基于电致化学发光技术的纳米生物传感器的设计与研究
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摘要
核酸、蛋白质及酶是组成生命的主要生物大分子。基因组核酸承载着传递遗传信息、编码生命功能的重要任务,基因的功能或表达则决定生命。蛋白质则贯穿所有的生命活动过程。酶在DNA合成中起重要作用,且在保持基因组的完整性方面也是必不可少的重要物质。核酸的复制、连接、修复等操作是重要的生命过程,DNA/RNA的结构片段,作为生物大分子的基本“砖块”,是生命活动的重要参与者,是重要的细胞信号传导物。基因的相关功能的完成需要蛋白质、酶及生物活性小分子化合物的协同参与,宏观上,这一系列生物分子的配合与作用构成生长、发育、繁殖、遗传和代谢等生命现象的基础。对功能基因组、蛋白质组及相关生物活性分子的研究正是后基因组时代所关注的热点。
随着科学家们逐渐揭开疾病的机理,并将其用于疾病的医疗诊断和治疗之中,对特定序列的DNA,相关蛋白、酶及生物活性小分子的检测日益受到重视。在传统的分析方法中,核酸与蛋白质的研究都采用放射性标记、凝胶电泳和放射性自显影等技术。这些方法过程复杂,周期长,特别是放射性标记,存在放射性污染。而非放射性标记法如荧光、化学发光和生物素标记法,标记过程繁琐、复杂,难以实现自动化且仪器价格昂贵。而生命科学的迅速、深入发展迫切需要新的手段和方法,以更灵敏、更真实、更快速地研究生命过程。
电致化学发光(ECL),是由电化学反应直接或间接引发的化学发光现象,是电化学和化学发光相结合的产物,兼具二者的优点。其方便快捷、灵敏度高、动力学响应范围宽、检出限低、可控性与选择性好、容易实现实时化和集成化,可进行原位检测。集以上优点于一身的电致化学发光技术近几年在分析化学,尤其在生物分析领域的应用引起了人们的极大关注,为生命科学进入到分子水平领域提供了有力的研究手段与方法。
当物质小到纳米数量级时,会产生独特的表面效应、体积效应、量子尺寸效应和宏观量子隧道效应等,其电学、磁学、光学和化学性质也相应地发生显著的变化,呈现出常规材料不具备的优越性能。因此,纳米材料在催化、电子材料、微器件、增强材料及传感器材料等方面有着广阔的应用前景。电化学过程与电极材料的表面性质有关,如果将纳米材料修饰在电极的表面,基于其比表面积大、催化活性高、亲和力强的特性,其能活化电极表面,增大电流响应,降低检测限,大大提高检测的灵敏度,同时最大限度地保持相关生物分子的生物活性。将纳米技术应用于生物活性分子的电致化学发光分析研究,是一个崭新的领域,有利于创新性地建立一些新理论、新技术和新方法。
本论文将纳米技术、生物分子识别元件的特异性及电致化学发光技术的灵敏性相结合,发展具有高灵敏度和高选择性的DNA、蛋白质及酶的新型纳米生物传感器。开拓生物传感器在后基因时代的研究领域,为研究多种生物分子提供新的手段与方法。论文分五个部分,共八章,具体内容如下:
Ⅰ.绪论(第一章)
本章系统阐述了电致化学发光的原理和特点,重点介绍了ECL两大发光体系的反应机理及其在分析中的应用。介绍了DNA与核酸适配体电致化学发光生物传感器的构造原理、分类特点及研究进展。介绍了各种纳米材料在电致化学发光生物传感器中的应用。最后阐述了本论文的目的和意义,指出论文的创新之处及主要研究内容。
Ⅱ.基于Ru(bpy)_3~(2+)-SiO_2 NPs的核酸适配体传感器实现凝血酶蛋白质电致化学发光特异性检测(第二、三章)
第二章基于Ru(bpy)_3~(2+)-SiO_2 NPs的单核酸适配体传感器经目标诱导靶替换实现凝血酶蛋白质特异性检测
利用反相微乳法合成了SiO_2包裹的电致化学发光活性物2,2＇-联吡啶钌(Ru(bpy)_3~(2+))纳米颗粒(Ru(bpy)_3~(2+)-SiO_2 NPs),以此作为DNA标记物,结合替换反应发展了一种简单有效的凝血酶蛋白质电致化学发光特异性检测的方法。首先,在金电极的表面组装凝血酶核酸适配体aptamer,将其与标记有Ru(bpy)_3~(2+)-SiO_2NPs并与aptamer部分序列完全互补的ssDNA探针进行杂交,测得第一个ECL信号(I_(ECL1))。然后将电极与凝血酶蛋白进行培育,凝血酶与aptamer特异性结合,将探针替换下来,此时测得第二个ECL信号(I_(ECL2))。利用前后两个ECL信号的差值△I_(ECL)(I_(ECL1)-I_(ECL2))来定量测定凝血酶。非特异性识别的蛋白不干扰测定,凝血酶在1.0×10~(-14)mol/L～1.0×10~(-11)mol/L范围内有良好的线性响应,检测限为1.0×10~(-15)mol/L(S/N=3,n=11)。方法可应用于实际血浆中凝血酶的检测。
第三章基于Ru(bpy)_3~(2+)-SiO_2 NPs的核酸适配体传感器经三明治传感系统实现凝血酶蛋白质特异性检测
利用凝血酶的两段核酸适配体(aptamerⅠ,aptamerⅡ)与凝血酶的高亲和识别作用结合电致化学发光技术设计了一种具有高灵敏度和高选择性的蛋白质生物传感器。将一段15个碱基的aptamerⅠ通过Au-S键自组装到金电极表面,形成生物识别层,特异性“捕捉”目标蛋白质—凝血酶,进而结合另一段标记有Ru(bpy)_3~(2+)-SiO_2NPs的29个碱基的aptamerⅡ探针构建三明治结构,在含有TPrA的检测液中通过检测Ru(bpy)_3~(2+)的ECL信号对凝血酶进行定量检测。此传感器对非特异性识别的蛋白如牛血清白蛋白、牛血红蛋白不产生ECL响应,凝血酶在2.0×10~(-15)mol/L～2.0×10~(-12)mol/L范围内有良好的线性响应,检测限为1.0×10~(-15)mol/L(S/N=3,n=11)。
Ⅲ.基于二茂铁标记分子灯塔构象变化的可控固相Ru(bpy)_3~(2+)-电致化学发光膜及其特异性识别DNA、蛋白质及连接酶(第四、五和六章)
第四章基于二茂铁标记分子灯塔构象变化的可控固相Ru(bpy)_3~(2+)-电致化学发光膜及其特异性识别DNA的研究
