基于Ru（bpy）_3~（2+）的电致化学发光生物传感器的研制
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摘要
核酸适配体(aptamer)是一种能特异性地结合如小分子、蛋白质甚至是细胞等目标物的单链DNA片段。与传统的抗体相比较,aptamer在对分析物检测上具有化学稳定性高、实用性强、基团修饰简便以及在生物传感器设计上能高度适应兼容。近十年来,aptamer已经在药物诊断、新药筛选、蛋白质分析、生物传感器和分子开关研制领域成为有着广泛应用前景的识别元件。将aptamer与荧光、化学发光和毛细管电泳等检测方法联用可分别实现对原癌蛋白血小板源性生长因子、可卡因和IgE等多种物质的灵敏检测。将aptamer与各种分析技术联用来实现对特定序列DNA、蛋白、酶及生物活性小分子的检测将对功能基因组、蛋白质组、癌症诊断研究起到至关重要的作用。
电致化学发光(ECL)是在化学发光的基础上发展起来的一种新的分析方法,它是化学发光与电化学互相渗透的产物,这种方法不但可以利用电化学分析的特长,而且可以发挥化学发光分析的优点,具有高灵敏度、高选择性、操作快速简便和仪器简单等特点,已成为分析领域,尤其是生物分析领域工作者感兴趣的重要课题。Ru(bpy)32+作为电致化学发光试剂,已经广泛被应用于传统液相ECL体系中,但由于其价格昂贵,故而如何成功有效地将其固定到电极上制成固相电致化学发光传感器以减少试剂消耗就显得尤为重要。
壳聚糖是由甲壳素经脱乙酰基过程制备的一种富含氨基的多聚糖,在自然界中含量十分丰富。由于壳聚糖具有良好的生物相溶性、生物可降解性,可成膜性及高效的生物活性等特点,现已广泛应用到医药、化工、饲料、农业、环保和生物技术等各个领域。近年来,壳聚糖作为一种优良的膜材料,也越来越受到人们的重视。当壳聚糖溶液的pH值小于6.3时呈现可溶状态,当pH值大于6.3时,此时壳聚糖形成凝胶状,故而可通过对pH值的调控将壳聚糖膜制成特殊的固定化载体。
本论文将生物分子识别元件与电致化学发光技术的高灵敏度特性相结合,构建具有高选择性的新型固相电致化学发光生物传感器,实现对凝血酶的检测。此外,还利用壳聚糖膜的特征,将Ru(bpy)32+选择性地电沉积到电极上,开发了一种全新的Ru(bpy)32+高效固定方法。
本论文主要内容如下：
第一章绪论
本章简要介绍了电致化学发光(ECL)分析方法的概念、原理和特点,以及各种ECL反应类型及其在分析化学中的应用,展望了ECL分析的未来发展前景。介绍了DNA与核酸适配体生物传感器的构造原理、分类特点和分析应用。介绍了壳聚糖的理化特性、壳聚糖膜的优势及其在生物技术方面的应用。最后阐述了本论文的目的和意义,指出论文的创新之处。
第二章基于二茂铁标记分子灯塔适配体的固相ECL传感器实现对凝血酶的检测
本章利用标记有二茂铁(Fc)的核酸适配体,结合分子灯塔特殊茎环结构,构建了全新的固相电致化学发光(ECL)生物传感器,并对凝血酶成功实现检测。这种特殊结构的生物传感器包含了ECL发光基底以及ECL信号开关两部分。金胶钌混聚物(Ru(bpy)32+-AuNPs)通过Au-S键固定到金电极上构成ECL发光基底,而标记有二茂铁的分子灯塔适配体则作为ECL信号开关,其环部由能够与凝血酶发生特异性结合的一段特殊序列构成。当凝血酶与核酸适配体发生反应时,分子灯塔的茎部打开,此时标记在一端的Fc远离发光基底,Fc对Ru(bpy)32+的猝灭作用降低,从而使得ECL信号增强。
第三章基于无标记适配体和分子灯塔构型变换的固相ECL传感器实现对凝血酶的检测
本章报道了一种利用非标记适配体与凝血酶之间的特异性结合作用以及分子灯塔的构象变化,通过检测前后的ECL信号变化来定量反映目标蛋白——凝血酶的含量。将与凝血酶适配aptamer杂交的标记有二茂铁(Fc)的分子灯塔(Fc-dsDNA)固定到ECL发光层上,当aptamer与凝血酶反应后,单链分子灯塔回环,二茂铁端接近发光层,ECL信号猝灭。利用前后两次ECL信号变化对凝血酶的浓度进行定量测定。此传感器具有高灵敏度和高选择性。
第四章阵列电极上选择性固定Ru(bpy)32+的固相电致化学发光传感器构建
本章基于将包裹三-(2,2’-联吡啶)钌(II)(Ru(bpy)32+)的硅纳米颗粒(RuDS NPs)/壳聚糖复合膜电沉积到电极表面这一原理,开发了一种有效将Ru(bpy)32+选择性固定在目标电极上的方法,即将Ru(bpy)32+选择性固定到阵列电极的任意电极上。实验结果不仅证明了能有效将Ru(bpy)32+选择性固定到阵列电极上的可能性,而且能多通道选择性固定不同的复合膜。故而此方法在生物分析、毛细管电泳和药物筛选上都有很重要的应用前景。实验采用紫外可见光谱、场发射扫描电子显微镜(FE-SEM)和透射电子显微镜(TEM)来表征RuDS NPs以及复合膜。基于复合膜修饰电极的电致化学发光传感器对三丙胺(TPrA)的检测呈现良好的重现性、稳定性和灵敏度。
Aptamers are synthetic single-strand nucleic acids with high specificity and affinity to some given targets ranging from small molecules to large proteins and even cells. Apatmers have been demonstrated to have advantages over antibodies with regard to chemical stability, readily availability, simple modifiability and high flexibility in biosensor desigh for analyte detection. In the last decade, aptamers have been widely reported as highly promising recognition probes for disease diagnosis, new drug screening, protein analysis, biosensor and molecular switch development, etc. Onco protein platelet-derived growth factor, cocaine and IgE, for example, can be sensitively determined by fluorescence anisotropy, electrogenerated chemiluminescence and affinity pro capillary electrophoresis, respectively. When combined with all other analysis techniques, aptamer can be utilized to detect DNA sequence, correlative protein, enzyme and small molecules, which is of great significance on the research of functional gene, protein, cancer diagnose in the postgenome era.
