凝血酶和可卡因电化学适体传感器的研究
摘要
现代生命科学、环境科学、材料科学的迅速发展,为分析化学的发展带来了新的机遇和挑战。随着人类基因组计划的顺利完成,从基因水平去认识疾病,认识基因对人类生老病死的控制过程等取得了很大进展。核酸和蛋白质是组成生命的主要生物大分子,核酸具有传递遗传信息等功能,蛋白质则贯穿所有的生命活动过程。核酸与蛋白质之间相互作用的信息的获得,将有助于对一些生命过程的研究,进而掌握生命活动和信息传递规律并实施调节及调控。因此,蛋白质、核酸和药物的检测对人类疾病的预防、诊断和治疗具有重要的现实意义。生物传感器是简单、快速和廉价免疫检测和核酸检测的重要工具。在生物传感器生物分子识别元件中,抗体由于其制备比较繁琐、固定化易失活、保存时间有限等,因此,亟需探索新型高特异高稳定的分子识别物质,建立高选择性高灵敏的生物传感技术。利用体外筛选技术获得的适体,由于其独特的优点,成为新一代生物识别分子,适体传感器的研究也受到了人们的极大关注。电化学生物传感器具有灵敏度高、响应快、操作简便、价格低廉等优点。因此,电化学适体传感器的研究成为继免疫电化学传感器和DNA电化学传感器后新的研究热点。
纳米技术的出现为纳米材料在分析化学领域的发展和应用开辟了新的方向。纳米材料的优异性能例如比表面积大、反应活性高等为分析化学开辟了新的研究途径,纳米粒子独特的性能为生物检测奠定了基础。将纳米粒子和高特异性的适体,高灵敏的电化学检测技术相结合,提高了电化学检测的灵敏度,扩大了纳米材料应用范围,为生物分析化学开辟了新的领域。
本论文的研究目的是将纳米材料与高特异性的分子识别物质适体,高灵敏的电化学检测技术相结合,建立测定蛋白质、小分子药物的新方法和电化学适体传感器。发展具有高灵敏高选择性的电化学适体传感器,开拓生物传感器的研究和应用范围,为蛋白与小分子药物的疾病诊断和临床治疗提供具有潜在应用价值的分析器件。主要通过两个途径:(1)基于纳米粒子为电极修饰物、信号标记物或信号物质载体,放大了检测信号,提高了检测灵敏度;(2)利用电化学阻抗技术,建立非标记型凝血酶蛋白质和小分子药物可卡因检测的新方法。本论文分为两部分:第一部分为综述部分,第二部分为研究报告部分。
第一章为引言。引言部分首先简要地介绍了适体的体外筛选方法、适体的特点、适体与配体的相互作用和适体的化学修饰;总结了适体传感器和电化学适体传感器的分类,适体的固定化方法以及相关的电化学分析技术;重点评述了标记型和非标记型电化学适体传感器的研究进展,对电化学适体传感器的发展进行了展望。最后阐述了本论文的选题背景、研究意义、研究目的和研究内容。
第二章为标记型凝血酶电化学适体传感器的研究。基于碳纳米管的多负载作用,设计了以碳纳米管负载纳米银标记适体,夹心法检测凝血酶的电化学方法。以凝血酶为蛋白质的研究模型,利用碳纳米管负载纳米银标记适体Ⅲ以及固定在金电极上的适体Ⅰ与凝血酶结合形成金电极/适体Ⅰ/凝血酶/碳纳米管负载纳米银标记适体Ⅲ的三明治夹心式结构,通过检测碳纳米管负载纳米银上银的电化学信号,对凝血酶进行定量测定。氧化电流与凝血酶浓度在0.012～10nmol/L呈良好的线性关系,检出限可以达到0.005 nmol/L。该方法对凝血酶具有高的特异性识别能力,其检测不受其他蛋白质的干扰。利用碳纳米管表面负载多个纳米银粒子,对电化学信号起到了放大作用,提高了检测凝血酶的灵敏度。使用脉冲伏安法直接检测,无需对纳米粒子进行酸化和溶出,检出电位低,可能克服血液中还原性物质的干扰。该传感器有望用于实际样品中凝血酶的检测。
第三章为非标记型凝血酶电化学适体传感器的研究。基于适体与凝血酶结合前后电子传递电阻的变化,构建了非标记适体检测凝血酶的交流阻抗电化学传感器。利用电化学沉积方法制备金纳米粒子修饰玻碳电极,将末端带巯基的适体通过分子自组装作用固定在金纳米粒子修饰玻碳电极上。通过扫描电镜、X-射线能谱和原子力显微镜等技术对沉积在电极上的金纳米粒子进行了形貌的表征。研究了以三种不同序列适体为分子识别物质,测定凝血酶的线性范围和检出限,比较了三者灵敏度的差异。考察和讨论了不同序列适体与凝血酶的动力学行为,利用动力学法和浓度平衡法计算了适体与凝血酶形成的结合常数及适体与凝血酶复合物的解离常数。对两种方法获得的适体与凝血酶复合物的解离常数进行了比较和讨论。本方法简单,无需对适体进行标记,金纳米粒子的多负载作用,提高了凝血酶检测的灵敏度。本研究对适体-目标蛋白质之间结合过程的机理有一个更深的理解,将有助于我们进一步揭示核酸与蛋白这两种生命中最关键物质之间的相互作用和关系,对于我们更好地理解基本的生物过程和预测设计适体生物检测方法,发展用于疾病诊断的方法有着重要的意义。