基于二茂铁(Fc)对电致化学发光活性物Ru(bpy)_3~(2+)的ECL的高效猝灭作用发展了一种可控的固相Ru(bpy)_3~(2+)-ECL膜。修饰电极的ECL强度与Fc和固定在电极表面的Ru(bpy)_3~(2+)的距离直接相关,这个距离由标记有Fc的分子灯塔(Fc-MB)的构象控制。我们尝试通过不同的途径改变Fc-MB的构象。Fc-MB与其环部碱基互补的DNA杂交,或者升温将Fc-MB及杂交得到的dsDNA解旋变性,都可以很好的改变Fc-MB的构象,进而改变修饰电极的ECL强度。系统研究了温度诱导二茂铁标记分子灯塔及其系列杂交产物的构象转变过程,成功预言了相关生物分子的熔化温度。为深入研究分子灯塔键合平衡常数和茎环构象的热力学参数提供了一种新思路。同时,此固相Ru(bpy)_3~(2+)-ECL膜可以特异性、高灵敏的测定靶DNA。
第五章基于二茂铁标记分子灯塔适配体的固相电致化学发光传感器特异性识别凝血酶蛋白质
通过纳米金胶将Ru(bpy)_3~(2+)固定,利用二茂铁对Ru(bpy)_3~(2+)的电致化学发光的高效猝灭,结合分子灯塔适配体的特殊茎环结构建立一种高灵敏的固相电致化学发光检测凝血酶的方法。通过凝血酶与灯塔环部核酸适配体的特异性结合来调整分子灯塔适配体的构象,即打开灯塔的茎部互补碱基对,改变Fc与固定在电极上的Ru(bpy)_3~(2+)的距离,通过调整前后修饰电极ECL信号的差值(△I_(ECL))来定量测定凝血酶。非特异性识别的蛋白不干扰测定,凝血酶在1.0×10~(-14)mol/L～1.0×10~(-11)mol/L范围内有良好的线性响应,检测限为1.0×10~(-15)mol/L(S/N=3,n=11),并应用于混合蛋白样品的检测。
第六章基于二茂铁标记分子灯塔的固相电致化学发光传感器特异性识别T4DNA连接酶
利用二茂铁对Ru(bpy)_3~(2+)的电致化学发光的高效猝灭,结合分子灯塔的特殊茎环结构及核酸连接的模板建立一种高灵敏的固相电致化学发光检测T4 DNA连接酶的方法。通过T4 DNA连接酶连接修复与Fc标记的分子灯塔(Fc-MB)两端环部碱基完全互补的两条短链DNA,形成与Fc-MB完全互补的长链DNA,将Fc-MB的茎部互补碱基对彻底打开,改变Fc与固定在电极上的Ru(bpy)_3~(2+)的距离,引起修饰电极的ECL信号的改变,通过前后ECL信号的差值(△I_(ECL))来定量测定T4DNA连接酶。该酶传感器具有高的灵敏度和选择性,对T4 DNA连接酶在5.0×10~(-3)U/μL～5.0×10~(-6)U/μL范围内有良好的线性响应,检测限为2.5×10~(-6)U/μL(S/N=3,n=11)。
Ⅳ.基于核酸适配体构象转换应用固相电致化学发光传感平台超灵敏检测腺苷(第七章)
基于腺苷-核酸适配体构象转换机制研制出一种高灵敏的固相电致化学发光检测腺苷的新型传感系统。把对Ru(bpy)_3~(2+)的电致化学发光有高效猝灭作用的二茂铁分子标记在腺苷-核酸适配体上,腺苷-核酸适配体与腺苷特异性结合后,其构象会发生变化,引起标记物Fc与Ru(bpy)_3~(2+)的距离变化,进而使得修饰电极的ECL信号发生变化,通过腺苷-核酸适配体构象转换前后修饰电极的ECL信号的差值来定量测定小分子腺苷。非特异性识别的其它小分子不干扰测定,腺苷在1.0×10~(-8)mol/L～1.0×10~(-5)mol/L范围内有良好的线性响应,检测限为5.0×10~(-9)mol/L(S/N=3,n=11)。回收试验结果满意。简易、快速、低耗及良好的响应性能使本传感系统在构建基于核酸适配体传感器检测小分子的研究方法中体现出较好的发展前景。
Ⅴ.应用MWNTs-Ru(bpy)_3~(2+)混聚体经酶放大电致化学发光特异性识别p53基因(第八章)
尝试通过多壁羧基化的碳纳米管(COOH-MWNTs)将Ru(bpy)_3~(2+)固定,经吡咯膜(PPy)把MWNTs-Ru(bpy)_3~(2+)混聚体与纳米金胶标记的DNA分析物相连,将葡萄糖脱氢酶参与的专一性的酶促反应与高灵敏的电致化学发光反应偶联,建立一种直接、准确的固相电致化学发光检测人类p53肿瘤抑制基因的方法。用此法检测目标p53野生型序列,检测限为1.0×10~(-13)mol/L,在相同条件下,单碱基错配的p53突变型序列和p53野生型序列的区分度达56.3%,其特异性、低灵敏度的检测为癌症的早期诊断提供了可能。
Nucleic acid,protein and enzyme are the most important biomolecules in life. Nucleic acid is in charge of transferring genetic information and coding other biomolecules,while protein has run through the whole life process.Enzyme plays an important role in DNA synthesis and maintaining the integrity of genome.The replication,repair and recombination of nucleic acids are essential life processes. DNA/RNA structure fragment,as "bricks" of the biomacromolecules,is an important participant in the activities of life and is an important signal transduction of cells.The completion of the related function of gene needs the cooperation of the protein, enzyme and the small bioactivity molecules.Actually,the matching and the effect between these biomolecules construct the basic of the vital phenomena,such as growth, generation and metabolism.The study of functional gene,protein and the related bioactivity molecules is the hot spots of postgenome era.