Electrochemiluminescence (ECL) is developed based on chemiluminescence (CL) and electrochemistry (EC) with a large number of advantages, such as high sensitivity and selectivity, rapid and convenient operation and relatively simple instrumentation system. Due to these inherent advantages, ECL method has attracted much attention from all analytical fields, especaily from biochemical analysis. As an important ECL substrate, Ru(bpy)32+ has been widely used in the traditiona solution-state ECL system. However, owning to the high price of Ru(bpy)32+, it becames significant to effectively immobilize Ru(bpy)32+ onto the electrode so as to reduce the consumption of expensive reagent.
Chitosan is an amine-rich polysaccharide derived by deacetylation of chitin, which is abundant in nature. It has been wildly used in scientific and industial fields due to its excellent properties, such as biocompatibility, biodegradability, ability to form films and high reactivity. As an outstanding film material, chitosan has attracted more attention. The pKa of chitosan is about 6.3. Therefore, when the pH is lower than 6.3, most of the amino groups are protonated and positively charged so that chitosan is soluble in aqueous solution. As the pH is higher than 6.3, the amine groups become deprotonated and uncharged, thus making chitosan insoluble in water. According to its special characteristic, chitosan can function as a matrix to immobilize biomolecules by adjusting the pH value of the solution.
The goal of the paper is to develop a novel solid-state ECL biosensor which combines the specific recognition of molecular recognition elements and electrochemiluminescence technique to detect thrombin with high sensitivity and selectivity. Moreover, the characteristics of chitosan is also used to selectively electrodeposite Ru(bpy)32+onto the array electrodes, which means a brand-new immobilization method of Ru(bpy)32+is structured.
The dissertation is composed of four chapters as following:
Chapter 1:Give a brief introduction on the principle and application of ECL, as well as several types of ECL reactions and ECL sensors in the field of analytical chemistry. DNA sensors including their principles, advantages and disadvantages are reviewed. Then, chitosan including its characteristics, advantages and application in biotechnology are detailed on the paper. At the end of chapter 1, the purpose of the dissertation is pointed out.
