基于电化学扫描显微镜的生物传感技术的设计与研究
摘要
自从1953年Watson和Crick发现DNA的双螺旋结构以来,各学科掀起了DNA研究的热潮,随着分子生物学学科逐步建立,这个热潮直到现在仍然持续高涨。人类基因组草图的绘制完成更是标志着生命科学的发展正式进入了后基因组时代,它让我们从一个崭新的角度去认识疾病以及理解人类的起源与进化。而怎样去研究如此众多的基因在生命过程中所扮演的角色成了全球生命科学的共同课题。所以,建立一种微型化的、快速的、高灵敏度、高通量的检测技术就显得格外重要了。这些客观需求推动了DNA芯片的诞生和发展。
电化学扫描显微镜(SECM)是由Bard于1989年提出的一种具有高空间分辨度的新型扫描探针显微镜。由于其具有“化学灵敏性”,所以不仅可以表征导体和绝缘体的表面形貌,而且可以分辨基底表面的电化学活性,研究固定在基底上的能够产生电活性物质或者本身带有电荷的生物分子和细胞。SECM弥补了其他扫描技术,例如扫描隧道显微镜(STM)和原子力显微镜(AFM),不能直接提供有关电化学活性信息的不足。此外利用SECM探针和基底之间的相互作用,还可以对基底表面进行微米级的加工,并可延伸至其他方面的应用性研究。
本论文将电化学扫描显微镜微加工技术,阵列芯片制作技术以及纳米颗粒信号放大技术相结合,研制具有高灵敏度,高选择性的新型DNA生物传感器、免疫传感器以及DNA芯片阵列,成功的实现了对DNA,免疫球蛋白等目标物的测定。本论文共分为六章,具体内容如下：
第一章绪论
本章系统的介绍了电化学扫描显微镜的工作原理和特点,以及目前的应用现状。接着重点总结和归纳了SECM在生物体系检测方面的发展和研究现状。最后阐述了本论文的设计思路和研究意义。
第二章电化学扫描显微镜生物传感技术平台的构建
本章以SECM的探针作为工具,在组装有十二硫醇的金基底上将通过金巯键修饰的十二硫醇局部蚀刻,构建出微米级的生物传感器平台。烷基硫醇可以在金基底的表面形成一层有序的疏水的单分子层,由于正十二硫醇有足够长的碳链,所以几乎在金的表面覆盖上了一层绝缘层。本实验通过在基底和探针之间施加特定的负电位脉冲,使得正对着探针的金基底表面上修饰的正十二硫醇的金巯键断裂,从而完成局部蚀刻。我们通过调节脉冲电位、脉冲圈数以及探针和基底之间的距离可以控制蚀刻范围的大小。为了考察蚀刻的稳定性,我们蚀刻出了E,C,N,U字母的图案。
第三章利用电化学扫描显微镜反馈模式检测金基底上的DNA片段
本章在己构建的生物传感器平台上组装上修饰有巯基的单链DNA,并且通过反馈模式检测出了组装的单链DNA,确定了我们构建的生物传感器平台用来构建DNA传感器的可行性。在反馈模式中,DNA的检测通过带有不同电荷的氧化还原电解质溶液与带负电的DNA磷酸骨架之间的库仑力的作用来实现检测的。带正电的氧化还原电解质溶液能够穿过DNA单分子膜在金基底表面实现正反馈,使得逼近曲线的电流增加。带负电的氧化还原电解质溶液则受到带负电的DNA单分子膜的排斥,不能到达金基底表面实现循环,使得逼近曲线的电流降低,得到负反馈。
第四章基于HRP/SiO2纳米颗粒信号放大的电化学扫描显微镜DNA生物传感技术
本章在已构建的生物传感器平台上组装了一个“三明治结构”的DNA传感器。三明治结构的DNA传感器是由固定在金基底上的修饰有巯基的固定探针、修饰有生物素(biotin)的指示探针以及能和这两种探针杂交的目标DNA组成。链酶亲和素(streptavidin)和辣根过氧化酶(HRP)包裹的二氧化硅纳米颗粒作为信号指示元件通过生物素和亲和素的相互作用链接到DNA三明治结构上。在双氧水存在的条件下,溶液中的对苯二酚被固定在DNA传感器上的HRP氧化成苯醌,而产生的苯醌可以在施加有负电位的SECM探针上还原,从而实现DNA的检测。这个检测体系有着良好的灵敏度和选择性,检测限达到0.8pM。
第五章电化学扫描显微镜DNA芯片阵列生物传感技术
本章以已经构建的DNA三明治结构传感器为基础,构建了DNA芯片阵列,实现了高通量的多种DNA序列的同时检测,检测效果良好。DNA芯片阵列是由纳米机器人点样技术将修饰有氨基的DNA固定探针通过醛胺反应固定到烷基化的玻璃基底表面。本实验在大范围的探针扫描过程中,在电解质溶液中加入一定量的表面活性剂,防止了探针表面的钝化,稳定了基底电流,得到了很好的检测效果。我们分别对DNA阵列芯片的选择性,重复性以及灵敏度进行了评估,其检测效果良好,目标DNA的浓度检测范围为10-7-10-12M。这项工作有希望应用于基因组测序。
第六章电化学扫描显微镜免疫传感技术
本章在玻璃基底表面上直接滴涂固定羊抗人IgG抗体,并且与人IgG以及标记有兔抗人IgG抗体和HRP的二氧化硅纳米颗粒之间发生特异性免疫反应,形成三明治结构,成功建立了用于检测人IgG的SECM免疫技术。在此SECM免疫传感器中,SECM探针的检测模式依然是产生/收集模式。在双氧水的存在下,对苯二酚在修饰有HRP的传感器表面被氧化成苯醌,产生的苯醌则被施加有负电位的探针还原检测。本实验实现了人IgG的定量检测,检测限达到10pg/L。效果令人满意。
第七章总结与展望
本章总结了此毕业论文的主要内容和取得的成果和意义,并对SECM电化学生物传感器今后的发展做出了展望。
Since the discovery of DNA double helix structure by Watson and Crick in1953, a hot wave of DNA research raised between each subject. As molecular biology subject gradually been established, the boom continue rising until now. Plotted of the draft of human genome marked the development of life science entered the post genome era, which make us recognize and understand the origin and evolution of human disease from a new angle.And how to study so many genes'role in the process of life became a global joint project of the life science. So, to establish a kind of miniaturization, rapid, high sensitivity, high throughput detection technology is especially important. These demands promoted the birth and development of DNA chips.
