基于核酸适配体化学发光检测新技术的研究
摘要
核酸适配体是近年来发展起来的一类经体外人工合成筛选出的单链寡核苷酸,能高效、特异性地结合各种生物目标分子,故它的出现为化学生物学界和生物医学界提供了一种新的高效快速识别的研究平台。目前生物分子检测通常采用抗原抗体特异相互作用识别模式,但由于受到抗体易失活、制备时间较长等因素的影响,在一定程度上限制了抗体检测技术的广泛应用。相比之下,核酸适配体自身稳定性好、制备合成相对简单、快速、易获得、易功能化修饰与标记,且在生物传感器设计中应用灵活等优点,近几年在生物分析检测方面备受关注。目前已经成为临床诊断、环境监测、药学研究等许多领域中的研究热点。
化学发光(CL)分析法具有不需光源,避免了杂散光的干扰,仪器设备简单、操作简便,具有极高的灵敏度,较宽的检测范围,可实现全自动化等特点,正逐渐成为分析检测中极为有用的工具,随着与众多学科交叉研究和应用领域的扩展,目前已成功地应用在药学、生物学、分子生物学、临床医学和环境学等诸多领域。在本论文中,我们采用化学发光分析法,利用核酸适配体对目标分子的高分辨识别,发展了多种具有创新意义的化学发光适配体生物传感器,也实现了同一份样品中双组分的同时检测。整个论文由以下五部分构成:
第一章:绪论
本绪论由两节构成,第一节介绍了核酸适配体技术检测生物分子的研究进展,包括了三部分。第一部分中简单介绍了核酸适配体的制备、特点、优势以及在分析领域中的应用;第二部分中介绍了基于核酸适配体识别模式的单组分检测技术的研究进展及其意义,主要内容包括:光检测、电化学检测以及其他检测方法,并列举了近年来分析领域中的部分典型示例;第三部分中介绍了基于核酸适配体识别模式的多组分检测技术的研究进展及其意义,也列举了近年来它们在该分析领域中的部分典型示例。第二节阐述了化学发光多组分酶检测研究进展以及本课题研究的目的、意义、主要研究内容以及创新之处,即核酸适配体在化学发光领域中应用与展望。
第二章:基于核酸适配体的化学发光无标记检测腺苷的新技术
由于目标分子在适配体上精确的结合位点与构象变化通常并不十分清楚,直接导致合适标记核酸适配体存在一定的难度,因此,适配体的无标记型检测技术已成为近年来的研究热点,尤其在生物检测、环境监控等领域无标记简单快速检测具有非常重要的意义。本章以腺苷为研究对象,采用羧基修饰的磁性微球作为分离载体,基于3,4,5-三甲氧基苯甲酰甲醛(TMPG)与鸟嘌呤(G)碱基之间的瞬时化学发光衍生反应,实现了生物小分子腺苷的无标记检测。本章包括以下两种腺苷检测原理的设计,具体实验步骤如下:(1)活化磁性微球,固定捕获探针序列;(2)方法A:一定量的适配体先与不同量的腺苷特异性结合,随后剩余的自由腺苷适配体与捕获探针序列在磁性微球表面进行杂交反应,从而连接在磁性微球上;方法B:适配体先与捕获探针序列进行杂交反应,随后加入不同量的腺苷,导致部分适配体序列脱离磁性微球表面,与溶液中腺苷形成复合物;(3)磁性分离后,TMPG直接检测结合在磁性微球表面的适配体中G碱基产生的CL信号,进行腺苷间接定量。结果表明:该两种方法均具有准确可靠、重现性和选择性好的特点。第一种方法的最低腺苷检测限为8×10~(-8)M,腺苷浓度在4×10~(-7)-1×10_(-5)M范围内,CL信号呈线性增加(R~2=0.9852);第二种方法的腺苷浓度在4×10~(-2)5×10(_5)M范围内,CL信号呈线性增加(R~=0.9764)。综合而言:本章发展的无标记检测生物小分子腺苷的CL新技术,具有简单,快速,灵敏度高等特点,有望在临床诊断、药学研究以及环境监测等领域发挥作用。
第三章:基于核酸适配体的无标记化学发光检测PDGF-BB的新技术
血小板源细胞衍生化生长因子(PDGF)是血清中由多种细胞所产生的能刺激增生平滑肌细胞、胶质细胞等的一种多肽,具有广泛的生理活性。PDGF-BB作为PDGF的主要亚型之一,近年来研究发现其含量对细胞转化和肿瘤生长有直接的影响,对PDGF-BB的检测在生物学上具有重要的意义。本章构建了一种基于抗体-抗原-核酸适配体的夹心反应模式,检测PDGF-BB的化学发光新技术。检测步骤如下:通过羧基氨基反应将PDGF-BB抗体固定在羧基磁性微球上,加入PDGF-BB、核酸适配体,形成抗体-抗原-核酸适配体的夹心复合物。洗涤后,利用TMPG直接检测磁性微球表面结合的PDGF-BB适配体上G碱基,无标记直接检测生物大分子PDGF-BB。随后,在此基础上构建了G_(20)放大检测技术,即在生物素化的适配体序列一端连接富含G碱基的DNA片段,借助链霉亲合素作为放大载体,构建了一种高效的放大检测技术。不放大技术的最低可检测浓度为1×10~(-8)M,目标蛋白浓度在1×10~(-8)-2×10_(-7)M范围内,CL信号呈线性增加(R~2=0.9901);G_(20)放大技术最低可检测浓度为4×10~(-10)M,较不放大技术灵敏度提高25倍,目标蛋白浓度在4×10~(-10)-2×10~(-8)M范围内,CL信号呈线性增加(R~2=0.9954),重现性良好,操作简便,成本低,适合进一步拓展。综上所述,此种无标记PDGF-BB化学发光检测技术,有望为临床应用领域的定量分析提供有价值的手段。
