基于功能寡核苷酸分子的蛋白质检测新方法研究
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摘要
自人类进入后基因组时代以来,建立简单、灵敏、快速、特异性强、高通量的蛋白质检测方法,已成为分析科学的研究重点之一。蛋白质参与生命体每一步反应和活动,与许多人类疾病有着直接的关系,因此实现蛋白质准确灵敏的检测,对疾病诊治均具重要意义。
适配体及脱氧核酶是近年来经体外指数富集配基的系统进化技术(SELEX)筛选出的两类寡核苷酸功能分子。适配体识别机制与抗体相似,但与靶分子间作用的特异性和亲和力更高,且具有靶分子范围更加广泛、无免疫原性、稳定性强、可构筑良好的纳米功能器件等诸多优点；脱氧核酶则为具有酶催化活性的小分子单链DNA (ssDNA),可在辅酶因子如金属离子、氨基酸的作用下,发挥高效的催化活性,具有易于合成和修饰及稳定性好等特点,作为新型的亲和分子,适配体和脱氧核酶在识别传感与检测领域备受关注。
本研究论文则鉴于适配体与靶蛋白高亲和力高特异性的结合性质及脱氧核酶的高催化活性,构建了基于适配体或适配体与脱氧核酶有机整合的新型分子识别与分析检测体系,从而实现对于靶蛋白高灵敏度、高特异性检测的目的。本论文共分5章。
第一章为前言部分,围绕着适配体及脱氧核酶在分析化学领域的应用这一重点,从适配体的定义与特点、适配体在分离分析及生物传感领域中的应用、脱氧核酶的定义与功能分类、脱氧核酶(包括变构脱氧核酶)在生物传感领域的应用等诸方面展开综述,共引用文献86篇。亦简要介绍了本论文的创新点与研究工作；
第二章利用磁珠作为分离介质,构建了一种基于适配体夹心法检测凝血酶的比色传感新方法。该方法简便易行,可用裸眼直接观测。利用磁珠比表面积大,可磁性分离的优点以及适配体与凝血酶特异性结合的性质,可实现5%及50%血清中凝血酶的比色检测,在5%血清中,凝血酶线性范围为10-80 nM,检测限(LOD)为10nM。G15D适配体5’末端连接臂的长短对凝血酶检测灵敏度具有显著影响,在其5'末端引入6T连接臂能显著提高与凝血酶的结合能力；在对适配体连接臂长度优化后,适配体G15D及60-18[29]都可作为捕获适配体,实现凝血酶的定量检测；
第三章基于新型适配体荧光探针5F-39mer,建立了一种定量检测rHuEPO-α的亲和探针毛细管电泳-激光诱导荧光检测(APCE/LIF)新方法,并探讨了适配体与rHuEPO-α间的相互作用机制。各种APCE条件的优化表明,溶液微环境中存在的Na+离子是5F-39mer-rHuEPO-α复合物存在与否的关键调控因素,运行缓冲液中Na+的存在可显著提高复合物及与游离适配体之间的分离度,而样品溶液中的Na+则可使复合物峰强度显著升高；另外,适当的电压和样品溶液添加剂(牛血清白蛋白)也是复合物稳定存在的必需条件。在最优的实验条件下,该方法可成功用于生理缓冲液、人工尿液及血清基质中rHuEPO-α的高灵敏,高特异性的检测,线性范围为0.2-100nM, LOD为0.2 nM,,也可用于实际样品(人促红细胞生成素rHuEPO-α注射液)中rHuEPO-α的准确定量。利用APCE/LIF技术还探讨了N连接位点的糖基在适配体与rHuEPO-α结合时所发挥的作用；
第四章中成功实现了生物素化的脱氧核酶-适配体连接体在链霉亲和素(STV)包被的磁珠或毛细管柱端的自组装,结合磁珠、CE等分离技术,建立了一类基于适配体分子特异性捕获富集、脱氧核酶催化剪切洗脱及检测蛋白质的新方法。首先详细考察了Cu2+依赖型的CA3II-43mer型脱氧核酶在自由溶液、固相介质如磁珠表面的催化活性的影响因素；在此基础上,把凝血酶适配体序列引入到CA3II-43mer底物链的5'端,将其固定于STV包被的磁珠表面对凝血酶进行捕获,通过测定脱氧核酶的催化切割活性实现凝血酶的定量检测；结合溴化己二甲铵(HDB)/STV层层组装技术则可成功实现脱氧核酶-适配体连接体在毛细管柱端的自组装,通过监测脱氧核酶切割产生的凝血酶-适配体复合物的荧光强度实现凝血酶的高灵敏检测,检测限可达nM级；
第五章利用磁珠作为分离介质,首次将依赖Cu2+的脱氧核酶与凝血酶适配体结合,构建了一种基于适配体型变构脱氧核酶定量检测凝血酶的新型传感体系。通过优化脱氧核酶Ⅰ区碱基配对数目、脱氧核酶Ⅰ区与适配体互补碱基数目、脱氧核酶与适配体浓度比例等主要因素,实现了适配体对酶切割活性的抑制,而凝血酶的加入则可促使脱氧核酶切割活性的恢复,通过监测酶切割产生的荧光信号实现凝血酶的定量检测。该体系简单、成本低,特异性好,无需标记适配体,普适性强,LOD为5.1 nM,线性范围为0-100 nM,为蛋白质的检测提供了新的分析平台。
With the implement of the human post-genome project, the development of protein detection methods with simple, rapid, high-sensitive, high-specific and high-throughput has attracted considerable attention in the analytical science. Proteins are involved in every physiological process and activity, and directly related to many human diseases. It is of great importance to develop methods for diagnosis and therapy of diseases with high sensitivity and accuracy.
