基于共价键固定探针的电化学发光适体传感器的研究

【作者】 孙波；

【导师】 张成孝；

【作者基本信息】 陕西师范大学 ， 分析化学， 2010， 硕士

【摘要】 电化学发光法兼有电化学和化学发光法的双重优点,具有极低的检测限、极宽的线性范围等优点；相对于荧光法,无光漂白,不需要光源和分光系统等优点；相对于电化学检测,具有检测限低和受电极污染影响小的优点,因此被广泛应用到免疫分析,DNA杂交检测,食品和水的测试,还有生物战争试剂和爆炸材料的检测等。适体是特异结合目标分子的核酸(DNA或RNA)片段。由于它具有高的亲合力、特异性强、目标范围广、分子量小、体外筛选,化学合成简单、稳定性好,可复性等,所以可以作为一类特异性识别分子应用在传感器的构建中。电化学发光适体传感器兼有电化学发光的高灵敏度和适体特异性强的优点,因此,受到研究工作者的极大兴趣。到目前为止,所有电化学发光适体传感器均采用金电极上巯基自组装固定电化学发光探针,因此传感器不可避免的存在稳定性差和保存时间短等缺点。从这个意义上说,如何构建稳定性好、保存时间长的电化学发光适体传感器是一项具有挑战性的工作。本论文的研究目的是探索制备稳定性好、保存时间长电化学发光传感器的新方法,进而研制高灵敏、可重复利用的电化学发光适体传感器,为以后进一步研究电化学发光或者电化学传感器提供一些基础研究资料。本论文研究工作是在国家自然科学基金“糖/凝集素电化学发光生物传感器的研究”(No.20975065),“电化学发光适配体生物传感器”(No.20775046)和陕西师范大学研究生创新基金“基于共价键固定探针的电化学发光适配体传感器的研究”(No.2009CXS012)的资助下完成的。本论文研制了两种高灵敏、可重复利用检测小分子物质的电化学发光传感器；研制了四种高灵敏、可重复利用的可卡因、三磷酸腺苷的电化学发光适体传感器。本论文由5章组成。第1章为引言。引言部分介绍了电化学发光的基本原理；重点介绍了电化学发光适体传感器的发展、存在的问题及其发展趋势；最后介绍了本论文的目的和意义及其主要研究内容。第2章介绍了“双共价键固定化探针的可卡因电化学发光适体传感器的研究”,利用双共价法制备了高灵敏和重复利用的电化学发光化学传感器检测季胺盐类物质和电化学发光适体传感器检测可卡因。电化学发光传感器是将一端带有氨基的钌联吡啶衍生物或者它标记的可卡因适体和对氨基苯磺酸共价修饰的石墨电极相连制备的。本实验分别以三丙胺和胃复安为模型分子来评价电化学发光化学传感器。传感器显示了高的灵敏度稳定性和重复性。检测三丙胺和胃复安的检出限分别是30和2×10-9mol/L,分别对50和200×10-9mol/L的三丙胺和胃复安重复扫描90和45个循环,相对标准偏差分别为2.1%和2.6%。电化学发光适体传感器检测可卡因的检出限为10×10-12mol/L,并且对可卡因、海洛因和咖啡因有很好的分辨能力。电化学发光适体传感器的检出限比基于光学和电化学交流循环伏安法建立的适体可卡因生物传感器低4-6个数量级。此传感器再生7次的相对标准偏差为2.8%,并且有长时间的保存稳定性(21天后电化学发光信号变化为原来的96.8%)。基于厄文·朗缪尔等温线的方法用电化学发光曲线估计可卡因和适体的结合常数为4.6×109mol/L-1。此工作解决了电化学发光传感器稳定性差、保存时间短等问题。第3章介绍了“基于目标诱导替换的三磷酸腺苷电化学发光体传感器的研究”,利用一端含有氨基的捕获探针和对氨基苯磺酸修饰的石墨电极共价连接。当没有加入三磷酸腺苷时,标记有钌联吡啶衍生物的三磷酸腺营适体链能够和捕获探针很好的结合,电化学发光信号很强；当加入三磷酸腺苷后,电化学发光探针解离,电化学发光信号降低,此为信号抑制型传感器。而对于信号增强型传感器,当没有加入三磷酸腺苷时,三磷酸腺苷适体链能够和捕获探针很好的结合,电化学发光信号很弱；当加入三磷酸腺苷后,三磷酸腺苷适体链解离,然后加入标记有钌联吡啶衍生物的三磷酸腺苷适体链杂交,电化学发光信号增强。信号抑制型传感器和信号增强型传感器的线性范围分别为1.0×10-10-5.0×10-8 mol/L和1.0×10-12-5.0×10-9 mol/L,检出限为3.0×10-11 mol/L和1.0×10-12 mol/L (S/N=3)。该工作证明了信号增强型传感器的性能要比信号抑制型传感器好,这些结论对以后的研究有一定的促进作用。第4章介绍了“三磷酸腺苷夹心式电化学发光适体传感器的研究”,设计了一种基于夹心式检测三磷酸腺苷的电化学发光适体传感器。碱基序列为5’-NH2-(CH2)6-ACC TGG GGG AGTAT-3’捕获探针通过与酰胺氯交联共价键合到修饰了对氨基苯磺酸的石墨电极表面。在没有三磷酸腺苷时,碱基序列为5’-TGC GGA GGA AGG T-(CH2)2-NH2-Rul-3’的电化学发光探针与捕获探针相互作用很弱,电化学发光信号小。当加入三磷酸腺苷后,电化学发光探针与捕获探针间的结合能力增强使得电化学发光信号增加。传感器的线性范围为5.0×10-11-5.0×10-8 mol/L,检出限为1.0×10-11 mol/L (S/N=3),此模型也可以用于血清中溶解细胞中三磷酸腺苷的检测。第5章利用离子液体为粘合剂制作碳糊电极,采用高分子聚合法,合成包埋有Ru(bpy)2 (dcbpy)2+的高分子聚合物,将钌聚合物掺杂于离子液体碳糊电极中,制作电化学发光传感器。结果表明该传感器具有很好的电化学和电化学发光特性,与石蜡油为粘合剂制作的电化学发光传感器相比,离子液体为粘合剂的电化学发光传感器检测三丙胺的检出限降低1个数量级。海洛因对电化学发光传感器的发光信号有很好的增强作用,基此建立了高灵敏度检测海洛因的电化学发光分析法,海洛因浓度与电化学发光信号在2.0×10-9 mol/L-2.0×10-5 mol/L范围内呈良好的线性关系,检出限为8×10-10 mol/L (S/N=3)。将电化学发光传感器在5.0×10-9 mol/L海洛因溶液中采用线性循环电位连续扫描60圈,相对标准偏差小于5%。该方法用于血清中海洛因的检测,回收率在94-101%之间。更多还原

