基于信号放大核酸适配体和G-四链体探针的生化分析新方法
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摘要
生物传感器具有灵敏度高、操作简便、选择性好、分析速度快、检测成本低等特点,在生化分析、环境监测、临床诊断和药物筛选领域有着广阔的应用前景。同时,由于核酸具有合成容易、稳定性好、设计简单、生物相容性好、信号机制灵活等诸多优势,它是构建生物传感器的重要素材。另外,它还可以借助各种信号放大方法实现对目标物高效快速的高灵敏度检测。本论文借助核酸适配体、分子信标、G-四链体、金纳米粒子和脱氧核酶的优良的特性和全新的设计思路,依据不同的分析物的检测建立了各种高效的检测方法。和传统的检测方法相比,本论文所建立的检测方法具有灵敏度高、操作简便、分析成本低廉的特点,这为生物传感技术的快速发展提供了一定程度的借鉴意义。
免疫球蛋白E (IgE)的测定在过敏性鼻炎和过敏性哮喘等相关疾病的临床诊断中具有重要作用。在第2章中,搭建了基于电子通道开关核酸适配体电化学传感器用于IgE的检测。在该传感检测中,免疫球蛋白E绑定到核酸适配体上导致覆盖碱性磷酸酶(ALP)。同时,由于IgE是非传导性介质,使得ALP不能催化1-萘基磷酸酯水解产物-萘酚生成电活性物质。因此,IgE与核酸适配体结合阻碍电子转移,导致电流强度下降。该电化学传感器的信号产生不依赖于核酸适配体探针的构象变化,它克服传统的电化学核酸适配体传感器遇到的困难。并且该方法具有较高的灵敏度,其检测下限达至4.44×10-6μg·mL-1(22.7fM)。在复杂环境中,该检测方法同样表现出了良好的分析性能,血清样品中的回收实验结果令人满意。
赭曲霉素A(OTA)是对人类和动物的健康有极大危害的元素,包括肾脏损害、致畸性、致癌性、细胞毒性和遗传毒性等。第3章中,针对核酸适配体电化学传感器检测小分子性能有待提高的情况,将核酸适配体序列在恰当的部位剪切成两段,构建电信号增强型核酸适配体传感探针。该方法灵敏度较高、选择性好、操作简单和低成本,同时为基于核酸适配体探针检测其它生物分子提供了一条有效的途径。
三磷酸腺苷(ATP)是生物体内细胞一切生命活动所需能量的直接来源,参与体内脂肪、糖、蛋白质和核苷酸的合成。迅速而准确地检测ATP,对于研究生物体的新陈代谢过程和疾病的临床诊断等方面都有非常重要的意义。第4章中,以ATP为模型分析物,将ATP核酸适配体剪切成合适的两部分,同时结合杂交连锁反应(hybridization chain reaction,HCR)信号放大技术,构建了一种基于核酸适配体-HCR的电化学传感器用于ATP的检测。两条剪切的核酸适配体片段中的一条巯基标记,另一条核酸适配体片段延长核苷酸序列,且延长的核酸序列能引发后续的HCR,从而使得大量的碱性磷酸酶通过生物素-亲和素作用存在于电极表面,实现对ATP的高灵敏检测,其检测下限为0.2nM。
较多文献指出传统的分离式信号读出方式在链置换扩增信号放大检测方法中存在“信号耗损”现象。针对该现象我们提出了一种集成式信号读出生化分析传感技术。第5章中,在同一条核酸序列上将信号传导探针与目标识别探针通过适当的剪切而揉和成一体,同时借助核酸外切酶Ⅲ信号放大方式,提高了G-四链体-血红素脱氧核酶催化H2O2氧化2,2'-联氮基双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)检测p53基因的效率,实现对p53基因定量比色分析。把G-四链体核酶的核苷酸序列与目标识别序列揉和成一体,并且设计成分子信标,这种模式具有极大的优越性。此外,分子信标对G-四链体的形成有协助功能,该方案目标物响应信号的生成更快,检测范围更宽,检测灵敏度更好,其检测下限达到1pM。第6章中,同样基于集成式信号读出方式,当检测物p53基因与未形成G-四链体的分子信标杂交时,富G碱基序列被释放,生成G-四链体,同时形成新的分子信标,使得聚合酶链置换扩增反应发生,生成大量的G-四链体-血红素脱氧核酶催化H202氧化ABTS,完成对p53基因超灵敏检测,其检测下限达到25fM。
第7章中,基于纳米金团聚策略,提出了一种G-四链体为传导媒介可视化分析方法用于肿瘤突变p53基因测定。在该比色传感器中,与靶DNA的杂交使得富G碱基发夹结构的核酸链被强制打开,促进高级结构的同向平行G-四链体的形成。由于设计的巧妙,导致生成一维或多维的G-四链体分子器件。当捕获探针修饰的金纳米颗粒加入时,由于捕获探针DNA与G-四链体的末端DNA片段杂交,从而使得金纳米颗粒彼此靠近而发生团聚现象。本论文提出的比色传感器不仅能提供纳摩尔水平的检测能力,也可以可视化判断突变的p53基因。所建立的检测方法为肿瘤相关的临床检测、药物筛选和DNA分子器件设计提供了潜在的应用价值。
Biosensors have wide application in biochemical analysis, environmental monitoring, clinical diagnostics and drug screening, because of their high sensitivity, easy operation, excellent selectivity, short analysis time and low-cost. Meanwhile, with the advantages of easy to synthesize, good stability, simple design, good biocompatibility, flexibility and signaling mechanisms, nucleic acid has important significance in the construction of biosensors. In addition, biosensors can realize the detection of targets rapidly, efficiently and high sensitively depending on a variety