In recent years, two kinds of functional oligonucleotides, aptamers and DNAzymes have been generated by an in vitro selection technique named SELEX (Systematic Evolution of Ligands by EXponential enrichment). The molecular recognition formats of aptamer-target and antibody-antigen are similar, beyond that the aptamers have several advantages over antibodies, such as higher specificity and affinity, allowing a wide range of targets, low or no immunogenicity, extreme stability and can be fabricated into functional nanostructures; DNAzymes refer to single-stranded DNA molecules with high catalytic capabilities under the action of coenzyme factor such as metal ion and amino acid, and featured as ease of synthesis and modification, and high stability. Emerging as two kinds of novel recognition modules, aptamer and DNAzyme raised great attention in the recognition, sensing and detection fields.
In combination of high specifity, high affinity of aptamer, and high catalytic activity of DNAzyme, this dissertation focused on the construction of new analytical methods for the detection of target proteins with high sensitivity and high specificity. Five chapters are included.
The first chapter is the introduction. Focused on the wide applications of aptamer and DNAzyme in the field of analytical chemistry, a review composed of the definition and features of aptamer, the applications of aptamer in separation and biosensing fields, the definition and functional classification of DNAzyme, the biosensing application of DNAzyme (including allosteric DNAzyme) is summarized.86 articles are cited. The innovation concepts and researches of this dissertation are briefly introduced.
In chaper 2, using magnetic beads as separation medium, a novel colorimetric sensing system for thrombin detection was developed with two different aptamers as recognition molecular for sandwich assay. This method was simple and the detection of thrombin can be carried out with naked eyes. Benefited from magnetic beads with large surface area and can be magnetic separated and aptamer specific to the target protein, the thrombin can be detected in 5% or 50% human serum. A linear range was obtained from 10 to 80 nM in 5% human serum, the limit of detection (LOD) was 10 nM. The linker length of G15D has great impact on the detection sensitivity, and the attachment of linker (6 thymidine unit) at the 5'-end of the aptamer can increase thrombin binding capacity significantly. Under the optimized linker length, both aptamers (G15D and 60-18[29]) can be used as capturing modules for quantification of thrombin, respectively.
In chapter 3, based on a new fluorescence aptamer probe 5F-39mer, a novel affinity probe capillary electrophoresis-laser induced fluorescence detection (APCE/LIF) method for quantification of recombinant human erythropoietin-a (rHuEPO-a) was establilshed. The mechanism of binding interaction between aptamer and rHuEPO-a was also investigated. After optimization of APCE conditions, the results showed that the existence of Na+in the micro-environment solution was the key factor for the formation of 5F-39mer-rHuEPO-a complex. The presence of Na+in the running buffer significantly improved the resolution between free aptamer and complex. Meanwhile, the existence of Na+in the sample buffer increased the intensity of complex. Additionally, suitable electric voltage and sample buffer additives (bovine serum albumin) were necessary to stabilze the complex. Under the optimized conditions the method was successfully applied for the quantification of rHuEPO-a in physiological buffer, artificial urine and human serum with high sensitivity and high specificity.The linear range of rHuEPO-a covered from 0.2 to 100 nM with a LOD of 0.2 nM. The concentration of rHuEPO-a in the real sample (commercial rHuEPO-a injection) could also be quantified precisely. Using such an aptamer-based APCE/LIF method, we investigated the the role of N-oligosaccharide moiety contribute to the specific aptamer-rHuEPO-a recognition.
