DNA和凝血酶荧光分析新方法的研究
摘要
核酸是生命的遗传物质，蛋白质是生命的体现者。人类许多遗传疾病与DNA碱基序列的变异有关，人体中特定蛋白质浓度也可以作为疾病诊断的依据，因此，建立高灵敏度、高选择性、简单、快速检测特定序列DNA和蛋白质的分析方法，对疾病诊断和预防具有重要的意义。荧光分析方法因其高灵敏度、高选择性、简单、快速等优点，已在生命分析中得到广泛的应用。
本论文由综述和研究报告两部分组成。第一部分介绍了荧光分析方法的原理及特点，简要评述了DNA、蛋白质检测方法的研究进展。以荧光纳米粒子为重点介绍了常见的荧光标记物，最后阐述了本论文的研究目的和内容。
第二部分为研究报告，由两部分组成。第一部分以异硫氰酸罗丹明B嵌合二氧化硅纳米粒子为荧光信号物质构建纳米荧光核酸探针，使其与固定在磁性纳米粒子上的目标单链DNA杂交，经磁场分离后测量沉淀物的荧光强度。实验结果表明，该荧光强度与目标单链DNA的浓度在1.3×10~(-14)～2.5×10~(-13) mol／L范围内呈线性关系，线性方程为I=40.1C+10.1(C的单位为10~(-13) mol／L)，相关系数为0.9968。对6.7×10~(-14) mol／L浓度的目标单链DNA进行11次测定，相对标准偏差为2.6％。方法的检出限为1.6×10~(-15) mol／L。本研究结合纳米技术及生物磁场分离技术通过磁性材料标记生物分子实现了磁性分离，结合分子识别技术，通过磁性材料标记生物分子实现了磁性分离，提高了分离的选择性。建立了一种高灵敏的DNA杂交荧光检测方法，对DNA分析方法的研究具有一定的参考价值。
研究报告的第二部分提出了基于竞争反应的非标记荧光蛋白质检测方法。以凝血酶为检测对象，使用能够专一结合凝血酶的核酸适体作为识别分子，设计了含AP位点的适体的互补DNA，建立了高选择性荧光检测凝血酶的新方法。没有凝血酶时，核酸适体可以和内含AP位点单链DNA最大程度地杂交形成双链DNA，荧光分子氨氯吡咪嵌合在双链DNA中的AP位点处并与AP位点对面碱基T结合，使得氨氯吡咪的荧光信号下降；当凝血酶存在时，凝血酶竞争结合其适体，释放出双链中嵌在碱基缺失位点(AP site)的荧光报告分子氨氯吡咪，荧光信号增强，并且荧光强度的增强程度与凝血酶浓度呈线性关系。据此，可基于荧光强度的增强来对凝血酶进行定量测定。在选定实验条件下，检测凝血酶的线性范围为4.2×10~(-9)～4.2×10~(-8) mol／L，线性方程为△I=0.0204C-0.0251(C的单位：10~(-9) mol／L)，相关系数为0.9962，对3.0×10~(-8) mol／L凝血酶进行11次测定，相对标准偏差为3.2％。方法的检出限为1.3×10~(-9) mol／L。实验结果表明，利用凝血酶与其核酸适体的互补链的竞争作用，释放出嵌在双链DNA中AP位点的荧光分子的原理可建立高选择性、均相、非标记检测凝血酶的分析方法。该方法的建立为基于含核酸适体和AP位点双链DNA非标记测定蛋白质提供了一条新思路。
Deoxyribonucleic acid is an important hereditary material. The basevariation of DNA is tightly related to genetic diseases. Protein is one of the importantparts of organism. The quantity changes of protein in the human body may lead topathological changes, so it is important to detect DNA sequence and protein sensitivelyfor disease diagnosis and clinical applications.
Fluorescence is becoming increasingly promising method for biomedical andenvironmental analysis because of its advantages such as simple instrumentation, highsensitivity, simplicity and rapidity.