With the increasing knowledge about human diseases,the specific sequence of DNA,correlative protein,enzyme and small bioactivity molecule detection received increasing attention.Many detection techniques of DNA sequence and protein have been developed in recent years,but there is still a lot of dissatisfaction.The radioactive labels present many problems such as a potential hazard to analyst and environment. Non-radioactive labels such as fluorescent,chemiluminescent and biotin-avidin label probes also present many shortcomings such as low sensitivity or complex equipment or others.So it is necessary to develop another method for the more sensitive, easy-to-use,fast,inexpensive detection of correlative bioactivity molecules to adapt to wide-scale genetic and protein testing requires.
Electrochemiluminescence(ECL),the generation of an optical signal triggered by an electrochemical reaction,has attracted much attention during the past several decades due to its versatility,simplified optical setup,very low background signal, good temporal and spatial control,and has become an important and valuable technique for immunosensing,DNA hybridization assays and proteinic biosensors.It also provides a powerful research tools and methods for the life sciences entering into the molecular level field.
Today,nano-science and technology has entered the limelight and has been investigated extensively by governments and scientists all over the world.It is out of question that the revolution of nanotechnology is coming.In recent work,it has been discovered that materials in the nano-size scale(1～100 nm)display size-dependent optical,magnetic,electronic and chemical properties.Except for these,nanomaterials have unknown chemical and physical properties that differ greatly from the bulk substances.Therefore,nanoparticles can be applied to many fields,such as optical devices,electronic devices,catalysis,sensor technology,and biomolecular labeling, etc.,which is also the reason that a burst of research activities have been focused on nanoparticles.Modified electrodes based on nanomaterials combined with high surface area and good electrocatalytic abilities that can largely improve electrical responses and the detection sensitivity.At the same time,the bioactivity of the biomolecules can be remained preferably.Today,many wonderful nanostructured materials have been applied in electroanalytical chemistry and some important progresses along these topics have recently achieved.
The goal of the present study is to design and research novel DNA,protein and enzyme nano-biosensor with high sensitivity and selectivity.This paper combines the excellent characteristics of nanoparticles,the specific recognition of molecular recognition elements and the electrochemiluminescence technique.It would broaden the research field of post-genetic time.This paper altogether divided into five parts. The details are given as follows:
Part one:Preface(Chapter 1)
In this chapter,we elaborated review from the principles,characteristics of the electrochemiluminescence,the two most primary types of ECL reaction and their application in analytical chemistry field.The principles,characteristics and the research progress of DNA and the aptamer electrochemiluminescent biosensor all had been introducded.Nanoparticles,especially the application of the variety of nanomaterials in the electrochemiluminescent biosensor were presented.Finally, expounded the aim and the significance,pointed out the research content and the innovation in this paper.
Part two:Detection of thrombin using electrochemiluminescence based on Ru(bpy)_3~(2+)-doped silica nanoparticle aptasensor(Chapter 2,3)
Tri(2,2'-bipyridyl)ruthenium(Ⅱ)-doped silica nanoparticles(Ru(bpy)_3~(2+)-SiO_2 NPs) were prepared by water-in-oil(W/O)microemulsion method.A great deal of Ru(bpy)_3~(2+)was immobilized inside the nanoparticle,which could greatly enhance the ECL response and result in the increased sensitivity.
In chapter 2,sensitive and selective aptasensor using Ru(bpy)_3~(2+)-SiO_2 NPs as DNA tags for detection of thrombin was developed based on the target protein-induced strand displacement of the DNA probe.For the proposed aptasensor,the aptamer was assembled on the surface of the Au electrode through Au-S binding.The hybridization event between the DNA probe labeled by the Ru(bpy)_3~(2+)-SiO_2 NPs and the aptamer was evaluated by ECL measurements.Then,the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again.The difference of ECL intensity(ΔI_(ECL))of the two events could be used to quantify the thrombin.Other proteins,such as bovine serum albumin and bovine hemoglobin,had almost negligibleΔI_(ECL).Under the optimal conditions,theΔI_(ECL)was linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)_3~(2+)molecules were labeled on the DNA probe.
