非标记型电化学适配体传感器的构建及其在蛋白质检测中的应用
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摘要
蛋白质是生命的重要组成物质之一，也是整个生命活动的主要参与者。因此，蛋白质的检测特别是疾病状态下蛋白表达水平的检测将有助于我们更深入的了解生命的活动过程，对人类疾病的预防、诊断和治疗具有重要的现实意义。生物传感器是以生物分子作为识别元件(Recognition Element)的检测方法，因其具有高特异性、高灵敏度和操作便捷等特点成为目前最具有前景的蛋白质检测方法。抗体是目前应用最为广泛的生物分子识别元件，但是因其制备周期长、易失活、对检测环境要求较高等缺点，限制了其在生物传感器中的进一步发展。近年来越来越多的新型识别元素正在被引入到生物传感器的制备中，其中核酸适配体就是最为引人瞩目的一员。适配体是利用SELEX体外筛选技术从庞大的核酸分子文库中筛选获得的单链DNA或RNA，它们除了具有类似抗体的高亲和性结合与高特异性识别特性外，还表现出许多优于抗体的特性如分子量小、靶分子广泛、易于重复合成、易于标记、保存稳定且可以变性复性等，因此适配体正逐渐成为是一种新的理想的生物传感器识别元件。电化学生物传感器是一类通过生物分子识别元件将待测物质的浓度转变为可检测的电信号的检测传感器，其具有高灵敏度、不受溶液颜色影响，响应速度快、操作便捷、价格低廉、易于微型化等优点，将其与核酸适配体相结合，将成为继免疫电化学传感器和DNA电化学传感器后新的研究热点。
本课题的研究目的是建立一种高灵敏度、高特异性、操作便捷的蛋白质电化学适配体生物传感器。方式是将体外筛选得到的高特异性核酸适配体作为电化学生物传感器的分子识别元件，通过基于酶信号放大的设计策略，建立能够非标记地检测蛋白质的具有高灵敏度、高特异性的新型电化学适配体传感器，并将其应用于血小板生长因子及蛇毒种类鉴别检测的研究中。论文内容包括以下几部分：
第一章引言简要的介绍了核酸适配体的筛选原理、优越性及利用核酸适配体作为识别元件的方法学研究进展；详细的介绍了电化学传感器的原理及应用，以及基于酶信号放大的电化学适配体传感器的研究进展；着重论述了非标记电化学适配体传感器的设计策略和研究进展，并同时介绍了蛋白质检测的意义及方法学现状。
第二章基于酶放大设计策略的非标记电化学适配体传感器的构建近年来，许多氧化还原酶类的直接电子传递(DET)得到了较多的重视和应用。然而，由于固载基质的不稳定以及酶活性中心深埋在分子内，如果将酶分子直接固定到固体电极上，其DET是很难实现的。为了克服这一缺陷，有不少工作提出将酶固定于纳米材料修饰的电极表面，通过纳米材料的优点来实现酶的DET。其中，石墨烯(Gra)作为一种理想的材料，由于其优异的物理化学性能，如大的比表面积、好的导电性和易于功能化等，已经得到广泛的应用。另一方面，纳米金(GNPs)具有良好的生物相容性、强吸附能力和好的导电性，也被看作一种优良的纳米材料。这些性能也就使得PDDA保护的石墨烯-纳米金(P-Gra-GNPs)复合物成为更优异的固载基质，它不仅能加快活性中心与电极之间的DET，更为酶的固定提供了一个良好的微环境。由此本研究在二甲基二烯丙基氯化铵均聚物(PDDA)存在下，采用肼将氧化石墨烯还原，得到PDDA保护的还原石墨烯(P-Gra)。P-Gra表面带正电，通过静电吸附大量的纳米金(AuNPs)，制得纳米金功能化的石墨烯复合物(P-Gra-AuNPs)。利用酶的催化特性来放大识别过程中产生的电化学信号。基于此，我们利用葡萄糖氧化酶(GOD)的直接电子传递(DET)首次构建了一种新型无试剂无媒介的电化学适体传感器，并用于对血小板源性生长因子(PDGF)的检测。
第三章基于电化学适配体传感器的血小板源性生长因子的检测血小板源性生长因子(Platelet derived growth factor，PDGF)是一种低分子量促细胞分裂的肽类调节因子，因正常生理状态下存在于血小板的α颗粒内而得名，当血液凝固时由破裂的血小板释放出来并且被激活，具有刺激停滞于G0/G1期的成纤维细胞、神经胶质细胞、平滑肌细胞等进入分裂增殖周期的功能。血小板源性生长因子的检测在纤维化及癌症等疾病的诊断、预后评估中具有重要的临床意义。
我们利用血小板源性生长因子适配体构建了基于酶放大的电化学核酸适配体传感器，经过对电化学传感器修饰电极的表征，可以看到传感器的氧化还原峰电流减小值与待测PDGF浓度成正比。该适体传感器显示出良好的电化学响应，峰电流的减小值与浓度对数的增加值在0.005-60nM的范围内呈良好的线性，检测限为1.7pM。该传感器同样具有高的选择性，好的重现性和稳定性，也为其他基于适配体的蛋白检测提供了可行的依据。
第四章蛇毒种属特异性核酸适配体的筛选蛇伤的鉴别诊断需要准确的检测蛇毒的类型，目前蛇伤的实验室诊断主要依赖以蛇毒抗体的免疫学诊断方法，但是蛇毒抗体目前存在特异性差、毒性强依靠免疫动物制备困难等缺陷。受核酸适配体筛选原理(SELEX)中反筛原理的启发，我们通过在适配体经典筛选流程中引入多个鉴别靶标的反筛流程，来获取与目标靶标更高特异性结合的种属特异性适配体，我们将该技术称之为消减指数富集配体系统进化技术(Deplete systematic evolution of ligands byexponential enrichment, Deplete-SELEX)。我们在蛇毒种属特异性适配体筛选中引入了该方法，利用该技术筛选出用于鉴别中华眼镜蛇毒与国内常见的三种种毒蛇(尖吻蝮蛇、江浙蝮蛇、烙铁头)的中华眼镜蛇毒种属特异性核酸适配体，以利用于后续建立基于电化学适配体生物传感器的蛇伤现场快速检测方法的研究。
第五章基于电化学适配体传感器的蛇毒快速检测方法的研究目前蛇伤的诊断主要依靠临床症状(如伤口、局部体征等)、常规化验(血红蛋白、尿胆红素等)或捕获的蛇来确诊，但由于蛇伤早期临床症状往往不明显、不典型，且大多数的蛇伤发生在晚间或山林草地中很难看清蛇种，导致野外条件下蛇伤不能得到及时准确的诊断，无法正确指导抗蛇毒血清的使用以至延误抢救治疗良机。多年来，也有研究者应用免疫电泳、酶联免疫以及放射免疫等方法检测病人标本中各种毒蛇的种属特异性蛇毒成分，进行蛇伤免疫学鉴别诊断方法的研究，但这些方法的敏感度不够理想，阳性检出率受检材的种类、取材的时间、方法、诊断抗体的质量等因素影响较大。电化学适配体生物传感器因其检测速度快，灵敏度高，特异性强，简单便携成为蛇毒诊断与现场快速检测的理想检测手段。我们利用筛选得到的蛇毒种属特异性核酸适配体与电化学生物传感器相结合，构建了用于检测毒素的电化学适配体生物传感器，结果显示该传感器在毒素种属的鉴别检测中具有很好的特异性与灵敏度，可能成为蛇伤检测中的重要检测手段。
综上所述，我们采用GOD的直接电化学方法，成功构建了一个基于核酸适配体的高灵敏和高选择性的非标记电化学适配体传感器，用于对血小板生长因子及蛇毒特异性蛋白的检测研究。该传感器主要有以下优点：首先，P-Gra-GNPs复合物膜具有比表面积大和优良的生物相容性，它不仅能增加GOD的固载量，还能保持生物分子的活性，提高传感器的稳定性。其次，GNCs膜为PBA的固定提供了一个稳定和多孔的表面，进一步增强电化学信号。另外，双层GOD膜作为示踪剂带来更高的电流响应和高的灵敏度。基于以上的原因，该传感器显示出较低的检测限，满意的选择性，良好的稳定性和重现性。并且该方法也为其他蛋白质和生物标记物的检测提供了一定的参考。同时我们建立了适配体Deplete-SELEX筛选技术，并成功筛选到了中华眼镜蛇蛇毒种属特异性核酸适配体，成功构建了基于电化学适配体传感器的蛇毒快速检测新方法。
As a major component of life, proteins have played an important role in entire lifeactivities and regulations. Therefore, Detection of proteins expression particular in diseasestate will help us to better understand of the life and to prevent, diagnosis and treatment ofdiseases significantly.
Biosensor based on biological molecules as recognition elements, becomes the mostpromising methods for protein detection with its high specificity, high sensitivity, andconvenient operation. Currently antibodies are the most widely used biomolecularrecognition element, but its further application on biosensors was limited because of theirlong preparation period, instability, Stringent environmental requirements and othershortcomings. In recent years, more and more new recognition elements are introduced intothe preparation of biosensor, wherein the nucleic acid aptamer is the most remarkable one.Aptamers are single-stranded DNA or RNA screened from a large single nucleotide libraryby “Systemic Evolution of Ligands by Exponential Enrichment technique”(SELEX). Inaddition to their high affinity binding ability and high specific recognition characteristicscompare with antibodies, they also exhibit many other distinctive advantages such assmaller molecular weight, wide range of target, accurate synthesis, ease of labeling，stability and can be refolding after denatured, etc. Therefore aptamer is becoming a newideal biosensor recognition element. Electrochemical biosensors are a class of detectionsensor which convert biological concentration of the substance into a detectable signal byrecognition element with characters of high sensitivity, without affected by color of thesolution, quickly response, convenient operation, inexpensive, portable, etc., Combinedwith the aptamer, electrochemical aptasensors become a new hotspot beyond research ofimmune electrochemical sensors and DNA electrochemical sensors.
This project aims to establish a high-sensitivity, high specificity, and convenientoperation electrochemical aptamer biosensor for protein detectation. We set the aptamer as highly specific molecular recognition element of biosensor， and construction highsensitivity, high specificity of label free aptamers electrochemical sensors based onenzyme-based amplification strategies for protein detection. And study the application ofaptasensor in platelet growth factors (PDGF) and snake venom detection. Works containthe following sections:
1. Review section. After general introducing of principle of SELEX and theadvateges of aptamer, the electrochemical sensor and its applications are highlighted.Moreover, the strategy of label-free base on enzyme-aplification in electrochemicalaptasensor was presented. And at the same time, this paper introduces the significance ofprotein detection and the present development of protein detection methodology
2. Construction of label free electrochemical aptasensor base on enzyme-basedamplification strategies. Recently, there has been substantial attention in the directelectron transfer (DET) of many redox enzymes. However, DET of most enzymes on baresolid electrodes is difficult to achieve because of the instability of matrix and the deeplyembedded active sites in enzyme molecules. In order to overcome this drawback, greateffort has been made to develop novel reagentless biosensor devices based on DET ofenzyme by immobilizing it on nanomaterials modified electrode. Thereinto, graphene (Gra)as an ideal base has been widely applied due to the unique physical and chemical properties,such as high surface area, excellent conductivity and ease of functionalization. On the otherhand, gold nanoparticles (GNPs) are considered as satisfactory nanomaterials because oftheir excellent biocompatibility, strong adsorption ability and good conductivity. Suchproperties make poly (diallyldimethylammonium chloride)(PDDA)-protected Gra-GNPs(P-Gra-GNPs) composite be more attractive as a host immobilization matrix, which notonly assist DET between active sites of enzyme and electrode, but also provide a favorablemicroenvironment for immobilizing enzymes. For this proposed aptasensor, poly(diallyldimethylammonium chloride)(PDDA)-protected graphene-gold nanoparticles(P-Gra-GNPs) composite was firstly coated on the glassy carbon electrode surface to formthe interface of biocompatibility and huge surface area for the adsorption of GOD layer.Subsequently, gold nanoclusters (GNCs) was deposited on the surface of GOD to capturePDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to blockthe remaining active sites of the GNCs and avoid the nonspecific adsorption. Herein, based on the above observations, we first developed a novel reagentless and mediatorlesselectrochemical aptasensor based on the DET of glucose oxidase (GOD) for the detection ofplatelet-derived growth factor (PDGF).
3. Detection of PDGF based on electrochemical aptasensor. Platelet-derivedgrowth factor (PDGF) is a small molecular weight mitogenic peptides regulating factor,which named with its presentation in platelet α particles in normal physiological conditions.PDGF will be released from platelets when blood coagulation, and be activated to stimulatefibroblasts, glial cells, and smooth muscle cells from G0/G1phase into the proliferation cycle.Detection of platelet-derived growth factor plays an important role in fibrosis, cancerdiagnosis and prognostic evaluation. In this work, a new label-free electrochemicalaptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growthfactor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). With thedirect electron transfer of double layer GOD membranes, the aptasensor showed excellentelectrochemical response and the peak current decreased linearly with increasing logarithm ofPDGF concentration from0.005nM to60nM with a relatively low limit of detection of1.7pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-termstability, which provided a new promising technique for aptamer-based protein detection.