Chapter 2:A solid-state electrochemiluminescence (ECL) biosensing switch system based on special ferrocenelabeled molecular beacon aptamer (Fc-MBA) has been developed successfully for thrombin detection. Such special switch system includes two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate is made by modifying the complex of Au nanoparticle and Ruthenium (Ⅱ) tris-(bipyridine) (Ru(bpy)32+-AuNPs) onto Au electrode. A molecular beacon aptamer labeled by ferrocene acts as the ECL intensity switch. The loop bases of the ECL intensity switch are designed with special anti-thrombin aptamer sequence which can be combined with its target protein via the reaction between aptamer and thrombin. During the reaction, the molecular beacon aptamer opens its stem-loop, and the labeled Fc is, therefore, kept away from the ECL substrate. Such conformation adjustment results in an obvious ECL intensity increment due to the decreased quenching effect of Fc to the ECL substrate.
Chapter 3:A solid-state electrochemiluminescence (ECL) biosensor based on the switch system of ferrocene-labeled double-stranded DNA (Fc-dsDNA) has been developed for thrombin detection. This special switch system contains an ECL substrate which is made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)32+-AuNPs) onto Au electrode, and one dsDNA hybridized by a thrombin-aptamer and one ferrocene-labeled molecular beacon is used as the ECL signal switch. Before the reaction of thrombin and its aptamer, the molecular beacon opens its stem-loop and the labeled Fc, therefore, is kept away from the ECL substrate. After the specific recognition between aptamer and thrombin occurs, the Fc is pulled back onto the electrode so as to decline the ECL intensity due to the quenching effect of Fc to Ru(bpy)32+. The analysis results with sensitivity and specificity.
Chapter 4:An effective method for the selective immobilization of tris(2,2'-bipyridyl)ruthenium(Ⅱ) (Ru(bpy)32+) onto one target electrode surface based on the electrodeposition of RuDS NPs Ru(bpy)32+ doped silica nanoparticles)/chitosan composite film is presented in this paper. Ru(bpy)32+ is selectively deposited onto four individual electrodes in an Au electrode array. The result demonstrates the possibility of selective immobilization of Ru(bpy)32+onto array electrodes and selective immobilization of different ECL composite. The method shows great potential of application in the field of bioanalysis, capillary electrophoresis and drug screening. RuDS NPs and the resulting composite film were characterized by UV-vis spectroscopy, transmission electron microscopy (TEM) and Field emission-scanning electron microscope (FE-SEM). The electrochemiluminescence (ECL) sensor based on the composite film modified electrode exhibites excellent reproducibility, stability and sensitivity for the detection of tri-n-propylamine (TPrA).
电致化学发光(ECL)是在化学发光的基础上发展起来的一种新的分析方法,它是化学发光与电化学互相渗透的产物,这种方法不但可以利用电化学分析的特长,而且可以发挥化学发光分析的优点,具有高灵敏度、高选择性、操作快速简便和仪器简单等特点,已成为分析领域,尤其是生物分析领域工作者感兴趣的重要课题。Ru(bpy)32+作为电致化学发光试剂,已经广泛被应用于传统液相ECL体系中,但由于其价格昂贵,故而如何成功有效地将其固定到电极上制成固相电致化学发光传感器以减少试剂消耗就显得尤为重要。