In this paper, we combined the electrochemical scanning microscope micro processing technology, the array chip production techniques and nanoparticles signal amplification technology, and developed the new DNA micro biosensor, immune sensor and DNA chip with high sensitivity and high selectivity. This thesis was divided into six chapters, and the concrete content is as follows:
Scanning electrochemical microscopy (SECM), which presented by the Bard in1989, is a new type of scanning probe microscope with high spatial resolution.Since it has "chemical sensitivity", SECM can not only characterize the surface topography of conductor and insulator, but also distinguish electrochemical activity on the substrate surface. It also could research the biological molecules and cells, which generated electroactive substances or were charged themselves. It is different from other scanning techniques, such as scanning tunneling microscope (STM) and atomic force microscope (AFM), SECM could directly provide information of electrochemical activity. Moreover micro surface processing could be achieved using the interaction between SECM probe and substrate, and this can extend to other aspects of applied research.
Chapter1Introduction
In this chapter, we systematically introduced the work principle and characteristics of scanning electrochemical microscope (SECM), and observed the current application of SECM. Then we emphatically summarized the SECM using in DNA detection and immune detection. Finally we expounded the design thinking and research significance of this paper.
Chapter2Scanning electrochemical microscope building micro biosensor platform
In this chapter, we successfully built a micro biosensor platform on the1-dodecanethiolmodified gold substrate surface using SECM probe as a tool.1-dodecanethiol can be modified on the gold substrate and orderly formed a hydrophobic monolayer. As there is sufficiently long carbon chain in this monolayer, the modified substrate is insulating. In this experiment, we applied a negative potential pulse between the SECM probe and modified substrate, broken the SH bond of1-dodecanethiol monolayer beneath the SECM probe, thus complete the etching. By adjusting the pulse voltage, pulse cycle and the distance between the probe and substrate, we can control the size of the range of etching. In order to investigate the stability of etching, E, C, N, U pattern of letters were etched out.