4. Screening of Snake venom species-specific aptamer. Snakebite diagnosisrequires accurate identification of the type of snake venom. Current laboratory diagnosis ofsnakebite venom relies mainly on immunological diagnostic methods based on antibodies.But its poor specificity, complex preparation process limit futher application. The principleof aptamer selection (SELEX) inspires us that we can screen target species-specific aptamerfrom ssDNA library by introducing several deplete-screening processs for depleting crosstargets. We call this technology as deplete systematic evolution of ligands by exponentialenrichment (Deplete-SELEX). Using of Deplete-SELEX we obtain Naja naja atra venomspecies-specific nucleic acid aptamers to identify Agkistrodon acutus Guenther venom,Agkistrodon halysvenom, and Trimeresurus mucrosquamatus Canto venom to takeadvantage of subsequent establishment of aptamer-based electrochemical biosensors forsnak venom identification.
5. Identification of snake venom by electrochemical aptasensor. Currentlysnakebite diagnosis relies on clinical symptoms (such as wound, local signs, etc.), routine laboratory tests (hemoglobin, urine bilirubin, etc.) or confirms the species of captured snake.But in the early clinical symptoms of snakebites are often not obvious, and even because ofthe majority of snakebites occur in the evening or in the mountains it is difficult to identifysnake species, which leading to false diagnosis, and miss guidance of the use of antivenintreatment. Over the years, methods such as immuno electrophoresis, ELISA and RIA areused to detect species-specific snake venom components from patient specimens bite byvarious snakes. Affected by material types, diagnostic antibodies quality and other factorsthese methods show low sensitive. Electrochemical aptamer biosensor with high sensitivity,specificity and portable characters will become ideal venom rapid detection means. Bycombining venom species-specificity aptamers with electrochemical biosensor we constructan electrochemical aptamer biosensor for snake venom detection. The sensor shows goodspecificity and sensitivity in snake venom identification, and may become an importantmeans for venom detection.
In summary, we have successfully fabricated a highly sensitive and selectiveaptasensor for PDGF and snake venom detection by using the direct electrochemistry ofGOD on P-Gra-GNPs composite matrix. Moreover, the proposed method holds greatpromise for other proteins or biomarkers detection. Meanwhile we have establishedDeplete-SELEX screening technology, and successfully obtained Naja naja atra venomspecies-specific aptamers. The advantages of the proposed aptasensor were shown asfollows: Firstly, the P-Gra-GNPs composite film possessed large specific surface area andexcellent biocompatibility, which not only increased the immobilization of GOD, but alsoretained the active of immobilized biomolecules and enhanced the stability of aptasensor.Secondly, the GNCs film provided a stable and porous surface for PBA immobilization andfurther amplified the electrochemical signal. In addition, the double layer GOD membranesas tracer brought about higher current response and higher sensitivity. On the basis of theabove reasons, the proposed aptasensor showed relatively low detection limit, satisfactoryselectivity, acceptable stability and reproducibility.
本课题的研究目的是建立一种高灵敏度、高特异性、操作便捷的蛋白质电化学适配体生物传感器。方式是将体外筛选得到的高特异性核酸适配体作为电化学生物传感器的分子识别元件，通过基于酶信号放大的设计策略，建立能够非标记地检测蛋白质的具有高灵敏度、高特异性的新型电化学适配体传感器，并将其应用于血小板生长因子及蛇毒种类鉴别检测的研究中。论文内容包括以下几部分：
第一章引言简要的介绍了核酸适配体的筛选原理、优越性及利用核酸适配体作为识别元件的方法学研究进展；详细的介绍了电化学传感器的原理及应用，以及基于酶信号放大的电化学适配体传感器的研究进展；着重论述了非标记电化学适配体传感器的设计策略和研究进展，并同时介绍了蛋白质检测的意义及方法学现状。
第二章基于酶放大设计策略的非标记电化学适配体传感器的构建近年来，许多氧化还原酶类的直接电子传递(DET)得到了较多的重视和应用。然而，由于固载基质的不稳定以及酶活性中心深埋在分子内，如果将酶分子直接固定到固体电极上，其DET是很难实现的。为了克服这一缺陷，有不少工作提出将酶固定于纳米材料修饰的电极表面，通过纳米材料的优点来实现酶的DET。其中，石墨烯(Gra)作为一种理想的材料，由于其优异的物理化学性能，如大的比表面积、好的导电性和易于功能化等，已经得到广泛的应用。另一方面，纳米金(GNPs)具有良好的生物相容性、强吸附能力和好的导电性，也被看作一种优良的纳米材料。这些性能也就使得PDDA保护的石墨烯-纳米金(P-Gra-GNPs)复合物成为更优异的固载基质，它不仅能加快活性中心与电极之间的DET，更为酶的固定提供了一个良好的微环境。由此本研究在二甲基二烯丙基氯化铵均聚物(PDDA)存在下，采用肼将氧化石墨烯还原，得到PDDA保护的还原石墨烯(P-Gra)。P-Gra表面带正电，通过静电吸附大量的纳米金(AuNPs)，制得纳米金功能化的石墨烯复合物(P-Gra-AuNPs)。利用酶的催化特性来放大识别过程中产生的电化学信号。基于此，我们利用葡萄糖氧化酶(GOD)的直接电子传递(DET)首次构建了一种新型无试剂无媒介的电化学适体传感器，并用于对血小板源性生长因子(PDGF)的检测。
第三章基于电化学适配体传感器的血小板源性生长因子的检测血小板源性生长因子(Platelet derived growth factor，PDGF)是一种低分子量促细胞分裂的肽类调节因子，因正常生理状态下存在于血小板的α颗粒内而得名，当血液凝固时由破裂的血小板释放出来并且被激活，具有刺激停滞于G0/G1期的成纤维细胞、神经胶质细胞、平滑肌细胞等进入分裂增殖周期的功能。血小板源性生长因子的检测在纤维化及癌症等疾病的诊断、预后评估中具有重要的临床意义。
我们利用血小板源性生长因子适配体构建了基于酶放大的电化学核酸适配体传感器，经过对电化学传感器修饰电极的表征，可以看到传感器的氧化还原峰电流减小值与待测PDGF浓度成正比。该适体传感器显示出良好的电化学响应，峰电流的减小值与浓度对数的增加值在0.005-60nM的范围内呈良好的线性，检测限为1.7pM。该传感器同样具有高的选择性，好的重现性和稳定性，也为其他基于适配体的蛋白检测提供了可行的依据。
第四章蛇毒种属特异性核酸适配体的筛选蛇伤的鉴别诊断需要准确的检测蛇毒的类型，目前蛇伤的实验室诊断主要依赖以蛇毒抗体的免疫学诊断方法，但是蛇毒抗体目前存在特异性差、毒性强依靠免疫动物制备困难等缺陷。受核酸适配体筛选原理(SELEX)中反筛原理的启发，我们通过在适配体经典筛选流程中引入多个鉴别靶标的反筛流程，来获取与目标靶标更高特异性结合的种属特异性适配体，我们将该技术称之为消减指数富集配体系统进化技术(Deplete systematic evolution of ligands byexponential enrichment, Deplete-SELEX)。我们在蛇毒种属特异性适配体筛选中引入了该方法，利用该技术筛选出用于鉴别中华眼镜蛇毒与国内常见的三种种毒蛇(尖吻蝮蛇、江浙蝮蛇、烙铁头)的中华眼镜蛇毒种属特异性核酸适配体，以利用于后续建立基于电化学适配体生物传感器的蛇伤现场快速检测方法的研究。
第五章基于电化学适配体传感器的蛇毒快速检测方法的研究目前蛇伤的诊断主要依靠临床症状(如伤口、局部体征等)、常规化验(血红蛋白、尿胆红素等)或捕获的蛇来确诊，但由于蛇伤早期临床症状往往不明显、不典型，且大多数的蛇伤发生在晚间或山林草地中很难看清蛇种，导致野外条件下蛇伤不能得到及时准确的诊断，无法正确指导抗蛇毒血清的使用以至延误抢救治疗良机。多年来，也有研究者应用免疫电泳、酶联免疫以及放射免疫等方法检测病人标本中各种毒蛇的种属特异性蛇毒成分，进行蛇伤免疫学鉴别诊断方法的研究，但这些方法的敏感度不够理想，阳性检出率受检材的种类、取材的时间、方法、诊断抗体的质量等因素影响较大。电化学适配体生物传感器因其检测速度快，灵敏度高，特异性强，简单便携成为蛇毒诊断与现场快速检测的理想检测手段。我们利用筛选得到的蛇毒种属特异性核酸适配体与电化学生物传感器相结合，构建了用于检测毒素的电化学适配体生物传感器，结果显示该传感器在毒素种属的鉴别检测中具有很好的特异性与灵敏度，可能成为蛇伤检测中的重要检测手段。
综上所述，我们采用GOD的直接电化学方法，成功构建了一个基于核酸适配体的高灵敏和高选择性的非标记电化学适配体传感器，用于对血小板生长因子及蛇毒特异性蛋白的检测研究。该传感器主要有以下优点：首先，P-Gra-GNPs复合物膜具有比表面积大和优良的生物相容性，它不仅能增加GOD的固载量，还能保持生物分子的活性，提高传感器的稳定性。其次，GNCs膜为PBA的固定提供了一个稳定和多孔的表面，进一步增强电化学信号。另外，双层GOD膜作为示踪剂带来更高的电流响应和高的灵敏度。基于以上的原因，该传感器显示出较低的检测限，满意的选择性，良好的稳定性和重现性。并且该方法也为其他蛋白质和生物标记物的检测提供了一定的参考。同时我们建立了适配体Deplete-SELEX筛选技术，并成功筛选到了中华眼镜蛇蛇毒种属特异性核酸适配体，成功构建了基于电化学适配体传感器的蛇毒快速检测新方法。
As a major component of life, proteins have played an important role in entire lifeactivities and regulations. Therefore, Detection of proteins expression particular in diseasestate will help us to better understand of the life and to prevent, diagnosis and treatment ofdiseases significantly.
Biosensor based on biological molecules as recognition elements, becomes the mostpromising methods for protein detection with its high specificity, high sensitivity, andconvenient operation. Currently antibodies are the most widely used biomolecularrecognition element, but its further application on biosensors was limited because of theirlong preparation period, instability, Stringent environmental requirements and othershortcomings. In recent years, more and more new recognition elements are introduced intothe preparation of biosensor, wherein the nucleic acid aptamer is the most remarkable one.Aptamers are single-stranded DNA or RNA screened from a large single nucleotide libraryby “Systemic Evolution of Ligands by Exponential Enrichment technique”(SELEX). Inaddition to their high affinity binding ability and high specific recognition characteristicscompare with antibodies, they also exhibit many other distinctive advantages such assmaller molecular weight, wide range of target, accurate synthesis, ease of labeling，stability and can be refolding after denatured, etc. Therefore aptamer is becoming a newideal biosensor recognition element. Electrochemical biosensors are a class of detectionsensor which convert biological concentration of the substance into a detectable signal byrecognition element with characters of high sensitivity, without affected by color of thesolution, quickly response, convenient operation, inexpensive, portable, etc., Combinedwith the aptamer, electrochemical aptasensors become a new hotspot beyond research ofimmune electrochemical sensors and DNA electrochemical sensors.
This project aims to establish a high-sensitivity, high specificity, and convenientoperation electrochemical aptamer biosensor for protein detectation. We set the aptamer as highly specific molecular recognition element of biosensor， and construction highsensitivity, high specificity of label free aptamers electrochemical sensors based onenzyme-based amplification strategies for protein detection. And study the application ofaptasensor in platelet growth factors (PDGF) and snake venom detection. Works containthe following sections:
1. Review section. After general introducing of principle of SELEX and theadvateges of aptamer, the electrochemical sensor and its applications are highlighted.Moreover, the strategy of label-free base on enzyme-aplification in electrochemicalaptasensor was presented. And at the same time, this paper introduces the significance ofprotein detection and the present development of protein detection methodology
2. Construction of label free electrochemical aptasensor base on enzyme-basedamplification strategies. Recently, there has been substantial attention in the directelectron transfer (DET) of many redox enzymes. However, DET of most enzymes on baresolid electrodes is difficult to achieve because of the instability of matrix and the deeplyembedded active sites in enzyme molecules. In order to overcome this drawback, greateffort has been made to develop novel reagentless biosensor devices based on DET ofenzyme by immobilizing it on nanomaterials modified electrode. Thereinto, graphene (Gra)as an ideal base has been widely applied due to the unique physical and chemical properties,such as high surface area, excellent conductivity and ease of functionalization. On the otherhand, gold nanoparticles (GNPs) are considered as satisfactory nanomaterials because oftheir excellent biocompatibility, strong adsorption ability and good conductivity. Suchproperties make poly (diallyldimethylammonium chloride)(PDDA)-protected Gra-GNPs(P-Gra-GNPs) composite be more attractive as a host immobilization matrix, which notonly assist DET between active sites of enzyme and electrode, but also provide a favorablemicroenvironment for immobilizing enzymes. For this proposed aptasensor, poly(diallyldimethylammonium chloride)(PDDA)-protected graphene-gold nanoparticles(P-Gra-GNPs) composite was firstly coated on the glassy carbon electrode surface to formthe interface of biocompatibility and huge surface area for the adsorption of GOD layer.Subsequently, gold nanoclusters (GNCs) was deposited on the surface of GOD to capturePDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to blockthe remaining active sites of the GNCs and avoid the nonspecific adsorption. Herein, based on the above observations, we first developed a novel reagentless and mediatorlesselectrochemical aptasensor based on the DET of glucose oxidase (GOD) for the detection ofplatelet-derived growth factor (PDGF).