第四章非标记型可卡因电化学适体传感器的研究。基于荷正电的六氨合钌离子与DNA骨架的静电结合作用,构建了检测可卡因的非标记型电化学适体传感器。将末端带巯基的可卡因适体与部分互补的单链DNA杂交,杂交溶液通过自组装作用固定在金电极上。未加入可卡因时,部分互补双链与六氨合钌离子静电结合,导纳值较大;加入可卡因后,可卡因适体与可卡因结合,构象发生变化,部分互补单链DNA解旋脱离电极表面,适体上静电结合的六氨合钌离子数量降低,导纳值减小。通过检测六氨合钌离子导纳的抑制值对可卡因进行定量测定。导纳虚部的峰值与可卡因的浓度在2～100μmol/L之间呈良好的线性关系,检出限为0.6μmol/L。对30.0μmol/L可卡因进行7次平行测定,相对标准偏差为4.0%。
第五章标记型可卡因电化学适体传感器的研究。基于金纳米粒子多负载作用,构建了一种新型的检测可卡因的电化学适体传感器。首先将金纳米粒子通过双巯基自组装作用固定在金电极上。利用自组装作用将末端带巯基的二茂铁标记可卡因适体固定到金纳米粒子修饰金电极上。研究发现,氧化电流与可卡因的浓度在1.0～15.0μmol/L范围呈良好的线性关系,检出限为0.5μmol/L。对15.0μmol/L可卡因进行7次平行测定,相对标准偏差为3.5%。与裸金电极相比较,金纳米粒子修饰金电极可以提高适体的负载量,使测定可卡因的灵敏度提高了10倍。研究了氧化还原过程中的二茂铁的表观异相电子转移速率常数k_s,金纳米粒子修饰金电极上二茂铁的表观异相电子转移速率常数k_s大于裸金电极上二茂铁的表观异相电子转移速率常数k_s。金纳米修饰电极可以加速氧化还原反应的电子转移速度。本方法可望用于其他小分子药物的检测。
本论文以适体为分子识别物质,以凝血酶和可卡因作为研究模型,以纳米粒子为电极修饰材料、信号物质载体或信号物质,设计了两种高灵敏的标记型电化学适体传感器;利用电化学交流阻抗技术,构建了两种非标记型电化学适体传感器。本论文所研制的电化学适体传感器,为蛋白质和小分子药物的灵敏、简单、快速检测提供了良好的分析器件和分析新方法。电化学适体传感器的研究将不仅为电化学适体传感器的设计提供新概念和思路,而且还将为临床疾病诊断、基因检测和药物筛选提供具有潜在应用价值的分析器件,对生命科学和生物传感器的发展具有重要意义。
Determination of protein has been a topic of significant interest with the accomplishment of Human Genome Project and the rapid progress of proteomic strategies.Intense research activities are carried out to develop rapid,simple,specific and sensitive detection devices for protein in medical diagnostics and biomedical research application,while the detection methodologies for protein based on antibodies cannot meet the demand.Aptamers have been emerging as a new protein recognition element in wide range of bioassays.Aptamers have attracted a considerable attention due to their ability to bind target protein with high affinity and specificity and have many advantages over antibodies.These properties of aptamers make aptamers ideal candidates as protein recognition elements in a wide range of bioassays and for the development of diseases diagnostics. Electrochemical aptamer-based biosensors have attracted significant interest due to its simple, sensitive and low-cost detection capability for applications including clinical genetic analysis, environment monitoring,and forensic identification.
