基于MWCNT和IL阪崎肠杆菌免疫传感器的研究

发布时间：2018-01-26 03:16

本文关键词： 免疫传感器离子液体多壁碳纳米管阪崎肠杆菌快速检测　出处：《浙江工商大学》2012年硕士论文　论文类型：学位论文

【摘要】：阪崎肠杆菌曾名为黄色阴沟肠杆菌,阪崎肠杆菌被认为是严重危害特定人群,危害生命或导致慢性实质性后遗症的致病菌,其致死率为40%-80%。电化学免疫传感器具有高特异性、简易低廉、易于携带等优点而被广泛应用于临床诊断、环境分析和食品产业等领域。应用新材料,例如多壁碳纳米管(multi-wall carbon nanotubes, MWCNTs)和离子液体(i)nic liquid,IL)制作传感器是当前生物传感器研究领域的热点。本论文主要围绕以下几个方面开展了研究工作： 1阪崎肠杆菌抗原的制备本试验成功制备了以0.4%(v/v)甲醛灭活12h的阪崎肠杆菌抗原,此抗原具有良好的生物活性和特异性,为后续研究工作的顺利开展奠定了基础。 2基于多壁碳纳米管/硫堇阪崎肠杆菌免疫传感器的研究为了研制一种灵敏度高、电化学性能稳定的免疫传感器,构建准确检测阪崎肠杆菌的电化学方法,将硫堇、辣根过氧化物酶标记的阪崎肠杆菌抗体依次自组装固定于多壁碳纳米管/十二烷基苯磺酸钠修饰的四通道丝网印刷电极上,制得一次性免疫传感器。采用原子力显微镜表征电极修饰和孵育抗原后的表而形态,用循环伏安法考察不同修饰电极的电化学特性,根据还原峰电流的变化对阪崎肠杆菌进行测定。在优化的试验条件下,该方法检测阪崎肠杆菌的线性范围为102～108CFU/mL,检测限为5.7×101CFU/mL(S/N=3)。该免疫传感器灵敏度高,具有较好的特异性、重现性(RSD=6.3%)、稳定性(4℃无菌容器中放置15d后电流响应为初始值的93.24%)和准确性(与GB/T4789.40-2010符合率为96.67%),具有一定的应用潜力。 3基于多壁碳纳米管/离子液体/硫堇的阪崎肠杆菌免疫传感器首次研发了基于多壁碳纳米管/离子液体/硫堇修饰丝网印刷电极的免疫传感器,提高了免疫传感器的贮存稳定性。用原子力显微镜表征电极各层修饰后的表面形态,用循环伏安法考察修饰电极的电化学特性,十二烷基苯磺酸钠降低了多壁碳纳米管/离子液体胶体的背景电流。包埋在离子液体中的阪崎肠杆菌酶标抗体表现出较高的活性和稳定性((4℃无菌容器中放置30d后电流响应为初始值的96.30%)。在最优条件下,该方法检测阪崎肠杆菌的线性范围为103～109CFU/mL,在磷酸盐缓冲溶液检出限为7.7×101CFU/mL(S/N=3),模拟样品检出限为8.3×102CFU/mL 4基于MWCNT/Nafion和离子液体[BMIM]PFF6的免疫传感器研究为改善基于MWCNT/Nafion生物传感器电化学信号及储存的稳定性,采用[BMIM]PF6/Nafion复合物将辣根过氧化物酶标抗体包埋固定于MWCNT/Nafion修饰的丝网印刷碳电极上,构建了一种新的免疫传感器。用原子力显微镜表征电极各层修饰后的表面形态,用循环伏安法和交流阻抗法考察修饰电极的电化学特性,利用还原峰电流变化对被检物进行测定。本试验选用阪崎肠杆菌为目标抗原,在优化的检测条件下,该方法检测阪崎肠杆菌的线性范围为103～109CFU/mL,检出限为3.4×102CFU/mL(S/N=3)。该免疫传感器的储存稳定性有明显改善,4℃放置30d后电流响应值仍为初始值的95.50%,且具有较高的灵敏度,较好的重现性(RSD6%)、特异性(非目标抗原的△Ipc0.3μA)和准确性(与GB/T4789.40-2010符合率为100%),操作简便、成本低,有很好的潜在应用价值。
[Abstract]:Sakazakii Enterobacteriaceae, once known as yellow cloaca, Enterobacter sakazakii, is considered to be a serious risk to specific populations, life-threatening or chronic sequelae, and its mortality is 40%-80%..
The electrochemical immunosensor with high specificity, simple and inexpensive, easy to carry advantages and is widely used in clinical diagnosis, environmental analysis and food industry and other fields. The application of new materials, such as multi walled carbon nanotubes (multi-wall carbon, nanotubes, MWCNTs) and ionic liquid (I) nic liquid, IL) is a hotspot in biological sensor the sensor research field. This thesis mainly focuses on the following research work has been carried out:
1 Preparation of Enterobacter sakazakii antigen
In this experiment, we successfully prepared the antigen of Enterobacter sakazakii, which was inactivated by 0.4% (v/v) formaldehyde. The antigen has good biological activity and specificity, which laid the foundation for the subsequent research work to carry out smoothly. The 12h antigen was successfully prepared.
2 based on multi wall carbon nanotube / Enterobacter osakazaki immunosensor