Chapter3Detection of DNA sequences modified on the gold substrate using SECM feedback mode
In this chapter, thiol-sequenced DNA was assembled on the prepared micro biosensor platform and detected by SECM using feedback mode, which confirmed the feasibility of construct DNA sensors in this mode. In feedback mode, the detection of DNA was achieved by the interaction between the different charged mediator and negative charged DNA phosphate backbone. Positive charged mediator could pass though the DNA molecule and recycled at the gold substrate, thus the current of the approach curve was increased. While the electrostatic repulsion between negative charged mediator and DNA phosphate backbone prevent the mediator diffusing to the gold substrate, and the current of the approach curve was decreased.
Chapter4Qualitative and quantitative detection of DNA amplified withHRP-modified SiO2nanoparticles using scanningelectrochemical microscopy
In this chapter, a"sandwich" DNA structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture probe), assembled on the prepared micro biosensor platform, with target DNA and biotinylated indicator probe. HRP (horseradish peroxidase)-wrapped SiO2nanoparticles were linked to the sandwich structure through biotin-streptavidin interaction. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface where sequence-specific hybridization had occurred through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. The detection limit of complementary DNAwas as low as0.8pM. This technique is promising for the application on electrochemical DNA chip.
Chapter5Scanning Electrochemical Microscopy of DNA Hybridization on DNA Microarrays Enhanced by HRP-Modified SiO2Nanoparticles
In this chapter, based on the sandwich structured DNA sensor, we reported a DNA microarray method. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. DNA arrays were fabricated by robotic printing of capture DNA probes onto a glass substrate. The capture probe (CP) sequences were amine-tethered oligodeoxynucleotides that could be immobilized on the aldehyde-covered glass substrate through the amine-aldehyde interaction. Passivation of the tip during large scale scanning was avoided by adding surfactant to the solution, which keeps the tip clean and the background of the tip current was stable during scanning. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10-7-10-12M). This technique may find applications in genomic sequencing.
Chapter6Detection of Human IgG using GC mode of SECM
In this chapter, sheep-anti-human IgG was directly dropped on the glass substrate, then reacted with human IgG and Indicating element, which formed by rabbit-anti-human IgG-HRP wrapped SiO2nanoparticles, forming a sandwich-typed immunocomplex. In this SECM immune sensor, detecting mode is still generation/collection. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. The detection limit of human IgG was as low as10pg/L.