第四章:基于适配体特异性结合与磁性微球富集的化学发光检测ATP新技术
免疫磁性微球富集法是一种高效、简单快速的筛选方法。在从复杂样品中富集和纯化低含量的样品方面,免疫磁性微球具有独特的优势:一,它的不规则形状使其拥有较大的比表面积,富集效率高,磁性微球表面结合探针数目多,能偶合大量的目标物;二,优质的磁性微球具有高度的均一性和良好的顺磁性,在与复杂的生物样品反应时受到颗粒性杂质等的影响较小;三,移液器吸去没有反应的生物分子时,由于表面吸附原因,看似吸干的磁性微球上存有少量的液体,这有利于在操作过程中暂时保护生物分子的活性。本章我们在免疫磁性微球载体表面化学偶联了三磷酸腺苷(ATP)核酸适配体,这种磁性微球能够在混合体系中特异性捕获ATP分子,用CL方法测定免疫磁性微球特异性捕获目标分子的富集效果。结果表明磁性微球均能特异性富集目标分子,纯化分离ATP分子,并实现对其的准确可靠分析,其最低检测限1×10~(-8)M,在1×10~(-8)-1×10~(-5)M范围内,CL信号和浓度有良好的线性关系(R~2=0.9918)。综合而言,免疫磁性微球高效的富集性和适配体高度的特异性,在生物分子的纯化分离和检测工作中,将会显示出良好的开发应用前景。
第五章:基于核酸适配体的双组分化学发光生物传感器新技术
鉴于第二章中腺苷检测技术取得的成果,本章尝试将这种新型的适配体分辨CL技术拓宽至两种小分子的同时检测。为此,本章以腺苷和可卡因作为分析物,采用碱性磷酸酯酶(ALP)和辣根过氧化物酶(HRP)作为双组分标记物,实现了两种小分子的同时检测。整个分析过程由以下三步构成:(1)以羧基修饰的磁性微球作为载体,活化后固定捕获探针序列,随后分别与两条核酸适配体序列杂交,再与生物素和地高辛修饰的两条报告序列反应,制备两种不同的核酸适配体生物传感器;(2)加入目标分子腺苷和可卡因混合物,由于适配体和目标分子的结合反应,导致部分报告序列脱离磁性微球表面;(3)依据两种标记物的性质差异加入相应的酶进行反应,结束后分离洗涤,将磁性微球载体均分两份,用相应的酶底物试剂盒进行检测。研究结果表明:该法腺苷和可卡因的最低检测浓度均达到10~(-8)M。综合而言,该实验利用核酸适配体差异分辨,进行单载体、双标记实现两种小分子腺苷和可卡因的同时检测;具有仪器设备简单、操作简便的特点,有望在多组分生物小分子的分析测定等方面发挥重要的作用。
DNA aptamers are synthetic single-strand nucleic acids discriminated by screening in vitro,which are of high affinity and specificity to many given targets ranging from small molecules to large proteins and even cells.Some new methods for efficient and fast detection in biochemical and biomedical fields are being developed based on the molecular recognition of aptamers.Aptamers have been demonstrated to have advantages over antibodies with regard to chemical stability,readily availability, simple modifiability,and high flexibility in biosensor design for analyte detection.As a novel functional molecular,aptamers have gained considerable interest in bio-analytical application in many fields such as clinical diagnosis,environmental supervision,pharmacal study,etc.
