【作者】 封科军；

【导师】 俞汝勤； 沈国励；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 博士

【摘要】 随着人类基因组计划的完成和功能蛋白质组学的研究进展,建立简单、灵敏、快速、特异性强、高通量的蛋白质和基因检测方法,已成为分析科学家们研究的重点之一。核酸碱基的突变与人类许多疾病有着直接的关系,因此实现单核苷酸突变准确灵敏的检测对疾病的早期诊断也有着重要的意义。核酸适配体是近年来发展起来的一类经体外人工进化程序筛选出的寡聚核苷酸。由于其不但和抗体一样能与配体高效、特异性地结合,而且具有许多抗体无法比拟的优点,因此将核酸适配体应用于生物传感器实现对包括蛋白质、小分子等目标物的检测有着巨大的发展潜力。鉴于电化学及压电检测技术所需仪器简单、检测成本低、易于实现微型化,且灵敏度较高等优点,本研究论文针对当前传感器设计中的探针固定化和检测方法两个关键技术,在电化学及压电DNA传感器用于基因检测和点突变识别以及电化学适配体传感器两方面开展了一些新方法研究工作。具体内容如下:(1)在第2章中,研制出一种新型的基于纳米多孔CeO2/壳聚糖复合膜的固定基质。用它来固定单链DNA探针,构建了检测与结肠癌高度相关的基因序列的电化学DNA生物传感器。该固定基质制备方法简单,成本低,结合了CeO2和壳聚糖的优点,具有良好的生物兼容性,无毒性和优越的电子传导性。将其用于固定结肠癌基因的互补探针序列,能够显著增强ssDNA探针在电极表面的负载量。制得的传感器检测目标的线性范围在1.6×10-11-1.2×10-7 M,检测下限为1.0×10-11 M,具有识别完全互补目标序列和四碱基错配序列的能力。(2)在第3章中,基于连接酶的高保真性和生物催化沉积反应,提出了用于高特异性识别单碱基突变的压电检测方法。实验中,目标DNA链先与连接在石英晶振上的DNA捕获探针杂交,再与生物素标记的等位特异性DNA检测探针杂交。只有在目标DNA链和检测探针完全匹配时,连接反应才能发生,即将捕获探针和检测探针相连接。否则,即使只有一个等位错配的基因,连接反应也不会发生。经过高温热处理,形成的双链解开,只有与目标链完全匹配的检测探针保留在电极表面。生物素化的检测探针继而与链酶亲和素化的辣根过氧化物(SA-HRP)绑定,辣根过氧化物催化过氧化氢氧化底物中的3, 3–二氨基联苯胺(DAB)在电极表面生成不溶性沉淀物,使压电频率响应显著增大。用该方法对β-地中海贫血基因-28位密码子的突变情况进行了检测,使突变型和野生型得到了很好的区分,对目标的检测线性范围为0.7-100 nM,检测下限达0.1 nM,是一个低成本高效率的检测方法。(3)在第4章中,基于等位基因特异性延伸法,结合酶催化银沉积的放大体系,提出了用于单碱基突变的电化学检测方法。首先,在金电极表面固定的等位特异性捕获探针。由于其与野生型目标链完全互补,在聚合酶的作用下能够发生延伸反应;而突变型目标链由于3’端与捕获探针发生错配,延伸反应不能进行,从而实现对突变型和野生型基因的区分。用1 M NaOH进行解链处理后,双链结构解开,目标链从电极表面脱落。在电极表面滴加与探针延伸部分相互补的生物素化的检测探针后,只有发生延伸反应的捕获探针链会进一步与生物素化的检测探针杂交,并引入链亲和素化的碱性磷酸酶,继而发生酶催化沉积银的反应。通过线性扫描伏安法可以检测出电极表面沉积的银。该方法成功用于β-地中海贫血基因-28位密码子单碱基突变的区分和定量检测,简单、快速、灵敏度高,线性范围为3.0×10-16-3.0×10-8 M,检测下限为1.0×10-16 M。(4)在第5章中,报道了基于目标物诱导置换适配体的无标记电化学传感器,用于以腺苷为分析物模型的目标物检测。该传感器使用1,6-巯基己醇自组装层修饰的金电极为传感基底,以巯基己醇为媒介组装金纳米颗粒层,能够增加巯基捕获探针的表面负载量,提高信号强度。含有腺苷适配体序列的一段寡核苷酸序列一部分可与捕获探针杂交,在电极表面形成捕获探针与适配体的双链复合物。含有腺苷的分析物的加入,把腺苷适配体序列置换下来,使其从电极表面解离,从而减少了电极表面核酸链的数量,使与核酸作用的电化学活性指示剂亚甲基蓝的量相应减少。指示剂的还原电流的大小能够反映被分析物的浓度。构造的传感器简单、快速、灵敏、选择性好,线形范围为5-1000 nM,检测下限为1 nM,同时具有简便快速的再生能力。(5)在第6章中,报道了基于抗体与生物素标记的适配体新型夹心的酶放大电化学免疫传感器,用于免疫球蛋白E(IgE)的检测。IgE多克隆抗体作为捕获探针通过半胱胺自组装层以及交联剂戊二醛的作用共价固定在金电极表面,与目标物发生免疫反应后,继续捕获生物素化的IgE适配体的检测探针,从而形成一种新型的夹心结构。之后,通过生物素与亲和素的特异性亲和作用,亲和素标记的碱性磷酸酯酶能与电极表面适配体上的生物素相结合,促使碱性磷酸酯酶催化底物抗坏血酸磷酸酯水解成强还原剂抗坏血酸,并将底液中的银离子还原成单质银,沉积到电极表面。沉积的银的量与电极表面捕获的目标IgE的量成正比,可以用溶出伏安法来定量。结果表明,该免疫传感器结合了抗体和适配体的双重特异性,并利用了酶催化银沉积体系的高灵敏性,检测目标蛋白的灵敏度高、特异性好,线性范围为0.1-100 nM,检测下限为0.02 nM。(6)第7章,同样基于抗体与适配体的新型夹心结构,构建了纳米金/壳聚糖电化学沉积复合膜的新型固定基质用于抗体的固定,采用亚甲基蓝为电活性指示剂,报道了检测凝血酶的无标记电化学方法。该复合膜结合了纳米金和壳聚糖独特的性能,提高了传感器的导电性能以及负载量。实验考察了纳米金/壳聚糖电化学沉积条件,并用扫描电子显微镜对该膜进行了表征。在这个工作中,在不改变适配体与目标物结合作用的基础上,对原凝血酶的适配体序列进行了适当延伸,以提高亚甲基蓝嵌入适配体中的量,增强信号响应。传感器构造方法简单、价格低廉,无需对探针进行任何修饰,凝血酶的线性响应范围是1-60 nM,检测下限为0.5 nM。更多还原

【Abstract】 With the implement of the Human Genome Project and the development of the functional proteomics research, it’s an important domain that high sensitive assay methods were developed for the detection of the proteins and DNA. Because point mutations in genomic DNA have direct link with human diseases,there has been considerable interest in developing rapid and sensitive methods to detect point mutations.DNA aptamers are short nucleic acid ligands artificially