【作者】 孙晨虎；

【导师】 俞汝勤；

【作者基本信息】 湖南大学 ， 分析化学， 2010， 硕士

【摘要】 核酸适体,核酸酶和适体酶统称为功能型核酸,其能力超越了传统意义上核酸的遗传作用。功能型核酸具有适用于生物传感器的优越性质,包括高度的稳定性,低成本,易于合成与修饰等,一般认为能够用于检测任何分析物。因此,功能型核酸在生物传感领域起着越来越重要的作用。本文开发了三种类型的功能型核酸电化学传感器,首先研究了核酸适体传感器,再进一步研究了DNA酶传感器和适体酶传感器,具体内容如下：在本文第二章,报道了一种基于目标物诱导置换适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。该传感器使用金纳米颗粒层修饰的金电极为传感基底。腺苷适体可与捕获探针杂交,在电极表面形成双链复合物。腺苷加入后把适体置换下来,使其从电极表面解离,从而减少了适体／捕获探针双链的数量,同时使与核酸双链结合的电化学活性指示剂亚甲基兰从电极表面解吸。指示剂氧化还原电流的大小能够反映被分析物的浓度。该传感器具有高度的灵敏性和选择性。在本文第三章,报道了一种基于邻近DNA杂交的电化学DNA酶传感器用于检测DNA。目标DNA的部分序列与识别探针杂交,形成一个稳定的杂交体。杂交体一侧目标DNA与识别探针其余序列邻近,形成一个完整的10-23 DNA酶结构,能够切断发卡型底物序列中的RNA碱基位点,产生两条单链DNA片段。其中一条标记有生物素,作为信号探针被电极表面的巯基探针捕获,再与标记碱性磷酸酯酶的链霉亲和素反应。最后通过测量1-萘磷酸盐经过酶反应后得到的1-萘酚的量,考察目标DNA的浓度。实验结果表明该传感器对检测幽门螺旋菌的片段互补序列具有高度的灵敏性,并能够特异性区分错配序列。在本文第四章,报道了一种基于适体酶的电化学传感器用于检测腺苷。通过在8-17 DNA酶序列的一段连接一条包含腺苷适体的核酸抑制链,来调节适体酶的活性。没有腺苷时,DNA酶的部分序列与抑制链杂交,不能形成催化结构。引入腺苷与适体结合后,抑制链从DNA酶序列解离,使DNA酶与发卡型底物杂交,在铅离子存在下切断RNA碱基位点,产生两条单链DNA片段。其中一条标记有二茂铁,作为信号探针被电极表面巯基探针捕获产生电化学信号。该传感器具有高度的灵敏性和选择性,在构建合理的适体酶传感器设计与腺苷的临床诊断方面具有广阔的应用前景。更多还原

【Abstract】 Aptamers, nucleic acid enzymes, and aptazymes are collectively called functional nucleic acids (FNAs), whose functions are beyond the conventional genetic roles of nucleic acids. FNAs, which have superior properties as excellent sensors, including high stability, low cost, and ease of synthesis and modification, are generally believed to be able to target any analyte of choice. Hence, FNAs have taken an increasingly significant role in biosensing events. The topics presented in this paper focus on three signaling methods developed to construct electrochemical FNA sensors. Aptamer-based sensors are described first, followed by DNAzyme and aptazyme sensors. The details are summarized as follows:In Chapter 2, a label-free electrochemical sensor based on target-induced displacement was reported with adenosine as the model analyte. The sensing substrate was prepared using a gold electrode modified with a gold nanoparticle film. An aptamer for adenosine was applied to hybridizing with the capture probe. The interaction of adenosine with the aptamer displaced the aptamer sequence and caused it to dissociate from the interface. This resulted in a decrease in the amount of aptamer/capture probe duplex form, and accordingly, the desorption of methylene blue from the electrode. Then, the redox current of the indicator could reflect the concentration of the analyte. The fabricated sensor was shown to exhibit high sensitivity, desirable selectivity and a three-decade wide linear range.In Chapter 3, a novel electrochemical DNAzyme sensor was reported for the detection of nucleic acids based on proximity-dependent DNA ligation assays. When the target DNA was introduced into the system, part of it was complementary to 5’-end of the recognition probe, resulting in the ligation of a stable duplex. This duplex containing a complete 10-23 DNAzyme structure could cleave the purine-pyrimidine cleavage site of the hairpin substrate, which resulted in the fragmentation of the hairpin structure and the release of two single-stranded nucleic acids, one of which was biotinylated and acted as the signal probe. An immobilized thiolated capture probe could bind with the signal probe, using biotin as a tracer in the signal probe, and streptavidin-alkaline phosphatase (SA-ALP) as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 5 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The results revealed that the sensor showed a sensitive response to complementary target sequences of H. pylori. In addition, the sensing system could discriminate the complementary sequence from mismatched sequences.In Chapter 4, an aptazyme-based electrochemical biosensor for the detection of adenosine was reported. Aptazyme activity was modulated by appending an "inhibitor" oligonucleiotide strand containing a 32-base adenosine aptamer to the 8-17 DNAzyme. In the absence of adenosine, the DNAzyme could not form appropriate catalytic structure due to the binding with the inhibitor strand. Upon adenosine binding to the aptamer, the inhibitor strand was dissociated from the DNAzyme sequence. This allowed the DNAzyme to open and bind with the hairpin substrate, cleaving the substrate at its ribonucleotide site in the presence of Pb2+. Cleavage of the substrate yields two single-stranded products, one of which was ferrocene-tagged and acted as the signal probe. The thiolated probe modified on the gold electrode could capture the signal probe. As a result, the ferrocene moiety was brought in close proximity to the electrode surface and the Faradaic current was observed. The fabricated sensor is shown to exhibit high sensitivity and desirable selectivity, which might be promising for the rational construction of aptazyme-based biosensors and the determination of adenosine in clinical examination.更多还原