.5, the activator were EDC/NHS, the antibody dosage was 70 μg, and the coupling reaction time was 2 hours. The experiments showed that the surface modification of nanoparticles by using EDC/NHS activator could effectively improve the coupling ability of nanoparticles and CRP antibody. After contacting with CRP antibody, the activated carboxyl beads can covalently couple with the CRP antibody. After coupling the CRP antibody, the particle size of magnetic beads was increased. Changes in the size of nanoparticles before and after antibody coupling were well correlated with CRP antibody concentration. Moreover, the prepared immunomagnetic beads can specifically identify and react with CRP antigens in the liquid, to achieve the enrichment of CRP, and the enrichment rate can reach more than 99%. A platform based on multi-functional magnetic nanomaterials was established to separate and detect CRP, which provide a theoretical basis for the subsequent development of new CRP detection kit and automatic detection instruments for CRP.

C反应蛋白 磁性纳米材料 免疫磁珠 C反应蛋白抗体

C reactive protein magnetic nanomaterial immunomagnetic beads C reactive protein antibody

中文

联合培养

南方科技大学

汪珍玉. 基于新型多功能磁性纳米材料平台高效检测C反应蛋白[D]. 深圳. 哈尔滨工业大学,2020.