电化学扫描显微镜(SECM)是由Bard于1989年提出的一种具有高空间分辨度的新型扫描探针显微镜。由于其具有“化学灵敏性”,所以不仅可以表征导体和绝缘体的表面形貌,而且可以分辨基底表面的电化学活性,研究固定在基底上的能够产生电活性物质或者本身带有电荷的生物分子和细胞。SECM弥补了其他扫描技术,例如扫描隧道显微镜(STM)和原子力显微镜(AFM),不能直接提供有关电化学活性信息的不足。此外利用SECM探针和基底之间的相互作用,还可以对基底表面进行微米级的加工,并可延伸至其他方面的应用性研究。
本论文将电化学扫描显微镜微加工技术,阵列芯片制作技术以及纳米颗粒信号放大技术相结合,研制具有高灵敏度,高选择性的新型DNA生物传感器、免疫传感器以及DNA芯片阵列,成功的实现了对DNA,免疫球蛋白等目标物的测定。本论文共分为六章,具体内容如下：
第一章绪论
本章系统的介绍了电化学扫描显微镜的工作原理和特点,以及目前的应用现状。接着重点总结和归纳了SECM在生物体系检测方面的发展和研究现状。最后阐述了本论文的设计思路和研究意义。
第二章电化学扫描显微镜生物传感技术平台的构建
本章以SECM的探针作为工具,在组装有十二硫醇的金基底上将通过金巯键修饰的十二硫醇局部蚀刻,构建出微米级的生物传感器平台。烷基硫醇可以在金基底的表面形成一层有序的疏水的单分子层,由于正十二硫醇有足够长的碳链,所以几乎在金的表面覆盖上了一层绝缘层。本实验通过在基底和探针之间施加特定的负电位脉冲,使得正对着探针的金基底表面上修饰的正十二硫醇的金巯键断裂,从而完成局部蚀刻。我们通过调节脉冲电位、脉冲圈数以及探针和基底之间的距离可以控制蚀刻范围的大小。为了考察蚀刻的稳定性,我们蚀刻出了E,C,N,U字母的图案。
第三章利用电化学扫描显微镜反馈模式检测金基底上的DNA片段
本章在己构建的生物传感器平台上组装上修饰有巯基的单链DNA,并且通过反馈模式检测出了组装的单链DNA,确定了我们构建的生物传感器平台用来构建DNA传感器的可行性。在反馈模式中,DNA的检测通过带有不同电荷的氧化还原电解质溶液与带负电的DNA磷酸骨架之间的库仑力的作用来实现检测的。带正电的氧化还原电解质溶液能够穿过DNA单分子膜在金基底表面实现正反馈,使得逼近曲线的电流增加。带负电的氧化还原电解质溶液则受到带负电的DNA单分子膜的排斥,不能到达金基底表面实现循环,使得逼近曲线的电流降低,得到负反馈。
第四章基于HRP/SiO2纳米颗粒信号放大的电化学扫描显微镜DNA生物传感技术
本章在已构建的生物传感器平台上组装了一个“三明治结构”的DNA传感器。三明治结构的DNA传感器是由固定在金基底上的修饰有巯基的固定探针、修饰有生物素(biotin)的指示探针以及能和这两种探针杂交的目标DNA组成。链酶亲和素(streptavidin)和辣根过氧化酶(HRP)包裹的二氧化硅纳米颗粒作为信号指示元件通过生物素和亲和素的相互作用链接到DNA三明治结构上。在双氧水存在的条件下,溶液中的对苯二酚被固定在DNA传感器上的HRP氧化成苯醌,而产生的苯醌可以在施加有负电位的SECM探针上还原,从而实现DNA的检测。这个检测体系有着良好的灵敏度和选择性,检测限达到0.8pM。
第五章电化学扫描显微镜DNA芯片阵列生物传感技术
本章以已经构建的DNA三明治结构传感器为基础,构建了DNA芯片阵列,实现了高通量的多种DNA序列的同时检测,检测效果良好。DNA芯片阵列是由纳米机器人点样技术将修饰有氨基的DNA固定探针通过醛胺反应固定到烷基化的玻璃基底表面。本实验在大范围的探针扫描过程中,在电解质溶液中加入一定量的表面活性剂,防止了探针表面的钝化,稳定了基底电流,得到了很好的检测效果。我们分别对DNA阵列芯片的选择性,重复性以及灵敏度进行了评估,其检测效果良好,目标DNA的浓度检测范围为10-7-10-12M。这项工作有希望应用于基因组测序。
第六章电化学扫描显微镜免疫传感技术
本章在玻璃基底表面上直接滴涂固定羊抗人IgG抗体,并且与人IgG以及标记有兔抗人IgG抗体和HRP的二氧化硅纳米颗粒之间发生特异性免疫反应,形成三明治结构,成功建立了用于检测人IgG的SECM免疫技术。在此SECM免疫传感器中,SECM探针的检测模式依然是产生/收集模式。在双氧水的存在下,对苯二酚在修饰有HRP的传感器表面被氧化成苯醌,产生的苯醌则被施加有负电位的探针还原检测。本实验实现了人IgG的定量检测,检测限达到10pg/L。效果令人满意。
第七章总结与展望
本章总结了此毕业论文的主要内容和取得的成果和意义,并对SECM电化学生物传感器今后的发展做出了展望。
Since the discovery of DNA double helix structure by Watson and Crick in1953, a hot wave of DNA research raised between each subject. As molecular biology subject gradually been established, the boom continue rising until now. Plotted of the draft of human genome marked the development of life science entered the post genome era, which make us recognize and understand the origin and evolution of human disease from a new angle.And how to study so many genes'role in the process of life became a global joint project of the life science. So, to establish a kind of miniaturization, rapid, high sensitivity, high throughput detection technology is especially important. These demands promoted the birth and development of DNA chips.