of signal amplification methods. In this thesis, using excellent features of aptamers, molecular beacons, G-quadruplex, gold nanoparticles and deoxyribozymes, new design ideas have been deconstructed for detection of different analysis objects. Compared with the traditional detection methods, the proposed detection methods are highly sensitive, easy to operate, low cost, and high speed of analysis.
Immunoglobulin E (IgE) detection of allergic rhinitis, allergic asthma and other related diseases is important in clinical diagnosis. In chapter2, an electronic channel switching-based (ECS) aptasensor was developed for ultra-sensitive protein detection. In the detection mechanism of sensor, the hairpin structure of aptamer was designed to pull electroactive species towards electrode surface and use the surface-immobilized IgE to serve as a barrier that separated enzyme from its substrate. As a result, the IgE binding to the aptamer has been shown capable of causing the decrease in peak current intensity. In the presence of target IgE, the aptamer could specifically "capture" its taget ligand that served as separator between ALP and1-NP, inhibiting the enzymatic reaction. Moreover, the formation of dielectric layer of IgE could impede the subsequent oxidation of naphthol. For this biosensor, the achievement of electrochemical signal did not depend on the conformational change of aptamer probe, and no other oligonucleotide probes were involved, these features can overcome the difficulties encountered by the conventional electrochemical aptasensors. The method had high sensitivity, the detection limit reached4.44×10-6μg·mL-1(22.7fM). It also exhibited good recoveries in diluted serum samples.
Ochratoxin A (OTA) is a hazard element for human and animal health, including nephrotoxicity, teratogenicity, carcinogenicity, cytotoxicity, genotoxicity and so on. In chapter3, combination of the high specificity of aptamer, using OTA as a model analyte, an electrochemical biological detection method was designed based on the reconstructive aptamer platform. Two parts of split aptamer can specifically recognize adenosine together, alkaline phosphatase (ALP) could then stained in the electrode by affinity of biotin and avidin. ALP plays enzymatic role which enzymatic conversion of1-Naphthyl phosphate (1-NP) into an electroactive naphthol, leading to the electrochemical signal generation. This method possesses high sensitivity, good selectivity and simplicity in operation. It also provides an efficient way for the detection of other biomolecules using the methods of cleft aptamers.