【Abstract】 Electrogenerated chemiluminescence (also called electrochemiluminescence, abbreviated as ECL) is the process where species generated at electrodes undergo electron-transfer reactions to form excited states that emit light. ECL method has many distinct advantages over fluorescence method because it does not involve a light source and avoids the attendant problems of scattered light and impurities luminescent. Moreover, the specificity of the ECL reaction associated with the ECL label and the coreactant species decreases problems with side reactions and is characterized by good spatial and temporal resolution. ECL has been widely used in many chemical and biochemical related applications over the past several years, which include immunoassay, DNA hybridization detection, food and water testing, as well as biowarfare agent or explosive material detection. Aptamers are nucleic acid (both DNA and RNA) receptors that bind to specific target molecules. Aptamers exhibit a strong and specific binding affinity towards their targets with a remarkable stability, simply synthesized via cost-effective and readily automated routes and so on. With regard to this, aptamers can be used as new recognition entities for specific sensing of a variety of analyte targets in biosensor. Some ECL aptamer-based biosensor about the combination of high sensitive ECL with aptamer have been attracted much attention in recent years. Up to date, all the ECL aptamer-based biosensor are broadly used thiolated SAMs on Au for the attachment of ruthenium complexes, this approach can only allow for the anodic ECL measurements conducted under potentials not positive than-0.80 V vs Ag/AgCl so that the thiol layer’s damage can be avoided. Additionally, because gradual degradation of the SAMs in air or in a phosphate saline buffer (PBS) solution often occurs, the long-term storage stability of the ECL detector or the biosensor is a challenge.The aim of this thesis is to design reusable and long storage stability ECL biosensors in order to provide a new strategy in fabricating various ECL or electrochemical chemical sensors or biosensors. The research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No.20775046, No.20975065) and the innovation Funds for Graduate Programs of Shaanxi Normal University (No.2009CXS012).The thesis includes two parts. First part, chapter 1 is general introduction while second part consisting of four chapters, is a research report. In Chapter 1, general introduction to the principle of ECL, ECL aptamer-based biosensors including their principles and research development, and the purpose of this research work were presented. In Chapter 2, a double covalent coupling method for the fabrication of highly sensitive and reusable ECL chemical sensor for the detection of tertiary amines and ECL aptamer-based (ECL-AB) biosensor for the detection of cocaine was reported. The ECL sensors were constructed by covalent coupling of Ru(bpy)32+derivatives (Ru1,Ru(bpy)32+=tris(2,2’-bipyridyl)ruthenium(Ⅱ)) or cocaine aptamer-Ru1 to the surface of paraffin-impregnated graphite electrode (PIGE) that had been covalently modified with a monolayer of 4-aminobenzene sulfonic acid via electrochemical oxidations. ECL performance of the newly developed chemical sensors was evaluated using tri-n-propylamine (TPrA) and metoclopramide (MCP) as model analytes. The sensors exhibited excellent sensitivity, stability, and reproducibility with a detection limit of 30 nmol/L for TPrA and 2.0 nmol/L for MCP, and relative standard deviations (RSD) of 2.1% over 90 cyclic potential cycles (0 to 1.50 V vs Ag/AgCl) and 2.6% over 45 