3. Detection of PDGF based on electrochemical aptasensor. Platelet-derivedgrowth factor (PDGF) is a small molecular weight mitogenic peptides regulating factor,which named with its presentation in platelet α particles in normal physiological conditions.PDGF will be released from platelets when blood coagulation, and be activated to stimulatefibroblasts, glial cells, and smooth muscle cells from G0/G1phase into the proliferation cycle.Detection of platelet-derived growth factor plays an important role in fibrosis, cancerdiagnosis and prognostic evaluation. In this work, a new label-free electrochemicalaptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growthfactor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). With thedirect electron transfer of double layer GOD membranes, the aptasensor showed excellentelectrochemical response and the peak current decreased linearly with increasing logarithm ofPDGF concentration from0.005nM to60nM with a relatively low limit of detection of1.7pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-termstability, which provided a new promising technique for aptamer-based protein detection.
4. Screening of Snake venom species-specific aptamer. Snakebite diagnosisrequires accurate identification of the type of snake venom. Current laboratory diagnosis ofsnakebite venom relies mainly on immunological diagnostic methods based on antibodies.But its poor specificity, complex preparation process limit futher application. The principleof aptamer selection (SELEX) inspires us that we can screen target species-specific aptamerfrom ssDNA library by introducing several deplete-screening processs for depleting crosstargets. We call this technology as deplete systematic evolution of ligands by exponentialenrichment (Deplete-SELEX). Using of Deplete-SELEX we obtain Naja naja atra venomspecies-specific nucleic acid aptamers to identify Agkistrodon acutus Guenther venom,Agkistrodon halysvenom, and Trimeresurus mucrosquamatus Canto venom to takeadvantage of subsequent establishment of aptamer-based electrochemical biosensors forsnak venom identification.
5. Identification of snake venom by electrochemical aptasensor. Currentlysnakebite diagnosis relies on clinical symptoms (such as wound, local signs, etc.), routine laboratory tests (hemoglobin, urine bilirubin, etc.) or confirms the species of captured snake.But in the early clinical symptoms of snakebites are often not obvious, and even because ofthe majority of snakebites occur in the evening or in the mountains it is difficult to identifysnake species, which leading to false diagnosis, and miss guidance of the use of antivenintreatment. Over the years, methods such as immuno electrophoresis, ELISA and RIA areused to detect species-specific snake venom components from patient specimens bite byvarious snakes. Affected by material types, diagnostic antibodies quality and other factorsthese methods show low sensitive. Electrochemical aptamer biosensor with high sensitivity,specificity and portable characters will become ideal venom rapid detection means. Bycombining venom species-specificity aptamers with electrochemical biosensor we constructan electrochemical aptamer biosensor for snake venom detection. The sensor shows goodspecificity and sensitivity in snake venom identification, and may become an importantmeans for venom detection.
In summary, we have successfully fabricated a highly sensitive and selectiveaptasensor for PDGF and snake venom detection by using the direct electrochemistry ofGOD on P-Gra-GNPs composite matrix. Moreover, the proposed method holds greatpromise for other proteins or biomarkers detection. Meanwhile we have establishedDeplete-SELEX screening technology, and successfully obtained Naja naja atra venomspecies-specific aptamers. The advantages of the proposed aptasensor were shown asfollows: Firstly, the P-Gra-GNPs composite film possessed large specific surface area andexcellent biocompatibility, which not only increased the immobilization of GOD, but alsoretained the active of immobilized biomolecules and enhanced the stability of aptasensor.Secondly, the GNCs film provided a stable and porous surface for PBA immobilization andfurther amplified the electrochemical signal. In addition, the double layer GOD membranesas tracer brought about higher current response and higher sensitivity. On the basis of theabove reasons, the proposed aptasensor showed relatively low detection limit, satisfactoryselectivity, acceptable stability and reproducibility.
引文
1Osborne, S. E., Ellington, A. E. Nucleic acid selection and the challenge of combinatorial chemistry. ChemRev97(1997),349-370.
2Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo I Biotech74(2000),5-13.
3Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
4Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.
5Ellington A D, Szostak J W. Invitro selection of RNA molecules that bind specific ligands.Nature,1990,346(6287):818-822.
6Robertson D L, Joyee G E. Selection invitro of an RNA enzyme that specially cleaves singl-stranded DNA.Nature,1990,344(6265):467-68.
1Tuerk C, Gold L. Systematic evolution of ligands by exponential enriehment: RNA ligands to bacteriophage T4DNAPolymerase. Seience,1990,249(4968):505-510.
2Darfeuille. P., Reigacias, S., Hansen, J.B., Orum, H., Di Primo, C., Toulme, J. J. Aptamers targeted to an RNA hairpinshow improved specificity compared to that of complementary oligonucleotides. Biochemistry45(2006),12076-12082.
1Cox, J.C., Hayhurst, A., Hesselberth, J., Bayer, T.S., Georgiou, G., Ellmgton, A. D.. Automated selection of aptamersagainst protein targets translated in vitro: from gene to aptamer. Nucleic Acids Res30(2002), el08.
2Jayasena SD,Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem.1999Sep;45(9):1628-50.
3S.L. Clark, V.T. Remcho. Aptamers as analytical reagents. Electrophoresis23(2002)1335.
4Willner, M. Zayats, Angew. Electronic aptamer-based sensors. Chem. Int. Ed. Engl.46(2007)6408.
5T. Mairal, V. Cengiz Ozalp, P. Lozano Sanchez, M. Mir, I. Katakis,C.K. O Sullivan. Aptamers: molecular tools foranalytical applications. Anal. Bioanal. Chem.(2007).
6S. Tombelli, M. Minunni, M. Mascini. Analytical applications of aptamers Biosens Biosens. Bioelectron.20(2005)2424
7Lu, Y.(2002). New transition-metal-dependent DNA-zymes as efficient endonucleases and as selective metalbiosensors. Chem Eur J8,4588-4596.
8Fiammengo, R., Jaschke, A.(2005). Nude'ic add enzymes. Curr Opin Biotechnol16,614-621.
9Silverman, S. K.(2004). Deoxyribozymes: DNA catalysts for bioorganic chemistry. Org Biomol Chem2,2701-2706.
1Tombelli S, Minunni M, Mascini M. Aptamers-based assays for diagnostics, environmental and food analysis. BiomolEng.2007Jun;24(2):191-200. Epub2007Mar23.
2Patolsky, F., Lichtenstein, A., Willner, I.(2003). Highly sensitive amplified electronic detection of DNA bybiocatalyzed precipitation of an insoluble product onto electrodes. Chem Eur J9,1137-1145.
1Polsky, R., Gill, R., Willner, I.(2006). Nucleic acid-functionalized Pt nanoparticles: catalytic labels for the amplifiedelectrochemical detection of biomolecules. AnaZ Chern78,
2Floch, F.L., Ho, H.A., Leclerc, M.(2006). Label-free electrochemical detection of protein based on a ferrocene-bearingcationic polythiophene and aptamer. AnaZ Chem76,4727-4731.
3Wang, J., Xu, D., Kawde, A-N., Polsky, R.(2001). Metal nanoparticle-based electrochemical stripping potentionietricdetection of DNA hybridization. AnaZ Chem73,5576-5581.
4Tasset, D.M., Kubik, M.F., Steiner, W.(1997). Oligonucleotide inhibitors of human thrombin that bind distinct epitopes.J Mol Biol272,688-698.
5Ikebukuro, K., Kiyohara, C., Sode, K.(2005). Novel electrochemical sensor system for protein using the aptamers insandwich manner. Biosens Bioelectron20,2168-2172.
12H.J. David, Bunka, G. Peter, Stockley, Aptamers come of age-at last, Nature4(2006)588-596.
M.N. Stojanovic, D.W. Landry, Aptamer-based colorimetric probe for cocaine, J. Am. Chem. Soc.124(2002)9678-9679.
3J.K. Herr, J.E. Smith, C.D. Medley, D. Shangguan, W. Tan, Aptamer-conjugated nanoparticles for selective collectionand detection of cancer cells, Anal. Chem.78(2006)2918-2924.
4K. Stadtherr, H. Wolf, P. Lindner, An aptamer-based protein biochip, Anal. Chem.77(2005)3437-3443.
5Willner and M. Zayats, Electronic aptamer-based sensors, Angew. Chem. Int. Ed.46(2007)6408-6418.
6L.J. Bai, R. Yuan, Y.Q. Chai, Y.L. Yuan, Y. Zhuo, L. Mao, Bi-enzyme functionalized hollow PtCo nanochains as labelsfor an electrochemical aptasensor, Biosens. Bioelectron.26(2011)4331-4336.
7E.E. Ferapontova, E.M. Olsen, K.V. Gothelf, An RNA aptamer-based electrochemical biosensor for detection oftheophylline in serum, J. Am. Chem. Soc.130(2008)4256-4258.
8X.M. Li, J.M. Liu, S.S. Zhang, Electrochemical analysis of two analytes based on a dual-functional aptamer DNAsequence, Chem. Commun.46(2010)595-597.
9X.L. Zuo, Y. Xiao, K.W. Plaxco, High specificity, electrochemical sandwich assays based on single aptamer sequencesand suitable for the direct detection of small-molecule targets in blood and other complex matrices, J. Am. Chem. Soc.131(2009)6944-6945.
10Y. Du, B.L. Li, F. Wang, S.J. Dong, Au nanoparticles grafted sandwich platform used amplified small molecule1e1lectrochemical aptasensor, Biosens. Bioelectron.24(2009)1979-1983.
11E. Katz and I. Willner, Probing biomolecular interactions at conductive and semiconductive surfaces by impedancespectroscopy: Routes to impedimetric immunosensors, DNA-sensors, and enzyme biosensors, Electroanalysis15(2003)913-947.
12Y. Kang, K.J. Feng, J.W. Chen, J.H. Jiang, G.L. Shen, R.Q. Yu, Electrochemical detection of thrombin by sandwichapproach using antibody and aptamer, Bioelectrochemistry73(2008)76-81.
1M.A. Rahman, H.B. Noh, Y.B. Shim, Direct electrochemistry of laccase immobilized on Au nanoparticlesencapsulated-dendrimer bonded conducting polymer: Application for a catechin sensor, Anal. Chem.80(2008)8020-8027.
2X.Y. He, L. Zhou, E.P. Nesterenko, P.N. Nesterenko, B. Paull, J.O. Omamogho, J.D. Glennon, J.H.T. Luong, Porousgraphitized carbon monolith as an electrode material for probing direct bioelectrochemistry and selective detection ofhydrogen peroxide, Anal. Chem.84(2012)2351-2357.
3A.A. Abdelwahab, W.C.A. Koh, H.B. Noh, Y.B. Shim, A selective nitric oxide nanocomposite biosensor based on directelectron transfer of microperoxidase: Removal of interferences by co-immobilized enzymes, Biosens. Bioelectron.26(2010)1080-1086.
4L. Meng, J. Jin, G.X. Yang, T.H. Lu, H. Zhang, C.X. Cai, Nonenzymatic electrochemical detection of glucose based onpalladium-single-walled carbon nanotube hybrid nanostructures, Anal. Chem.81(2009)7271-7280.
5Q. Xie, Y.Y. Zhao, X. Chen, H.M. Liu, D.G. Evans, W.S. Yang, Nanosheet-based titania microspheres with hollowcore-shell structure encapsulating horseradish peroxidase for a mediator-free biosensor, Biomaterials32(2011)6588-6594.
6P. Si, S.J. Ding, J. Yuan, X.W. Lou, D.H. Kim, Hierarchically structured one-dimensional TiO2for proteinimmobilization, direct electrochemistry, and mediator-free glucose sensing, ACS Nano5(2011)1272-1277.