In chapter 4, self-assembled immobilization of biotinylated DNAzyme-aptamer intergrater was accomplished on the streptavidin (STV)-coated magnetic beads or capillary wall. Using the separation technique such as magnetic bead or CE technique, a novel detection method for proteins based on the specific capturing and enrichment of aptamer, and the catalytic cleavage of DNAzyme was established. First, we thoroughly investigated the factors that influenced the cleavage activity of Cu2+-dependent CA3Ⅱ-43mer in the free solution and solid support (the surface of magnetic beads.Then the anti-thrombin aptamer was attached to the 5'-end of the substrate chain of CA3Ⅱ-43mer, and immobilization of CA3Ⅱ-43mer-aptamer was accomplished on the STV-coated magnetic beads, the capture and detection of thrombin was fulfilled by determining the self-cleavage activity of DNAzyme. Furthermore, the immobilization of DNAzyme-aptamer intergrater was successfully accomplished on the capillary wall using hexadimethrine bromide (HDB)/STV layer-by-layer techniques, and the fluorescecce intensity of the thrombin-aptamer complex that produced by DNAzyme self-cleavage was employed for detection of thrombin down to nM concentration.
In the last chapter, with magnetic beads as the separation medium, a novel sensing method for quantification of thrombin protein was constructed on the basis of allosterically aptameric DNAzyme (aptazyme), which composed of an anti-thrombin aptamer and a Cu2+-dependent DNAzyme sequences. After optimizing the main factors, such as the base pair number of stem I, the complementary bases between aptamer and stem I, and the concentration ratio of DNAzyme and aptamer, the self-cleavage activity of DNAzyme had been inhibited by aptamer. In the presence of the thrombin, the formation of a thrombin-aptamer complex resulted in restoration of the catalytic activity of the aptazyme. The self-cleavage activity was coupled to the generation of a fluorescent signal, permitting the quantification of thrombin. This novel sensing system was simple, cost-effective, highly specific and label-free, a linear range was obtained from 0 to 100 nM, LOD was 5.1 nM. It will provide a new analytical platform for the protein detection.
适配体及脱氧核酶是近年来经体外指数富集配基的系统进化技术(SELEX)筛选出的两类寡核苷酸功能分子。适配体识别机制与抗体相似,但与靶分子间作用的特异性和亲和力更高,且具有靶分子范围更加广泛、无免疫原性、稳定性强、可构筑良好的纳米功能器件等诸多优点；脱氧核酶则为具有酶催化活性的小分子单链DNA (ssDNA),可在辅酶因子如金属离子、氨基酸的作用下,发挥高效的催化活性,具有易于合成和修饰及稳定性好等特点,作为新型的亲和分子,适配体和脱氧核酶在识别传感与检测领域备受关注。
本研究论文则鉴于适配体与靶蛋白高亲和力高特异性的结合性质及脱氧核酶的高催化活性,构建了基于适配体或适配体与脱氧核酶有机整合的新型分子识别与分析检测体系,从而实现对于靶蛋白高灵敏度、高特异性检测的目的。本论文共分5章。
第一章为前言部分,围绕着适配体及脱氧核酶在分析化学领域的应用这一重点,从适配体的定义与特点、适配体在分离分析及生物传感领域中的应用、脱氧核酶的定义与功能分类、脱氧核酶(包括变构脱氧核酶)在生物传感领域的应用等诸方面展开综述,共引用文献86篇。亦简要介绍了本论文的创新点与研究工作；
