基于纳米复合物的金黄色葡萄球菌肠毒素B适体传感器的研究

发布时间：2018-03-27 17:28

本文选题：金黄色葡萄球菌肠毒素B　切入点：金纳米粒子　出处：《重庆医科大学》2015年硕士论文

【摘要】：目的：金黄色葡萄球菌是引起人类感染的一种重要致病菌,其在自然界中分布广泛,耐药谱广,耐药性强,因此在体外能够很好的存活。金黄色葡萄球菌通过产生和分泌一系列毒素来攻击人体的防御系统。其中SEB是最致命的毒素,SEB耐热、耐酸,性质稳定,能够引起许多人类疾病,例如,爆发性紫癜、高热、过敏性皮炎、坏死性肺炎。因此,采用快速、敏感的方法对低浓度的SEB进行检测是我们研究的方向。本研究充分发挥纳米复合材料的优势,构建了一种灵敏度高、特异性强的电化学适体传感器。该传感器采取目标物介导适体释放策略,来实现对SEB的检测,具有良好的分析性能和实际应用价值。方法：1.GNPs-ZrO2-Chits复合物的制备：利用Chits的还原性及生物相容性将HAuCl4原位还原成GNPs,并对进行ZrO2分散溶解,制备成GNPs-ZrO2-Chits复合物。2.SEB适体传感器的制备：采用制备所得GNPs-ZrO2-Chits复合物修饰金电极表面,捕获探针通过Au-S键固定于电极表面纳米材料复合层,通过碱基互补配对原则,与适体链进行杂交反应。利用目标物介导适体释放策略,定量检测样本中的SEB。3.优化适体传感器检测的实验条件：孵育时间、工作溶液pH值、SA-HRP和H2O2浓度等。4.考察电化学适体传感器性能：线性范围、最低检测限、选择性、重现性和稳定性等。5.实际标本的检测：本方法与ELISA分别检测13份血清标本中的SEB,进行方法学比较。结果：1.对制备所得的复合物分别进行形态学、光谱学和电化学分析,在透射电镜图中可见GNPs大小形态均匀的分散在Zr02表面,形成明显的壳核结构,成像清晰；UV-vis及EDX进一步证实壳核结构的形成,表明GNPs-ZrO2-Chits复合物制备成功。循环伏安法和电化学交流阻抗共同证实了修饰电极的步骤。2.在实验优化条件下,将制备好的适体传感器对SEB进行检测,其电流响应达到最好。在此条件下,对不同浓度的目标物SEB进行检测,在浓度2～512 ng/mL范围内与DPV信号呈线性相关,相关系数为0.992,最低检测限为0.24 ng/mL(S/N=3)。3.该传感器性能优越：4℃干燥情况下储存15天后,电流响应仅下降了8.2%,稳定性好；能特异的分辨SEA, SEC和凝血酶,选择性好；相同条件下制备一批6个适体传感器,分别检测浓度32,64,128,256,和512 ng/mL目标物,得到相对标准偏差(RSD)分别为4.0%,5.6%,4.8%,5.2%和3.8%,重现性好。4.用所制备的适体传感器和ELISA分别检测实际样本中的SEB,进行方法学比较,结果显示两种检测方法的相关系数为0.998,表明该适体传感器可以应用于实际标本的检测。结论：本研究结果表明,将纳米金-氧化锆-壳聚糖膜修饰在电极上不仅能增加电极的导电性和有效表面积,并且可以增加核酸分子的固定量。更重要的是,采取目标物介导适体释放的策略,使该适体传感器拥有较宽的检测范围,较低的检测限和较高的特异性。本研究的方法具有通用性,在置换相应的适体后,可以用于SEA、SEC等其他肠毒素的检测,因此,能够用于临床上对肠毒素感染等相关疾病进行筛查。
[Abstract]:Objective: Staphylococcus aureus is an important cause of human infection of pathogenic bacteria, which is widely distributed in nature, broad resistance spectrum, strong resistance, so in vitro can survive well. Staphylococcus aureus through produce and secrete a series of toxins to attack the body's defense system in which SEB is the most deadly. SEB toxin, heat resistance, acid resistance, stable property, can cause many human diseases, for example, purpura fulminans, high fever, allergic dermatitis, necrotizing pneumonia. Therefore, the rapid and sensitive method for detection of low concentration of SEB is the direction of our research. This study give full play to nano composite material advantage, construct a high sensitivity, specificity and strong electrochemical aptamer sensor. The sensor to the target aptamer mediated release strategy, to achieve the detection of the SEB, has good performance analysis and practical application value. The party Method: the preparation of 1.GNPs-ZrO2-Chits composite by reducing Chits and biocompatibility of HAuCl4 in situ reduction GNPs, and dissolution of ZrO2, preparation of GNPs-ZrO2-Chits composite.2.SEB aptamer sensor preparation: the prepared GNPs-ZrO2-Chits composite modified gold electrode surface of the system, the capture probes immobilized on the electrode surface nano material composite layer by Au-S bond, through complementary base pairing, hybridization reaction with the aptamer. The target aptamer mediated release strategy, the SEB.3. optimal experimental conditions of body sensor detection quantitative detection of samples: the incubation time, solution pH value, SA-HRP and H2O2 concentration of.4. the electrochemical performance of suitable body sensor: the linear range, detection limit, selectivity, reproducibility and stability of.5. detection and ELISA: the actual samples were detected 13 serum specimens The SEB method was compared. Results: 1. of the complexes prepared respectively, morphology, and electrochemical spectroscopy analysis, TEM, visible GNPs in size and shape evenly dispersed on the surface of Zr02, the formation of core-shell structure, clear clear imaging; UV-vis and EDX further confirmed the formation of core-shell structure, show GNPs-ZrO2-Chits composite was prepared successfully. Cyclic voltammetry and electrochemical impedance confirmed by modified electrode process.2. under the optimized conditions, the prepared aptamer sensor for the detection of SEB, the current response to the best. Under these conditions, to detect different concentrations of target SEB in concentration in the range of 2~512 ng/mL and DPV signal was linearly correlated, the correlation coefficient was 0.992, the lowest detection limit was 0.24 ng/mL (S/N=3).3. performance of the sensor location: 4 C under dry condition after 15 days of storage, electric Flow response only decreased by 8.2%, the stability is good; the specific resolution SEA, SEC and thrombin, good selectivity; preparation of a batch of 6 aptasensors were detected under the same conditions, the concentration of 32,64128256, and 512 ng/mL target, relative standard deviation (RSD) were 4%, 5.6%, 4.8%, and 5.2% 3.8%, good reproducibility of.4. with the preparation of the aptamer and ELISA were detected in real samples SEB, methodology, results showed that the correlation coefficient of the two detection methods was 0.998, showed that the aptamer sensor can be applied to the actual specimen test. Conclusion: the results of this study show that the nano gold - zirconia - chitosan film modified electrode on the electrode can not only increase the conductivity and the effective surface area, and can increase the fixed amount of nucleic acid molecules. More importantly, take the target aptamer mediated release strategy, make the aptasensors. It has wide detection range, low detection limit and high specificity. The method of this research is universal. After replacement of corresponding aptamers, it can be used for other enterotoxin detection such as SEA and SEC, so it can be used for screening enterotoxin infection and other related diseases in clinic.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5;TP212.3

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