7C.S. Shan, H.F. Yang, J.F. Song, D.X. Han, A. Ivaska, L. Niu, Direct electrochemistry of glucose oxidase andbiosensing for glucose based on graphene, Anal. Chem.81(2009)2378-2382.
8C.X. Guo, X.T. Zheng, S.R. Ng, Y.C. Lai, Y. Lei, C.M. Li, In situ molecular detection of ischemic cells by enhancedprotein direct electron transfer on a unique horseradish peroxidase-Au nanoparticles-polyaniline nanowires biofilm,
9Chem. Commun.47(2011)2652-2654.L.J. Bai, R. Yuan, Y.Q. Chai, Y. Zhuo, Y.L. Yuan, Y. Wang, Simultaneous electrochemical detection of multiple analytesbased on dual signal amplification of single-walled carbon nanotubes and multi-labeled graphene, Biomaterials33(2012)1090-1096.
10B.I. Ipe, K. Yoosaf, K.G. Thomas, Functionalized gold nanoparticles as phosphorescent nanomaterials and sensors, J.Am. Chem. Soc.128(2006)1907-1913.
1L.L. Li, K.P. Liu, G.H. Yang, C.M. Wang, J.R. Zhang, J.J. Zhu, Fabrication of graphene-quantum dots composites forsensitive electrogenerated chemiluminescence immunosensing, Adv. Funct. Mater.21(2011)869-878.
1J.C. Meyer, A.K. Geim, M.I. Katsnelson, K.S. Novoselov, T.J. Booth, S. Roth, The structure of suspended graphenesheets, Nature446(2007)60-63.
1B.I. Ipe, K. Yoosaf, K.G. Thomas, Functionalized gold nanoparticles as phosphorescent nanomaterials and sensors, J.Am. Chem. Soc.128(2006)1907-1913.
1Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor [J]. Physiol Rev,1999,79(4):1283-1316.
2Thieringer F, MaassT, Czochra P, et a.l Spon taneous hepatic fibros is in transgenic mice overexpressing PDGF-A [J].Gen e,2008,423(1):23-28.
3Campbell JS, Hughes SD, Gibertson DG, et a.l Platelet-derived growth factor C induces liver fibrosis, steatosis, andhepatocellular carcinoma [J]. P roc Nat lAcad S ciU S A,2005,102(9):3389-3394.
4Zhang M, Zhang BH, Chen L, An W. Overexpression of heme oxygenase-1protects smooth muscle cells againstoxidative injury and inhibits cell proliferation [J]. Cell Res,2002,12:12-32.
5Henriksen R, Funa K, Wilander E, et al. Expression and prognostic significance of platelet-derived growth factor b andits receptors in epithelial ovarian neoplasms[J]. Cancer Res,1993,53(19):4550-4554.
6FredrikssonL, Li H, Eriksson U. The PDGF family: four gene Produets form five dimeric isoforms. Cytokine GrowthFaetor Rev2004,15:197-204.
7Ustach CV, Kim HR. Platelet-derived growth faetor D is activated by urokinase plasminogen activator in Prostatecarcinoma cells. Mol Cell Biol2005,25:6279一6288.
1FredrikssonL, Li H, Eriksson U. The PDGF family: four gene Produets form five dimeric isoforms. Cytokine GrowthFaetor Rev2004,15:197-204.
2Ustach CV, Kim HR. Platelet-derived growth faetor D is activated by urokinase plasminogen activator in Prostatecarcinoma cells. Mol Cell Biol2005,25:6279一6288.
1周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
2Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin. Toxicol.41,(3)277–290;317–327.
3Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987. Immunodiagnosis of snake venom poisoning. AsianPacific J. Allerg.Immunol.5,187–190.
4Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snake venoms, toxins and venom antibodies,review on recent trends1987–1997. Toxicon37,565–586.
5Theakston, R.D.G.,1983. The application of immunoassay techniques, including enzyme-linked immunosorbent assay(ELISA), to snake venom research. Toxicon21,341–352.
1Willner, I., Zayats, M.(2007). Electronic aptamer-based sensors. Ange-w Chem Int Ed Engl46,2-13.
2Bang, G. S., Cho, S., Kim, B. G. A novel electrochemical detection method for aptamer biosensors. Biosens andBioelectron (2005)21,863-870.
1T.E. Lin, W.H. Chen, Y.C. Shiang, C.C. Huang, H.T. Chang, Colorimetric detection of platelet-derived growth factorsthrough competitive interactions between proteins and functional gold nanoparticles, Biosens. Bioelectron.29(2011)204-209.
2Li, H. X., Rothberg, L. Colorimetric detection of DNA sequences based on electrostatic interactions with unmodifiedgold nanoparticles. Proc Natl Acad Sci USA.(2004).101,14036-14039.
1Z.S. Wu, S.B. Zhang, H. Zhou, G.L. Shen, R.Q. Yu, Universal aptameric system for highly sensitive detection ofprotein based on structure-switching-triggered rolling circle amplification, Anal. Chem.82(2010)2221-2227.
2Z.S. Wu, H. Zhou, S.B. Zhang, G.L. Shen, R.Q. Yu, Electrochemical aptameric recognition system for a sensitiveprotein assay based on specific target binding-induced rolling circle amplification, Anal. Chem.82(2010)2282-2289.
3D.B. Zhu, X.M. Zhou, D. Xing, A new kind of aptamer-based immunomagnetic electrochemiluminescence assay forquantitative detection of protein, Biosens. Bioelectron.26(2010)285-288
1Balamurugan, S., Obubuafo, A., Soper, S. A., McCarley, R. L., Spivak, D. A.(2006). Designing highly specificbiosensing surfaces using aptamer monolayers on gold. Langmuir,2006(22).6446-6453.
1周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
2Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin.Toxicol.41,(3)277–290;317–327.
3Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987. Immunodiagnosis of snake venom poisoning. AsianPacific J. Allerg.Immunol.5,187–190.
4Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snake venoms, toxins and venom antibodies,review on recent trends1987–1997. Toxicon37,565–586.
5Theakston, R.D.G.,1983. The application of immunoassay techniques, including enzyme-linked immunosorbent assay(ELISA), to snake venom research. Toxicon21,341–352.
1Pukrittayakamee, S., Ratcli.e, P.J., McMichael, A.J., Warrell, D.A., Bunnag, D.,1987. A competitiveradioimmunoassay using a monoclonal antibody to detect the factor_activator of Russell's viper venom. Toxicon25,721-729.
2Theakston, R.D.G.,1983. The application of immunoassay techniques, including enzyme-linked immuno-sorbent assay(ELISA), to snake venom research. Toxicon21,341-352.
3Nakamura, M., Hanashiro, K., Kosugi, T.,1993. A modi cation of the ELISA-double sandwich method or estimatingthe concentration of habutobin. Toxicon31,1325-1328.
4Lomonte, B., Gutierrez, J.M., Rojas, G., Calderon, L.,1991. Quantitation by enzyme-immunoassay of antibodiesagainst Bothrops myotoxins in four commercially-available antivenoms. Toxicon29,695-702.
1Liu Minghua, Yao Jie, Xiang Qiang, Zhang Bo, Fu Weiling. Study on piezoelectric quartz crystalm icroarrayimmunosensor for the detection of Agkistrodon acutus venom. Acta academiae medicine militaristertiae.2009(31):1158-1160.
2Selvanayagam, Z.E., Gopalakrishnakone, P., Sridhar, U., Neuzil, P., Sing, M., Ho, L.C., Immobilisation of antibodiesonto silicon wafers and its potential application in the development of ISFET immunosensors. Proceedings of4th
3National Symposium on Progress in Materials Research, National University of Singapore, Singapore,27March1998.Osborne, S. E., Ellington, A. E. Nucleic acid selection and the challenge of combinatorial chemistry.ChemRev97(1997),349-370.
4Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo I Biotech74(2000),5-13.
5Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
6Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.
[1] Osborne, S. E., Ellington, A. E. Nucleic acid selection and the challenge ofcombinatorial chemistry.ChemRev97(1997),349-370.
[2] Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo IBiotech74(2000),5-13.
[3] Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
[4] Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.
[5] Ellington A D, Szostak J W. Invitro selection of RNA molecules that bind specificligands. Nature,1990,346(6287):818-822.
[6] Robertson D L, Joyee G E. Selection invitro of an RNA enzyme that speciallycleaves singl-stranded DNA. Nature,1990,344(6265):467-68.
[7] Tuerk C, Gold L. Systematic evolution of ligands by exponential enriehment: RNAligands to bacteriophage T4DNA Polymerase. Seience,1990,249(4968):505-510.
[8] Darfeuille. P., Reigacias, S., Hansen, J.B., Orum, H., Di Primo, C., Toulme, J. J.Aptamers targeted to an RNA hairpin show improved specificity compared to thatof complementary oligonucleotides. Biochemistry45(2006),12076-12082.
[9] Cox, J.C., Hayhurst, A., Hesselberth, J., Bayer, T.S., Georgiou, G., Ellmgton, A. D..Automated selection of aptamers against protein targets translated in vitro: fromgene to aptamer. Nucleic Acids Res30(2002), el08.
[10] Jayasena SD,Aptamers: an emerging class of molecules that rival antibodies indiagnostics. Clin Chem.1999Sep;45(9):1628-50.
[11] S.L. Clark, V.T. Remcho. Aptamers as analytical reagents. Electrophoresis23(2002)1335.
[12] Willner, M. Zayats, Angew. Electronic aptamer-based sensors. Chem. Int. Ed. Engl.46(2007)6408.
[13] T. Mairal, V. Cengiz Ozalp, P. Lozano Sanchez, M. Mir, I. Katakis,C.K. O Sullivan.Aptamers: molecular tools for analytical applications. Anal. Bioanal. Chem.(2007).
[14] S. Tombelli, M. Minunni, M. Mascini. Analytical applications of aptamers BiosensBiosens. Bioelectron.20(2005)2424
[15] Lu, Y.(2002). New transition-metal-dependent DNA-zymes as efficientendonucleases and as selective metal biosensors. Chem Eur J8,4588-4596.
[16][67] Fiammengo, R., Jaschke, A.(2005). Nude'ic add enzymes. Curr OpinBiotechnol16,614-621.
[17][68] Silverman, S. K.(2004). Deoxyribozymes: DNA catalysts for bioorganicchemistry. Org Biomol Chem2,2701-2706.
[18] Tombelli S, Minunni M, Mascini M. Aptamers-based assays for diagnostics,environmental and food analysis. Biomol Eng.2007Jun;24(2):191-200. Epub2007Mar23.
[19] Patolsky, F., Lichtenstein, A., Willner, I.(2003). Highly sensitive amplifiedelectronic detection of DNA by biocatalyzed precipitation of an insoluble productonto electrodes. Chem Eur J9,1137-1145.
[20] Polsky, R., Gill, R., Willner, I.(2006). Nucleic acid-functionalized Pt nanoparticles:catalytic labels for the amplified electrochemical detection of biomolecules. AnaZChern78,
[21] Floch, F.L., Ho, H.A., Leclerc, M.(2006). Label-free electrochemical detection ofprotein based on a ferrocene-bearing cationic polythiophene and aptamer. AnaZChem76,4727-4731.
[22] Wang, J., Xu, D., Kawde, A-N., Polsky, R.(2001). Metal nanoparticle-basedelectrochemical stripping potentionietric detection of DNA hybridization. AnaZChem73,5576-5581.
[23] Tasset, D.M., Kubik, M.F., Steiner, W.(1997). Oligonucleotide inhibitors of humanthrombin that bind distinct epitopes. J Mol Biol272,688-698.
[24] Ikebukuro, K., Kiyohara, C., Sode, K.(2005). Novel electrochemical sensor systemfor protein using the aptamers in sandwich manner. Biosens Bioelectron20,2168-2172.
[25] H.J. David, Bunka, G. Peter, Stockley, Aptamers come of age-at last, Nature4(2006)588-596.
[26] M.N. Stojanovic, D.W. Landry, Aptamer-based colorimetric probe for cocaine, J.Am. Chem. Soc.124(2002)9678-9679.
[27] J.K. Herr, J.E. Smith, C.D. Medley, D. Shangguan, W. Tan, Aptamer-conjugatednanoparticles for selective collection and detection of cancer cells, Anal. Chem.78(2006)2918-2924.
[28] K. Stadtherr, H. Wolf, P. Lindner, An aptamer-based protein biochip, Anal. Chem.77(2005)3437-3443.
[29] Willner and M. Zayats, Electronic aptamer-based sensors, Angew. Chem. Int. Ed.46(2007)6408-6418.
[30] L.J. Bai, R. Yuan, Y.Q. Chai, Y.L. Yuan, Y. Zhuo, L. Mao, Bi-enzymefunctionalized hollow PtCo nanochains as labels for an electrochemical aptasensor,Biosens. Bioelectron.26(2011)4331-4336.
[31] E.E. Ferapontova, E.M. Olsen, K.V. Gothelf, An RNA aptamer-basedelectrochemical biosensor for detection of theophylline in serum, J. Am. Chem. Soc.130(2008)4256-4258.