Chemiluminescence(CL)has been exploited within a wide range of applications in various fields,due to their extremely high sensitivity along with their extra advantages such as needless special light,simple instrument,wide calibration ranges, and suitability for automatical operation in analytical chemistry.Nowadays,based on cross-research with other disciplines,CL has been successfully served for different applications.Our study developed a series of novel detection technologies on the strength of the high specifically binding between aptamers and its corresponding ligands in the thesis.And simultaneous detection of two molecules in one sample has been made grounded on aptamer switch.Description of research in the thesis is presented as follows:
Chapter 1:Introduction
This chapter is composed of two phases.The first phase addresses the source,traits, and advantages of aptamers and outlines aptamer-based detection techniques,then presents single analysis of current studies based on aptamer in detail,including optical aptasensors,electrochemical aptasensors,and other mass sensitive aptasensors. Following that,the multiplex analysis has been also stated in the development of aptamer-based biosensors and bioassay methods.The second phase of this chapter reviews current simultaneous multi-analytes detection by substrate-resolved CL technology exampled HRP and ALP in assay field.With that,objectives,significance, contents of this CL research based on aptamer are summarized.
Chapter 2:Label-free aptamer-based chemiluminescence detection of adenosine
Owing to no elucidation for the binding property of aptamer,it has difficult to label on aptamer and the label process would affect the binding affinity between the targets and their aptamers to a greater or lesser degree.Consequently,some label-free detection methods have been focused to develop,especially in some fileds like bioassay and environmental supervision.In this chapter,employing magnetic bead as a special biomolecule separating carrier,we demonstrate a sensitive CL aptasensor for label-free adenosine assay based on 3,4,5-trimethoxyl-phenylglyoxal(TMPG)as the signal molecule,which reacts specially and instantaneously with guanine nucleobases of adenosine-binding aptamer strands.We designed two different experimental routes for assay and the sketchy experiment steps were described as follows.(1)The magnetic beads were activated and the capture DNA immobilized on magnetic beads by the force of NH_2-COOH covalent bind.(2)Route A:The excess and quantitative adenosine aptamers reacted with different amounts of adenosine and formed an adenosine-aptamer complex.Then the rest free aptamer,along with the complex,were hybridized with capture DNA immobiled on magnetic beads.Route B:the aptamers first hybridized with capture DNA-magnetic bead conjugates and then reacted with adenosine.When adenosine as a specific competitor appeared,parts of aptamer were compelled to take apart from the magnetic beads and formed an adenosine-aptamer complex.(3)After speration by magnetic force,The CL signal was gotten from the raction between TMPG and guanine nucleobases of aptamer strands on magnetic beads.Using this system,adenosine could be specifically detected and a relatively low detection limit(8×10~(-8)M)as well as a wide linear dynamic range(4×10~(-7)-1×10~(-5) M)could be achieved with route A(R~2=0.9852).With route B,adenosine can be detected in a shorter range from 4×10~(-7)M to 5×10~(-5)M(R~2=0.9764).Overall,the label-free protocol described here may have value in a variety of clinical,pharmacal and environmental applications for which the simple,fast and accurate quantitative analysis of biomolecular adenosine is desired.