Adenosine triphosphate (ATP) is the direct source of energy with which all biological cells in vivo participate in the life activities, including the synthesis of adipose, sugar, protein and nucleotide synthesis. Detecting ATP rapidly and accurately has very important significance in researching metabolic processes and clinical diagnosis. In chapter4, using ATP as the model analyte, aptamer is split into two parts. Simultaneously, hybridization chain reaction (HCR) is combined with the above platform to accomplish the goal of signal amplification technology. It can be said that the procedure is easy. Aptamer is split two fragments, with one part to mark with mercapto and other part to extend with bases. Lots of ALP attached the electrode for HCR which contribute to high sensitivity for ATP detection, and the detection limit of0.2nM.
Literature reports demonstrate that there is a "signal misreading" behavior in existing machines where the target recognition process and signal transduction is separated from each other. In chapter5, we established an integrated signal transduction-based autonomous machine, in which the recognition element and signal reporter are integrated into the same DNA strand. This new biosensing machine can execute the amplification of target-induced signal. Using exonuclease III to execute signal amplification method, which generated a large number of G-quadruplex-heme as catalyzer in the system of H2O2and ABTS. The machine was employed to detect the p53gene in a more ascendant fashion, and improved assay characteristics are achieved, including dynamic response range and sensitivity. The proposed strategy is also selective and sensitive with a detection limit of1pM. However, we hope that the proposed platform of the p53gene detection is more sensitive. Thus, in chapter6, strand displacement amplification (SDA) was executed signal amplification based on the integrated signal readout mode. The present strategy is highly selective, possessing wide dynamic range and sensitive for p53gene detection with a detection limit of25fM. Moreover, the evaluation of p53gene using this colorimetric method was also successfully demonstrated.
In chapter7, multidimensional devices of G-rich oligonucleotides were designed and applied in gold nanoparticle aggregation-based colorimetric sensor for cancer diagnosis. When p53genes hybridize with molecular beacons embedded G-rich strand, multidimensional devices of G-quadruplex form for particular DNA. Simultaneously, when multidimensional devices were added in solution, it happened aggregation of gold nanoparticles (AuNPs) modified with capture probe. And the colorimetric system exhibited an obvious red-to-purple color change within10-min hybridization. The colorimetric sensor can not only provide nanomolar level of detection capability but also visualize the mutant p53gene. The method provides potential application for the detection of tumor clinical diagnosis, drug screening and DNA nanodevice design.
