纳米金棒的优化聚合及应用于血吸虫病检测的基础研究

发布时间：2018-03-29 14:54

本文选题：纳米金棒　切入点：血吸虫病　出处：《大理大学》2017年硕士论文

【摘要】：目的纳米技术目前已成为多种学科研究的热点问题和前沿方向,纳米金作为一种性能优良的纳米材料在疾病的早期诊断、检测以及治疗方面得到广泛研究。本研究对纳米金棒的制备条件进行了优化,选择最具有所需光学性质的纳米金棒,并成功将其功能化,制备了实验室条件下可针对日本血吸虫病检测的高特异性、敏感性的纳米金生物探针。方法通过种子介导的生长法聚合纳米金棒,通过对增长媒质中不同成分的比例、反应时间、温度等控制,系统的研究纳米金棒最佳的聚合条件,最大程度的提高单个纳米颗粒的生物功能,聚合具有所需的形貌和波长可控的纳米金棒。制备日本血吸虫尾蚴和成虫可溶性抗原,并将抗原与纳米金棒结合,构建NanoSPR生物探针,将NanoSPR生物探针功能化,通过表面等离子共振峰的LSPR峰变化来探索抗原包被的最佳浓度。将功能化的NanoSPR生物探针检测不同浓度梯度的感染日本血吸虫阳性血清抗体,并与ELISA检测结果做对照分析研究,探索日本血吸虫NanoSPR生物芯片的高特异性。将功能化的NanoSPR生物探针检测感染日本血吸虫前1-8周的兔血清抗体,并与ELISA检测结果做对照分析研究,探索日本血吸虫NanoSPR生物芯片的高敏感性。结果通过对GNRs在制备过程中还原剂、活性剂、温度等条件的优化,系统的研究了GNRs的最佳实验制备条件。在增长媒质中CTAB的最佳浓度为0.095mol/L,AA的最佳浓度为0.640m mol/L,AgNO3的最佳浓度为72μmol/L,种子的最佳生长温度为28°C,种子溶液在增长媒质中生长12h基本趋于稳定,各项指标基本不再发生变化。在成功制备NanoSPR生物探针后,同过对NanoSPR生物探针不同的实验分析得出:包被浓度为20μg/m L时包被前后LSPR峰出现了最大程度的红移达到18nm且峰形正常,确定为最佳包被浓度;对比包被两种不同抗原的GNRs的检测结果,其中NanoSPR-SCA生物探针相对于NanoSPR-AWA的识别日本血吸虫感染早期的血清具有一定的优势;相比于ELISA检测结果的对比发现,通过纳米金标记对抗原抗体反应产生的放大作用,使NanoSPR-AWA生物探针对血清中血吸虫抗体的检测更具有灵敏性,最低检测限可达到稀释倍数的80倍。相比于传统的ELISA检测法,NanoSPR生物探针针对日本血吸虫感染早期的血清体现出了更强的识别能力。结论利用种子生长法通过探索最佳CTAB、AgNO3、AA等试剂浓度和影响金棒制备因素的实验条件,制备了长径比较高、等离子体共振峰位置和强度可有效调控的纳米金棒。并对基于纳米金标记的免疫传感器NanoSPR生物探针进行了实验室阶段的基础研究,证实基于纳米金标记的传感器是一种可以针对日本血吸虫病具有更高特异性,更灵敏的检测方法和工具,并为丰富日本血吸虫病的检测方法提供了技术支持的新思路。
[Abstract]:Objective Nano-technology has become a hot topic and a leading direction in many disciplines. As an excellent nano-material, nano-gold can be used in the early diagnosis of disease. In this study, the preparation conditions of gold nanorods were optimized, and the nanocrystalline gold rods with the most needed optical properties were selected, and the nanocrystalline gold rods were successfully functionalized. A highly specific and sensitive nanoscale gold bioprobe for detection of schistosomiasis japonicum was prepared under laboratory conditions. Methods the nanocrystalline gold rods were polymerized by seeded growth method and by the proportion of different components in the growth medium. Under the control of reaction time, temperature and so on, the optimum polymerization conditions of nanocrystalline gold rods were studied systematically, and the biological function of single nanoparticles was maximized. The soluble antigens of cercariae and adults of Schistosoma japonicum were prepared by polymerizing the gold nanorods with the desired morphology and wavelength. The NanoSPR biological probes were constructed by combining the antigens with gold nanorods, and the NanoSPR bioprobes were functionalized. The best concentration of antigen coating was explored by the change of LSPR peak of surface plasmon resonance peak. The positive serum antibody of different concentration gradient infected Schistosoma japonicum was detected by functional NanoSPR biological probe and compared with the result of ELISA detection. To explore the high specificity of NanoSPR biochip of Schistosoma japonicum, the functional NanoSPR bioprobe was used to detect the serum antibodies of rabbits before 1 to 8 weeks of infection with Schistosoma japonicum, and the results were compared with the results of ELISA. To explore the high sensitivity of NanoSPR biochip of Schistosoma japonicum. Results by optimizing the conditions of reducing agent, active agent, temperature and so on during the preparation of GNRs, The optimum preparation conditions of GNRs were systematically studied. The optimum concentration of CTAB was 0.095 mol / L, the optimum concentration of CTAB was 0.640 mmol / L, the optimum concentration of Agno _ 3 was 72 渭 mol / L, the optimum growth temperature of seed was 28 掳C, and the seed solution was stable in growth medium for 12 h. After the successful preparation of NanoSPR biological probe, different experimental analysis on NanoSPR bioprobe showed that the maximum redshift of the LSPR peak before and after the coating was 20 渭 g / mL, and the peak shape was normal, and the maximum red shift of the LSPR peak was observed when the coating concentration was 20 渭 g / mL. Compared with the results of GNRs coated with two different antigens, NanoSPR-SCA biological probe has some advantages over NanoSPR-AWA in identifying the early serum of Schistosoma japonicum infection, and compared with the results of ELISA detection. Through the amplification of the antigen-antibody reaction by nano-gold labeling, the NanoSPR-AWA biological probe is more sensitive to the detection of schistosomiasis antibody in serum. The minimum detection limit can reach 80 times of dilution multiple. Compared with the traditional ELISA method, the detection of ELISA biological probe for early infection of Schistosoma japonicum showed a stronger ability to recognize the serum. Conclusion the seed growth method can be used to explore the method of detection of Schistosoma japonicum in the early stage of infection. The optimum concentration of CTABX Agno _ 3H _ 3AA and the experimental conditions affecting the preparation of gold rods were studied. Nanocrystalline gold rods with high length and high plasma resonance peak position and intensity were prepared. The basic research of NanoSPR biosensor based on nanoscale gold labeling was carried out in laboratory. It is proved that the sensor based on gold nanoparticles is a more specific and sensitive method and tool for detection of schistosomiasis japonicum, and provides a new idea for enriching the detection methods of schistosomiasis japonicum.
【学位授予单位】：大理大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R532.21

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