This thesis includes a review and a research section. In the review section, thebasic principles, characteristics and the applications of fluorescence in DNA and proteinanalysis are introduced. Development of various kinds of fluorescence labels is mainlyreviewed. Finally, the purpose and content of the research work in this thesis arepresented.
The research section contains two subunits. In the first subunit, the fluorescencesignal amplification of DNA hybridization was studied, which was based on dye-dopedsilica nanoparticles used as fluorescence probe. The fluorescence emission enhancementwas significantly observed upon DNA probe hybriding with target DNA which wasattached on the surface of magnetic nanoparticles. It was found that DNA could bequantified in good linearity range of 1.2×10~(-14)～2.5×10~(-13) mol/L with a low detectionlimit of 1.6×10~(-15) mol/L, and the relative standard deviation for 6.7×10~(-14)mol/L DNAwas 2.6%(n=11). The method developed was sensitive, simple and suitable for thedetermination of DNA sequence.
In the second subunit, a new strategy of fluorescence assay for protein based onDNA duplex containing AP site was proposed. In this work, a novel label freefluorescence method for homogeneous detection of thrombin, which chosen as a modelprotein, was developed on basis of dsDNA containing AP site and aptamer. Aptameragainst thrombin and amiloride was used as a molecular recognizer and a fluorescencereporter, respectively. In the absence of thrombin, the aptamer could hybridize with cDNA containing AP site in the highest degree and amiloride could bind to thymineopposite AP site in the DNA duplexes, resulting in fluorescence quenching. In theprescence of thrombin, amiloride exhibits enhancement of its fluorescence and theenhance degree was relative to the increase of the concentration of thrombin. Accordingto the fluorescence signal change, thrombin could be determined rapidly and sensitively.It was found that under optimal conditions the enhanced fluorescence intensity had agood linearity with the concentration of thrombin in the range from 4.2×10~(-9) mol/L to4.2×10~(-8) mol/L with a detection limit of 1.3×10~(-9) mol/L. The relative standard deviation(n=11) was 3.2% at 3.0×10~(-8) mol/L thrombin. This work demonstrated that the proposedmethod provides a promising strategy of fluorescence assay for protein based on DNAduplex containing AP site and an aptamer as molecular recognizer.
本论文由综述和研究报告两部分组成。第一部分介绍了荧光分析方法的原理及特点，简要评述了DNA、蛋白质检测方法的研究进展。以荧光纳米粒子为重点介绍了常见的荧光标记物，最后阐述了本论文的研究目的和内容。
第二部分为研究报告，由两部分组成。第一部分以异硫氰酸罗丹明B嵌合二氧化硅纳米粒子为荧光信号物质构建纳米荧光核酸探针，使其与固定在磁性纳米粒子上的目标单链DNA杂交，经磁场分离后测量沉淀物的荧光强度。实验结果表明，该荧光强度与目标单链DNA的浓度在1.3×10~(-14)～2.5×10~(-13) mol／L范围内呈线性关系，线性方程为I=40.1C+10.1(C的单位为10~(-13) mol／L)，相关系数为0.9968。对6.7×10~(-14) mol／L浓度的目标单链DNA进行11次测定，相对标准偏差为2.6％。方法的检出限为1.6×10~(-15) mol／L。本研究结合纳米技术及生物磁场分离技术通过磁性材料标记生物分子实现了磁性分离，结合分子识别技术，通过磁性材料标记生物分子实现了磁性分离，提高了分离的选择性。建立了一种高灵敏的DNA杂交荧光检测方法，对DNA分析方法的研究具有一定的参考价值。
研究报告的第二部分提出了基于竞争反应的非标记荧光蛋白质检测方法。以凝血酶为检测对象，使用能够专一结合凝血酶的核酸适体作为识别分子，设计了含AP位点的适体的互补DNA，建立了高选择性荧光检测凝血酶的新方法。没有凝血酶时，核酸适体可以和内含AP位点单链DNA最大程度地杂交形成双链DNA，荧光分子氨氯吡咪嵌合在双链DNA中的AP位点处并与AP位点对面碱基T结合，使得氨氯吡咪的荧光信号下降；当凝血酶存在时，凝血酶竞争结合其适体，释放出双链中嵌在碱基缺失位点(AP site)的荧光报告分子氨氯吡咪，荧光信号增强，并且荧光强度的增强程度与凝血酶浓度呈线性关系。据此，可基于荧光强度的增强来对凝血酶进行定量测定。在选定实验条件下，检测凝血酶的线性范围为4.2×10~(-9)～4.2×10~(-8) mol／L，线性方程为△I=0.0204C-0.0251(C的单位：10~(-9) mol／L)，相关系数为0.9962，对3.0×10~(-8) mol／L凝血酶进行11次测定，相对标准偏差为3.2％。方法的检出限为1.3×10~(-9) mol／L。实验结果表明，利用凝血酶与其核酸适体的互补链的竞争作用，释放出嵌在双链DNA中AP位点的荧光分子的原理可建立高选择性、均相、非标记检测凝血酶的分析方法。该方法的建立为基于含核酸适体和AP位点双链DNA非标记测定蛋白质提供了一条新思路。