[32] X.M. Li, J.M. Liu, S.S. Zhang, Electrochemical analysis of two analytes based on adual-functional aptamer DNA sequence, Chem. Commun.46(2010)595-597.
[33] X.L. Zuo, Y. Xiao, K.W. Plaxco, High specificity, electrochemical sandwich assaysbased on single aptamer sequences and suitable for the direct detection ofsmall-molecule targets in blood and other complex matrices, J. Am. Chem. Soc.131(2009)6944-6945.
[34] Y. Du, B.L. Li, F. Wang, S.J. Dong, Au nanoparticles grafted sandwich platformused amplified small molecule electrochemical aptasensor, Biosens. Bioelectron.24(2009)1979-1983.
[35] E. Katz and I. Willner, Probing biomolecular interactions at conductive andsemiconductive surfaces by impedance spectroscopy: Routes to impedimetricimmunosensors, DNA-sensors, and enzyme biosensors, Electroanalysis15(2003)913-947.
[36] Y. Kang, K.J. Feng, J.W. Chen, J.H. Jiang, G.L. Shen, R.Q. Yu, Electrochemicaldetection of thrombin by sandwich approach using antibody and aptamer,Bioelectrochemistry73(2008)76-81.
[37] M.A. Rahman, H.B. Noh, Y.B. Shim, Direct electrochemistry of laccaseimmobilized on Au nanoparticles encapsulated-dendrimer bonded conductingpolymer: Application for a catechin sensor, Anal. Chem.80(2008)8020-8027.
[38] X.Y. He, L. Zhou, E.P. Nesterenko, P.N. Nesterenko, B. Paull, J.O. Omamogho,J.D. Glennon, J.H.T. Luong, Porous graphitized carbon monolith as an electrodematerial for probing direct bioelectrochemistry and selective detection of hydrogenperoxide, Anal. Chem.84(2012)2351-2357.
[39] A.A. Abdelwahab, W.C.A. Koh, H.B. Noh, Y.B. Shim, A selective nitric oxidenanocomposite biosensor based on direct electron transfer of microperoxidase:Removal of interferences by co-immobilized enzymes, Biosens. Bioelectron.26(2010)1080-1086.
[40] L. Meng, J. Jin, G.X. Yang, T.H. Lu, H. Zhang, C.X. Cai, Nonenzymaticelectrochemical detection of glucose based on palladium-single-walled carbonnanotube hybrid nanostructures, Anal. Chem.81(2009)7271-7280.
[41] Q. Xie, Y.Y. Zhao, X. Chen, H.M. Liu, D.G. Evans, W.S. Yang, Nanosheet-basedtitania microspheres with hollow core-shell structure encapsulating horseradishperoxidase for a mediator-free biosensor, Biomaterials32(2011)6588-6594.
[42] P. Si, S.J. Ding, J. Yuan, X.W. Lou, D.H. Kim, Hierarchically structuredone-dimensional TiO2for protein immobilization, direct electrochemistry, andmediator-free glucose sensing, ACS Nano5(2011)1272-1277.
[43] C.S. Shan, H.F. Yang, J.F. Song, D.X. Han, A. Ivaska, L. Niu, Directelectrochemistry of glucose oxidase and biosensing for glucose based on graphene,Anal. Chem.81(2009)2378-2382.
[44] C.X. Guo, X.T. Zheng, S.R. Ng, Y.C. Lai, Y. Lei, C.M. Li, In situ moleculardetection of ischemic cells by enhanced protein direct electron transfer on a uniquehorseradish peroxidase-Au nanoparticles-polyaniline nanowires biofilm, Chem.Commun.47(2011)2652-2654.
[45] L.J. Bai, R. Yuan, Y.Q. Chai, Y. Zhuo, Y.L. Yuan, Y. Wang, Simultaneouselectrochemical detection of multiple analytes based on dual signal amplification ofsingle-walled carbon nanotubes and multi-labeled graphene, Biomaterials33(2012)1090-1096.
[46] B.I. Ipe, K. Yoosaf, K.G. Thomas, Functionalized gold nanoparticles asphosphorescent nanomaterials and sensors, J. Am. Chem. Soc.128(2006)1907-1913.
[47] L.L. Li, K.P. Liu, G.H. Yang, C.M. Wang, J.R. Zhang, J.J. Zhu, Fabrication ofgraphene-quantum dots composites for sensitive electrogeneratedchemiluminescence immunosensing, Adv. Funct. Mater.21(2011)869-878.
[48] J.C. Meyer, A.K. Geim, M.I. Katsnelson, K.S. Novoselov, T.J. Booth, S. Roth, Thestructure of suspended graphene sheets, Nature446(2007)60-63.
[49] Heldin CH, Westermark B. Mechanism of action and in vivo role ofplatelet-derived growth factor [J]. Physiol Rev,1999,79(4):1283-1316.
[50] Thieringer F, MaassT, Czochra P, et a.l Spon taneous hepatic fibros is in transgenicmice overexpressing PDGF-A [J]. Gen e,2008,423(1):23-28.
[51] Campbell JS, Hughes SD, Gibertson DG, et a.l Platelet-derived growth factor Cinduces liver fibrosis, steatosis, and hepatocellular carcinoma [J]. P roc Nat lAcadS ciU S A,2005,102(9):3389-3394.
[52] Zhang M, Zhang BH, Chen L, An W. Overexpression of heme oxygenase-1protects smooth muscle cells against oxidative injury and inhibits cell proliferation[J]. Cell Res,2002,12:12-32.
[53] Henriksen R, Funa K, Wilander E, et al. Expression and prognostic significance ofplatelet-derived growth factor b and its receptors in epithelial ovarian neoplasms[J].Cancer Res,1993,53(19):4550-4554.
[54] FredrikssonL, Li H, Eriksson U. The PDGF family: four gene Produets form fivedimeric isoforms. Cytokine Growth Faetor Rev2004,15:197-204.
[55] Ustach CV, Kim HR. Platelet-derived growth faetor D is activated by urokinaseplasminogen activator in Prostate carcinoma cells. Mol Cell Biol2005,25:6279一6288.
[56] Wang Z, Kong D, Bane S, et al. Down-regulation of Platelet-derived growthfaetor-D inhibits cell growth and angiogenesis through inactivation of Notch-1andnuclear factor-kappa B signaling. Cancer Res2007,67:11377一11385.
[57] Xu L,Tong R, Coehran DM, et al. Blocking patelet-derived growth faetor-D/platelet-derived growth factor receptor beta signaling inhibits human renal cellcarcinoma progress in an orthotopic mouse model. Caneer Res2005.65:5711一5719.
[58]周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
[59] Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin. Toxicol.41,(3)277–290;317–327.
[60] Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987.Immunodiagnosis of snake venom poisoning. Asian Pacific J. Allerg.Immunol.5,187–190.
[61] Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snakevenoms, toxins and venom antibodies, review on recent trends1987–1997. Toxicon37,565–586.
[62] Theakston, R.D.G.,1983. The application of immunoassay techniques, includingenzyme-linked immunosorbent assay (ELISA), to snake venom research. Toxicon21,341–352.
[63] Gold, L., Polisky, B., Uhlenbeck,0., Yarus, M. Diversity of oligonucleotidefunctions. Annu Rev Biochem64(1995),763-797.
[1] Baker, B. R.. I..,, R. Y.. Wood, M. s.. Doctor, E. H., Heeger, A. J., Plaxco, K. w. AnElectronic, Aptamer-Based Small-Molecule Sensor for the Rapid, Label-FreeDetection of Cocaine in Adulterated Samples and Biological Fluids. J AmChem Soc128,3138-3139.
[2] Lee, M., Wait, D. R.(2000). A fiber-optic microarray biosensor using aptamers asreceptors. Anal Biochem282,142-146.
[3] Liss, M., Petersen, B., Wolf, H., Prohaska, E.. An aptamer-based quartz crystalprotein biosensor. AnalChemlA,(2002)4488-4495.
[4] Tombelli, S., Minunni, A., Luzi, E., Mascini, M. Aptamer-based biosensors for thedetection of HIV-1Tat protein. Bioelectrochemistry (2005)67,135-141.
[5] Nutiu, R., Mei, S., Liu, Z. J., Li, Y. F. Engineering DNA aptamers and DNA enzymeswith fluorescence-signaling properties. Pure Appl Chem (2004)76,1547-1561.
[6] Pavlov, V., Shlyahovsky, B., Willner, I. Fluorescence detection of DNA by thecatalytic activation of an aptamer/thrombin complex. J Am Chem Soc (2005)127,6522-6523.
[7] Yang, C. J., Jockusch, S., Vicens, M., Turro, N. J., Tan, W. H.. Light-switchingexcimer probes for rapid protein monitoring in complex biological fluids. Proc NatlAcad Sci USA (2005)102,17278-17283.
[8] Rupcich, N., Nutiu, R.y Li, Y. F., Brennan, J. D. Solid-phase enzyme activity assayutilizing an entrapped fluorescence-signaling DNA aptamer. Ange-w Chern Int EdEngl (2006)45,3295-3299.
[9] Lu, Y., Liu, J. W. Functional DNA nanotechnology: emerging applications ofDNAzymes and aptamers. Curr Opin Biotechno (2006)17,580-588.
[10] Lu, Y., Liu, J. W. Smart nanomaterials inspired by biology: dynamic assembly oferror-free manomaterials in response to multiple chemical and biological stimuli. AceChemRes (2007).40,315-323.
[11] Ravelet, C., Grosset, C., Peyrin, E. Liquid chromatography, electrochromatographyand capillary electrophoresis applications of DNA and RNA aptamers.|J ChromatogrA (2006)1117,1-10.
[12] Willner, I., Zayats, M.. Electronic aptamer-based sensors. Ange-w Chem Int Ed Engl(2007)46,2-13. I
[13] Willner, I., Zayats, M.(2007). Electronic aptamer-based sensors. Ange-w Chem IntEd Engl46,2-13.
[14] Bang, G. S., Cho, S., Kim, B. G. A novel electrochemical detection method foraptamer biosensors. Biosens and Bioelectron (2005)21,863-870.
[15] G.S. Bang, S. Cho, B.G. Kim, A novel electrochemical detection method for aptamerbiosensors. Biosens and Bioelectron.21(2005)863.
[16] F. Le Floch, H.A. Ho, M. Leclerc. Label-free electrochemical detection of proteinbased on a ferrocene-bearing cationic polythiophene and aptamer. Anal. Chem.78(2006)4727.
[17] A.K. Cheng, B. Ge, H.Z. Yu. A Review: Electrochemical Aptasensors with VariousDetection Strategies. Anal. Chem.79(2007)5158.
[18] H.M. So, K. Won, Y.H. Kim, B.K. Kim, B.H. Ryu, P.S. Na, H. Kim,J.O. Lee, J. Am.Single-walled carbon nanotube biosensors using aptamers as molecular recognitionelements. Chem. Soc.127(2005)11906.
[19] K. Maehashi, T. Katsura, K. Kerman, Y. Takamura, K. Matsumoto, E. Tamiya.Label-Free Protein Biosensor Based on Aptamer-Modified Carbon NanotubeField-Effect Transistors. Anal. Chem.79(2007)782
[20] Stojanovic, M. N., Kolpashchikov, D. M.(2004). Modular aptameric sensors. J AmC/iem Soc126,9266-9270.
[21] T.E. Lin, W.H. Chen, Y.C. Shiang, C.C. Huang, H.T. Chang, Colorimetric detection ofplatelet-derived growth factors through competitive interactions between proteins andfunctional gold nanoparticles, Biosens. Bioelectron.29(2011)204-209.
[22] Li, H. X., Rothberg, L. Colorimetric detection of DNA sequences based onelectrostatic interactions with unmodified gold nanoparticles. Proc Natl Acad SciUSA.(2004).101,14036-14039.
[23] Balamurugan, S., Obubuafo, A., Soper, S. A., McCarley, R. L., Spivak, D. A.(2006).Designing highly specific biosensing surfaces using aptamer monolayers on gold.Langmuir,2006(22).6446-6453.
[24] Z.S. Wu, S.B. Zhang, H. Zhou, G.L. Shen, R.Q. Yu, Universal aptameric system forhighly sensitive detection of protein based on structure-switching-triggered rollingcircle amplification, Anal. Chem.82(2010)2221-2227.
[25] Z.S. Wu, H. Zhou, S.B. Zhang, G.L. Shen, R.Q. Yu, Electrochemical aptamericrecognition system for a sensitive protein assay based on specific targetbinding-induced rolling circle amplification, Anal. Chem.82(2010)2282-2289.
[26] D.B. Zhu, X.M. Zhou, D. Xing, A new kind of aptamer-based immunomagneticelectrochemiluminescence assay for quantitative detection of protein, Biosens.Bioelectron.26(2010)285-288
[1]周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
[2] Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin.Toxicol.41,(3)277–290;317–327.