Nanoparticles are of considerable interest owing to their unique physical and chemical properties, and offer excellent prospects for biosensor.The power and scope of nanoparticles can be greatly enhanced by coupling them with the high specificity of the aptamer and the high sensitivity of the electrochemical recognitions.
The aim of the present work is to develop novel electrochemical biosensors based on nanoparticles and aptamer.Labeled and label free electrochemical aptamer-based biosensors were designed for the determination of biological molecules such as protein and drug with high sensitivity and selectivity.
This thesis consists of two parts.First part,chapter 1,is general introduction while second part, consisting of four chapters,is research report.
Chapter 1-General introduction.In this chapter,the concepts of aptamer,including in vitro selection of aptamer,the characteristic of aptamer,the interactions between aptamer and target, chemical modification of aptamers,were described;the principle of aptamer-based biosensor and electrochemical aptamer-based biosensor were presented;the recent developments of labeled and label-free electrochemical aptamer-based biosensor were reviewed;the purpose and the significance of this research work were simply presented.
Chapter 2-Labeled electrochemical aptamer-based biosensor for the determination of thrombin. A highly sensitive electrochemical method for the detection of thrombin based on the two thrombin-binding-aptamers(TBAⅠand TBAⅢ) has been developed.TBAⅠwith a thiol group at the 3′-terminal by self-assembled onto the surface of a gold electrode was employed as an electrochemical probe for capturing thrombin.TBAⅢlabeled with carbon-nanotubes(CNTs) tagged with silver nanoparticles(CNTs-Ag) was used for the detection of thrombin.CNTs were decorated with a large number of silver nanoparticles to form CNTs-Ag.Binding events were monitored by differential pulse voltammetric(DPV) signal of silver nanoparticles.There was a linear relation between the peak current at+0.20 V(vs.Ag/AgCl) and thrombin concentration over the range from 0.012 to 10 nmol/L with a detection limit of 0.005 nmol/L of thrombin.The detection processes are simple due to no requirement of acid-dissolving the label metal,the presence of other protein,as BSA did not affect the detection.This work demonstrated that the probe labeled with CNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of protein.
Chapter 3-Label free electrochemical aptamer-based biosensor for the determination of thrombin. A simple and highly sensitive electrochemical impedance spectroscopy(EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin.The signal enhancement was achieved by using gold nanoparticles(GNPs),which was electrodeposited onto a glassy carbon electrode(GCE),as a platform for the immobilization of the thiolated aptamer.In the measurement of thrombin,the change in interfacial electron transfer resistance of the biosensor using a redox couple of[Fe(CN)_6]~(3-/4-) as the probe was monitored.The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nmol/L to 30 nmol/L.The association and dissociation rate constants of the immobilized aptamer-thrombin complex were 6.7×10~3 M~(-1) s~(-1) and 1.0×10~(-4) s~(-1), respectively.The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed.This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein.This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.The obtained kinetics parameter and equilibrium constants will direct us to find out the basic principle for designing the aptamer based biosensor and to deeply understand the properties of the aptamer toward protein,and it will be great importance of developing new approaches for disease diagnosis.