Chapter 3:Label-free aptamer-based chemiluminescence detection of PDGF-BB
Platelet-derived growth factor(PDGF)is a ubiquitous mitogen and chemotactic polypeptide present in serum for stimulating cells of mesenchymal origin and elicits multifunctional actions with a variety of cells.PDGF-BB,one of the important isoforms of PDGF,has been directly implicated in the cell transformation process and in tumor growth and progression.And the detection of PDGF-BB is of very valuable in biological fields.In this chapter,a“sandwich-type”detection strategy was employed in our design to make PDGF-BB detection.The procedure was as follows. Anti-PDGF-BB antibody immobilized on the surface of carboxyl terminated magnetic beads and DNA aptamers,both of which flanked PDGF-BB.After washing,the CL signal was obtained via the instantaneous derivatization reaction between TMPG and guanines nucleotides of aptamer to detect target protein without any label procedure. On this ground,an amplified CL approach was investigated employing G-rich sequence linked with aptamer as the amplification unit.The streptavidin was employed as an amplification platform to develop a high effective CL approach. Nonamplified approach achieved a good linear correlation from 1×10~(-8)to 2×10~(-7)M with the lowest detection concentration(1×10~(-8)M)and correlation coefficient (R~2=0.9901).In amplified approach,an excellent linearity was found within the range of 4×l0~(-10)-2×10~(-8)M(R~2=0.9954)with the lowest detection concentration of 4×10~(10), which was 25-fold improvement on nonamplified methods.The result demonstrated that this assay was reproducible,speedy,easy to use and suitable to make further development.In a word,this novel lable-free CL approach provided great promise for PDGF-BB assay in clinical field.
Chapter 4:ATP CL determination coupled the enrichment of magnetic beads with the specific binding of aptamer
The enrichment of immuno-magnetic beads is one of simple,speedy and high efficient screening methods.For one complicate sample with trace quantities of targets,it has unique merits following as:First,The irregular shape of the magnetic beads allows for much greater surface area.The large surface area results in high binding capacities,allowing much more targets captured with increasing capture probes on magnetic beads.Second,magnetic beads with high quality have great homogenicity and paramagnetism,which guarantee smoothly reaction with biomolecular without any intervention from any particle impurity.Third,Most of the rinsing solution was removed by pipet,but some buffer liquid(~3μL)remained around the magnetic beads because of the force of superficial adsorption.It was almost impossible to dry the sample completely and this benefited the prolonged active of biomolecular.Greater magnetic responsiveness results in faster magnetic separations especially on high throughput automated platforms.In this chapter,we employed adenosine triphosphate(ATP)aptamer as capture probe to covalently bind on magnetic beads.ATP present in trace quantities were enriched from the mixed sample on the magnetic beads and could be rapidly isolated from the sample by employing an external magnetic field in order to make CL detection.Experimental results confirm that it has good sensitivity with a detection limit of 1×10~(-8)M for ATP. ATP could be specifically isolated and detected and a wide linear dynamic range (1×10~(-8)-1×10~(-5)M)could be achieved(R~2=0.9918).On the whole,by using magnetic beads with great enrichment and aptamer with high discrimination,this method may show good perceptive on the purification and detection of biomolecular.
Chapter 5:Simultaneous detection founded on sptasensors by CL technology
A novel aptamer-based CL biosensing platform for the simultaneous detection of two small molecules as exemplified by adenosine and cocaine has been developed. Herein,we employed magnetic bead as the special biomolecule immobilizing carrier and made CL detection catalyzed by two labeled enzymes of alkaline phosphatase (ALP)and horseradish peroxidase(HRP).The principles of homogeneous substrate-resolved technology for CL detection have three steps:(1)preparation of adenosine and cocain aptamer-linked biosensors based on magnetic beads as carriers; (2)binding with a mixture of adenosine and cocaine to initiate target-aptamer reactions;(3)reacting with a mixed solution of Digoxin-ALP and Strepavidin-HRP followed by magnetic force separation of the magnetic beads conjugates and parallel detection of CL signals from HRP and ALP with their corresponding assay kits. Experimental results confirm that this CL immunosensing platform has good sensitivity with the lowest detection concentration of 1×10~(-8)M for adenosine and cocaine.On the whole,by using different aptamers,this method may offer a new direction in designing high-performance CL aptasensors for sensitive simultaneous determination of small molecules.And the technique would play an important role in multiplex analysis for biomoleculars with simple instrument and convenient procedure.
