DNA荧光生物传感器的制备及水溶性量子点在DNA分析中的应用研究
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摘要
核酸是生命的遗传物质,人类许多遗传疾病与DNA碱基序列的变异有关,因此,建立高灵敏度、高选择性、简单、快速检测特定序列DNA的分析方法,对疾病诊断和预防具有重要的意义。量子点具有优异的光学性质,与传统的有机染料相比,量子点具有激发光谱宽,发射光谱可调、发射峰较窄,斯托克斯位移较大和不易光解等优点,己在生命分析中得到广泛的应用。本论文主要分为以下四个部分:
(1)处理过的光纤为探针固载基质,以酶催化底物放大杂交信号为检测方法,制备了一种新型的DNA荧光生物传感器。在光纤端面,DNA探针与生物素修饰的靶DNA杂交形成双链DNA,再共价接入亲和素修饰的辣根过氧化物酶(streptavidin-HRP)。通过检测酶催化产物联二对羟基苯乙酸(DBDA)的荧光强度,进行定量检测靶DNA。实验结果表明,检测DNA的线性范围为1.69-169 pM,检测限为1 pM(S/N=3)。用尿素溶液处理有利于传感器的再生,三次循环之后,传感器仍能保持70%的信号。
(2)直接合成水溶性CdSe和CdSe/ZnS量子点。针对有机相中合成量子点耗时、反应条件苛刻、非水溶性等问题,研究了以NaHSe为前躯体、巯基乙酸为稳定剂以及在无氧、搅拌等氛围下直接合成了水溶性量子点。同时研究了反应温度、时间、pH值、稳定剂及其用量等因素对量子点荧光的影响,使用透射电镜、荧光光谱法和紫外光谱法等对量子点进行了表征。实验结果表明,该方法能在简单的条件下快速合成稳定性好、荧光性能良好的CdSe和CdSe/ZnS量子点。以NaHTe为前躯体、巯基乙酸为稳定剂以及在无氧、搅拌等氛围下直接合成了水溶性CdTe量子点。用此法合成的量子点具有更窄的半峰宽,更加对称的发射峰和更高的量子产率。CdTe量子点放置三个月后量子产率达可达到45%,而且实验条件简便、成本低廉、可重复性强,因而是水相合成高质量量子点的一种较成功的方法。同时还具备制备具有近红外荧光量子点的可能性,为量子点在生化分析领域的应用提供了更多的机会。
(3)基于切口酶和靶DNA循环,以CdTe量子点为荧光探针,磁珠为DNA探针固载和分离基质,发展了一种新型的、高灵敏度荧光检测DNA的方法。该方法具有很好的选择性,能够精确的区分单碱基错配DNA和靶DNA。在最佳实验条件下,该方法测定DNA的线性范围是5.0 fM到500 fM。除了具有很高的灵敏度和选择性,该方法还可以结合一些传统的检测方法及其外延技术,从而实现对核酸、核酸适体、RNA和金属离子的检测。该方法也可以用于核苷酸多态性(SNP)及混合分析,例如,选用不同粒径的量子点可同时检测一系列的DNA和蛋白质分子。
(4)基于Hg2+可以嵌插到DNA的T-T错配碱基中形成T-Hg2+-T桥和单链DNA与SWNTs对靶DNA的竞争结合作用,建立了一种方便、快捷、灵敏的荧光检测Hg2+的方法。在最佳实验条件下,该体系测定Hg2+的线性范围是4.52×10-8 -7.21×10-7 M,检测限可达到0.1 nM,并具有良好的选择性。
Deoxyribonucleic acid is an important hereditary material, and the base variation of DNA is tightly related to genetic diseases. So it is important to detect DNA sequence for disease diagnosis and clinical applications. Compared with the traditional organic fluorescent dye, the quantum dots exhibit remarkably important advantages, including broad excitation spectrum, the tunable and narrow emission spectrum, large stokes displacement, high photobleaching threshold and excellent photostability. Quantum dots is becoming an increasingly promising method for biomedical analysis. This masteral dissertation constitutes by four parts besides the preface:
(1) A novel DNA fluorescent biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacet- ic acid (DBDA) generated from the substrate 4-hydroxyphenylaceticacid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It was discovered that the sensor can retain 70 % of its original activity after three detection-regeneration cycles.
