發(fā)布時(shí)間：2018-03-18 10:26

  本文選題：聚吡咯/鏈霉親和素　切入點(diǎn)：PAMAM-Au　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的基于聚吡咯/親和素膜和Au-PAMAM-CP生物納米復(fù)合物探針材料,擬構(gòu)建一種DNA生物傳感器用于神經(jīng)管畸形相關(guān)基因VANGL1的單核苷酸多態(tài)性(SNP)(rs4839469 c.346GA p.Ala116Thr)位點(diǎn)的檢測(cè)。方法(1)利用聚吡咯(PPy)良好的生物相容性和優(yōu)異的導(dǎo)電性,采用恒電位沉積法將聚吡咯和鏈霉親和素一步共沉積到金電極表面。通過三維激光掃描、掃描電子顯微鏡(SEM)、紅外可見吸收光譜(FT-IR)和循環(huán)伏安法(CV)對(duì)合成的聚吡咯/鏈霉親和素膜進(jìn)行表征;(2)合成聚酰胺胺樹狀大分子包裹的金納米粒子(Au-PAMAM),設(shè)計(jì)與制備特異性檢測(cè)VANGL1基因SNP位點(diǎn)的莖環(huán)探針作為傳感器的捕獲探針(CP),以Au-PAMAM作為載體固載CP,制備Au-PAMAM-CP生物納米復(fù)合物探針。同時(shí)通過透射電鏡、高分辨率透射電鏡、FT-IR和紫外可見吸收光譜(UV-Vis)表征Au-PAMAM和Au-PAMAM-CP驗(yàn)證Au-PAMAM-CP是否成功合成;(3)瓊脂糖凝膠電泳驗(yàn)證目標(biāo)DNA和CP是否雜交成功;(4)傳感器構(gòu)建過程采用循環(huán)伏安法和電化學(xué)交流阻抗法表征;(5)采用差分脈沖伏安法(DPV)對(duì)聚吡咯/鏈霉親和素的電沉積時(shí)間、CP和目標(biāo)DNA的雜交溫度和雜交時(shí)間以及電極捕獲時(shí)間進(jìn)行優(yōu)化,以提高傳感器靈敏度、特異性和準(zhǔn)確性;(6)利用傳感器檢測(cè)0.1,1,10,50和100 n M不同濃度目標(biāo)DNA,通過差分脈沖伏安法進(jìn)行表征分析獲得傳感器的檢測(cè)范圍和檢測(cè)限;(7)驗(yàn)證傳感器的特異性、重現(xiàn)性和穩(wěn)定性;(8)驗(yàn)證傳感器的基質(zhì)效應(yīng),并通過標(biāo)準(zhǔn)加樣測(cè)試其檢測(cè)性能。結(jié)果(1)表征分析顯示聚吡咯和鏈霉親和素膜合成成功;(2)Au-PAMAM-CP生物納米復(fù)合物探針合成成功;(3)目標(biāo)DNA和CP雜交成功;(4)DNA生物傳感器構(gòu)建成功;(5)優(yōu)化后的檢測(cè)條件為:聚吡咯/鏈霉親和素的電沉積時(shí)間4 min,CP和目標(biāo)DNA的雜交時(shí)間30 min,捕獲時(shí)間40 min,CP和目標(biāo)DNA的雜交溫度35℃;(6)在最佳實(shí)驗(yàn)條件下,VANGL1基因在0.1-100 n M濃度范圍內(nèi)與峰電流的變化值I呈良好的線性關(guān)系,最低檢出限為0.033 n M(S/N=3);(7)該傳感器能有效識(shí)別目標(biāo)DNA、單堿基錯(cuò)配DNA、三堿基錯(cuò)配DNA和非互補(bǔ)DNA,具有較好的特異性。對(duì)10 n M VANGL1基因平行測(cè)定5次,相對(duì)標(biāo)準(zhǔn)偏差(RSD)為3.1%,具有較好的重現(xiàn)性。傳感器在4℃下儲(chǔ)存兩周后仍保持生物活性,電流響應(yīng)顯示沒有顯著變化,具有較好的穩(wěn)定性;(8)該測(cè)定法可以忽略實(shí)際樣品檢測(cè)時(shí)基質(zhì)的影響,研究所提出的信號(hào)放大策略對(duì)真實(shí)樣品有效,人體血樣加標(biāo)回收率為103%~105%。結(jié)論本研究成功構(gòu)建的DNA生物傳感器,具有簡(jiǎn)單、成本低、靈敏度高、重現(xiàn)性好等優(yōu)點(diǎn),可用于檢測(cè)VANGL1基因SNP位點(diǎn)。該方法為神經(jīng)管畸形的基因診斷提供了新的思路。
[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，

本文編號(hào)：1629196