In chapter 3,a new electrochemiluminescent detection system for protein using the aptamers was developed.Two different aptamers,which recognize different positions of thrombin,were chosen to construct sandwich type sensing system for protein,and one was immobilized onto the gold electrode for capturing thrombin onto the electrode and the other was used for detection:To obtain the signal,the aptamer for detection was labeled with Ru(bpy)_3~(2+)-SiO_2 NPs.The increase of the ECL signal generated by Ru(bpy)_3~(2+)-SiO_2 NPs was observed in dependent manner on the concentration of thrombin added.Bovine serum albumin and bovine hemoglobin had almost negligible responses.The ECL signal was linearly related to the concentration of the thrombin anatyte in the range of 2.0 fM to 2.0 pM and the assay allowed detection at levels as low as 1.0 fM of the thrombin.
Part three:A controllable solid-state Ru(bpy)_3~(2+)-electrochemiluminescence film for detection of biomolecules based on conformation change of ferrocene-labeled molecular beacon(Chapter 4,5 and 6)
In chapter 4,a controllable solid-state electrochemiluminescence film based on efficient and stable quenching of ECL of Ru(bpy)_3~(2+)by oxidizing ferrocene(Fc)at the electrode was developed.The ECL intensity was correlated to the distance which was controlled by the conformation of the ferrocene-labeled DNA molecular beacon (Fc-MB)between the Fc and Ru(bpy)_3~(2+)immobilized on the electrode.The conformation adjustment was conducted via complementary DNA's hybridizing with the bases in the loop of the Fc-MB and changing the temperature of the Fc-MB and the resultant double strand DNA(dsDNA).Those events all resulted in change of the ECL intensity.Therefore,the melting temperature of the relevant biomolecules was predicted successfully.With such characteristics,the solid-state Ru(bpy)_3~(2+)-ECL film had the potential to calculate thermodynamic parameters of equilibrium constants of MB binding and the stem-loop formation.Such controllable solid-state Ru(bpy)_3~(2+)-ECL film could detect target ssDNA,and the analysis results were sensitive and specific.
In chapter 5,we utilized the special solid-state Ru(bpy)_3~(2+)-ECL film to develop solid-state electrochemiluminescence biosensing switch.Thrombin as one important physiological protease in blood had been chosen as the target for investigating the application of the biosensing switch system to special protein.Herein,the loop bases of the Fc-MB were designed with the specific anti-thrombin aptamer sequence,which was expected to transform into G-quartet structure to combine with thrombin. Consequently,the Fc was far away the electrode surface and the ECL intensity was increased significantly.The enhanced ECL signal was expected to quantify the thrombin.Other proteins,such as bovine serum albumin and bovine hemoglobin,had almost negligibleΔI_(ECL).TheΔI_(ECL)was linearly related to the concentration of the thrombin analyte in the range of 10 fM to 10 pM and the assay allowed detection at levels as low as 1.0 fM of the thrombin.The biosensing switch system could apply to the mixed protein samples.
In chapter 6,we further utilized the special solid-state Ru(bpy)_3~(2+)-ECL film to develop solid-state electrochemiluminescence biosensing switch.T4 DNA ligase was chosen as the target herein for exploiting the switch system's application for biosensing ligase.The ligation system was composed of a DNA ligase,two oligos to be ligated and a MB,in which the combined sequences of the two oligos were complementary to the loop sequence of the MB.In the beginning,each oligo was hybridized to one-half of the loop of the MB to form a DNA complex with a nick.This would not open the MB stem completely,but would slightly destabilize the stem.When ligase was added, the ligation reaction closed the nick to form a longer DNA strand that was complementary to the MB,resulting in the MB opening completely and the Fc being pulled away from the electrode.Thus,the difference of ECL intensity before and after the ligation(ΔI_(ECL))could use to quantify the T4 DNA ligase with sensitivity and selectivity and the detection limit was down to 2.5 U/L.
Part four:Ultrasensitive detection of adenosine using solid-state electrochemiluminescent sensing platform based on structure-switching signaling aptamer(Chapter 7)
In chapter 7,in the present study,a solid-state electrochemiluminescent sensing platform based on structures-witching signaling aptamers for highly sensitive detection of small molecules wss developed using adenosine as a model analyte.A gold electrode was first modified with cysteamine and Ru(bpy)_3~(2+)-AuNPs composite.Then, ferrocene-labeled aptamer was assembled onto the modified electrode surface via Au-S interaction.The hybridization event between the ferrocene-labeled aptamer and the complementary ssDNA sequence was evaluated by ECL measurements.Then,the introduction of adenosine triggered structure switching of the aptamer.As a result,the complementary ssDNA sequence forced to dissociate from the sensing interface, resulting in a decrease in ECL intensity.The decrement of ECL signal(ΔI_(ECL))of the two events was proportional to the amount of adenosine.The present sensing system could provide both a wide linear dynamic range(10 nM to 10μM)and a low detection limit(5.0 nM).In addition,high selectivity,good reproducibility,stability,and reusability were achieved.The recovery test demonstrated the feasibility of the designed sensing system for an adenosine assay.
Part five:Enzyme-amplified electrochemiluminescence detection of p53 sequences using MWNTs-Ru(bpy)_3~(2+)aggregates(Chapter 8)
In chapter 8,an effective solid-state electrochemiluminescent biosensor had been developed for the detection of human tumor suppression p53 gene.Positively charged Ru(bpy)_3~(2+)could be immobilized stably on the electrode surface with negatively charged carboxyl of multi-wall carbon nanotubes(COOH-MWNTs)in the form of aggregate via electrostatic interaction.MWNTs-Ru(bpy)_3~(2+)composite and ssDNA labeled by the AuNPs were linked together through pyrrole polymer membrane(PPy). AuNPs were favorable candidates for the immobilization of enzymes because amine groups and cysteine residues in the enzymes were known to bind strongly with AuNPs. Moreover,MWNTs and PPy could act as tiny conduction centers to facilitate the transfer of electrons.Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection,and it displayed wide linear range,high sensitivity and a low detection limit(0.1 pM).The mutant type p53 DNA sequence could be obviously distinguished from the wide type p53 DNA sequence.