壳聚糖是由甲壳素经脱乙酰基过程制备的一种富含氨基的多聚糖,在自然界中含量十分丰富。由于壳聚糖具有良好的生物相溶性、生物可降解性,可成膜性及高效的生物活性等特点,现已广泛应用到医药、化工、饲料、农业、环保和生物技术等各个领域。近年来,壳聚糖作为一种优良的膜材料,也越来越受到人们的重视。当壳聚糖溶液的pH值小于6.3时呈现可溶状态,当pH值大于6.3时,此时壳聚糖形成凝胶状,故而可通过对pH值的调控将壳聚糖膜制成特殊的固定化载体。
本论文将生物分子识别元件与电致化学发光技术的高灵敏度特性相结合,构建具有高选择性的新型固相电致化学发光生物传感器,实现对凝血酶的检测。此外,还利用壳聚糖膜的特征,将Ru(bpy)32+选择性地电沉积到电极上,开发了一种全新的Ru(bpy)32+高效固定方法。
本论文主要内容如下：
第一章绪论
本章简要介绍了电致化学发光(ECL)分析方法的概念、原理和特点,以及各种ECL反应类型及其在分析化学中的应用,展望了ECL分析的未来发展前景。介绍了DNA与核酸适配体生物传感器的构造原理、分类特点和分析应用。介绍了壳聚糖的理化特性、壳聚糖膜的优势及其在生物技术方面的应用。最后阐述了本论文的目的和意义,指出论文的创新之处。
第二章基于二茂铁标记分子灯塔适配体的固相ECL传感器实现对凝血酶的检测
本章利用标记有二茂铁(Fc)的核酸适配体,结合分子灯塔特殊茎环结构,构建了全新的固相电致化学发光(ECL)生物传感器,并对凝血酶成功实现检测。这种特殊结构的生物传感器包含了ECL发光基底以及ECL信号开关两部分。金胶钌混聚物(Ru(bpy)32+-AuNPs)通过Au-S键固定到金电极上构成ECL发光基底,而标记有二茂铁的分子灯塔适配体则作为ECL信号开关,其环部由能够与凝血酶发生特异性结合的一段特殊序列构成。当凝血酶与核酸适配体发生反应时,分子灯塔的茎部打开,此时标记在一端的Fc远离发光基底,Fc对Ru(bpy)32+的猝灭作用降低,从而使得ECL信号增强。
第三章基于无标记适配体和分子灯塔构型变换的固相ECL传感器实现对凝血酶的检测
本章报道了一种利用非标记适配体与凝血酶之间的特异性结合作用以及分子灯塔的构象变化,通过检测前后的ECL信号变化来定量反映目标蛋白——凝血酶的含量。将与凝血酶适配aptamer杂交的标记有二茂铁(Fc)的分子灯塔(Fc-dsDNA)固定到ECL发光层上,当aptamer与凝血酶反应后,单链分子灯塔回环,二茂铁端接近发光层,ECL信号猝灭。利用前后两次ECL信号变化对凝血酶的浓度进行定量测定。此传感器具有高灵敏度和高选择性。
第四章阵列电极上选择性固定Ru(bpy)32+的固相电致化学发光传感器构建
本章基于将包裹三-(2,2’-联吡啶)钌(II)(Ru(bpy)32+)的硅纳米颗粒(RuDS NPs)/壳聚糖复合膜电沉积到电极表面这一原理,开发了一种有效将Ru(bpy)32+选择性固定在目标电极上的方法,即将Ru(bpy)32+选择性固定到阵列电极的任意电极上。实验结果不仅证明了能有效将Ru(bpy)32+选择性固定到阵列电极上的可能性,而且能多通道选择性固定不同的复合膜。故而此方法在生物分析、毛细管电泳和药物筛选上都有很重要的应用前景。实验采用紫外可见光谱、场发射扫描电子显微镜(FE-SEM)和透射电子显微镜(TEM)来表征RuDS NPs以及复合膜。基于复合膜修饰电极的电致化学发光传感器对三丙胺(TPrA)的检测呈现良好的重现性、稳定性和灵敏度。
Aptamers are synthetic single-strand nucleic acids with high specificity and affinity to some given targets ranging from small molecules to large proteins and even cells. Apatmers have been demonstrated to have advantages over antibodies with regard to chemical stability, readily availability, simple modifiability and high flexibility in biosensor desigh for analyte detection. In the last decade, aptamers have been widely reported as highly promising recognition probes for disease diagnosis, new drug screening, protein analysis, biosensor and molecular switch development, etc. Onco protein platelet-derived growth factor, cocaine and IgE, for example, can be sensitively determined by fluorescence anisotropy, electrogenerated chemiluminescence and affinity pro capillary electrophoresis, respectively. When combined with all other analysis techniques, aptamer can be utilized to detect DNA sequence, correlative protein, enzyme and small molecules, which is of great significance on the research of functional gene, protein, cancer diagnose in the postgenome era.
Electrochemiluminescence (ECL) is developed based on chemiluminescence (CL) and electrochemistry (EC) with a large number of advantages, such as high sensitivity and selectivity, rapid and convenient operation and relatively simple instrumentation system. Due to these inherent advantages, ECL method has attracted much attention from all analytical fields, especaily from biochemical analysis. As an important ECL substrate, Ru(bpy)32+ has been widely used in the traditiona solution-state ECL system. However, owning to the high price of Ru(bpy)32+, it becames significant to effectively immobilize Ru(bpy)32+ onto the electrode so as to reduce the consumption of expensive reagent.