Chapter 4-Label free electrochemical aptasensor for the determination of cocaine.A simple and highly sensitive impedance spectroscopy biosensor based on aptamer as molecular recognition element was developed for the determination of cocaine.The solution of the modified aptamer for cocaine and the complementary short DNA were first mixed in the buffer in equal more quantities to treat a half-duplex,and then the half-duplex aptamers were immobilized onto a gold electrode surface via thiol-Au interactions.The electroactive complex,[Ru(NH_3)_6]~(3+),which can bind to anionic phosphates of aptamer and DNA strands completely through electrostatic interactions,serves as a signaling probe.[Ru(NH_3)_6]~(3+) is adsorbed on a half-duplex aptamers backbone,giving a clear detection signal in admittance.After binding with cocaine,the complementary short DNA were released together with the[Ru(NH_3)_6]~(3+) that was electrostatically binding to them,resulting in a lower admittance signal for the[Ru(NH_3)_6]~(3+) confined on the electrode surface.The admittance response exhibits a linear relationship to the logarithm of the target concentration in the concentration range from 2μmol/L to 100μmol/L.This device combines the selectivity of the aptmer recognition layer with the sensitivity of admittance to the determination of cocaine.
Chapter 5-Labeled electrochemical aptamer-based biosensor for the determination of cocaine.A novel electrochemical self-assembled onto the surface of a gold electrode incorporating a signal enhancement for the determination of cocaine was designed.Gold nanoparticles were self-assembled onto the surface of a gold electrode through 1,6-hexanedithiol.A bifunctional derivative of the 32-base cocaine-binding aptamer with a redox-active ferrocene moiety and a thiol linker group at the termini of the strand was self-assembled onto the surface of gold nanoparticles.The oxidation peak current is linearly related to the concentration of cocaine from 1.0 to 15.0μmol/L with a detection limit of 0.5μmol/L.It was found that the sensitivity of the aptamer-based biosensor with gold nanoparticles modification is～10-folds higher than that of the aptamer-based biosensor without gold nanoparticles modification.The apparent heterogeneous electron transfer rate of ferrocene was determined to be 67 s~(-1).Compared with the apparent heterogeneous electron transfer rate of ferrocene at bare gold electrode,the apparent heterogeneous electron transfer rate of ferrocene increased at gold nanoparticles modified gold electrode.This work demonstrates that gold nanoparticles-assembled gold electrode provides a promising platform for immobilizing aptamer and enhancing the sensitivity.
纳米技术的出现为纳米材料在分析化学领域的发展和应用开辟了新的方向。纳米材料的优异性能例如比表面积大、反应活性高等为分析化学开辟了新的研究途径,纳米粒子独特的性能为生物检测奠定了基础。将纳米粒子和高特异性的适体,高灵敏的电化学检测技术相结合,提高了电化学检测的灵敏度,扩大了纳米材料应用范围,为生物分析化学开辟了新的领域。