selected for their high specificity and affinity for various targets including proteins, small molecules and even cells. Because of their numerous advantages over antibodies such as adaptability to various targets, ease in synthesis and storage, and versatility in labeling, immobilization, signaling and regeneration, the widespread application of aptamers to biosensors is expected to hold potential in the detection of various proteins and small molecules.Electrochemical and piezoelectric biosensors attract significant attention and become the research hotspot for their low-cost, fast response, being simple, sensitive and compatible with microfabrication technologies. Aiming at the key technique including probe immobilization method and detection method used in electrochemical biosensor, several novel methods been developed for electrochemical, piezoelectric DNA biosensors and electrochemical aptasensor. The detailed content is described as follows.1) In chapter 2,CeO2/Chitosan composite matrix was firstly developed for the single-stranded DNA (ssDNA) probe immobilization and the fabrication of DNA biosensor related to the colorectal cancer gene. The preparation method is quite simple and inexpensive. Such matrix combined the advantages of CeO2 and chitosan, with good biocompatibility, nontoxicity and excellent electronic conductivity. The matrix was used to immobilize completely complementary ssDNA probe with the target sequence and showed the enhanced loading of ssDNA probe on the surface of electrode. The established biosensor has high detection sensitivity, a relatively wide linear range from 1.6×10-11-1.2×10-7 M and the ability to discriminate completely complementary target sequence and four-base-mismatched sequence. The limit of the detection is 1.0×10-11 M.2) In chapter 3,a novel biosensing technique for highly specific identification of gene with single base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3, 3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single base mutation in -28 site of theβ-thalassemia gene. The target gene can be determined in the range from 0.7 nM to 100 nM with a detection limit of 0.1 nM.3) In chapter 4,a novel electrochemical method for SNP detection is proposed based on allele-specific extension and enzymatic-induced silver deposition. Briefly, allele-specific capture probe firstly immobilized on the gold electrode, which perfectly matches with wild gene and can be extended, whereas mismatching with the mutant gene at 3’ terminal bases cannot be extended. After denaturation with 1M NaOH, the formed duplexes unfold and target sequences dissociate from the electrode surface. Because the sequence extended perfectly match with biotin-modified detection probe, hybridization take place, and then streptavidin-conjugated alkaline phosphatase can be captured to the electrode surface due to the specific interaction of biotin and streptavidin. The enzyme-induced silver deposition is used to amplify the response. The electrochemical signal of