Deoxyribonucleic acid is an important hereditary material. The basevariation of DNA is tightly related to genetic diseases. Protein is one of the importantparts of organism. The quantity changes of protein in the human body may lead topathological changes, so it is important to detect DNA sequence and protein sensitivelyfor disease diagnosis and clinical applications.
Fluorescence is becoming increasingly promising method for biomedical andenvironmental analysis because of its advantages such as simple instrumentation, highsensitivity, simplicity and rapidity.
This thesis includes a review and a research section. In the review section, thebasic principles, characteristics and the applications of fluorescence in DNA and proteinanalysis are introduced. Development of various kinds of fluorescence labels is mainlyreviewed. Finally, the purpose and content of the research work in this thesis arepresented.
The research section contains two subunits. In the first subunit, the fluorescencesignal amplification of DNA hybridization was studied, which was based on dye-dopedsilica nanoparticles used as fluorescence probe. The fluorescence emission enhancementwas significantly observed upon DNA probe hybriding with target DNA which wasattached on the surface of magnetic nanoparticles. It was found that DNA could bequantified in good linearity range of 1.2×10~(-14)～2.5×10~(-13) mol/L with a low detectionlimit of 1.6×10~(-15) mol/L, and the relative standard deviation for 6.7×10~(-14)mol/L DNAwas 2.6%(n=11). The method developed was sensitive, simple and suitable for thedetermination of DNA sequence.
In the second subunit, a new strategy of fluorescence assay for protein based onDNA duplex containing AP site was proposed. In this work, a novel label freefluorescence method for homogeneous detection of thrombin, which chosen as a modelprotein, was developed on basis of dsDNA containing AP site and aptamer. Aptameragainst thrombin and amiloride was used as a molecular recognizer and a fluorescencereporter, respectively. In the absence of thrombin, the aptamer could hybridize with cDNA containing AP site in the highest degree and amiloride could bind to thymineopposite AP site in the DNA duplexes, resulting in fluorescence quenching. In theprescence of thrombin, amiloride exhibits enhancement of its fluorescence and theenhance degree was relative to the increase of the concentration of thrombin. Accordingto the fluorescence signal change, thrombin could be determined rapidly and sensitively.It was found that under optimal conditions the enhanced fluorescence intensity had agood linearity with the concentration of thrombin in the range from 4.2×10~(-9) mol/L to4.2×10~(-8) mol/L with a detection limit of 1.3×10~(-9) mol/L. The relative standard deviation(n=11) was 3.2% at 3.0×10~(-8) mol/L thrombin. This work demonstrated that the proposedmethod provides a promising strategy of fluorescence assay for protein based on DNAduplex containing AP site and an aptamer as molecular recognizer.
引文
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