免疫球蛋白E (IgE)的测定在过敏性鼻炎和过敏性哮喘等相关疾病的临床诊断中具有重要作用。在第2章中,搭建了基于电子通道开关核酸适配体电化学传感器用于IgE的检测。在该传感检测中,免疫球蛋白E绑定到核酸适配体上导致覆盖碱性磷酸酶(ALP)。同时,由于IgE是非传导性介质,使得ALP不能催化1-萘基磷酸酯水解产物-萘酚生成电活性物质。因此,IgE与核酸适配体结合阻碍电子转移,导致电流强度下降。该电化学传感器的信号产生不依赖于核酸适配体探针的构象变化,它克服传统的电化学核酸适配体传感器遇到的困难。并且该方法具有较高的灵敏度,其检测下限达至4.44×10-6μg·mL-1(22.7fM)。在复杂环境中,该检测方法同样表现出了良好的分析性能,血清样品中的回收实验结果令人满意。
赭曲霉素A(OTA)是对人类和动物的健康有极大危害的元素,包括肾脏损害、致畸性、致癌性、细胞毒性和遗传毒性等。第3章中,针对核酸适配体电化学传感器检测小分子性能有待提高的情况,将核酸适配体序列在恰当的部位剪切成两段,构建电信号增强型核酸适配体传感探针。该方法灵敏度较高、选择性好、操作简单和低成本,同时为基于核酸适配体探针检测其它生物分子提供了一条有效的途径。
三磷酸腺苷(ATP)是生物体内细胞一切生命活动所需能量的直接来源,参与体内脂肪、糖、蛋白质和核苷酸的合成。迅速而准确地检测ATP,对于研究生物体的新陈代谢过程和疾病的临床诊断等方面都有非常重要的意义。第4章中,以ATP为模型分析物,将ATP核酸适配体剪切成合适的两部分,同时结合杂交连锁反应(hybridization chain reaction,HCR)信号放大技术,构建了一种基于核酸适配体-HCR的电化学传感器用于ATP的检测。两条剪切的核酸适配体片段中的一条巯基标记,另一条核酸适配体片段延长核苷酸序列,且延长的核酸序列能引发后续的HCR,从而使得大量的碱性磷酸酶通过生物素-亲和素作用存在于电极表面,实现对ATP的高灵敏检测,其检测下限为0.2nM。
较多文献指出传统的分离式信号读出方式在链置换扩增信号放大检测方法中存在“信号耗损”现象。针对该现象我们提出了一种集成式信号读出生化分析传感技术。第5章中,在同一条核酸序列上将信号传导探针与目标识别探针通过适当的剪切而揉和成一体,同时借助核酸外切酶Ⅲ信号放大方式,提高了G-四链体-血红素脱氧核酶催化H2O2氧化2,2'-联氮基双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)检测p53基因的效率,实现对p53基因定量比色分析。把G-四链体核酶的核苷酸序列与目标识别序列揉和成一体,并且设计成分子信标,这种模式具有极大的优越性。此外,分子信标对G-四链体的形成有协助功能,该方案目标物响应信号的生成更快,检测范围更宽,检测灵敏度更好,其检测下限达到1pM。第6章中,同样基于集成式信号读出方式,当检测物p53基因与未形成G-四链体的分子信标杂交时,富G碱基序列被释放,生成G-四链体,同时形成新的分子信标,使得聚合酶链置换扩增反应发生,生成大量的G-四链体-血红素脱氧核酶催化H202氧化ABTS,完成对p53基因超灵敏检测,其检测下限达到25fM。
第7章中,基于纳米金团聚策略,提出了一种G-四链体为传导媒介可视化分析方法用于肿瘤突变p53基因测定。在该比色传感器中,与靶DNA的杂交使得富G碱基发夹结构的核酸链被强制打开,促进高级结构的同向平行G-四链体的形成。由于设计的巧妙,导致生成一维或多维的G-四链体分子器件。当捕获探针修饰的金纳米颗粒加入时,由于捕获探针DNA与G-四链体的末端DNA片段杂交,从而使得金纳米颗粒彼此靠近而发生团聚现象。本论文提出的比色传感器不仅能提供纳摩尔水平的检测能力,也可以可视化判断突变的p53基因。所建立的检测方法为肿瘤相关的临床检测、药物筛选和DNA分子器件设计提供了潜在的应用价值。
Biosensors have wide application in biochemical analysis, environmental monitoring, clinical diagnostics and drug screening, because of their high sensitivity, easy operation, excellent selectivity, short analysis time and low-cost. Meanwhile, with the advantages of easy to synthesize, good stability, simple design, good biocompatibility, flexibility and signaling mechanisms, nucleic acid has important significance in the construction of biosensors. In addition, biosensors can realize the detection of targets rapidly, efficiently and high sensitively depending on a variety of signal amplification methods. In this thesis, using excellent features of aptamers, molecular beacons, G-quadruplex, gold nanoparticles and deoxyribozymes, new design ideas have been deconstructed for detection of different analysis objects. Compared with the traditional detection methods, the proposed detection methods are highly sensitive, easy to operate, low cost, and high speed of analysis.