化学发光(CL)分析法具有不需光源,避免了杂散光的干扰,仪器设备简单、操作简便,具有极高的灵敏度,较宽的检测范围,可实现全自动化等特点,正逐渐成为分析检测中极为有用的工具,随着与众多学科交叉研究和应用领域的扩展,目前已成功地应用在药学、生物学、分子生物学、临床医学和环境学等诸多领域。在本论文中,我们采用化学发光分析法,利用核酸适配体对目标分子的高分辨识别,发展了多种具有创新意义的化学发光适配体生物传感器,也实现了同一份样品中双组分的同时检测。整个论文由以下五部分构成:
第一章:绪论
本绪论由两节构成,第一节介绍了核酸适配体技术检测生物分子的研究进展,包括了三部分。第一部分中简单介绍了核酸适配体的制备、特点、优势以及在分析领域中的应用;第二部分中介绍了基于核酸适配体识别模式的单组分检测技术的研究进展及其意义,主要内容包括:光检测、电化学检测以及其他检测方法,并列举了近年来分析领域中的部分典型示例;第三部分中介绍了基于核酸适配体识别模式的多组分检测技术的研究进展及其意义,也列举了近年来它们在该分析领域中的部分典型示例。第二节阐述了化学发光多组分酶检测研究进展以及本课题研究的目的、意义、主要研究内容以及创新之处,即核酸适配体在化学发光领域中应用与展望。
第二章:基于核酸适配体的化学发光无标记检测腺苷的新技术
由于目标分子在适配体上精确的结合位点与构象变化通常并不十分清楚,直接导致合适标记核酸适配体存在一定的难度,因此,适配体的无标记型检测技术已成为近年来的研究热点,尤其在生物检测、环境监控等领域无标记简单快速检测具有非常重要的意义。本章以腺苷为研究对象,采用羧基修饰的磁性微球作为分离载体,基于3,4,5-三甲氧基苯甲酰甲醛(TMPG)与鸟嘌呤(G)碱基之间的瞬时化学发光衍生反应,实现了生物小分子腺苷的无标记检测。本章包括以下两种腺苷检测原理的设计,具体实验步骤如下:(1)活化磁性微球,固定捕获探针序列;(2)方法A:一定量的适配体先与不同量的腺苷特异性结合,随后剩余的自由腺苷适配体与捕获探针序列在磁性微球表面进行杂交反应,从而连接在磁性微球上;方法B:适配体先与捕获探针序列进行杂交反应,随后加入不同量的腺苷,导致部分适配体序列脱离磁性微球表面,与溶液中腺苷形成复合物;(3)磁性分离后,TMPG直接检测结合在磁性微球表面的适配体中G碱基产生的CL信号,进行腺苷间接定量。结果表明:该两种方法均具有准确可靠、重现性和选择性好的特点。第一种方法的最低腺苷检测限为8×10~(-8)M,腺苷浓度在4×10~(-7)-1×10_(-5)M范围内,CL信号呈线性增加(R~2=0.9852);第二种方法的腺苷浓度在4×10~(-2)5×10(_5)M范围内,CL信号呈线性增加(R~=0.9764)。综合而言:本章发展的无标记检测生物小分子腺苷的CL新技术,具有简单,快速,灵敏度高等特点,有望在临床诊断、药学研究以及环境监测等领域发挥作用。
第三章:基于核酸适配体的无标记化学发光检测PDGF-BB的新技术
血小板源细胞衍生化生长因子(PDGF)是血清中由多种细胞所产生的能刺激增生平滑肌细胞、胶质细胞等的一种多肽,具有广泛的生理活性。PDGF-BB作为PDGF的主要亚型之一,近年来研究发现其含量对细胞转化和肿瘤生长有直接的影响,对PDGF-BB的检测在生物学上具有重要的意义。本章构建了一种基于抗体-抗原-核酸适配体的夹心反应模式,检测PDGF-BB的化学发光新技术。检测步骤如下:通过羧基氨基反应将PDGF-BB抗体固定在羧基磁性微球上,加入PDGF-BB、核酸适配体,形成抗体-抗原-核酸适配体的夹心复合物。洗涤后,利用TMPG直接检测磁性微球表面结合的PDGF-BB适配体上G碱基,无标记直接检测生物大分子PDGF-BB。随后,在此基础上构建了G_(20)放大检测技术,即在生物素化的适配体序列一端连接富含G碱基的DNA片段,借助链霉亲合素作为放大载体,构建了一种高效的放大检测技术。不放大技术的最低可检测浓度为1×10~(-8)M,目标蛋白浓度在1×10~(-8)-2×10_(-7)M范围内,CL信号呈线性增加(R~2=0.9901);G_(20)放大技术最低可检测浓度为4×10~(-10)M,较不放大技术灵敏度提高25倍,目标蛋白浓度在4×10~(-10)-2×10~(-8)M范围内,CL信号呈线性增加(R~2=0.9954),重现性良好,操作简便,成本低,适合进一步拓展。综上所述,此种无标记PDGF-BB化学发光检测技术,有望为临床应用领域的定量分析提供有价值的手段。