本论文的研究目的是将纳米材料与高特异性的分子识别物质适体,高灵敏的电化学检测技术相结合,建立测定蛋白质、小分子药物的新方法和电化学适体传感器。发展具有高灵敏高选择性的电化学适体传感器,开拓生物传感器的研究和应用范围,为蛋白与小分子药物的疾病诊断和临床治疗提供具有潜在应用价值的分析器件。主要通过两个途径:(1)基于纳米粒子为电极修饰物、信号标记物或信号物质载体,放大了检测信号,提高了检测灵敏度;(2)利用电化学阻抗技术,建立非标记型凝血酶蛋白质和小分子药物可卡因检测的新方法。本论文分为两部分:第一部分为综述部分,第二部分为研究报告部分。
第一章为引言。引言部分首先简要地介绍了适体的体外筛选方法、适体的特点、适体与配体的相互作用和适体的化学修饰;总结了适体传感器和电化学适体传感器的分类,适体的固定化方法以及相关的电化学分析技术;重点评述了标记型和非标记型电化学适体传感器的研究进展,对电化学适体传感器的发展进行了展望。最后阐述了本论文的选题背景、研究意义、研究目的和研究内容。
第二章为标记型凝血酶电化学适体传感器的研究。基于碳纳米管的多负载作用,设计了以碳纳米管负载纳米银标记适体,夹心法检测凝血酶的电化学方法。以凝血酶为蛋白质的研究模型,利用碳纳米管负载纳米银标记适体Ⅲ以及固定在金电极上的适体Ⅰ与凝血酶结合形成金电极/适体Ⅰ/凝血酶/碳纳米管负载纳米银标记适体Ⅲ的三明治夹心式结构,通过检测碳纳米管负载纳米银上银的电化学信号,对凝血酶进行定量测定。氧化电流与凝血酶浓度在0.012～10nmol/L呈良好的线性关系,检出限可以达到0.005 nmol/L。该方法对凝血酶具有高的特异性识别能力,其检测不受其他蛋白质的干扰。利用碳纳米管表面负载多个纳米银粒子,对电化学信号起到了放大作用,提高了检测凝血酶的灵敏度。使用脉冲伏安法直接检测,无需对纳米粒子进行酸化和溶出,检出电位低,可能克服血液中还原性物质的干扰。该传感器有望用于实际样品中凝血酶的检测。
第三章为非标记型凝血酶电化学适体传感器的研究。基于适体与凝血酶结合前后电子传递电阻的变化,构建了非标记适体检测凝血酶的交流阻抗电化学传感器。利用电化学沉积方法制备金纳米粒子修饰玻碳电极,将末端带巯基的适体通过分子自组装作用固定在金纳米粒子修饰玻碳电极上。通过扫描电镜、X-射线能谱和原子力显微镜等技术对沉积在电极上的金纳米粒子进行了形貌的表征。研究了以三种不同序列适体为分子识别物质,测定凝血酶的线性范围和检出限,比较了三者灵敏度的差异。考察和讨论了不同序列适体与凝血酶的动力学行为,利用动力学法和浓度平衡法计算了适体与凝血酶形成的结合常数及适体与凝血酶复合物的解离常数。对两种方法获得的适体与凝血酶复合物的解离常数进行了比较和讨论。本方法简单,无需对适体进行标记,金纳米粒子的多负载作用,提高了凝血酶检测的灵敏度。本研究对适体-目标蛋白质之间结合过程的机理有一个更深的理解,将有助于我们进一步揭示核酸与蛋白这两种生命中最关键物质之间的相互作用和关系,对于我们更好地理解基本的生物过程和预测设计适体生物检测方法,发展用于疾病诊断的方法有着重要的意义。
第四章非标记型可卡因电化学适体传感器的研究。基于荷正电的六氨合钌离子与DNA骨架的静电结合作用,构建了检测可卡因的非标记型电化学适体传感器。将末端带巯基的可卡因适体与部分互补的单链DNA杂交,杂交溶液通过自组装作用固定在金电极上。未加入可卡因时,部分互补双链与六氨合钌离子静电结合,导纳值较大;加入可卡因后,可卡因适体与可卡因结合,构象发生变化,部分互补单链DNA解旋脱离电极表面,适体上静电结合的六氨合钌离子数量降低,导纳值减小。通过检测六氨合钌离子导纳的抑制值对可卡因进行定量测定。导纳虚部的峰值与可卡因的浓度在2～100μmol/L之间呈良好的线性关系,检出限为0.6μmol/L。对30.0μmol/L可卡因进行7次平行测定,相对标准偏差为4.0%。
第五章标记型可卡因电化学适体传感器的研究。基于金纳米粒子多负载作用,构建了一种新型的检测可卡因的电化学适体传感器。首先将金纳米粒子通过双巯基自组装作用固定在金电极上。利用自组装作用将末端带巯基的二茂铁标记可卡因适体固定到金纳米粒子修饰金电极上。研究发现,氧化电流与可卡因的浓度在1.0～15.0μmol/L范围呈良好的线性关系,检出限为0.5μmol/L。对15.0μmol/L可卡因进行7次平行测定,相对标准偏差为3.5%。与裸金电极相比较,金纳米粒子修饰金电极可以提高适体的负载量,使测定可卡因的灵敏度提高了10倍。研究了氧化还原过程中的二茂铁的表观异相电子转移速率常数k_s,金纳米粒子修饰金电极上二茂铁的表观异相电子转移速率常数k_s大于裸金电极上二茂铁的表观异相电子转移速率常数k_s。金纳米修饰电极可以加速氧化还原反应的电子转移速度。本方法可望用于其他小分子药物的检测。
本论文以适体为分子识别物质,以凝血酶和可卡因作为研究模型,以纳米粒子为电极修饰材料、信号物质载体或信号物质,设计了两种高灵敏的标记型电化学适体传感器;利用电化学交流阻抗技术,构建了两种非标记型电化学适体传感器。本论文所研制的电化学适体传感器,为蛋白质和小分子药物的灵敏、简单、快速检测提供了良好的分析器件和分析新方法。电化学适体传感器的研究将不仅为电化学适体传感器的设计提供新概念和思路,而且还将为临床疾病诊断、基因检测和药物筛选提供具有潜在应用价值的分析器件,对生命科学和生物传感器的发展具有重要意义。
Determination of protein has been a topic of significant interest with the accomplishment of Human Genome Project and the rapid progress of proteomic strategies.Intense research activities are carried out to develop rapid,simple,specific and sensitive detection devices for protein in medical diagnostics and biomedical research application,while the detection methodologies for protein based on antibodies cannot meet the demand.Aptamers have been emerging as a new protein recognition element in wide range of bioassays.Aptamers have attracted a considerable attention due to their ability to bind target protein with high affinity and specificity and have