Immunoglobulin E (IgE) detection of allergic rhinitis, allergic asthma and other related diseases is important in clinical diagnosis. In chapter2, an electronic channel switching-based (ECS) aptasensor was developed for ultra-sensitive protein detection. In the detection mechanism of sensor, the hairpin structure of aptamer was designed to pull electroactive species towards electrode surface and use the surface-immobilized IgE to serve as a barrier that separated enzyme from its substrate. As a result, the IgE binding to the aptamer has been shown capable of causing the decrease in peak current intensity. In the presence of target IgE, the aptamer could specifically "capture" its taget ligand that served as separator between ALP and1-NP, inhibiting the enzymatic reaction. Moreover, the formation of dielectric layer of IgE could impede the subsequent oxidation of naphthol. For this biosensor, the achievement of electrochemical signal did not depend on the conformational change of aptamer probe, and no other oligonucleotide probes were involved, these features can overcome the difficulties encountered by the conventional electrochemical aptasensors. The method had high sensitivity, the detection limit reached4.44×10-6μg·mL-1(22.7fM). It also exhibited good recoveries in diluted serum samples.
Ochratoxin A (OTA) is a hazard element for human and animal health, including nephrotoxicity, teratogenicity, carcinogenicity, cytotoxicity, genotoxicity and so on. In chapter3, combination of the high specificity of aptamer, using OTA as a model analyte, an electrochemical biological detection method was designed based on the reconstructive aptamer platform. Two parts of split aptamer can specifically recognize adenosine together, alkaline phosphatase (ALP) could then stained in the electrode by affinity of biotin and avidin. ALP plays enzymatic role which enzymatic conversion of1-Naphthyl phosphate (1-NP) into an electroactive naphthol, leading to the electrochemical signal generation. This method possesses high sensitivity, good selectivity and simplicity in operation. It also provides an efficient way for the detection of other biomolecules using the methods of cleft aptamers.
Adenosine triphosphate (ATP) is the direct source of energy with which all biological cells in vivo participate in the life activities, including the synthesis of adipose, sugar, protein and nucleotide synthesis. Detecting ATP rapidly and accurately has very important significance in researching metabolic processes and clinical diagnosis. In chapter4, using ATP as the model analyte, aptamer is split into two parts. Simultaneously, hybridization chain reaction (HCR) is combined with the above platform to accomplish the goal of signal amplification technology. It can be said that the procedure is easy. Aptamer is split two fragments, with one part to mark with mercapto and other part to extend with bases. Lots of ALP attached the electrode for HCR which contribute to high sensitivity for ATP detection, and the detection limit of0.2nM.
Literature reports demonstrate that there is a "signal misreading" behavior in existing machines where the target recognition process and signal transduction is separated from each other. In chapter5, we established an integrated signal transduction-based autonomous machine, in which the recognition element and signal reporter are integrated into the same DNA strand. This new biosensing machine can execute the amplification of target-induced signal. Using exonuclease III to execute signal amplification method, which generated a large number of G-quadruplex-heme as catalyzer in the system of H2O2and ABTS. The machine was employed to detect the p53gene in a more ascendant fashion, and improved assay characteristics are achieved, including dynamic response range and sensitivity. The proposed strategy is also selective and sensitive with a detection limit of1pM. However, we hope that the proposed platform of the p53gene detection is more sensitive. Thus, in chapter6, strand displacement amplification (SDA) was executed signal amplification based on the integrated signal readout mode. The present strategy is highly selective, possessing wide dynamic range and sensitive for p53gene detection with a detection limit of25fM. Moreover, the evaluation of p53gene using this colorimetric method was also successfully demonstrated.
In chapter7, multidimensional devices of G-rich oligonucleotides were designed and applied in gold nanoparticle aggregation-based colorimetric sensor for cancer diagnosis. When p53genes hybridize with molecular beacons embedded G-rich strand, multidimensional devices of G-quadruplex form for particular DNA. Simultaneously, when multidimensional devices were added in solution, it happened aggregation of gold nanoparticles (AuNPs) modified with capture probe. And the colorimetric system exhibited an obvious red-to-purple color change within10-min hybridization. The colorimetric sensor can not only provide nanomolar level of detection capability but also visualize the mutant p53gene. The method provides potential application for the detection of tumor clinical diagnosis, drug screening and DNA nanodevice design.
引文
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