(2) CdSe and CdSe/ZnS quantum dots was directly synthesized in aqueous phase. In order to overcome the problems of time consuming, harsh reaction conditions and non water-solubility of quantum dots synthesis in organic solvent phase, we synthesize quantum dots directly under non-oxygen, stirring, and using the NaHSe as precursor and mercaptoacetic acid as stabilizer. We also study the effects of temperature, reaction time, pH, stabilizer and its usage to the quantum dots fluorescent. In addition, TEM, fluorescence and UV/Vis spectrum were used to characterize the prepared nanoparticles. These results clearly showed that this method could synthetize the good and stable fluorescence CdSe and CdSe/ZnS quantum dots quickly under the simple condition. We synthesize CdTe quantum dots directly under non-oxygen, stirring, and using the NaHSe as precursor and mercaptoacetic acid as stabilizer. As-prepared CdTe QDs were shown to have narrower full wave at half maximum(FWHM), higher photoluminescence quantum yields(PL QY),and symmetric fluorescent emission peak. The PLQY of CdTe QDs reaches 45% at room temperature after placing three months. To our best knowledge, the simple experimental conditions, low cost and good repeatability, make it become one of the most successful methods which can directly prepare CdTe QDs in aqueous solution with high quality. In addition, it showed feasibility to synthesize CdTe QDs with near infrared fluorescence that make it be able to apply widely to biochemical analysis in the near future.
(3) A novel ultrasensitive fluorescence detection method for DNA based on NEase and target recycles assisted QDs and MBs has been developed. The novel method can discriminate complementary target from single mismatch target accurately. The linear relationship between the analytical signal and the concentration of target is in the range from to 5.0 fM to 500 fM. Aside from high sensitivity and selectivity, this method, can be readily coupled to convention analytical techniques and extended useful for various applications involving the nucleic acids, aptamer-binding small molecules, RNA, metal ions. This system can also be used for SNP of a large number of genes and multiplexed analysis, for example, using different QDs to detect a series of DNA and proteins at the same time.
(4) Based on binding of Hg2+ to promote these T-T mismatches to form stable T-Hg2+-T base pairs and the binding rate of DNA and the nanotube was lower than DNA hybridization, we have developed a convenient, fast, sensitive method of fluorescence detection of Hg2+. Under the optimal assembling and detection conditions, a good linearity for simultaneous detection was obtained in the range from 4.52×10-8 to 7.21×10-7 M, and the detection limit was estimated to be 0.1 nM. The presence of other metal ions did not interfere the detection of Hg2+.
(1)处理过的光纤为探针固载基质,以酶催化底物放大杂交信号为检测方法,制备了一种新型的DNA荧光生物传感器。在光纤端面,DNA探针与生物素修饰的靶DNA杂交形成双链DNA,再共价接入亲和素修饰的辣根过氧化物酶(streptavidin-HRP)。通过检测酶催化产物联二对羟基苯乙酸(DBDA)的荧光强度,进行定量检测靶DNA。实验结果表明,检测DNA的线性范围为1.69-169 pM,检测限为1 pM(S/N=3)。用尿素溶液处理有利于传感器的再生,三次循环之后,传感器仍能保持70%的信号。
(2)直接合成水溶性CdSe和CdSe/ZnS量子点。针对有机相中合成量子点耗时、反应条件苛刻、非水溶性等问题,研究了以NaHSe为前躯体、巯基乙酸为稳定剂以及在无氧、搅拌等氛围下直接合成了水溶性量子点。同时研究了反应温度、时间、pH值、稳定剂及其用量等因素对量子点荧光的影响,使用透射电镜、荧光光谱法和紫外光谱法等对量子点进行了表征。实验结果表明,该方法能在简单的条件下快速合成稳定性好、荧光性能良好的CdSe和CdSe/ZnS量子点。以NaHTe为前躯体、巯基乙酸为稳定剂以及在无氧、搅拌等氛围下直接合成了水溶性CdTe量子点。用此法合成的量子点具有更窄的半峰宽,更加对称的发射峰和更高的量子产率。CdTe量子点放置三个月后量子产率达可达到45%,而且实验条件简便、成本低廉、可重复性强,因而是水相合成高质量量子点的一种较成功的方法。同时还具备制备具有近红外荧光量子点的可能性,为量子点在生化分析领域的应用提供了更多的机会。
(3)基于切口酶和靶DNA循环,以CdTe量子点为荧光探针,磁珠为DNA探针固载和分离基质,发展了一种新型的、高灵敏度荧光检测DNA的方法。该方法具有很好的选择性,能够精确的区分单碱基错配DNA和靶DNA。在最佳实验条件下,该方法测定DNA的线性范围是5.0 fM到500 fM。除了具有很高的灵敏度和选择性,该方法还可以结合一些传统的检测方法及其外延技术,从而实现对核酸、核酸适体、RNA和金属离子的检测。该方法也可以用于核苷酸多态性(SNP)及混合分析,例如,选用不同粒径的量子点可同时检测一系列的DNA和蛋白质分子。
(4)基于Hg2+可以嵌插到DNA的T-T错配碱基中形成T-Hg2+-T桥和单链DNA与SWNTs对靶DNA的竞争结合作用,建立了一种方便、快捷、灵敏的荧光检测Hg2+的方法。在最佳实验条件下,该体系测定Hg2+的线性范围是4.52×10-8 -7.21×10-7 M,检测限可达到0.1 nM,并具有良好的选择性。