silver is obtained by using linear sweep voltammetry. The present approach has been demonstrated with the identification of single-base mutation in -28 site (A to G) forβ-thalassemia gene and the wild type target can be determined in the range from with 3.0×10-16-3.0×10-8 M with a low detection limit of 1.0×10-16 M.4) In chapter 5 , a label-free electrochemical sensor using aptamer based on target-induced displacement is reported with adenosine as the model analyte. The sensing substrate is prepared using a gold electrode modified with a self-assembled monolayer of 1, 6-hexanedithiol that mediates the assembly of a gold nanoparticle film, which can increase the surface loading of capture probe and enhance the signal. An aptamer for adenosine is applied to hybridizing with the capture probe, yielding a double-stranded complex of the aptamer and the capture probe on the surface. The interaction of adenosine with the aptamer displaces the aptamer sequence and causes it to dissociate from the interface. This results in a decrease of absorption state of methylene blue. Then, the redox current of the indicator can reflect the concentration of the analyte. The fabricated sensor is shown to exhibit high sensitivity, desirable selectivity. The linear range for target detection is 5-1000 nM with a detection limit of 1 nM. The regeneration of the developed biosensor is simple and fast. 5) In chapter 6, an electrochemical immunosensor is reported by usingaptamer-based enzymatic amplification with immunoglobin E (IgE) as the model analyte. The IgE-antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target captured, the biotinlynated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinlynated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then, silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range from 0.1-100 nM with a low detection limit of 0.02 nM, which possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.6) In chapter 7, based on the principle of antibody-aptamer sandwich in chapter 6, we reported a label-free electrochemical biosensor for thrombin detection. A novel nanogold-chitosan composite film using the electrochemical deposition method was prepared to immobilize thrombin antibody. Combined the special properties of gold nanoparticles and chitosan, the composite film showed enhanced conductivity and loading ability. The electrochemical deposition conditions of the composite film was investigated and its surface morphology was characterized by SEM. Methylene blue (MB) as the electrochemical active marker intercalating in the probing aptamer was applied to offer the response signal by differential pulse voltammetry (DPV). With appropriate extended design of the aptamer sequence, the amount of MB intercalating in the aptamer was increased and the signal response was also enhanced. The fabricated sensor was simple and low-cost and without the need of labeling. The linear response range for thrombin detection covered 1-60 nM with a detection limit of 0.5nM.更多还原