随着科学家们逐渐揭开疾病的机理,并将其用于疾病的医疗诊断和治疗之中,对特定序列的DNA,相关蛋白、酶及生物活性小分子的检测日益受到重视。在传统的分析方法中,核酸与蛋白质的研究都采用放射性标记、凝胶电泳和放射性自显影等技术。这些方法过程复杂,周期长,特别是放射性标记,存在放射性污染。而非放射性标记法如荧光、化学发光和生物素标记法,标记过程繁琐、复杂,难以实现自动化且仪器价格昂贵。而生命科学的迅速、深入发展迫切需要新的手段和方法,以更灵敏、更真实、更快速地研究生命过程。
电致化学发光(ECL),是由电化学反应直接或间接引发的化学发光现象,是电化学和化学发光相结合的产物,兼具二者的优点。其方便快捷、灵敏度高、动力学响应范围宽、检出限低、可控性与选择性好、容易实现实时化和集成化,可进行原位检测。集以上优点于一身的电致化学发光技术近几年在分析化学,尤其在生物分析领域的应用引起了人们的极大关注,为生命科学进入到分子水平领域提供了有力的研究手段与方法。
当物质小到纳米数量级时,会产生独特的表面效应、体积效应、量子尺寸效应和宏观量子隧道效应等,其电学、磁学、光学和化学性质也相应地发生显著的变化,呈现出常规材料不具备的优越性能。因此,纳米材料在催化、电子材料、微器件、增强材料及传感器材料等方面有着广阔的应用前景。电化学过程与电极材料的表面性质有关,如果将纳米材料修饰在电极的表面,基于其比表面积大、催化活性高、亲和力强的特性,其能活化电极表面,增大电流响应,降低检测限,大大提高检测的灵敏度,同时最大限度地保持相关生物分子的生物活性。将纳米技术应用于生物活性分子的电致化学发光分析研究,是一个崭新的领域,有利于创新性地建立一些新理论、新技术和新方法。
本论文将纳米技术、生物分子识别元件的特异性及电致化学发光技术的灵敏性相结合,发展具有高灵敏度和高选择性的DNA、蛋白质及酶的新型纳米生物传感器。开拓生物传感器在后基因时代的研究领域,为研究多种生物分子提供新的手段与方法。论文分五个部分,共八章,具体内容如下:
Ⅰ.绪论(第一章)
本章系统阐述了电致化学发光的原理和特点,重点介绍了ECL两大发光体系的反应机理及其在分析中的应用。介绍了DNA与核酸适配体电致化学发光生物传感器的构造原理、分类特点及研究进展。介绍了各种纳米材料在电致化学发光生物传感器中的应用。最后阐述了本论文的目的和意义,指出论文的创新之处及主要研究内容。
Ⅱ.基于Ru(bpy)_3~(2+)-SiO_2 NPs的核酸适配体传感器实现凝血酶蛋白质电致化学发光特异性检测(第二、三章)
第二章基于Ru(bpy)_3~(2+)-SiO_2 NPs的单核酸适配体传感器经目标诱导靶替换实现凝血酶蛋白质特异性检测
利用反相微乳法合成了SiO_2包裹的电致化学发光活性物2,2＇-联吡啶钌(Ru(bpy)_3~(2+))纳米颗粒(Ru(bpy)_3~(2+)-SiO_2 NPs),以此作为DNA标记物,结合替换反应发展了一种简单有效的凝血酶蛋白质电致化学发光特异性检测的方法。首先,在金电极的表面组装凝血酶核酸适配体aptamer,将其与标记有Ru(bpy)_3~(2+)-SiO_2NPs并与aptamer部分序列完全互补的ssDNA探针进行杂交,测得第一个ECL信号(I_(ECL1))。然后将电极与凝血酶蛋白进行培育,凝血酶与aptamer特异性结合,将探针替换下来,此时测得第二个ECL信号(I_(ECL2))。利用前后两个ECL信号的差值△I_(ECL)(I_(ECL1)-I_(ECL2))来定量测定凝血酶。非特异性识别的蛋白不干扰测定,凝血酶在1.0×10~(-14)mol/L～1.0×10~(-11)mol/L范围内有良好的线性响应,检测限为1.0×10~(-15)mol/L(S/N=3,n=11)。方法可应用于实际血浆中凝血酶的检测。
第三章基于Ru(bpy)_3~(2+)-SiO_2 NPs的核酸适配体传感器经三明治传感系统实现凝血酶蛋白质特异性检测
利用凝血酶的两段核酸适配体(aptamerⅠ,aptamerⅡ)与凝血酶的高亲和识别作用结合电致化学发光技术设计了一种具有高灵敏度和高选择性的蛋白质生物传感器。将一段15个碱基的aptamerⅠ通过Au-S键自组装到金电极表面,形成生物识别层,特异性“捕捉”目标蛋白质—凝血酶,进而结合另一段标记有Ru(bpy)_3~(2+)-SiO_2NPs的29个碱基的aptamerⅡ探针构建三明治结构,在含有TPrA的检测液中通过检测Ru(bpy)_3~(2+)的ECL信号对凝血酶进行定量检测。此传感器对非特异性识别的蛋白如牛血清白蛋白、牛血红蛋白不产生ECL响应,凝血酶在2.0×10~(-15)mol/L～2.0×10~(-12)mol/L范围内有良好的线性响应,检测限为1.0×10~(-15)mol/L(S/N=3,n=11)。
Ⅲ.基于二茂铁标记分子灯塔构象变化的可控固相Ru(bpy)_3~(2+)-电致化学发光膜及其特异性识别DNA、蛋白质及连接酶(第四、五和六章)
第四章基于二茂铁标记分子灯塔构象变化的可控固相Ru(bpy)_3~(2+)-电致化学发光膜及其特异性识别DNA的研究
基于二茂铁(Fc)对电致化学发光活性物Ru(bpy)_3~(2+)的ECL的高效猝灭作用发展了一种可控的固相Ru(bpy)_3~(2+)-ECL膜。修饰电极的ECL强度与Fc和固定在电极表面的Ru(bpy)_3~(2+)的距离直接相关,这个距离由标记有Fc的分子灯塔(Fc-MB)的构象控制。我们尝试通过不同的途径改变Fc-MB的构象。Fc-MB与其环部碱基互补的DNA杂交,或者升温将Fc-MB及杂交得到的dsDNA解旋变性,都可以很好的改变Fc-MB的构象,进而改变修饰电极的ECL强度。系统研究了温度诱导二茂铁标记分子灯塔及其系列杂交产物的构象转变过程,成功预言了相关生物分子的熔化温度。为深入研究分子灯塔键合平衡常数和茎环构象的热力学参数提供了一种新思路。同时,此固相Ru(bpy)_3~(2+)-ECL膜可以特异性、高灵敏的测定靶DNA。