many advantages over antibodies.These properties of aptamers make aptamers ideal candidates as protein recognition elements in a wide range of bioassays and for the development of diseases diagnostics. Electrochemical aptamer-based biosensors have attracted significant interest due to its simple, sensitive and low-cost detection capability for applications including clinical genetic analysis, environment monitoring,and forensic identification.
Nanoparticles are of considerable interest owing to their unique physical and chemical properties, and offer excellent prospects for biosensor.The power and scope of nanoparticles can be greatly enhanced by coupling them with the high specificity of the aptamer and the high sensitivity of the electrochemical recognitions.
The aim of the present work is to develop novel electrochemical biosensors based on nanoparticles and aptamer.Labeled and label free electrochemical aptamer-based biosensors were designed for the determination of biological molecules such as protein and drug with high sensitivity and selectivity.
This thesis consists of two parts.First part,chapter 1,is general introduction while second part, consisting of four chapters,is research report.
Chapter 1-General introduction.In this chapter,the concepts of aptamer,including in vitro selection of aptamer,the characteristic of aptamer,the interactions between aptamer and target, chemical modification of aptamers,were described;the principle of aptamer-based biosensor and electrochemical aptamer-based biosensor were presented;the recent developments of labeled and label-free electrochemical aptamer-based biosensor were reviewed;the purpose and the significance of this research work were simply presented.
Chapter 2-Labeled electrochemical aptamer-based biosensor for the determination of thrombin. A highly sensitive electrochemical method for the detection of thrombin based on the two thrombin-binding-aptamers(TBAⅠand TBAⅢ) has been developed.TBAⅠwith a thiol group at the 3′-terminal by self-assembled onto the surface of a gold electrode was employed as an electrochemical probe for capturing thrombin.TBAⅢlabeled with carbon-nanotubes(CNTs) tagged with silver nanoparticles(CNTs-Ag) was used for the detection of thrombin.CNTs were decorated with a large number of silver nanoparticles to form CNTs-Ag.Binding events were monitored by differential pulse voltammetric(DPV) signal of silver nanoparticles.There was a linear relation between the peak current at+0.20 V(vs.Ag/AgCl) and thrombin concentration over the range from 0.012 to 10 nmol/L with a detection limit of 0.005 nmol/L of thrombin.The detection processes are simple due to no requirement of acid-dissolving the label metal,the presence of other protein,as BSA did not affect the detection.This work demonstrated that the probe labeled with CNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of protein.