[3] Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987. Immunodiagnosisof snake venom poisoning. Asian Pacific J. Allerg.Immunol.5,187–190.
[4] Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snake venoms,toxins and venom antibodies, review on recent trends1987–1997. Toxicon37,565–586.
[5] Theakston, R.D.G.,1983. The application of immunoassay techniques, includingenzyme-linked immunosorbent assay (ELISA), to snake venom research. Toxicon21,341–352.
[6] Pukrittayakamee, S., Ratcli.e, P.J., McMichael, A.J., Warrell, D.A., Bunnag, D.,1987.A competitive radioimmunoassay using a monoclonal antibody to detect thefactor_activator of Russell's viper venom. Toxicon25,721-729.
[7] Theakston, R.D.G.,1983. The application of immunoassay techniques, includingenzyme-linked immuno-sorbent assay (ELISA), to snake venom research. Toxicon21,341-352.
[8] Nakamura, M., Hanashiro, K., Kosugi, T.,1993. A modi cation of the ELISA-doublesandwich method or estimating the concentration of habutobin. Toxicon31,1325-1328.
[9] Lomonte, B., Gutierrez, J.M., Rojas, G., Calderon, L.,1991. Quantitation byenzyme-immunoassay of antibodies against Bothrops myotoxins in fourcommercially-available antivenoms. Toxicon29,695-702.
[10] Liu Minghua, Yao Jie, Xiang Qiang, Zhang Bo, Fu Weiling. Study on piezoelectricquartz crystalm icroarray immunosensor for the detection of Agkistrodon acutusvenom. Acta academiae medicine militaris tertiae.2009(31):1158-1160.
[11] Selvanayagam, Z.E., Gopalakrishnakone, P., Sridhar, U., Neuzil, P., Sing, M., Ho,L.C., Immobilisation of antibodies onto silicon wafers and its potential application inthe development of ISFET immunosensors. Proceedings of4th National Symposiumon Progress in Materials Research, National University of Singapore, Singapore,27March1998.
[12] Osborne, S. E., Ellington, A. E. Nucleic acid selection and the challenge ofcombinatorial chemistry.ChemRev97(1997),349-370.
[13] Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo IBiotech74(2000),5-13.
[14] Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
[15] Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.
2Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo I Biotech74(2000),5-13.
3Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
4Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.
5Ellington A D, Szostak J W. Invitro selection of RNA molecules that bind specific ligands.Nature,1990,346(6287):818-822.
6Robertson D L, Joyee G E. Selection invitro of an RNA enzyme that specially cleaves singl-stranded DNA.Nature,1990,344(6265):467-68.
1Tuerk C, Gold L. Systematic evolution of ligands by exponential enriehment: RNA ligands to bacteriophage T4DNAPolymerase. Seience,1990,249(4968):505-510.
2Darfeuille. P., Reigacias, S., Hansen, J.B., Orum, H., Di Primo, C., Toulme, J. J. Aptamers targeted to an RNA hairpinshow improved specificity compared to that of complementary oligonucleotides. Biochemistry45(2006),12076-12082.
1Cox, J.C., Hayhurst, A., Hesselberth, J., Bayer, T.S., Georgiou, G., Ellmgton, A. D.. Automated selection of aptamersagainst protein targets translated in vitro: from gene to aptamer. Nucleic Acids Res30(2002), el08.
2Jayasena SD,Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem.1999Sep;45(9):1628-50.
3S.L. Clark, V.T. Remcho. Aptamers as analytical reagents. Electrophoresis23(2002)1335.
4Willner, M. Zayats, Angew. Electronic aptamer-based sensors. Chem. Int. Ed. Engl.46(2007)6408.
5T. Mairal, V. Cengiz Ozalp, P. Lozano Sanchez, M. Mir, I. Katakis,C.K. O Sullivan. Aptamers: molecular tools foranalytical applications. Anal. Bioanal. Chem.(2007).
6S. Tombelli, M. Minunni, M. Mascini. Analytical applications of aptamers Biosens Biosens. Bioelectron.20(2005)2424
7Lu, Y.(2002). New transition-metal-dependent DNA-zymes as efficient endonucleases and as selective metalbiosensors. Chem Eur J8,4588-4596.
8Fiammengo, R., Jaschke, A.(2005). Nude'ic add enzymes. Curr Opin Biotechnol16,614-621.
9Silverman, S. K.(2004). Deoxyribozymes: DNA catalysts for bioorganic chemistry. Org Biomol Chem2,2701-2706.
1Tombelli S, Minunni M, Mascini M. Aptamers-based assays for diagnostics, environmental and food analysis. BiomolEng.2007Jun;24(2):191-200. Epub2007Mar23.
2Patolsky, F., Lichtenstein, A., Willner, I.(2003). Highly sensitive amplified electronic detection of DNA bybiocatalyzed precipitation of an insoluble product onto electrodes. Chem Eur J9,1137-1145.
1Polsky, R., Gill, R., Willner, I.(2006). Nucleic acid-functionalized Pt nanoparticles: catalytic labels for the amplifiedelectrochemical detection of biomolecules. AnaZ Chern78,
2Floch, F.L., Ho, H.A., Leclerc, M.(2006). Label-free electrochemical detection of protein based on a ferrocene-bearingcationic polythiophene and aptamer. AnaZ Chem76,4727-4731.
3Wang, J., Xu, D., Kawde, A-N., Polsky, R.(2001). Metal nanoparticle-based electrochemical stripping potentionietricdetection of DNA hybridization. AnaZ Chem73,5576-5581.
4Tasset, D.M., Kubik, M.F., Steiner, W.(1997). Oligonucleotide inhibitors of human thrombin that bind distinct epitopes.J Mol Biol272,688-698.
5Ikebukuro, K., Kiyohara, C., Sode, K.(2005). Novel electrochemical sensor system for protein using the aptamers insandwich manner. Biosens Bioelectron20,2168-2172.
12H.J. David, Bunka, G. Peter, Stockley, Aptamers come of age-at last, Nature4(2006)588-596.
M.N. Stojanovic, D.W. Landry, Aptamer-based colorimetric probe for cocaine, J. Am. Chem. Soc.124(2002)9678-9679.
3J.K. Herr, J.E. Smith, C.D. Medley, D. Shangguan, W. Tan, Aptamer-conjugated nanoparticles for selective collectionand detection of cancer cells, Anal. Chem.78(2006)2918-2924.
4K. Stadtherr, H. Wolf, P. Lindner, An aptamer-based protein biochip, Anal. Chem.77(2005)3437-3443.
5Willner and M. Zayats, Electronic aptamer-based sensors, Angew. Chem. Int. Ed.46(2007)6408-6418.
6L.J. Bai, R. Yuan, Y.Q. Chai, Y.L. Yuan, Y. Zhuo, L. Mao, Bi-enzyme functionalized hollow PtCo nanochains as labelsfor an electrochemical aptasensor, Biosens. Bioelectron.26(2011)4331-4336.
7E.E. Ferapontova, E.M. Olsen, K.V. Gothelf, An RNA aptamer-based electrochemical biosensor for detection oftheophylline in serum, J. Am. Chem. Soc.130(2008)4256-4258.
8X.M. Li, J.M. Liu, S.S. Zhang, Electrochemical analysis of two analytes based on a dual-functional aptamer DNAsequence, Chem. Commun.46(2010)595-597.
9X.L. Zuo, Y. Xiao, K.W. Plaxco, High specificity, electrochemical sandwich assays based on single aptamer sequencesand suitable for the direct detection of small-molecule targets in blood and other complex matrices, J. Am. Chem. Soc.131(2009)6944-6945.
10Y. Du, B.L. Li, F. Wang, S.J. Dong, Au nanoparticles grafted sandwich platform used amplified small molecule1e1lectrochemical aptasensor, Biosens. Bioelectron.24(2009)1979-1983.
11E. Katz and I. Willner, Probing biomolecular interactions at conductive and semiconductive surfaces by impedancespectroscopy: Routes to impedimetric immunosensors, DNA-sensors, and enzyme biosensors, Electroanalysis15(2003)913-947.
12Y. Kang, K.J. Feng, J.W. Chen, J.H. Jiang, G.L. Shen, R.Q. Yu, Electrochemical detection of thrombin by sandwichapproach using antibody and aptamer, Bioelectrochemistry73(2008)76-81.
1M.A. Rahman, H.B. Noh, Y.B. Shim, Direct electrochemistry of laccase immobilized on Au nanoparticlesencapsulated-dendrimer bonded conducting polymer: Application for a catechin sensor, Anal. Chem.80(2008)8020-8027.
2X.Y. He, L. Zhou, E.P. Nesterenko, P.N. Nesterenko, B. Paull, J.O. Omamogho, J.D. Glennon, J.H.T. Luong, Porousgraphitized carbon monolith as an electrode material for probing direct bioelectrochemistry and selective detection ofhydrogen peroxide, Anal. Chem.84(2012)2351-2357.
3A.A. Abdelwahab, W.C.A. Koh, H.B. Noh, Y.B. Shim, A selective nitric oxide nanocomposite biosensor based on directelectron transfer of microperoxidase: Removal of interferences by co-immobilized enzymes, Biosens. Bioelectron.26(2010)1080-1086.
4L. Meng, J. Jin, G.X. Yang, T.H. Lu, H. Zhang, C.X. Cai, Nonenzymatic electrochemical detection of glucose based onpalladium-single-walled carbon nanotube hybrid nanostructures, Anal. Chem.81(2009)7271-7280.
5Q. Xie, Y.Y. Zhao, X. Chen, H.M. Liu, D.G. Evans, W.S. Yang, Nanosheet-based titania microspheres with hollowcore-shell structure encapsulating horseradish peroxidase for a mediator-free biosensor, Biomaterials32(2011)6588-6594.
6P. Si, S.J. Ding, J. Yuan, X.W. Lou, D.H. Kim, Hierarchically structured one-dimensional TiO2for proteinimmobilization, direct electrochemistry, and mediator-free glucose sensing, ACS Nano5(2011)1272-1277.
7C.S. Shan, H.F. Yang, J.F. Song, D.X. Han, A. Ivaska, L. Niu, Direct electrochemistry of glucose oxidase andbiosensing for glucose based on graphene, Anal. Chem.81(2009)2378-2382.
8C.X. Guo, X.T. Zheng, S.R. Ng, Y.C. Lai, Y. Lei, C.M. Li, In situ molecular detection of ischemic cells by enhancedprotein direct electron transfer on a unique horseradish peroxidase-Au nanoparticles-polyaniline nanowires biofilm,
9Chem. Commun.47(2011)2652-2654.L.J. Bai, R. Yuan, Y.Q. Chai, Y. Zhuo, Y.L. Yuan, Y. Wang, Simultaneous electrochemical detection of multiple analytesbased on dual signal amplification of single-walled carbon nanotubes and multi-labeled graphene, Biomaterials33(2012)1090-1096.
10B.I. Ipe, K. Yoosaf, K.G. Thomas, Functionalized gold nanoparticles as phosphorescent nanomaterials and sensors, J.Am. Chem. Soc.128(2006)1907-1913.
1L.L. Li, K.P. Liu, G.H. Yang, C.M. Wang, J.R. Zhang, J.J. Zhu, Fabrication of graphene-quantum dots composites forsensitive electrogenerated chemiluminescence immunosensing, Adv. Funct. Mater.21(2011)869-878.
1J.C. Meyer, A.K. Geim, M.I. Katsnelson, K.S. Novoselov, T.J. Booth, S. Roth, The structure of suspended graphenesheets, Nature446(2007)60-63.
1B.I. Ipe, K. Yoosaf, K.G. Thomas, Functionalized gold nanoparticles as phosphorescent nanomaterials and sensors, J.Am. Chem. Soc.128(2006)1907-1913.
1Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor [J]. Physiol Rev,1999,79(4):1283-1316.
2Thieringer F, MaassT, Czochra P, et a.l Spon taneous hepatic fibros is in transgenic mice overexpressing PDGF-A [J].Gen e,2008,423(1):23-28.
3Campbell JS, Hughes SD, Gibertson DG, et a.l Platelet-derived growth factor C induces liver fibrosis, steatosis, andhepatocellular carcinoma [J]. P roc Nat lAcad S ciU S A,2005,102(9):3389-3394.
4Zhang M, Zhang BH, Chen L, An W. Overexpression of heme oxygenase-1protects smooth muscle cells againstoxidative injury and inhibits cell proliferation [J]. Cell Res,2002,12:12-32.
5Henriksen R, Funa K, Wilander E, et al. Expression and prognostic significance of platelet-derived growth factor b andits receptors in epithelial ovarian neoplasms[J]. Cancer Res,1993,53(19):4550-4554.
6FredrikssonL, Li H, Eriksson U. The PDGF family: four gene Produets form five dimeric isoforms. Cytokine GrowthFaetor Rev2004,15:197-204.
7Ustach CV, Kim HR. Platelet-derived growth faetor D is activated by urokinase plasminogen activator in Prostatecarcinoma cells. Mol Cell Biol2005,25:6279一6288.
