电化学DNA生物传感器的制备
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摘要
DNA电化学生物传感器是一门全新的DNA(基因)检测技术,它具有快速、灵敏、操作方便、无污染等特点,并具有分子识别功能,在分子生物学和生物工程医学等领域有巨大的应用前景,在临床基因诊断、抗癌药物的筛选等方面也起着越来越重要的作用。
本文利用自组装膜法,将人工合成的鼠疫单链DNA作为检测目标,将与其互补的单链DNA片段固定在金电极表面,制成分子探针,结合电化学活性物质醛基二茂铁构成夹心面包式DNA电化学传感器。在探针合成过程中,探讨了自组装膜的成膜条件,以及DNA固定和杂交机理,并考察了探针的电子传递性。利用合成的探针对溶液中互补DNA进行定性和定量分析。在对标准互补液的杂交检测过程中,探讨了杂交时间、杂交温度、信号DNA的合成条件等对DNA电化学传感器的影响。利用DNA电化学生物传感器检测标准DNA样品,测定传感器的检测范围、杂交特异性和寿命等基本性能。
本课题所研制的以亚甲基兰(MB)为电化学活性指示剂的嵌入式DNA生物传感器。其中寡聚DNA探针通过-SH与Au的反应而连到金电极表面,用线性扫描伏安法检测电极表面DNA是否组装完成。用与探针DNA完全互补的ssDNA溶液与探针进行杂交反应。杂交后可看到MB的峰电流减小,当目标DNA的浓度在5.0×10~(-10)到1.8×10~(-9)mol/L时,MB的峰电流与目标DNA浓度呈线性关系。检出限是5.0×10~(-10)mol/L。
纳米金自组装于金电极表面可以增强DNA在电极表面的固着量。用恒电位吸附的方式将探针DNA修饰到金电极上,并通过Co(phen)_3~(2+)表征修饰电极的电化学性能。比较了在纳米金修饰金电极上吸附的DNA电信号与在棵金电极上吸附DNA的电信号,前者有明显的增大,并考察了DNA修饰金电极的稳定性。探讨了ssDNA与dsDNA的吸附特性,同时,比较了DNA在不同电极基体上的吸附行为特性。
未来生物传感器在医学领域中将有很大的发展潜力,随着计算机技术、微制造技术和生物材料学的不断发展,生物传感器技术在医学领域的应用将越来越广泛,它将取代医学上一些传统的检测、化验方法,成为广泛普及的常规分析检测仪器。
Electrochemical DNA biosensor is a novel electronic technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being: simple, reliable, cheap, sensitive and selective for gene detection. And it can be compatible with DNA biochip. It has a broad prospect of application in the fields of clinic examination of inherited diseases and screening drug.The DNA electrochemical biosensor was prepared by self-assembling the designed ratbite fever DNA specific probe on the surface of Au electrode together with FCA(Ferrocenecarboxaldehyde) as electro-active indicator.In this paper, the condition of self-assembling、 immobility of DNA and hybridization mechanism were studied. The electron-transfer capacity of the DNA modified probe was also observed. The prepared DNA electrochemical sensor was used to determine the struture and quality of complementary DNA. While the hybridization of DNA probe with complementary DNA was detected, many affective factors such as: hybridization time、 hybridization temperature、 washing time and the synthetic condition of signal DNA were determined, and their affection to the final detection result was discussed. Some fundamental properties such as specificity、 life span、 detection scale were also tested.This supplied some reference for the pre-diagnose of diseases.A novel electrochemical DNA biosensor based on methylene blue (MB) as redox indicator for DNA hybridization detection was presented. Oligo nucleotide probes with -SH at the 5 ' of DNA end were attached onto the Au surface. Hybridization was induced by exposure of Au electrode to complementary ssDNA in solution. The decreases in the peak currents of MB, an electroactive label, were observed upon hybridization of probe with the target. The cathodic peak current (Δ Ip) of MB after hybridization with the target DNA was linearly related to the value of the target DNA concentration ranging from 2.25×10~(-10) to 2.25×10~(-9)mol/L. A detection limit of this method was 1.0×10~(-10) mol/L.Colloidal Au were used to enhance immobilization amount of DNA on a gold electrode. The DNA modified electrode were electrochemically characterirzed with Co(phen)_3~(2+), a electroactive DNA-binding complex, as an indicator.An increased absorb capacity for nucleic acid incurred on the electrode is due to self-assembly of
colloidal Au onto L-cysteine modified on gold electrode. The stability of the DNA-modified electrode was investigated. The difference between several materials was also discussed.Tomorrow biosensor will have more potential in medicine. With the development of computer technology, microfabrication technology, and biological material, biosensor will be widely used in medicine. The application of biosensor will replace the traditional methods of detection and testing and became widespread device of routine analysis and measuring.