Chitosan is an amine-rich polysaccharide derived by deacetylation of chitin, which is abundant in nature. It has been wildly used in scientific and industial fields due to its excellent properties, such as biocompatibility, biodegradability, ability to form films and high reactivity. As an outstanding film material, chitosan has attracted more attention. The pKa of chitosan is about 6.3. Therefore, when the pH is lower than 6.3, most of the amino groups are protonated and positively charged so that chitosan is soluble in aqueous solution. As the pH is higher than 6.3, the amine groups become deprotonated and uncharged, thus making chitosan insoluble in water. According to its special characteristic, chitosan can function as a matrix to immobilize biomolecules by adjusting the pH value of the solution.
The goal of the paper is to develop a novel solid-state ECL biosensor which combines the specific recognition of molecular recognition elements and electrochemiluminescence technique to detect thrombin with high sensitivity and selectivity. Moreover, the characteristics of chitosan is also used to selectively electrodeposite Ru(bpy)32+onto the array electrodes, which means a brand-new immobilization method of Ru(bpy)32+is structured.
The dissertation is composed of four chapters as following:
Chapter 1:Give a brief introduction on the principle and application of ECL, as well as several types of ECL reactions and ECL sensors in the field of analytical chemistry. DNA sensors including their principles, advantages and disadvantages are reviewed. Then, chitosan including its characteristics, advantages and application in biotechnology are detailed on the paper. At the end of chapter 1, the purpose of the dissertation is pointed out.
Chapter 2:A solid-state electrochemiluminescence (ECL) biosensing switch system based on special ferrocenelabeled molecular beacon aptamer (Fc-MBA) has been developed successfully for thrombin detection. Such special switch system includes two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate is made by modifying the complex of Au nanoparticle and Ruthenium (Ⅱ) tris-(bipyridine) (Ru(bpy)32+-AuNPs) onto Au electrode. A molecular beacon aptamer labeled by ferrocene acts as the ECL intensity switch. The loop bases of the ECL intensity switch are designed with special anti-thrombin aptamer sequence which can be combined with its target protein via the reaction between aptamer and thrombin. During the reaction, the molecular beacon aptamer opens its stem-loop, and the labeled Fc is, therefore, kept away from the ECL substrate. Such conformation adjustment results in an obvious ECL intensity increment due to the decreased quenching effect of Fc to the ECL substrate.
Chapter 3:A solid-state electrochemiluminescence (ECL) biosensor based on the switch system of ferrocene-labeled double-stranded DNA (Fc-dsDNA) has been developed for thrombin detection. This special switch system contains an ECL substrate which is made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)32+-AuNPs) onto Au electrode, and one dsDNA hybridized by a thrombin-aptamer and one ferrocene-labeled molecular beacon is used as the ECL signal switch. Before the reaction of thrombin and its aptamer, the molecular beacon opens its stem-loop and the labeled Fc, therefore, is kept away from the ECL substrate. After the specific recognition between aptamer and thrombin occurs, the Fc is pulled back onto the electrode so as to decline the ECL intensity due to the quenching effect of Fc to Ru(bpy)32+. The analysis results with sensitivity and specificity.
Chapter 4:An effective method for the selective immobilization of tris(2,2'-bipyridyl)ruthenium(Ⅱ) (Ru(bpy)32+) onto one target electrode surface based on the electrodeposition of RuDS NPs Ru(bpy)32+ doped silica nanoparticles)/chitosan composite film is presented in this paper. Ru(bpy)32+ is selectively deposited onto four individual electrodes in an Au electrode array. The result demonstrates the possibility of selective immobilization of Ru(bpy)32+onto array electrodes and selective immobilization of different ECL composite. The method shows great potential of application in the field of bioanalysis, capillary electrophoresis and drug screening. RuDS NPs and the resulting composite film were characterized by UV-vis spectroscopy, transmission electron microscopy (TEM) and Field emission-scanning electron microscope (FE-SEM). The electrochemiluminescence (ECL) sensor based on the composite film modified electrode exhibites excellent reproducibility, stability and sensitivity for the detection of tri-n-propylamine (TPrA).
引文
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