新型纳米材料修饰的DNA生物传感器用于VANGL1基因SNP位点检测

发布时间：2018-03-18 10:26

  本文选题：聚吡咯/链霉亲和素　切入点：PAMAM-Au　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的基于聚吡咯/亲和素膜和Au-PAMAM-CP生物纳米复合物探针材料,拟构建一种DNA生物传感器用于神经管畸形相关基因VANGL1的单核苷酸多态性(SNP)(rs4839469 c.346GA p.Ala116Thr)位点的检测。方法(1)利用聚吡咯(PPy)良好的生物相容性和优异的导电性,采用恒电位沉积法将聚吡咯和链霉亲和素一步共沉积到金电极表面。通过三维激光扫描、扫描电子显微镜(SEM)、红外可见吸收光谱(FT-IR)和循环伏安法(CV)对合成的聚吡咯/链霉亲和素膜进行表征;(2)合成聚酰胺胺树状大分子包裹的金纳米粒子(Au-PAMAM),设计与制备特异性检测VANGL1基因SNP位点的茎环探针作为传感器的捕获探针(CP),以Au-PAMAM作为载体固载CP,制备Au-PAMAM-CP生物纳米复合物探针。同时通过透射电镜、高分辨率透射电镜、FT-IR和紫外可见吸收光谱(UV-Vis)表征Au-PAMAM和Au-PAMAM-CP验证Au-PAMAM-CP是否成功合成;(3)琼脂糖凝胶电泳验证目标DNA和CP是否杂交成功;(4)传感器构建过程采用循环伏安法和电化学交流阻抗法表征;(5)采用差分脉冲伏安法(DPV)对聚吡咯/链霉亲和素的电沉积时间、CP和目标DNA的杂交温度和杂交时间以及电极捕获时间进行优化,以提高传感器灵敏度、特异性和准确性;(6)利用传感器检测0.1,1,10,50和100 n M不同浓度目标DNA,通过差分脉冲伏安法进行表征分析获得传感器的检测范围和检测限;(7)验证传感器的特异性、重现性和稳定性;(8)验证传感器的基质效应,并通过标准加样测试其检测性能。结果(1)表征分析显示聚吡咯和链霉亲和素膜合成成功;(2)Au-PAMAM-CP生物纳米复合物探针合成成功;(3)目标DNA和CP杂交成功;(4)DNA生物传感器构建成功;(5)优化后的检测条件为:聚吡咯/链霉亲和素的电沉积时间4 min,CP和目标DNA的杂交时间30 min,捕获时间40 min,CP和目标DNA的杂交温度35℃;(6)在最佳实验条件下,VANGL1基因在0.1-100 n M浓度范围内与峰电流的变化值I呈良好的线性关系,最低检出限为0.033 n M(S/N=3);(7)该传感器能有效识别目标DNA、单碱基错配DNA、三碱基错配DNA和非互补DNA,具有较好的特异性。对10 n M VANGL1基因平行测定5次,相对标准偏差(RSD)为3.1%,具有较好的重现性。传感器在4℃下储存两周后仍保持生物活性,电流响应显示没有显着变化,具有较好的稳定性;(8)该测定法可以忽略实际样品检测时基质的影响,研究所提出的信号放大策略对真实样品有效,人体血样加标回收率为103%~105%。结论本研究成功构建的DNA生物传感器,具有简单、成本低、灵敏度高、重现性好等优点,可用于检测VANGL1基因SNP位点。该方法为神经管畸形的基因诊断提供了新的思路。
[Abstract]:Objective based on polypyrrole / avidin film and Au-PAMAM-CP biological nanocomposite probe material, To construct a DNA biosensor for the detection of the single nucleotide polymorphisms (SNPs 4839469 c. 346GA p.Ala116Thr) of the neural tube deformation-associated gene VANGL1. The polypyrrole and streptavidin were codeposited on the surface of gold electrode by potentiostatic deposition. Characterization of Polypyrrole / Streptomyces Affinity Film synthesized by scanning Electron microscope (SEM), FT-IR (IR) and cyclic voltammetry (CVV). The stem ring probe of the SNP site of VANGL1 gene was used as the capture probe of the sensor, and the Au-PAMAM carrier was used as carrier to immobilize the Au-PAMAM-CP biological nanocomposite probe. At the same time, the probe was prepared by transmission electron microscope. High Resolution Transmission Electron Microscopy FT-IR and UV-Vis-UV absorption Spectroscopy characterization of Au-PAMAM and Au-PAMAM-CP to verify whether Au-PAMAM-CP was successfully synthesized or not) agarose gel electrophoresis was used to verify whether the target DNA and CP were hybridized successfully and the sensors were constructed by cyclic voltammetry and cyclic voltammetry. Electrochemical impedance spectroscopy (EIS) was used to optimize the electrodeposition time of polypyrrole / streptomycin (CP) and the hybridization time of target DNA and the electrode capture time by differential pulse voltammetry (DPV). In order to improve the sensitivity, specificity and accuracy of the sensor, the sensitivity, specificity and accuracy of the sensor were improved. The detection range and detection limit of the sensor were obtained by differential pulse voltammetry (differential pulse voltammetry) to verify the specificity of the sensor. Reproducibility and stability) to verify the matrix effect of the sensor, The results showed that the polypyrrole and Streptomyces avidin membrane were synthesized successfully by using the standard addition method. Results: a novel DNA and CP hybridization method was used to construct a novel DNA biosensor for the successful synthesis of polypyrrole and streptomycin membrane. The optimized detection conditions were as follows: the electrodeposition time of polypyrrole / Streptomycin was 4 mins CP and the hybridization time of target DNA was 30 min, the capture time was 40 min CP and the crossing temperature of target DNA was 35 鈩，

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