本文利用自组装膜法,将人工合成的鼠疫单链DNA作为检测目标,将与其互补的单链DNA片段固定在金电极表面,制成分子探针,结合电化学活性物质醛基二茂铁构成夹心面包式DNA电化学传感器。在探针合成过程中,探讨了自组装膜的成膜条件,以及DNA固定和杂交机理,并考察了探针的电子传递性。利用合成的探针对溶液中互补DNA进行定性和定量分析。在对标准互补液的杂交检测过程中,探讨了杂交时间、杂交温度、信号DNA的合成条件等对DNA电化学传感器的影响。利用DNA电化学生物传感器检测标准DNA样品,测定传感器的检测范围、杂交特异性和寿命等基本性能。
本课题所研制的以亚甲基兰(MB)为电化学活性指示剂的嵌入式DNA生物传感器。其中寡聚DNA探针通过-SH与Au的反应而连到金电极表面,用线性扫描伏安法检测电极表面DNA是否组装完成。用与探针DNA完全互补的ssDNA溶液与探针进行杂交反应。杂交后可看到MB的峰电流减小,当目标DNA的浓度在5.0×10~(-10)到1.8×10~(-9)mol/L时,MB的峰电流与目标DNA浓度呈线性关系。检出限是5.0×10~(-10)mol/L。
纳米金自组装于金电极表面可以增强DNA在电极表面的固着量。用恒电位吸附的方式将探针DNA修饰到金电极上,并通过Co(phen)_3~(2+)表征修饰电极的电化学性能。比较了在纳米金修饰金电极上吸附的DNA电信号与在棵金电极上吸附DNA的电信号,前者有明显的增大,并考察了DNA修饰金电极的稳定性。探讨了ssDNA与dsDNA的吸附特性,同时,比较了DNA在不同电极基体上的吸附行为特性。
未来生物传感器在医学领域中将有很大的发展潜力,随着计算机技术、微制造技术和生物材料学的不断发展,生物传感器技术在医学领域的应用将越来越广泛,它将取代医学上一些传统的检测、化验方法,成为广泛普及的常规分析检测仪器。
Electrochemical DNA biosensor is a novel electronic technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being: simple, reliable, cheap, sensitive and selective for gene detection. And it can be compatible with DNA biochip. It has a broad prospect of application in the fields of clinic examination of inherited diseases and screening drug.The DNA electrochemical biosensor was prepared by self-assembling the designed ratbite fever DNA specific probe on the surface of Au electrode together with FCA(Ferrocenecarboxaldehyde) as electro-active indicator.In this paper, the condition of self-assembling、 immobility of DNA and hybridization mechanism were studied. The electron-transfer capacity of the DNA modified probe was also observed. The prepared DNA electrochemical sensor was used to determine the struture and quality of complementary DNA. While the hybridization of DNA probe with complementary DNA was detected, many affective factors such as: hybridization time、 hybridization temperature、 washing time and the synthetic condition of signal DNA were determined, and their affection to the final detection result was discussed. Some fundamental properties such as specificity、 life span、 detection scale were also tested.This supplied some reference for the pre-diagnose of diseases.A novel electrochemical DNA biosensor based on methylene blue (MB) as redox indicator for DNA hybridization detection was presented. Oligo nucleotide probes with -SH at the 5 ' of DNA end were attached onto the Au surface. Hybridization was induced by exposure of Au electrode to complementary ssDNA in solution. The decreases in the peak currents of MB, an electroactive label, were observed upon hybridization of probe with the target. The cathodic peak current (Δ Ip) of MB after hybridization with the target DNA was linearly related to the value of the target DNA concentration ranging from 2.25×10~(-10) to 2.25×10~(-9)mol/L. A detection limit of this method was 1.0×10~(-10) mol/L.Colloidal Au were used to enhance immobilization amount of DNA on a gold electrode. The DNA modified electrode were electrochemically characterirzed with Co(phen)_3~(2+), a electroactive DNA-binding complex, as an indicator.An increased absorb capacity for nucleic acid incurred on the electrode is due to self-assembly of
colloidal Au onto L-cysteine modified on gold electrode. The stability of the DNA-modified electrode was investigated. The difference between several materials was also discussed.Tomorrow biosensor will have more potential in medicine. With the development of computer technology, microfabrication technology, and biological material, biosensor will be widely used in medicine. The application of biosensor will replace the traditional methods of detection and testing and became widespread device of routine analysis and measuring.