第五章基于二茂铁标记分子灯塔适配体的固相电致化学发光传感器特异性识别凝血酶蛋白质
通过纳米金胶将Ru(bpy)_3~(2+)固定,利用二茂铁对Ru(bpy)_3~(2+)的电致化学发光的高效猝灭,结合分子灯塔适配体的特殊茎环结构建立一种高灵敏的固相电致化学发光检测凝血酶的方法。通过凝血酶与灯塔环部核酸适配体的特异性结合来调整分子灯塔适配体的构象,即打开灯塔的茎部互补碱基对,改变Fc与固定在电极上的Ru(bpy)_3~(2+)的距离,通过调整前后修饰电极ECL信号的差值(△I_(ECL))来定量测定凝血酶。非特异性识别的蛋白不干扰测定,凝血酶在1.0×10~(-14)mol/L～1.0×10~(-11)mol/L范围内有良好的线性响应,检测限为1.0×10~(-15)mol/L(S/N=3,n=11),并应用于混合蛋白样品的检测。
第六章基于二茂铁标记分子灯塔的固相电致化学发光传感器特异性识别T4DNA连接酶
利用二茂铁对Ru(bpy)_3~(2+)的电致化学发光的高效猝灭,结合分子灯塔的特殊茎环结构及核酸连接的模板建立一种高灵敏的固相电致化学发光检测T4 DNA连接酶的方法。通过T4 DNA连接酶连接修复与Fc标记的分子灯塔(Fc-MB)两端环部碱基完全互补的两条短链DNA,形成与Fc-MB完全互补的长链DNA,将Fc-MB的茎部互补碱基对彻底打开,改变Fc与固定在电极上的Ru(bpy)_3~(2+)的距离,引起修饰电极的ECL信号的改变,通过前后ECL信号的差值(△I_(ECL))来定量测定T4DNA连接酶。该酶传感器具有高的灵敏度和选择性,对T4 DNA连接酶在5.0×10~(-3)U/μL～5.0×10~(-6)U/μL范围内有良好的线性响应,检测限为2.5×10~(-6)U/μL(S/N=3,n=11)。
Ⅳ.基于核酸适配体构象转换应用固相电致化学发光传感平台超灵敏检测腺苷(第七章)
基于腺苷-核酸适配体构象转换机制研制出一种高灵敏的固相电致化学发光检测腺苷的新型传感系统。把对Ru(bpy)_3~(2+)的电致化学发光有高效猝灭作用的二茂铁分子标记在腺苷-核酸适配体上,腺苷-核酸适配体与腺苷特异性结合后,其构象会发生变化,引起标记物Fc与Ru(bpy)_3~(2+)的距离变化,进而使得修饰电极的ECL信号发生变化,通过腺苷-核酸适配体构象转换前后修饰电极的ECL信号的差值来定量测定小分子腺苷。非特异性识别的其它小分子不干扰测定,腺苷在1.0×10~(-8)mol/L～1.0×10~(-5)mol/L范围内有良好的线性响应,检测限为5.0×10~(-9)mol/L(S/N=3,n=11)。回收试验结果满意。简易、快速、低耗及良好的响应性能使本传感系统在构建基于核酸适配体传感器检测小分子的研究方法中体现出较好的发展前景。
Ⅴ.应用MWNTs-Ru(bpy)_3~(2+)混聚体经酶放大电致化学发光特异性识别p53基因(第八章)
尝试通过多壁羧基化的碳纳米管(COOH-MWNTs)将Ru(bpy)_3~(2+)固定,经吡咯膜(PPy)把MWNTs-Ru(bpy)_3~(2+)混聚体与纳米金胶标记的DNA分析物相连,将葡萄糖脱氢酶参与的专一性的酶促反应与高灵敏的电致化学发光反应偶联,建立一种直接、准确的固相电致化学发光检测人类p53肿瘤抑制基因的方法。用此法检测目标p53野生型序列,检测限为1.0×10~(-13)mol/L,在相同条件下,单碱基错配的p53突变型序列和p53野生型序列的区分度达56.3%,其特异性、低灵敏度的检测为癌症的早期诊断提供了可能。
Nucleic acid,protein and enzyme are the most important biomolecules in life. Nucleic acid is in charge of transferring genetic information and coding other biomolecules,while protein has run through the whole life process.Enzyme plays an important role in DNA synthesis and maintaining the integrity of genome.The replication,repair and recombination of nucleic acids are essential life processes. DNA/RNA structure fragment,as "bricks" of the biomacromolecules,is an important participant in the activities of life and is an important signal transduction of cells.The completion of the related function of gene needs the cooperation of the protein, enzyme and the small bioactivity molecules.Actually,the matching and the effect between these biomolecules construct the basic of the vital phenomena,such as growth, generation and metabolism.The study of functional gene,protein and the related bioactivity molecules is the hot spots of postgenome era.