第四章:基于适配体特异性结合与磁性微球富集的化学发光检测ATP新技术
免疫磁性微球富集法是一种高效、简单快速的筛选方法。在从复杂样品中富集和纯化低含量的样品方面,免疫磁性微球具有独特的优势:一,它的不规则形状使其拥有较大的比表面积,富集效率高,磁性微球表面结合探针数目多,能偶合大量的目标物;二,优质的磁性微球具有高度的均一性和良好的顺磁性,在与复杂的生物样品反应时受到颗粒性杂质等的影响较小;三,移液器吸去没有反应的生物分子时,由于表面吸附原因,看似吸干的磁性微球上存有少量的液体,这有利于在操作过程中暂时保护生物分子的活性。本章我们在免疫磁性微球载体表面化学偶联了三磷酸腺苷(ATP)核酸适配体,这种磁性微球能够在混合体系中特异性捕获ATP分子,用CL方法测定免疫磁性微球特异性捕获目标分子的富集效果。结果表明磁性微球均能特异性富集目标分子,纯化分离ATP分子,并实现对其的准确可靠分析,其最低检测限1×10~(-8)M,在1×10~(-8)-1×10~(-5)M范围内,CL信号和浓度有良好的线性关系(R~2=0.9918)。综合而言,免疫磁性微球高效的富集性和适配体高度的特异性,在生物分子的纯化分离和检测工作中,将会显示出良好的开发应用前景。
第五章:基于核酸适配体的双组分化学发光生物传感器新技术
鉴于第二章中腺苷检测技术取得的成果,本章尝试将这种新型的适配体分辨CL技术拓宽至两种小分子的同时检测。为此,本章以腺苷和可卡因作为分析物,采用碱性磷酸酯酶(ALP)和辣根过氧化物酶(HRP)作为双组分标记物,实现了两种小分子的同时检测。整个分析过程由以下三步构成:(1)以羧基修饰的磁性微球作为载体,活化后固定捕获探针序列,随后分别与两条核酸适配体序列杂交,再与生物素和地高辛修饰的两条报告序列反应,制备两种不同的核酸适配体生物传感器;(2)加入目标分子腺苷和可卡因混合物,由于适配体和目标分子的结合反应,导致部分报告序列脱离磁性微球表面;(3)依据两种标记物的性质差异加入相应的酶进行反应,结束后分离洗涤,将磁性微球载体均分两份,用相应的酶底物试剂盒进行检测。研究结果表明:该法腺苷和可卡因的最低检测浓度均达到10~(-8)M。综合而言,该实验利用核酸适配体差异分辨,进行单载体、双标记实现两种小分子腺苷和可卡因的同时检测;具有仪器设备简单、操作简便的特点,有望在多组分生物小分子的分析测定等方面发挥重要的作用。
DNA aptamers are synthetic single-strand nucleic acids discriminated by screening in vitro,which are of high affinity and specificity to many given targets ranging from small molecules to large proteins and even cells.Some new methods for efficient and fast detection in biochemical and biomedical fields are being developed based on the molecular recognition of aptamers.Aptamers have been demonstrated to have advantages over antibodies with regard to chemical stability,readily availability, simple modifiability,and high flexibility in biosensor design for analyte detection.As a novel functional molecular,aptamers have gained considerable interest in bio-analytical application in many fields such as clinical diagnosis,environmental supervision,pharmacal study,etc.