第二章利用磁珠作为分离介质,构建了一种基于适配体夹心法检测凝血酶的比色传感新方法。该方法简便易行,可用裸眼直接观测。利用磁珠比表面积大,可磁性分离的优点以及适配体与凝血酶特异性结合的性质,可实现5%及50%血清中凝血酶的比色检测,在5%血清中,凝血酶线性范围为10-80 nM,检测限(LOD)为10nM。G15D适配体5’末端连接臂的长短对凝血酶检测灵敏度具有显著影响,在其5'末端引入6T连接臂能显著提高与凝血酶的结合能力；在对适配体连接臂长度优化后,适配体G15D及60-18[29]都可作为捕获适配体,实现凝血酶的定量检测；
第三章基于新型适配体荧光探针5F-39mer,建立了一种定量检测rHuEPO-α的亲和探针毛细管电泳-激光诱导荧光检测(APCE/LIF)新方法,并探讨了适配体与rHuEPO-α间的相互作用机制。各种APCE条件的优化表明,溶液微环境中存在的Na+离子是5F-39mer-rHuEPO-α复合物存在与否的关键调控因素,运行缓冲液中Na+的存在可显著提高复合物及与游离适配体之间的分离度,而样品溶液中的Na+则可使复合物峰强度显著升高；另外,适当的电压和样品溶液添加剂(牛血清白蛋白)也是复合物稳定存在的必需条件。在最优的实验条件下,该方法可成功用于生理缓冲液、人工尿液及血清基质中rHuEPO-α的高灵敏,高特异性的检测,线性范围为0.2-100nM, LOD为0.2 nM,,也可用于实际样品(人促红细胞生成素rHuEPO-α注射液)中rHuEPO-α的准确定量。利用APCE/LIF技术还探讨了N连接位点的糖基在适配体与rHuEPO-α结合时所发挥的作用；
第四章中成功实现了生物素化的脱氧核酶-适配体连接体在链霉亲和素(STV)包被的磁珠或毛细管柱端的自组装,结合磁珠、CE等分离技术,建立了一类基于适配体分子特异性捕获富集、脱氧核酶催化剪切洗脱及检测蛋白质的新方法。首先详细考察了Cu2+依赖型的CA3II-43mer型脱氧核酶在自由溶液、固相介质如磁珠表面的催化活性的影响因素；在此基础上,把凝血酶适配体序列引入到CA3II-43mer底物链的5'端,将其固定于STV包被的磁珠表面对凝血酶进行捕获,通过测定脱氧核酶的催化切割活性实现凝血酶的定量检测；结合溴化己二甲铵(HDB)/STV层层组装技术则可成功实现脱氧核酶-适配体连接体在毛细管柱端的自组装,通过监测脱氧核酶切割产生的凝血酶-适配体复合物的荧光强度实现凝血酶的高灵敏检测,检测限可达nM级；
第五章利用磁珠作为分离介质,首次将依赖Cu2+的脱氧核酶与凝血酶适配体结合,构建了一种基于适配体型变构脱氧核酶定量检测凝血酶的新型传感体系。通过优化脱氧核酶Ⅰ区碱基配对数目、脱氧核酶Ⅰ区与适配体互补碱基数目、脱氧核酶与适配体浓度比例等主要因素,实现了适配体对酶切割活性的抑制,而凝血酶的加入则可促使脱氧核酶切割活性的恢复,通过监测酶切割产生的荧光信号实现凝血酶的定量检测。该体系简单、成本低,特异性好,无需标记适配体,普适性强,LOD为5.1 nM,线性范围为0-100 nM,为蛋白质的检测提供了新的分析平台。
With the implement of the human post-genome project, the development of protein detection methods with simple, rapid, high-sensitive, high-specific and high-throughput has attracted considerable attention in the analytical science. Proteins are involved in every physiological process and activity, and directly related to many human diseases. It is of great importance to develop methods for diagnosis and therapy of diseases with high sensitivity and accuracy.
In recent years, two kinds of functional oligonucleotides, aptamers and DNAzymes have been generated by an in vitro selection technique named SELEX (Systematic Evolution of Ligands by EXponential enrichment). The molecular recognition formats of aptamer-target and antibody-antigen are similar, beyond that the aptamers have several advantages over antibodies, such as higher specificity and affinity, allowing a wide range of targets, low or no immunogenicity, extreme stability and can be fabricated into functional nanostructures; DNAzymes refer to single-stranded DNA molecules with high catalytic capabilities under the action of coenzyme factor such as metal ion and amino acid, and featured as ease of synthesis and modification, and high stability. Emerging as two kinds of novel recognition modules, aptamer and DNAzyme raised great attention in the recognition, sensing and detection fields.
In combination of high specifity, high affinity of aptamer, and high catalytic activity of DNAzyme, this dissertation focused on the construction of new analytical methods for the detection of target proteins with high sensitivity and high specificity. Five chapters are included.