1FredrikssonL, Li H, Eriksson U. The PDGF family: four gene Produets form five dimeric isoforms. Cytokine GrowthFaetor Rev2004,15:197-204.
2Ustach CV, Kim HR. Platelet-derived growth faetor D is activated by urokinase plasminogen activator in Prostatecarcinoma cells. Mol Cell Biol2005,25:6279一6288.
1周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
2Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin. Toxicol.41,(3)277–290;317–327.
3Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987. Immunodiagnosis of snake venom poisoning. AsianPacific J. Allerg.Immunol.5,187–190.
4Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snake venoms, toxins and venom antibodies,review on recent trends1987–1997. Toxicon37,565–586.
5Theakston, R.D.G.,1983. The application of immunoassay techniques, including enzyme-linked immunosorbent assay(ELISA), to snake venom research. Toxicon21,341–352.
1Willner, I., Zayats, M.(2007). Electronic aptamer-based sensors. Ange-w Chem Int Ed Engl46,2-13.
2Bang, G. S., Cho, S., Kim, B. G. A novel electrochemical detection method for aptamer biosensors. Biosens andBioelectron (2005)21,863-870.
1T.E. Lin, W.H. Chen, Y.C. Shiang, C.C. Huang, H.T. Chang, Colorimetric detection of platelet-derived growth factorsthrough competitive interactions between proteins and functional gold nanoparticles, Biosens. Bioelectron.29(2011)204-209.
2Li, H. X., Rothberg, L. Colorimetric detection of DNA sequences based on electrostatic interactions with unmodifiedgold nanoparticles. Proc Natl Acad Sci USA.(2004).101,14036-14039.
1Z.S. Wu, S.B. Zhang, H. Zhou, G.L. Shen, R.Q. Yu, Universal aptameric system for highly sensitive detection ofprotein based on structure-switching-triggered rolling circle amplification, Anal. Chem.82(2010)2221-2227.
2Z.S. Wu, H. Zhou, S.B. Zhang, G.L. Shen, R.Q. Yu, Electrochemical aptameric recognition system for a sensitiveprotein assay based on specific target binding-induced rolling circle amplification, Anal. Chem.82(2010)2282-2289.
3D.B. Zhu, X.M. Zhou, D. Xing, A new kind of aptamer-based immunomagnetic electrochemiluminescence assay forquantitative detection of protein, Biosens. Bioelectron.26(2010)285-288
1Balamurugan, S., Obubuafo, A., Soper, S. A., McCarley, R. L., Spivak, D. A.(2006). Designing highly specificbiosensing surfaces using aptamer monolayers on gold. Langmuir,2006(22).6446-6453.
1周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
2Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin.Toxicol.41,(3)277–290;317–327.
3Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987. Immunodiagnosis of snake venom poisoning. AsianPacific J. Allerg.Immunol.5,187–190.
4Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snake venoms, toxins and venom antibodies,review on recent trends1987–1997. Toxicon37,565–586.
5Theakston, R.D.G.,1983. The application of immunoassay techniques, including enzyme-linked immunosorbent assay(ELISA), to snake venom research. Toxicon21,341–352.
1Pukrittayakamee, S., Ratcli.e, P.J., McMichael, A.J., Warrell, D.A., Bunnag, D.,1987. A competitiveradioimmunoassay using a monoclonal antibody to detect the factor_activator of Russell's viper venom. Toxicon25,721-729.
2Theakston, R.D.G.,1983. The application of immunoassay techniques, including enzyme-linked immuno-sorbent assay(ELISA), to snake venom research. Toxicon21,341-352.
3Nakamura, M., Hanashiro, K., Kosugi, T.,1993. A modi cation of the ELISA-double sandwich method or estimatingthe concentration of habutobin. Toxicon31,1325-1328.
4Lomonte, B., Gutierrez, J.M., Rojas, G., Calderon, L.,1991. Quantitation by enzyme-immunoassay of antibodiesagainst Bothrops myotoxins in four commercially-available antivenoms. Toxicon29,695-702.
1Liu Minghua, Yao Jie, Xiang Qiang, Zhang Bo, Fu Weiling. Study on piezoelectric quartz crystalm icroarrayimmunosensor for the detection of Agkistrodon acutus venom. Acta academiae medicine militaristertiae.2009(31):1158-1160.
2Selvanayagam, Z.E., Gopalakrishnakone, P., Sridhar, U., Neuzil, P., Sing, M., Ho, L.C., Immobilisation of antibodiesonto silicon wafers and its potential application in the development of ISFET immunosensors. Proceedings of4th
3National Symposium on Progress in Materials Research, National University of Singapore, Singapore,27March1998.Osborne, S. E., Ellington, A. E. Nucleic acid selection and the challenge of combinatorial chemistry.ChemRev97(1997),349-370.
4Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo I Biotech74(2000),5-13.
5Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
6Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.
[1] Osborne, S. E., Ellington, A. E. Nucleic acid selection and the challenge ofcombinatorial chemistry.ChemRev97(1997),349-370.
[2] Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo IBiotech74(2000),5-13.
[3] Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
[4] Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.
[5] Ellington A D, Szostak J W. Invitro selection of RNA molecules that bind specificligands. Nature,1990,346(6287):818-822.
[6] Robertson D L, Joyee G E. Selection invitro of an RNA enzyme that speciallycleaves singl-stranded DNA. Nature,1990,344(6265):467-68.
[7] Tuerk C, Gold L. Systematic evolution of ligands by exponential enriehment: RNAligands to bacteriophage T4DNA Polymerase. Seience,1990,249(4968):505-510.
[8] Darfeuille. P., Reigacias, S., Hansen, J.B., Orum, H., Di Primo, C., Toulme, J. J.Aptamers targeted to an RNA hairpin show improved specificity compared to thatof complementary oligonucleotides. Biochemistry45(2006),12076-12082.
[9] Cox, J.C., Hayhurst, A., Hesselberth, J., Bayer, T.S., Georgiou, G., Ellmgton, A. D..Automated selection of aptamers against protein targets translated in vitro: fromgene to aptamer. Nucleic Acids Res30(2002), el08.
[10] Jayasena SD,Aptamers: an emerging class of molecules that rival antibodies indiagnostics. Clin Chem.1999Sep;45(9):1628-50.
[11] S.L. Clark, V.T. Remcho. Aptamers as analytical reagents. Electrophoresis23(2002)1335.
[12] Willner, M. Zayats, Angew. Electronic aptamer-based sensors. Chem. Int. Ed. Engl.46(2007)6408.
[13] T. Mairal, V. Cengiz Ozalp, P. Lozano Sanchez, M. Mir, I. Katakis,C.K. O Sullivan.Aptamers: molecular tools for analytical applications. Anal. Bioanal. Chem.(2007).
[14] S. Tombelli, M. Minunni, M. Mascini. Analytical applications of aptamers BiosensBiosens. Bioelectron.20(2005)2424
[15] Lu, Y.(2002). New transition-metal-dependent DNA-zymes as efficientendonucleases and as selective metal biosensors. Chem Eur J8,4588-4596.
[16][67] Fiammengo, R., Jaschke, A.(2005). Nude'ic add enzymes. Curr OpinBiotechnol16,614-621.
[17][68] Silverman, S. K.(2004). Deoxyribozymes: DNA catalysts for bioorganicchemistry. Org Biomol Chem2,2701-2706.
[18] Tombelli S, Minunni M, Mascini M. Aptamers-based assays for diagnostics,environmental and food analysis. Biomol Eng.2007Jun;24(2):191-200. Epub2007Mar23.
[19] Patolsky, F., Lichtenstein, A., Willner, I.(2003). Highly sensitive amplifiedelectronic detection of DNA by biocatalyzed precipitation of an insoluble productonto electrodes. Chem Eur J9,1137-1145.
[20] Polsky, R., Gill, R., Willner, I.(2006). Nucleic acid-functionalized Pt nanoparticles:catalytic labels for the amplified electrochemical detection of biomolecules. AnaZChern78,
[21] Floch, F.L., Ho, H.A., Leclerc, M.(2006). Label-free electrochemical detection ofprotein based on a ferrocene-bearing cationic polythiophene and aptamer. AnaZChem76,4727-4731.
[22] Wang, J., Xu, D., Kawde, A-N., Polsky, R.(2001). Metal nanoparticle-basedelectrochemical stripping potentionietric detection of DNA hybridization. AnaZChem73,5576-5581.
[23] Tasset, D.M., Kubik, M.F., Steiner, W.(1997). Oligonucleotide inhibitors of humanthrombin that bind distinct epitopes. J Mol Biol272,688-698.
[24] Ikebukuro, K., Kiyohara, C., Sode, K.(2005). Novel electrochemical sensor systemfor protein using the aptamers in sandwich manner. Biosens Bioelectron20,2168-2172.
[25] H.J. David, Bunka, G. Peter, Stockley, Aptamers come of age-at last, Nature4(2006)588-596.
[26] M.N. Stojanovic, D.W. Landry, Aptamer-based colorimetric probe for cocaine, J.Am. Chem. Soc.124(2002)9678-9679.
[27] J.K. Herr, J.E. Smith, C.D. Medley, D. Shangguan, W. Tan, Aptamer-conjugatednanoparticles for selective collection and detection of cancer cells, Anal. Chem.78(2006)2918-2924.
[28] K. Stadtherr, H. Wolf, P. Lindner, An aptamer-based protein biochip, Anal. Chem.77(2005)3437-3443.
[29] Willner and M. Zayats, Electronic aptamer-based sensors, Angew. Chem. Int. Ed.46(2007)6408-6418.
[30] L.J. Bai, R. Yuan, Y.Q. Chai, Y.L. Yuan, Y. Zhuo, L. Mao, Bi-enzymefunctionalized hollow PtCo nanochains as labels for an electrochemical aptasensor,Biosens. Bioelectron.26(2011)4331-4336.
[31] E.E. Ferapontova, E.M. Olsen, K.V. Gothelf, An RNA aptamer-basedelectrochemical biosensor for detection of theophylline in serum, J. Am. Chem. Soc.130(2008)4256-4258.
[32] X.M. Li, J.M. Liu, S.S. Zhang, Electrochemical analysis of two analytes based on adual-functional aptamer DNA sequence, Chem. Commun.46(2010)595-597.
[33] X.L. Zuo, Y. Xiao, K.W. Plaxco, High specificity, electrochemical sandwich assaysbased on single aptamer sequences and suitable for the direct detection ofsmall-molecule targets in blood and other complex matrices, J. Am. Chem. Soc.131(2009)6944-6945.
[34] Y. Du, B.L. Li, F. Wang, S.J. Dong, Au nanoparticles grafted sandwich platformused amplified small molecule electrochemical aptasensor, Biosens. Bioelectron.24(2009)1979-1983.
[35] E. Katz and I. Willner, Probing biomolecular interactions at conductive andsemiconductive surfaces by impedance spectroscopy: Routes to impedimetricimmunosensors, DNA-sensors, and enzyme biosensors, Electroanalysis15(2003)913-947.
[36] Y. Kang, K.J. Feng, J.W. Chen, J.H. Jiang, G.L. Shen, R.Q. Yu, Electrochemicaldetection of thrombin by sandwich approach using antibody and aptamer,Bioelectrochemistry73(2008)76-81.
[37] M.A. Rahman, H.B. Noh, Y.B. Shim, Direct electrochemistry of laccaseimmobilized on Au nanoparticles encapsulated-dendrimer bonded conductingpolymer: Application for a catechin sensor, Anal. Chem.80(2008)8020-8027.
[38] X.Y. He, L. Zhou, E.P. Nesterenko, P.N. Nesterenko, B. Paull, J.O. Omamogho,J.D. Glennon, J.H.T. Luong, Porous graphitized carbon monolith as an electrodematerial for probing direct bioelectrochemistry and selective detection of hydrogenperoxide, Anal. Chem.84(2012)2351-2357.
[39] A.A. Abdelwahab, W.C.A. Koh, H.B. Noh, Y.B. Shim, A selective nitric oxidenanocomposite biosensor based on direct electron transfer of microperoxidase:Removal of interferences by co-immobilized enzymes, Biosens. Bioelectron.26(2010)1080-1086.
[40] L. Meng, J. Jin, G.X. Yang, T.H. Lu, H. Zhang, C.X. Cai, Nonenzymaticelectrochemical detection of glucose based on palladium-single-walled carbonnanotube hybrid nanostructures, Anal. Chem.81(2009)7271-7280.
[41] Q. Xie, Y.Y. Zhao, X. Chen, H.M. Liu, D.G. Evans, W.S. Yang, Nanosheet-basedtitania microspheres with hollow core-shell structure encapsulating horseradishperoxidase for a mediator-free biosensor, Biomaterials32(2011)6588-6594.