Chemiluminescence(CL)has been exploited within a wide range of applications in various fields,due to their extremely high sensitivity along with their extra advantages such as needless special light,simple instrument,wide calibration ranges, and suitability for automatical operation in analytical chemistry.Nowadays,based on cross-research with other disciplines,CL has been successfully served for different applications.Our study developed a series of novel detection technologies on the strength of the high specifically binding between aptamers and its corresponding ligands in the thesis.And simultaneous detection of two molecules in one sample has been made grounded on aptamer switch.Description of research in the thesis is presented as follows:
Chapter 1:Introduction
This chapter is composed of two phases.The first phase addresses the source,traits, and advantages of aptamers and outlines aptamer-based detection techniques,then presents single analysis of current studies based on aptamer in detail,including optical aptasensors,electrochemical aptasensors,and other mass sensitive aptasensors. Following that,the multiplex analysis has been also stated in the development of aptamer-based biosensors and bioassay methods.The second phase of this chapter reviews current simultaneous multi-analytes detection by substrate-resolved CL technology exampled HRP and ALP in assay field.With that,objectives,significance, contents of this CL research based on aptamer are summarized.
Chapter 2:Label-free aptamer-based chemiluminescence detection of adenosine
Owing to no elucidation for the binding property of aptamer,it has difficult to label on aptamer and the label process would affect the binding affinity between the targets and their aptamers to a greater or lesser degree.Consequently,some label-free detection methods have been focused to develop,especially in some fileds like bioassay and environmental supervision.In this chapter,employing magnetic bead as a special biomolecule separating carrier,we demonstrate a sensitive CL aptasensor for label-free adenosine assay based on 3,4,5-trimethoxyl-phenylglyoxal(TMPG)as the signal molecule,which reacts specially and instantaneously with guanine nucleobases of adenosine-binding aptamer strands.We designed two different experimental routes for assay and the sketchy experiment steps were described as follows.(1)The magnetic beads were activated and the capture DNA immobilized on magnetic beads by the force of NH_2-COOH covalent bind.(2)Route A:The excess and quantitative adenosine aptamers reacted with different amounts of adenosine and formed an adenosine-aptamer complex.Then the rest free aptamer,along with the complex,were hybridized with capture DNA immobiled on magnetic beads.Route B:the aptamers first hybridized with capture DNA-magnetic bead conjugates and then reacted with adenosine.When adenosine as a specific competitor appeared,parts of aptamer were compelled to take apart from the magnetic beads and formed an adenosine-aptamer complex.(3)After speration by magnetic force,The CL signal was gotten from the raction between TMPG and guanine nucleobases of aptamer strands on magnetic beads.Using this system,adenosine could be specifically detected and a relatively low detection limit(8×10~(-8)M)as well as a wide linear dynamic range(4×10~(-7)-1×10~(-5) M)could be achieved with route A(R~2=0.9852).With route B,adenosine can be detected in a shorter range from 4×10~(-7)M to 5×10~(-5)M(R~2=0.9764).Overall,the label-free protocol described here may have value in a variety of clinical,pharmacal and environmental applications for which the simple,fast and accurate quantitative analysis of biomolecular adenosine is desired.