引文
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[2] Joshua T Jones, Jason W Myers, James E Ferrell, Jr et al. Probing the precision of the mitotic clock with a live-cell fluorescent biosensor. Nature biotechnology. 2004, 22(3): 306-312
[3] S. Rauf. J. J, Gooding, K. Akhtar et al. Electrochemical approach of anticancer drugs-DNA interaction. Journal of Pharmaceutical and Biomedical Analysis. 2005, 37: 205-217
[4] Xi-Liang Luo, Jing-Juan Xu, Qing Zhang et al. Electrochemically deposited chitosan hydrogel for horseradish peroxidase immobilization through gold self-assembly. Biosensors and Bioelectronics. 2005, 21: 190-196
[5] Kagan Kerman, Masaaki Kobayashi, Eiichi Tamiya. Recent trends in electrochemical DNA biosensor technology. Measurement science and technology. 2004, 15: R1-R11
[6] Junhoe Cha, Jung Im Han, Young Choi et al. DNA hybridization electrochemical sensor using conducting polymer. Biosensors and Bioelectronics. 2003, 18: 1241-1247
[7] Ana-Maria Chiorcea, Ana Maria Oliveira Brett. Atomic force microscopy characterization of an electrochemicalDNA-biosensor. Bioelectrochemistry. 2004, 63: 229-232
[8] J. J. PANCRAZIO, J. P. WHELAN, D. A. BORKHOLDER. Development and Application of Cell-Based Biosensors. Annals of Biomedical Engineering, 1999, 27: 697-711
[9] Wenonah Vercoutere, Mark Akeson. Biosensors for DNA sequence detection. Current Opinion in Chemical Biology. 2002, 6: 816-822
[10] Fernando Patolsky, Amir Lichtenstein, Itamar Willner. Detection of single-base DNA mutations by enzyme-amplified electronic transduction, nature biotechnology. 2001, 19: 253-258
[11] Ana Maria Oliveira Brett, Luis Anto^nio da Silva, Hideki Fujii et al. Detection of the damage caused to DNA by a thiophene-S- oxide using an electrochemical DNA-biosensor. Journal of Electroanalytical Chemistry . 2003,549: 91-99
[12] F. C. Abreu, M. 0. F. Goulart, A.M. Oliveira Brett. Detection of the damage caused to DNA by niclosamide using an electrochemical DNA-biosensor. Biosensors and Bioelectronics. 2002,17:913-919
[13]Hafsa Korri-Youssoufi , Bouchra Makrouf. Electrochemical biosensing of DNA hybridization by ferrocenyl groups functionalized polypyrrole. Analytica Chimica Acta. 2002, 469:85-92
[14] Hong Cai, Yanqing Wang, Pingang He et al. Electrochemical detection of DNA hybridization based on silver-enhanced gold nanoparticle label. Analytica Chimica Acta .2002,469:165-172
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