Chapter 3-Label free electrochemical aptamer-based biosensor for the determination of thrombin. A simple and highly sensitive electrochemical impedance spectroscopy(EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin.The signal enhancement was achieved by using gold nanoparticles(GNPs),which was electrodeposited onto a glassy carbon electrode(GCE),as a platform for the immobilization of the thiolated aptamer.In the measurement of thrombin,the change in interfacial electron transfer resistance of the biosensor using a redox couple of[Fe(CN)_6]~(3-/4-) as the probe was monitored.The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nmol/L to 30 nmol/L.The association and dissociation rate constants of the immobilized aptamer-thrombin complex were 6.7×10~3 M~(-1) s~(-1) and 1.0×10~(-4) s~(-1), respectively.The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed.This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein.This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.The obtained kinetics parameter and equilibrium constants will direct us to find out the basic principle for designing the aptamer based biosensor and to deeply understand the properties of the aptamer toward protein,and it will be great importance of developing new approaches for disease diagnosis.
Chapter 4-Label free electrochemical aptasensor for the determination of cocaine.A simple and highly sensitive impedance spectroscopy biosensor based on aptamer as molecular recognition element was developed for the determination of cocaine.The solution of the modified aptamer for cocaine and the complementary short DNA were first mixed in the buffer in equal more quantities to treat a half-duplex,and then the half-duplex aptamers were immobilized onto a gold electrode surface via thiol-Au interactions.The electroactive complex,[Ru(NH_3)_6]~(3+),which can bind to anionic phosphates of aptamer and DNA strands completely through electrostatic interactions,serves as a signaling probe.[Ru(NH_3)_6]~(3+) is adsorbed on a half-duplex aptamers backbone,giving a clear detection signal in admittance.After binding with cocaine,the complementary short DNA were released together with the[Ru(NH_3)_6]~(3+) that was electrostatically binding to them,resulting in a lower admittance signal for the[Ru(NH_3)_6]~(3+) confined on the electrode surface.The admittance response exhibits a linear relationship to the logarithm of the target concentration in the concentration range from 2μmol/L to 100μmol/L.This device combines the selectivity of the aptmer recognition layer with the sensitivity of admittance to the determination of cocaine.
Chapter 5-Labeled electrochemical aptamer-based biosensor for the determination of cocaine.A novel electrochemical self-assembled onto the surface of a gold electrode incorporating a signal enhancement for the determination of cocaine was designed.Gold nanoparticles were self-assembled onto the surface of a gold electrode through 1,6-hexanedithiol.A bifunctional derivative of the 32-base cocaine-binding aptamer with a redox-active ferrocene moiety and a thiol linker group at the termini of the strand was self-assembled onto the surface of gold nanoparticles.The oxidation peak current is linearly related to the concentration of cocaine from 1.0 to 15.0μmol/L with a detection limit of 0.5μmol/L.It was found that the sensitivity of the aptamer-based biosensor with gold nanoparticles modification is～10-folds higher than that of the aptamer-based biosensor without gold nanoparticles modification.The apparent heterogeneous electron transfer rate of ferrocene was determined to be 67 s~(-1).Compared with the apparent heterogeneous electron transfer rate of ferrocene at bare gold electrode,the apparent heterogeneous electron transfer rate of ferrocene increased at gold nanoparticles modified gold electrode.This work demonstrates that gold nanoparticles-assembled gold electrode provides a promising platform for immobilizing aptamer and enhancing the sensitivity.
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