Chapter 3:Label-free aptamer-based chemiluminescence detection of PDGF-BB
Platelet-derived growth factor(PDGF)is a ubiquitous mitogen and chemotactic polypeptide present in serum for stimulating cells of mesenchymal origin and elicits multifunctional actions with a variety of cells.PDGF-BB,one of the important isoforms of PDGF,has been directly implicated in the cell transformation process and in tumor growth and progression.And the detection of PDGF-BB is of very valuable in biological fields.In this chapter,a“sandwich-type”detection strategy was employed in our design to make PDGF-BB detection.The procedure was as follows. Anti-PDGF-BB antibody immobilized on the surface of carboxyl terminated magnetic beads and DNA aptamers,both of which flanked PDGF-BB.After washing,the CL signal was obtained via the instantaneous derivatization reaction between TMPG and guanines nucleotides of aptamer to detect target protein without any label procedure. On this ground,an amplified CL approach was investigated employing G-rich sequence linked with aptamer as the amplification unit.The streptavidin was employed as an amplification platform to develop a high effective CL approach. Nonamplified approach achieved a good linear correlation from 1×10~(-8)to 2×10~(-7)M with the lowest detection concentration(1×10~(-8)M)and correlation coefficient (R~2=0.9901).In amplified approach,an excellent linearity was found within the range of 4×l0~(-10)-2×10~(-8)M(R~2=0.9954)with the lowest detection concentration of 4×10~(10), which was 25-fold improvement on nonamplified methods.The result demonstrated that this assay was reproducible,speedy,easy to use and suitable to make further development.In a word,this novel lable-free CL approach provided great promise for PDGF-BB assay in clinical field.
Chapter 4:ATP CL determination coupled the enrichment of magnetic beads with the specific binding of aptamer
The enrichment of immuno-magnetic beads is one of simple,speedy and high efficient screening methods.For one complicate sample with trace quantities of targets,it has unique merits following as:First,The irregular shape of the magnetic beads allows for much greater surface area.The large surface area results in high binding capacities,allowing much more targets captured with increasing capture probes on magnetic beads.Second,magnetic beads with high quality have great homogenicity and paramagnetism,which guarantee smoothly reaction with biomolecular without any intervention from any particle impurity.Third,Most of the rinsing solution was removed by pipet,but some buffer liquid(~3μL)remained around the magnetic beads because of the force of superficial adsorption.It was almost impossible to dry the sample completely and this benefited the prolonged active of biomolecular.Greater magnetic responsiveness results in faster magnetic separations especially on high throughput automated platforms.In this chapter,we employed adenosine triphosphate(ATP)aptamer as capture probe to covalently bind on magnetic beads.ATP present in trace quantities were enriched from the mixed sample on the magnetic beads and could be rapidly isolated from the sample by employing an external magnetic field in order to make CL detection.Experimental results confirm that it has good sensitivity with a detection limit of 1×10~(-8)M for ATP. ATP could be specifically isolated and detected and a wide linear dynamic range (1×10~(-8)-1×10~(-5)M)could be achieved(R~2=0.9918).On the whole,by using magnetic beads with great enrichment and aptamer with high discrimination,this method may show good perceptive on the purification and detection of biomolecular.
Chapter 5:Simultaneous detection founded on sptasensors by CL technology
A novel aptamer-based CL biosensing platform for the simultaneous detection of two small molecules as exemplified by adenosine and cocaine has been developed. Herein,we employed magnetic bead as the special biomolecule immobilizing carrier and made CL detection catalyzed by two labeled enzymes of alkaline phosphatase (ALP)and horseradish peroxidase(HRP).The principles of homogeneous substrate-resolved technology for CL detection have three steps:(1)preparation of adenosine and cocain aptamer-linked biosensors based on magnetic beads as carriers; (2)binding with a mixture of adenosine and cocaine to initiate target-aptamer reactions;(3)reacting with a mixed solution of Digoxin-ALP and Strepavidin-HRP followed by magnetic force separation of the magnetic beads conjugates and parallel detection of CL signals from HRP and ALP with their corresponding assay kits. Experimental results confirm that this CL immunosensing platform has good sensitivity with the lowest detection concentration of 1×10~(-8)M for adenosine and cocaine.On the whole,by using different aptamers,this method may offer a new direction in designing high-performance CL aptasensors for sensitive simultaneous determination of small molecules.And the technique would play an important role in multiplex analysis for biomoleculars with simple instrument and convenient procedure.
引文
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