With the increasing knowledge about human diseases,the specific sequence of DNA,correlative protein,enzyme and small bioactivity molecule detection received increasing attention.Many detection techniques of DNA sequence and protein have been developed in recent years,but there is still a lot of dissatisfaction.The radioactive labels present many problems such as a potential hazard to analyst and environment. Non-radioactive labels such as fluorescent,chemiluminescent and biotin-avidin label probes also present many shortcomings such as low sensitivity or complex equipment or others.So it is necessary to develop another method for the more sensitive, easy-to-use,fast,inexpensive detection of correlative bioactivity molecules to adapt to wide-scale genetic and protein testing requires.
Electrochemiluminescence(ECL),the generation of an optical signal triggered by an electrochemical reaction,has attracted much attention during the past several decades due to its versatility,simplified optical setup,very low background signal, good temporal and spatial control,and has become an important and valuable technique for immunosensing,DNA hybridization assays and proteinic biosensors.It also provides a powerful research tools and methods for the life sciences entering into the molecular level field.
Today,nano-science and technology has entered the limelight and has been investigated extensively by governments and scientists all over the world.It is out of question that the revolution of nanotechnology is coming.In recent work,it has been discovered that materials in the nano-size scale(1～100 nm)display size-dependent optical,magnetic,electronic and chemical properties.Except for these,nanomaterials have unknown chemical and physical properties that differ greatly from the bulk substances.Therefore,nanoparticles can be applied to many fields,such as optical devices,electronic devices,catalysis,sensor technology,and biomolecular labeling, etc.,which is also the reason that a burst of research activities have been focused on nanoparticles.Modified electrodes based on nanomaterials combined with high surface area and good electrocatalytic abilities that can largely improve electrical responses and the detection sensitivity.At the same time,the bioactivity of the biomolecules can be remained preferably.Today,many wonderful nanostructured materials have been applied in electroanalytical chemistry and some important progresses along these topics have recently achieved.
The goal of the present study is to design and research novel DNA,protein and enzyme nano-biosensor with high sensitivity and selectivity.This paper combines the excellent characteristics of nanoparticles,the specific recognition of molecular recognition elements and the electrochemiluminescence technique.It would broaden the research field of post-genetic time.This paper altogether divided into five parts. The details are given as follows:
Part one:Preface(Chapter 1)
In this chapter,we elaborated review from the principles,characteristics of the electrochemiluminescence,the two most primary types of ECL reaction and their application in analytical chemistry field.The principles,characteristics and the research progress of DNA and the aptamer electrochemiluminescent biosensor all had been introducded.Nanoparticles,especially the application of the variety of nanomaterials in the electrochemiluminescent biosensor were presented.Finally, expounded the aim and the significance,pointed out the research content and the innovation in this paper.
Part two:Detection of thrombin using electrochemiluminescence based on Ru(bpy)_3~(2+)-doped silica nanoparticle aptasensor(Chapter 2,3)
Tri(2,2'-bipyridyl)ruthenium(Ⅱ)-doped silica nanoparticles(Ru(bpy)_3~(2+)-SiO_2 NPs) were prepared by water-in-oil(W/O)microemulsion method.A great deal of Ru(bpy)_3~(2+)was immobilized inside the nanoparticle,which could greatly enhance the ECL response and result in the increased sensitivity.
In chapter 2,sensitive and selective aptasensor using Ru(bpy)_3~(2+)-SiO_2 NPs as DNA tags for detection of thrombin was developed based on the target protein-induced strand displacement of the DNA probe.For the proposed aptasensor,the aptamer was assembled on the surface of the Au electrode through Au-S binding.The hybridization event between the DNA probe labeled by the Ru(bpy)_3~(2+)-SiO_2 NPs and the aptamer was evaluated by ECL measurements.Then,the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again.The difference of ECL intensity(ΔI_(ECL))of the two events could be used to quantify the thrombin.Other proteins,such as bovine serum albumin and bovine hemoglobin,had almost negligibleΔI_(ECL).Under the optimal conditions,theΔI_(ECL)was linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)_3~(2+)molecules were labeled on the DNA probe.
In chapter 3,a new electrochemiluminescent detection system for protein using the aptamers was developed.Two different aptamers,which recognize different positions of thrombin,were chosen to construct sandwich type sensing system for protein,and one was immobilized onto the gold electrode for capturing thrombin onto the electrode and the other was used for detection:To obtain the signal,the aptamer for detection was labeled with Ru(bpy)_3~(2+)-SiO_2 NPs.The increase of the ECL signal generated by Ru(bpy)_3~(2+)-SiO_2 NPs was observed in dependent manner on the concentration of thrombin added.Bovine serum albumin and bovine hemoglobin had almost negligible responses.The ECL signal was linearly related to the concentration of the thrombin anatyte in the range of 2.0 fM to 2.0 pM and the assay allowed detection at levels as low as 1.0 fM of the thrombin.