Deoxyribonucleic acid is an important hereditary material, and the base variation of DNA is tightly related to genetic diseases. So it is important to detect DNA sequence for disease diagnosis and clinical applications. Compared with the traditional organic fluorescent dye, the quantum dots exhibit remarkably important advantages, including broad excitation spectrum, the tunable and narrow emission spectrum, large stokes displacement, high photobleaching threshold and excellent photostability. Quantum dots is becoming an increasingly promising method for biomedical analysis. This masteral dissertation constitutes by four parts besides the preface:
(1) A novel DNA fluorescent biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacet- ic acid (DBDA) generated from the substrate 4-hydroxyphenylaceticacid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It was discovered that the sensor can retain 70 % of its original activity after three detection-regeneration cycles.
(2) CdSe and CdSe/ZnS quantum dots was directly synthesized in aqueous phase. In order to overcome the problems of time consuming, harsh reaction conditions and non water-solubility of quantum dots synthesis in organic solvent phase, we synthesize quantum dots directly under non-oxygen, stirring, and using the NaHSe as precursor and mercaptoacetic acid as stabilizer. We also study the effects of temperature, reaction time, pH, stabilizer and its usage to the quantum dots fluorescent. In addition, TEM, fluorescence and UV/Vis spectrum were used to characterize the prepared nanoparticles. These results clearly showed that this method could synthetize the good and stable fluorescence CdSe and CdSe/ZnS quantum dots quickly under the simple condition. We synthesize CdTe quantum dots directly under non-oxygen, stirring, and using the NaHSe as precursor and mercaptoacetic acid as stabilizer. As-prepared CdTe QDs were shown to have narrower full wave at half maximum(FWHM), higher photoluminescence quantum yields(PL QY),and symmetric fluorescent emission peak. The PLQY of CdTe QDs reaches 45% at room temperature after placing three months. To our best knowledge, the simple experimental conditions, low cost and good repeatability, make it become one of the most successful methods which can directly prepare CdTe QDs in aqueous solution with high quality. In addition, it showed feasibility to synthesize CdTe QDs with near infrared fluorescence that make it be able to apply widely to biochemical analysis in the near future.
(3) A novel ultrasensitive fluorescence detection method for DNA based on NEase and target recycles assisted QDs and MBs has been developed. The novel method can discriminate complementary target from single mismatch target accurately. The linear relationship between the analytical signal and the concentration of target is in the range from to 5.0 fM to 500 fM. Aside from high sensitivity and selectivity, this method, can be readily coupled to convention analytical techniques and extended useful for various applications involving the nucleic acids, aptamer-binding small molecules, RNA, metal ions. This system can also be used for SNP of a large number of genes and multiplexed analysis, for example, using different QDs to detect a series of DNA and proteins at the same time.
(4) Based on binding of Hg2+ to promote these T-T mismatches to form stable T-Hg2+-T base pairs and the binding rate of DNA and the nanotube was lower than DNA hybridization, we have developed a convenient, fast, sensitive method of fluorescence detection of Hg2+. Under the optimal assembling and detection conditions, a good linearity for simultaneous detection was obtained in the range from 4.52×10-8 to 7.21×10-7 M, and the detection limit was estimated to be 0.1 nM. The presence of other metal ions did not interfere the detection of Hg2+.
引文
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