In this paper, we combined the electrochemical scanning microscope micro processing technology, the array chip production techniques and nanoparticles signal amplification technology, and developed the new DNA micro biosensor, immune sensor and DNA chip with high sensitivity and high selectivity. This thesis was divided into six chapters, and the concrete content is as follows:
Scanning electrochemical microscopy (SECM), which presented by the Bard in1989, is a new type of scanning probe microscope with high spatial resolution.Since it has "chemical sensitivity", SECM can not only characterize the surface topography of conductor and insulator, but also distinguish electrochemical activity on the substrate surface. It also could research the biological molecules and cells, which generated electroactive substances or were charged themselves. It is different from other scanning techniques, such as scanning tunneling microscope (STM) and atomic force microscope (AFM), SECM could directly provide information of electrochemical activity. Moreover micro surface processing could be achieved using the interaction between SECM probe and substrate, and this can extend to other aspects of applied research.
Chapter1Introduction
In this chapter, we systematically introduced the work principle and characteristics of scanning electrochemical microscope (SECM), and observed the current application of SECM. Then we emphatically summarized the SECM using in DNA detection and immune detection. Finally we expounded the design thinking and research significance of this paper.
Chapter2Scanning electrochemical microscope building micro biosensor platform
In this chapter, we successfully built a micro biosensor platform on the1-dodecanethiolmodified gold substrate surface using SECM probe as a tool.1-dodecanethiol can be modified on the gold substrate and orderly formed a hydrophobic monolayer. As there is sufficiently long carbon chain in this monolayer, the modified substrate is insulating. In this experiment, we applied a negative potential pulse between the SECM probe and modified substrate, broken the SH bond of1-dodecanethiol monolayer beneath the SECM probe, thus complete the etching. By adjusting the pulse voltage, pulse cycle and the distance between the probe and substrate, we can control the size of the range of etching. In order to investigate the stability of etching, E, C, N, U pattern of letters were etched out.
Chapter3Detection of DNA sequences modified on the gold substrate using SECM feedback mode
In this chapter, thiol-sequenced DNA was assembled on the prepared micro biosensor platform and detected by SECM using feedback mode, which confirmed the feasibility of construct DNA sensors in this mode. In feedback mode, the detection of DNA was achieved by the interaction between the different charged mediator and negative charged DNA phosphate backbone. Positive charged mediator could pass though the DNA molecule and recycled at the gold substrate, thus the current of the approach curve was increased. While the electrostatic repulsion between negative charged mediator and DNA phosphate backbone prevent the mediator diffusing to the gold substrate, and the current of the approach curve was decreased.
Chapter4Qualitative and quantitative detection of DNA amplified withHRP-modified SiO2nanoparticles using scanningelectrochemical microscopy
In this chapter, a"sandwich" DNA structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture probe), assembled on the prepared micro biosensor platform, with target DNA and biotinylated indicator probe. HRP (horseradish peroxidase)-wrapped SiO2nanoparticles were linked to the sandwich structure through biotin-streptavidin interaction. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface where sequence-specific hybridization had occurred through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. The detection limit of complementary DNAwas as low as0.8pM. This technique is promising for the application on electrochemical DNA chip.
Chapter5Scanning Electrochemical Microscopy of DNA Hybridization on DNA Microarrays Enhanced by HRP-Modified SiO2Nanoparticles
In this chapter, based on the sandwich structured DNA sensor, we reported a DNA microarray method. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. DNA arrays were fabricated by robotic printing of capture DNA probes onto a glass substrate. The capture probe (CP) sequences were amine-tethered oligodeoxynucleotides that could be immobilized on the aldehyde-covered glass substrate through the amine-aldehyde interaction. Passivation of the tip during large scale scanning was avoided by adding surfactant to the solution, which keeps the tip clean and the background of the tip current was stable during scanning. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10-7-10-12M). This technique may find applications in genomic sequencing.
Chapter6Detection of Human IgG using GC mode of SECM
In this chapter, sheep-anti-human IgG was directly dropped on the glass substrate, then reacted with human IgG and Indicating element, which formed by rabbit-anti-human IgG-HRP wrapped SiO2nanoparticles, forming a sandwich-typed immunocomplex. In this SECM immune sensor, detecting mode is still generation/collection. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. The detection limit of human IgG was as low as10pg/L.
引文
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