cycles (0.60 to+1.30 V vs Ag/AgCl) at 400 mV/s for 50 nmol/L TPrA and 200 nmol/L MCP, respectively. For the ECL-AB biosensor, it showed an extremely low detection limit of 10 pM for cocaine, and offered a good selectivity towards cocaine, heroin, and caffeine. This detection limit was about 4-6 orders of magnitude lower than that reported on the basis of AC voltammetry and optical aptamer-based cocaine biosensors. Additionally, the ECL-AB biosensor was highly reusable (RSD=2.8%, n=7) and possessed long-term storage stability (96.8% initial ECL recovery over 21 days storage). A binding constant of 4.6±0.3×109M-1 between cocaine and its aptamer was estimated using an ECL based Langmuir isotherm approach. Wide applications of the presently reported strategy in fabricating various chemical sensors or biosensors are expected.In Chapter 3, ECL-AB biosensors based on target-induced aptamer displacement were developed for the determination of ATP as a model system. "Signal on" and "Signal off" ECL-AB biosensors were fabricated by covalently coupling the partial complementary single strand DNA-ATP binding aptamer to the surface of PIGE modified with 4-ABSA through a cross-linking reaction amine-acyl chloride. In the absence of ATP, the "Signal off" ECL-AB biosensor led to a great ECL signal. In the presence of ATP, target-induced strand release of a 3’-end label Ru(bpy)32+ derivatives tagged aptamer from the aptamer capture DNA duplex bound on an electrode, led to decrease ECL signal. Therefore, this ECL-AB biosensor can be used to detect of ATP. With regard to the "Signal on" ECL-AB biosensor’s principle is that the interaction between the target and the aptamer strand may induce the formation and subsequent dissociation of target-aptamer complex from an electrode surface, and consequently, the remaining DNA strand on the electrode surface can hybridize again with a ssDNA containing an ECL probe. The linear of "Signal on" and "Signal off" were in the range from 1.0×10-12 mol/L to 5.0×10-9mol/L and 1.0×10-10mol/L to 5.0×10-8mol/L with a detection limit were 1.0×10-12mol/L,1.0×10-10 mol/L, respectively.In Chapter 4, a simple ECL sensing platform for high specificity and sensitive sandwich assay for the detection of small molecule target ATP was demonstrated. A capture probe,5’-NH2-(CH2)6-ACC TGG GGG AGT AT-3’was covalently coupled to the surface of PIGE modified with r-aminobenzene sulfonic acid through a cross-linking reaction amine-acyl chloride. In the absence of ATP, a ECL probe 5’-TGC GGA GGA AGGT-(CH2)2-NH2-Rul-3’was weakly interaction between the capture probe, led to a small ECL signal. In the presence of ATP, ECL probe was strongly interaction between the capture probe, led to a great ECL signal.The ECL intensity is linear with the concentration of ATP in the range from 5.0×10-11mol/L to 5.0×10-8 mol/L and the detection limit is 3×10-11 mol/L (S/N=3).In Chapter 5, a sensitive ECL sensor for the determination of heroin was developed by employing ruthenium complex polymer as ECL signal and ionic liquid as binding reagent. ECL sensor was prepared by thoroughly mixing ruthenium complex polymer, graphite powder and ionic liquid, which show good electrochemical and ECL behaviors. The ECL intensity is linear with the concentration of heroin in the range from 2.0×10-9 mol/L to 2.0×10-5 mol/L and the detection limit is 8×10-10 mol/L. The ECL sensor exhibited a long-term stability, fine reproducibility with relative standard deviation less than 5% for 5.0×10-9 mol/L heroin in 60 continuous determinations. The developed method allows the detection of heroin in a serum sample with recovery in 94-101%.更多还原