[42] P. Si, S.J. Ding, J. Yuan, X.W. Lou, D.H. Kim, Hierarchically structuredone-dimensional TiO2for protein immobilization, direct electrochemistry, andmediator-free glucose sensing, ACS Nano5(2011)1272-1277.
[43] C.S. Shan, H.F. Yang, J.F. Song, D.X. Han, A. Ivaska, L. Niu, Directelectrochemistry of glucose oxidase and biosensing for glucose based on graphene,Anal. Chem.81(2009)2378-2382.
[44] C.X. Guo, X.T. Zheng, S.R. Ng, Y.C. Lai, Y. Lei, C.M. Li, In situ moleculardetection of ischemic cells by enhanced protein direct electron transfer on a uniquehorseradish peroxidase-Au nanoparticles-polyaniline nanowires biofilm, Chem.Commun.47(2011)2652-2654.
[45] L.J. Bai, R. Yuan, Y.Q. Chai, Y. Zhuo, Y.L. Yuan, Y. Wang, Simultaneouselectrochemical detection of multiple analytes based on dual signal amplification ofsingle-walled carbon nanotubes and multi-labeled graphene, Biomaterials33(2012)1090-1096.
[46] B.I. Ipe, K. Yoosaf, K.G. Thomas, Functionalized gold nanoparticles asphosphorescent nanomaterials and sensors, J. Am. Chem. Soc.128(2006)1907-1913.
[47] L.L. Li, K.P. Liu, G.H. Yang, C.M. Wang, J.R. Zhang, J.J. Zhu, Fabrication ofgraphene-quantum dots composites for sensitive electrogeneratedchemiluminescence immunosensing, Adv. Funct. Mater.21(2011)869-878.
[48] J.C. Meyer, A.K. Geim, M.I. Katsnelson, K.S. Novoselov, T.J. Booth, S. Roth, Thestructure of suspended graphene sheets, Nature446(2007)60-63.
[49] Heldin CH, Westermark B. Mechanism of action and in vivo role ofplatelet-derived growth factor [J]. Physiol Rev,1999,79(4):1283-1316.
[50] Thieringer F, MaassT, Czochra P, et a.l Spon taneous hepatic fibros is in transgenicmice overexpressing PDGF-A [J]. Gen e,2008,423(1):23-28.
[51] Campbell JS, Hughes SD, Gibertson DG, et a.l Platelet-derived growth factor Cinduces liver fibrosis, steatosis, and hepatocellular carcinoma [J]. P roc Nat lAcadS ciU S A,2005,102(9):3389-3394.
[52] Zhang M, Zhang BH, Chen L, An W. Overexpression of heme oxygenase-1protects smooth muscle cells against oxidative injury and inhibits cell proliferation[J]. Cell Res,2002,12:12-32.
[53] Henriksen R, Funa K, Wilander E, et al. Expression and prognostic significance ofplatelet-derived growth factor b and its receptors in epithelial ovarian neoplasms[J].Cancer Res,1993,53(19):4550-4554.
[54] FredrikssonL, Li H, Eriksson U. The PDGF family: four gene Produets form fivedimeric isoforms. Cytokine Growth Faetor Rev2004,15:197-204.
[55] Ustach CV, Kim HR. Platelet-derived growth faetor D is activated by urokinaseplasminogen activator in Prostate carcinoma cells. Mol Cell Biol2005,25:6279一6288.
[56] Wang Z, Kong D, Bane S, et al. Down-regulation of Platelet-derived growthfaetor-D inhibits cell growth and angiogenesis through inactivation of Notch-1andnuclear factor-kappa B signaling. Cancer Res2007,67:11377一11385.
[57] Xu L,Tong R, Coehran DM, et al. Blocking patelet-derived growth faetor-D/platelet-derived growth factor receptor beta signaling inhibits human renal cellcarcinoma progress in an orthotopic mouse model. Caneer Res2005.65:5711一5719.
[58]周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
[59] Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin. Toxicol.41,(3)277–290;317–327.
[60] Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987.Immunodiagnosis of snake venom poisoning. Asian Pacific J. Allerg.Immunol.5,187–190.
[61] Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snakevenoms, toxins and venom antibodies, review on recent trends1987–1997. Toxicon37,565–586.
[62] Theakston, R.D.G.,1983. The application of immunoassay techniques, includingenzyme-linked immunosorbent assay (ELISA), to snake venom research. Toxicon21,341–352.
[63] Gold, L., Polisky, B., Uhlenbeck,0., Yarus, M. Diversity of oligonucleotidefunctions. Annu Rev Biochem64(1995),763-797.
[1] Baker, B. R.. I..,, R. Y.. Wood, M. s.. Doctor, E. H., Heeger, A. J., Plaxco, K. w. AnElectronic, Aptamer-Based Small-Molecule Sensor for the Rapid, Label-FreeDetection of Cocaine in Adulterated Samples and Biological Fluids. J AmChem Soc128,3138-3139.
[2] Lee, M., Wait, D. R.(2000). A fiber-optic microarray biosensor using aptamers asreceptors. Anal Biochem282,142-146.
[3] Liss, M., Petersen, B., Wolf, H., Prohaska, E.. An aptamer-based quartz crystalprotein biosensor. AnalChemlA,(2002)4488-4495.
[4] Tombelli, S., Minunni, A., Luzi, E., Mascini, M. Aptamer-based biosensors for thedetection of HIV-1Tat protein. Bioelectrochemistry (2005)67,135-141.
[5] Nutiu, R., Mei, S., Liu, Z. J., Li, Y. F. Engineering DNA aptamers and DNA enzymeswith fluorescence-signaling properties. Pure Appl Chem (2004)76,1547-1561.
[6] Pavlov, V., Shlyahovsky, B., Willner, I. Fluorescence detection of DNA by thecatalytic activation of an aptamer/thrombin complex. J Am Chem Soc (2005)127,6522-6523.
[7] Yang, C. J., Jockusch, S., Vicens, M., Turro, N. J., Tan, W. H.. Light-switchingexcimer probes for rapid protein monitoring in complex biological fluids. Proc NatlAcad Sci USA (2005)102,17278-17283.
[8] Rupcich, N., Nutiu, R.y Li, Y. F., Brennan, J. D. Solid-phase enzyme activity assayutilizing an entrapped fluorescence-signaling DNA aptamer. Ange-w Chern Int EdEngl (2006)45,3295-3299.
[9] Lu, Y., Liu, J. W. Functional DNA nanotechnology: emerging applications ofDNAzymes and aptamers. Curr Opin Biotechno (2006)17,580-588.
[10] Lu, Y., Liu, J. W. Smart nanomaterials inspired by biology: dynamic assembly oferror-free manomaterials in response to multiple chemical and biological stimuli. AceChemRes (2007).40,315-323.
[11] Ravelet, C., Grosset, C., Peyrin, E. Liquid chromatography, electrochromatographyand capillary electrophoresis applications of DNA and RNA aptamers.|J ChromatogrA (2006)1117,1-10.
[12] Willner, I., Zayats, M.. Electronic aptamer-based sensors. Ange-w Chem Int Ed Engl(2007)46,2-13. I
[13] Willner, I., Zayats, M.(2007). Electronic aptamer-based sensors. Ange-w Chem IntEd Engl46,2-13.
[14] Bang, G. S., Cho, S., Kim, B. G. A novel electrochemical detection method foraptamer biosensors. Biosens and Bioelectron (2005)21,863-870.
[15] G.S. Bang, S. Cho, B.G. Kim, A novel electrochemical detection method for aptamerbiosensors. Biosens and Bioelectron.21(2005)863.
[16] F. Le Floch, H.A. Ho, M. Leclerc. Label-free electrochemical detection of proteinbased on a ferrocene-bearing cationic polythiophene and aptamer. Anal. Chem.78(2006)4727.
[17] A.K. Cheng, B. Ge, H.Z. Yu. A Review: Electrochemical Aptasensors with VariousDetection Strategies. Anal. Chem.79(2007)5158.
[18] H.M. So, K. Won, Y.H. Kim, B.K. Kim, B.H. Ryu, P.S. Na, H. Kim,J.O. Lee, J. Am.Single-walled carbon nanotube biosensors using aptamers as molecular recognitionelements. Chem. Soc.127(2005)11906.
[19] K. Maehashi, T. Katsura, K. Kerman, Y. Takamura, K. Matsumoto, E. Tamiya.Label-Free Protein Biosensor Based on Aptamer-Modified Carbon NanotubeField-Effect Transistors. Anal. Chem.79(2007)782
[20] Stojanovic, M. N., Kolpashchikov, D. M.(2004). Modular aptameric sensors. J AmC/iem Soc126,9266-9270.
[21] T.E. Lin, W.H. Chen, Y.C. Shiang, C.C. Huang, H.T. Chang, Colorimetric detection ofplatelet-derived growth factors through competitive interactions between proteins andfunctional gold nanoparticles, Biosens. Bioelectron.29(2011)204-209.
[22] Li, H. X., Rothberg, L. Colorimetric detection of DNA sequences based onelectrostatic interactions with unmodified gold nanoparticles. Proc Natl Acad SciUSA.(2004).101,14036-14039.
[23] Balamurugan, S., Obubuafo, A., Soper, S. A., McCarley, R. L., Spivak, D. A.(2006).Designing highly specific biosensing surfaces using aptamer monolayers on gold.Langmuir,2006(22).6446-6453.
[24] Z.S. Wu, S.B. Zhang, H. Zhou, G.L. Shen, R.Q. Yu, Universal aptameric system forhighly sensitive detection of protein based on structure-switching-triggered rollingcircle amplification, Anal. Chem.82(2010)2221-2227.
[25] Z.S. Wu, H. Zhou, S.B. Zhang, G.L. Shen, R.Q. Yu, Electrochemical aptamericrecognition system for a sensitive protein assay based on specific targetbinding-induced rolling circle amplification, Anal. Chem.82(2010)2282-2289.
[26] D.B. Zhu, X.M. Zhou, D. Xing, A new kind of aptamer-based immunomagneticelectrochemiluminescence assay for quantitative detection of protein, Biosens.Bioelectron.26(2010)285-288
[1]周正彦；李丕鹏；陆宇燕；董丙君；中国特有蛇类资源及保护建议.四川动物.01(2008)：44-47
[2] Lalloo, D.G., Theakston, R.D.,2003. Snake antivenoms, J. Toxicol. Clin.Toxicol.41,(3)277–290;317–327.
[3] Ratanabanangkoon, K., Billings, P.B., Matangkasombut, P.,1987. Immunodiagnosisof snake venom poisoning. Asian Pacific J. Allerg.Immunol.5,187–190.
[4] Selvanayagam, Z.E., Gopalakrishnakone, P.,1999. Test for detection of snake venoms,toxins and venom antibodies, review on recent trends1987–1997. Toxicon37,565–586.
[5] Theakston, R.D.G.,1983. The application of immunoassay techniques, includingenzyme-linked immunosorbent assay (ELISA), to snake venom research. Toxicon21,341–352.
[6] Pukrittayakamee, S., Ratcli.e, P.J., McMichael, A.J., Warrell, D.A., Bunnag, D.,1987.A competitive radioimmunoassay using a monoclonal antibody to detect thefactor_activator of Russell's viper venom. Toxicon25,721-729.
[7] Theakston, R.D.G.,1983. The application of immunoassay techniques, includingenzyme-linked immuno-sorbent assay (ELISA), to snake venom research. Toxicon21,341-352.
[8] Nakamura, M., Hanashiro, K., Kosugi, T.,1993. A modi cation of the ELISA-doublesandwich method or estimating the concentration of habutobin. Toxicon31,1325-1328.
[9] Lomonte, B., Gutierrez, J.M., Rojas, G., Calderon, L.,1991. Quantitation byenzyme-immunoassay of antibodies against Bothrops myotoxins in fourcommercially-available antivenoms. Toxicon29,695-702.
[10] Liu Minghua, Yao Jie, Xiang Qiang, Zhang Bo, Fu Weiling. Study on piezoelectricquartz crystalm icroarray immunosensor for the detection of Agkistrodon acutusvenom. Acta academiae medicine militaris tertiae.2009(31):1158-1160.
[11] Selvanayagam, Z.E., Gopalakrishnakone, P., Sridhar, U., Neuzil, P., Sing, M., Ho,L.C., Immobilisation of antibodies onto silicon wafers and its potential application inthe development of ISFET immunosensors. Proceedings of4th National Symposiumon Progress in Materials Research, National University of Singapore, Singapore,27March1998.
[12] Osborne, S. E., Ellington, A. E. Nucleic acid selection and the challenge ofcombinatorial chemistry.ChemRev97(1997),349-370.
[13] Brody, E. N., Gold, L.(). Aptamers as therapeutic and diagnostic agents. Re-u Mo IBiotech74(2000),5-13.
[14] Toulme, J-J. Aptamers: selected oligonucleotides for therapy. Curr Opin Mol Ther2(2000),318-324.
[15] Gopinath, S. C. Methods developed for SELEX. Anal Bioanal Chem387(2007),171-182.