Part three:A controllable solid-state Ru(bpy)_3~(2+)-electrochemiluminescence film for detection of biomolecules based on conformation change of ferrocene-labeled molecular beacon(Chapter 4,5 and 6)
In chapter 4,a controllable solid-state electrochemiluminescence film based on efficient and stable quenching of ECL of Ru(bpy)_3~(2+)by oxidizing ferrocene(Fc)at the electrode was developed.The ECL intensity was correlated to the distance which was controlled by the conformation of the ferrocene-labeled DNA molecular beacon (Fc-MB)between the Fc and Ru(bpy)_3~(2+)immobilized on the electrode.The conformation adjustment was conducted via complementary DNA's hybridizing with the bases in the loop of the Fc-MB and changing the temperature of the Fc-MB and the resultant double strand DNA(dsDNA).Those events all resulted in change of the ECL intensity.Therefore,the melting temperature of the relevant biomolecules was predicted successfully.With such characteristics,the solid-state Ru(bpy)_3~(2+)-ECL film had the potential to calculate thermodynamic parameters of equilibrium constants of MB binding and the stem-loop formation.Such controllable solid-state Ru(bpy)_3~(2+)-ECL film could detect target ssDNA,and the analysis results were sensitive and specific.
In chapter 5,we utilized the special solid-state Ru(bpy)_3~(2+)-ECL film to develop solid-state electrochemiluminescence biosensing switch.Thrombin as one important physiological protease in blood had been chosen as the target for investigating the application of the biosensing switch system to special protein.Herein,the loop bases of the Fc-MB were designed with the specific anti-thrombin aptamer sequence,which was expected to transform into G-quartet structure to combine with thrombin. Consequently,the Fc was far away the electrode surface and the ECL intensity was increased significantly.The enhanced ECL signal was expected to quantify the thrombin.Other proteins,such as bovine serum albumin and bovine hemoglobin,had almost negligibleΔI_(ECL).TheΔI_(ECL)was linearly related to the concentration of the thrombin analyte in the range of 10 fM to 10 pM and the assay allowed detection at levels as low as 1.0 fM of the thrombin.The biosensing switch system could apply to the mixed protein samples.
In chapter 6,we further utilized the special solid-state Ru(bpy)_3~(2+)-ECL film to develop solid-state electrochemiluminescence biosensing switch.T4 DNA ligase was chosen as the target herein for exploiting the switch system's application for biosensing ligase.The ligation system was composed of a DNA ligase,two oligos to be ligated and a MB,in which the combined sequences of the two oligos were complementary to the loop sequence of the MB.In the beginning,each oligo was hybridized to one-half of the loop of the MB to form a DNA complex with a nick.This would not open the MB stem completely,but would slightly destabilize the stem.When ligase was added, the ligation reaction closed the nick to form a longer DNA strand that was complementary to the MB,resulting in the MB opening completely and the Fc being pulled away from the electrode.Thus,the difference of ECL intensity before and after the ligation(ΔI_(ECL))could use to quantify the T4 DNA ligase with sensitivity and selectivity and the detection limit was down to 2.5 U/L.
Part four:Ultrasensitive detection of adenosine using solid-state electrochemiluminescent sensing platform based on structure-switching signaling aptamer(Chapter 7)
In chapter 7,in the present study,a solid-state electrochemiluminescent sensing platform based on structures-witching signaling aptamers for highly sensitive detection of small molecules wss developed using adenosine as a model analyte.A gold electrode was first modified with cysteamine and Ru(bpy)_3~(2+)-AuNPs composite.Then, ferrocene-labeled aptamer was assembled onto the modified electrode surface via Au-S interaction.The hybridization event between the ferrocene-labeled aptamer and the complementary ssDNA sequence was evaluated by ECL measurements.Then,the introduction of adenosine triggered structure switching of the aptamer.As a result,the complementary ssDNA sequence forced to dissociate from the sensing interface, resulting in a decrease in ECL intensity.The decrement of ECL signal(ΔI_(ECL))of the two events was proportional to the amount of adenosine.The present sensing system could provide both a wide linear dynamic range(10 nM to 10μM)and a low detection limit(5.0 nM).In addition,high selectivity,good reproducibility,stability,and reusability were achieved.The recovery test demonstrated the feasibility of the designed sensing system for an adenosine assay.
Part five:Enzyme-amplified electrochemiluminescence detection of p53 sequences using MWNTs-Ru(bpy)_3~(2+)aggregates(Chapter 8)
In chapter 8,an effective solid-state electrochemiluminescent biosensor had been developed for the detection of human tumor suppression p53 gene.Positively charged Ru(bpy)_3~(2+)could be immobilized stably on the electrode surface with negatively charged carboxyl of multi-wall carbon nanotubes(COOH-MWNTs)in the form of aggregate via electrostatic interaction.MWNTs-Ru(bpy)_3~(2+)composite and ssDNA labeled by the AuNPs were linked together through pyrrole polymer membrane(PPy). AuNPs were favorable candidates for the immobilization of enzymes because amine groups and cysteine residues in the enzymes were known to bind strongly with AuNPs. Moreover,MWNTs and PPy could act as tiny conduction centers to facilitate the transfer of electrons.Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection,and it displayed wide linear range,high sensitivity and a low detection limit(0.1 pM).The mutant type p53 DNA sequence could be obviously distinguished from the wide type p53 DNA sequence.
引文
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