The first chapter is the introduction. Focused on the wide applications of aptamer and DNAzyme in the field of analytical chemistry, a review composed of the definition and features of aptamer, the applications of aptamer in separation and biosensing fields, the definition and functional classification of DNAzyme, the biosensing application of DNAzyme (including allosteric DNAzyme) is summarized.86 articles are cited. The innovation concepts and researches of this dissertation are briefly introduced.
In chaper 2, using magnetic beads as separation medium, a novel colorimetric sensing system for thrombin detection was developed with two different aptamers as recognition molecular for sandwich assay. This method was simple and the detection of thrombin can be carried out with naked eyes. Benefited from magnetic beads with large surface area and can be magnetic separated and aptamer specific to the target protein, the thrombin can be detected in 5% or 50% human serum. A linear range was obtained from 10 to 80 nM in 5% human serum, the limit of detection (LOD) was 10 nM. The linker length of G15D has great impact on the detection sensitivity, and the attachment of linker (6 thymidine unit) at the 5'-end of the aptamer can increase thrombin binding capacity significantly. Under the optimized linker length, both aptamers (G15D and 60-18[29]) can be used as capturing modules for quantification of thrombin, respectively.
In chapter 3, based on a new fluorescence aptamer probe 5F-39mer, a novel affinity probe capillary electrophoresis-laser induced fluorescence detection (APCE/LIF) method for quantification of recombinant human erythropoietin-a (rHuEPO-a) was establilshed. The mechanism of binding interaction between aptamer and rHuEPO-a was also investigated. After optimization of APCE conditions, the results showed that the existence of Na+in the micro-environment solution was the key factor for the formation of 5F-39mer-rHuEPO-a complex. The presence of Na+in the running buffer significantly improved the resolution between free aptamer and complex. Meanwhile, the existence of Na+in the sample buffer increased the intensity of complex. Additionally, suitable electric voltage and sample buffer additives (bovine serum albumin) were necessary to stabilze the complex. Under the optimized conditions the method was successfully applied for the quantification of rHuEPO-a in physiological buffer, artificial urine and human serum with high sensitivity and high specificity.The linear range of rHuEPO-a covered from 0.2 to 100 nM with a LOD of 0.2 nM. The concentration of rHuEPO-a in the real sample (commercial rHuEPO-a injection) could also be quantified precisely. Using such an aptamer-based APCE/LIF method, we investigated the the role of N-oligosaccharide moiety contribute to the specific aptamer-rHuEPO-a recognition.
In chapter 4, self-assembled immobilization of biotinylated DNAzyme-aptamer intergrater was accomplished on the streptavidin (STV)-coated magnetic beads or capillary wall. Using the separation technique such as magnetic bead or CE technique, a novel detection method for proteins based on the specific capturing and enrichment of aptamer, and the catalytic cleavage of DNAzyme was established. First, we thoroughly investigated the factors that influenced the cleavage activity of Cu2+-dependent CA3Ⅱ-43mer in the free solution and solid support (the surface of magnetic beads.Then the anti-thrombin aptamer was attached to the 5'-end of the substrate chain of CA3Ⅱ-43mer, and immobilization of CA3Ⅱ-43mer-aptamer was accomplished on the STV-coated magnetic beads, the capture and detection of thrombin was fulfilled by determining the self-cleavage activity of DNAzyme. Furthermore, the immobilization of DNAzyme-aptamer intergrater was successfully accomplished on the capillary wall using hexadimethrine bromide (HDB)/STV layer-by-layer techniques, and the fluorescecce intensity of the thrombin-aptamer complex that produced by DNAzyme self-cleavage was employed for detection of thrombin down to nM concentration.
In the last chapter, with magnetic beads as the separation medium, a novel sensing method for quantification of thrombin protein was constructed on the basis of allosterically aptameric DNAzyme (aptazyme), which composed of an anti-thrombin aptamer and a Cu2+-dependent DNAzyme sequences. After optimizing the main factors, such as the base pair number of stem I, the complementary bases between aptamer and stem I, and the concentration ratio of DNAzyme and aptamer, the self-cleavage activity of DNAzyme had been inhibited by aptamer. In the presence of the thrombin, the formation of a thrombin-aptamer complex resulted in restoration of the catalytic activity of the aptazyme. The self-cleavage activity was coupled to the generation of a fluorescent signal, permitting the quantification of thrombin. This novel sensing system was simple, cost-effective, highly specific and label-free, a linear range was obtained from 0 to 100 nM, LOD was 5.1 nM. It will provide a new analytical platform for the protein detection.
引文
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