In order to develop a kind of high sensitivity, stable performance of the electrochemical immunosensor, electrochemical method to construct accurate detection of Enterobacter sakazakii, the thionine, Enterobacter sakazakii horseradish peroxidase labeled antibody were fixed on the self-assembled multi wall carbon nanotubes / twelve benzene sulfonic acid sodium channel four modified screen printed electrode, preparation have disposable immunosensor. By using atomic force microscopy characterization of modified electrode and incubated with antigen after surface morphology, electrochemical properties of different modified electrode by cyclic voltammetry, according to the change of the reduction peak current was measured for Enterobacter sakazakii. Under the optimum conditions, the linear range of the method for detection of Enterobacter sakazakii for 102 ~ 108CFU/mL, the detection limit is 5.7 * 101CFU/mL (S/N=3). The immune sensor has high sensitivity and has good specificity and reproducibility (RSD=6.3%), stability (4 C sterile container The current response after 15d is 93.24% of the initial value and the accuracy (the coincidence rate of GB/T4789.40-2010 is 96.67%). It has a certain application potential.
3 immunological sensors based on multi walled carbon nanotubes / ionic liquids / thio
For the first time developed immunosensor of multi walled carbon nanotube / ionic liquid / thionine modified screen printed electrode based on improved storage stability of immunosensor. The surface morphology of each layer were characterized by atomic force modified electrode, electrochemical properties of the modified electrode by cyclic voltammetry, twelve sodium reduced the background current multi wall carbon nanotube / ionic liquid gel. Enterobacter sakazakii enzyme buried in ionic liquid antibody showed higher activity and stability ((placed 4 C 30d sterile container after the current response of the initial value is 96.30%). Under optimal conditions, the linear range for the detection of Enterobacter sakazakii 103 ~ 109CFU/mL in phosphate buffer solution detection limit of 7.7 * 101CFU/mL (S/N=3), the simulated sample detection limit of 8.3 * 102CFU/mL
4 immune sensor based on MWCNT/Nafion and ionic liquid [BMIM]PFF6
In order to improve the stability of electrochemical MWCNT/Nafion biosensor based on signal and storage, using [BMIM]PF6/Nafion compound to HRP screen printed carbon electrode antibody immobilized on the MWCNT/Nafion modified on the construction of a new immune sensor. The surface morphology of the modified electrode layer was characterized by atomic force, electrochemical characteristics on modified electrode by using cyclic voltammetry and AC impedance method, the reduction peak current change of the detected objects were determined. The experiment of Enterobacter sakazakii as target antigen, detected in the optimum conditions, the linear range of the method for detection of Enterobacter sakazakii was 103 ~ 109CFU/mL, the detection limit of 3.4 * 102CFU/mL (S/N=3). Improved the storage stability of the immunosensor, 4 DEG C after placing 30d response is still the initial value of 95.50%, and has high sensitivity, good Reproducibility (RSD6%), specificity (non target antigen Ipc0.3 A (GB/T4789.40-2010) and the accuracy and the coincidence rate is 100%), simple operation, low cost, has good potential application value.

【学位授予单位】：浙江工商大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1;TP212

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