基于毛细管电泳的量子点生物探针分离检测新技术研究

发布时间：2018-07-26 14:58

【摘要】：随着生命科学的发展,待测生物样品的复杂性越来越高,从而对分析方法的检测灵敏度、效率、速度、通量及成本提出了更高要求。作为一种新型的荧光探针,量子点具有有机荧光染料和荧光蛋白无法比拟的光学性质,近年来基于量子点的荧光分析法已在生物分析领域得到了广泛应用。另一方面,作为一种高分辨、高灵敏、高速度、高通量及低样品消耗的微分离技术,毛细管电泳在生物分析领域也具有广阔的应用前景。基于此,本论文将上述两种分析方法进行了结合,建立了一种基于量子点探针和毛细管电泳的分析新方法(即利用量子点作为荧光探针,毛细管电泳荧光检测作为分析手段,),并重点研究了其在生物分析检测中应用。论文完成的工作如下： (1)采用毛细管筛分电泳对不同粒径的水溶性CdSe/ZnS核壳量子点进行了分离,并探讨了筛分介质的种类和浓度、缓冲溶液的浓度和pH、分离电压对量子点分离的影响。在这些因素优化条件下,四种不同粒径的量子点得到了有效分离,且重现性良好,迁移时间的最大相对标准偏差为1.01%,且量子点的电泳淌度和粒径间存在着很好的相关性(R2=0.997)。实验证实,这一方法可用于水溶性CdSe/ZnS核壳量子点的粒径测量。这一工作为量子点粒径的检测提供了一种新的方法,同时也为接下来基于毛细管电泳技术和量子点荧光探针的生物分析提供了重要参考。 (2)分别选用发射波长为532 nm的CdTe量子点和632 nm的CdSe/ZnS量子点作为荧光共振能量转移体系的供体和受体,通过共价偶联的方法将上述两种量子点分别标记上小鼠1gG和羊抗小鼠1gG,抗原和抗体间的免疫亲和作用拉近了两种量子点间的距离,从而导致供体和受体间荧光共振能量转移的发生。我们利用毛细管电泳对上述荧光共振能量转移进行了分析。实验中,为了同时检测两种量子点的荧光强度变化,我们利用两个固定检测波长通道分别对供体和受体的荧光强度进行了同时检测。在成功地通过毛细管电泳实现荧光共振能量转移体系与其它“多余”的荧光物得到有效分离的同时,准确测量了供体及受体的荧光强度,并计算了不同浓度受体情况下的荧光共振能量转移效率(38.56-69.58%),这一效率比常规的荧光强度测量法得到的荧光共振能量转移效率(12.77-52.37%)有一定程度的提高,而且对荧光共振能量转移的观察更加直观,灵敏度更高,样品消耗更少。这一工作为荧光共振能量转移的研究提供了一种新的分析方法。 (3)分别利用亲和素-生物素系统及直接共价偶联的方法将荧光发射波长为585和650 nm的CdTe量子点与两种具有不同碱基序列的分子信标进行了连接,构建得到了两种量子点-分子信标探针。量子点-分子信标探针与不同靶标杂交后的毛细管电泳结果表明,这两种量子点-分子信标探针都只与和其环状部分序列完全互补的靶标特异性杂交,且都具有对单碱基突变识别的能力。利用这两种量子点-分子信标探针,我们借助毛细管电泳成功地实现了对特定核酸序列两个位点单碱基突变的同时检测。这一研究不仅为今后多位点的核酸单碱基突变检测提供了重要手段,而且在诸如核酸高灵敏度检测及单核苷酸多态性研究领域中也有很广泛的应用前景。 (4)分别将CdSe/ZnS量子点及金纳米颗粒与不同碱基长度的互补DNA单链进行了偶联,并通过DNA链的互补杂交将量子点与金纳米颗粒连接到一起,构建得到了一个研究溶液中金属增强量子点荧光的模型,然后利用毛细管电泳考察了金纳米颗粒和量子点间的距离对量子点荧光增强的影响。实验结果表明溶液中的金纳米颗粒增强量子点荧光有很强的距离依赖性,只有与金纳米颗粒与量子点相距6.8-18.7nm时,量子点才出现荧光增强效应,其中11.9 nm是金纳米颗粒增强量子点荧光的最佳距离,此时量子点的荧光强度增强为原来的2.3倍。接下来,我们在量子点和纳米金颗粒相距11.9 nm的体系中加入与量子点偶联的DNA链相同碱基序列的目的DNA,利用目的DNA与量子点-DNA间的特异性竞争,实现了对19.6 pg(15 nM)目的DNA的检测。这一工作不仅为今后溶液中金属增强量子点荧光的研究提供了重要参考,而且在诸如DNA杂交分析及高灵敏度DNA检测等领域也有很广泛的应用前景。
[Abstract]:With the development of life science, the complexity of the biological samples to be measured is becoming more and more complex, which puts forward higher requirements for the detection sensitivity, efficiency, speed, flux and cost of the analytical methods. As a new type of fluorescence probe, the quantum dots have the optical properties incomparable with the organic fluorescent dyes and the fluorescent egg white. In recent years, the quantum dots are based on the quantum dots. Fluorescence analysis has been widely used in the field of biological analysis. On the other hand, as a differential separation technology with high resolution, high sensitivity, high speed, high throughput and low sample consumption, capillary electrophoresis has a broad application prospect in the field of biological analysis. Based on this, the above two analytical methods are combined and established in this paper. A new analytical method based on quantum dot probe and capillary electrophoresis (using quantum dots as a fluorescent probe, capillary electrophoresis fluorescence detection as an analytical method), and its application in bioanalysis and detection is studied. The work completed in this paper is as follows:
(1) the capillary sieving electrophoresis was used to separate the water soluble CdSe/ZnS core shell quantum dots with different particle sizes, and the types and concentrations of the sieve medium, the concentration of the buffer solution and the pH, the effect of the separation voltage on the separation of the quantum dots were discussed. Under these conditions, the quantum dots of four different particle sizes were effectively separated and reproduced. The maximum relative standard deviation of the migration time is 1.01%, and there is a good correlation between the electrophoretic mobility and the particle size of the quantum dots (R2=0.997). The experiment proves that this method can be used to measure the particle size of the water soluble CdSe/ZnS nuclear point quantum dots. It provides an important reference for bioanalysis based on capillary electrophoresis and quantum dots fluorescent probes.
(2) the CdTe quantum dots with emission wavelength of 532 nm and the CdSe/ZnS quantum dots of 632 nm are used as donors and receptors of the fluorescence resonance energy transfer system. The above two quantum dots are labeled on the mice 1gG and the Sheep anti mouse 1gG respectively by covalent coupling method, and the immune affinity between the antigen and the antibody is close to two quantum dots. Distance, which leads to the occurrence of fluorescence resonance energy transfer between the donor and the receptor. We analyzed the above fluorescence resonance energy transfer by capillary electrophoresis. In order to detect the fluorescence intensity changes of the two quantum dots at the same time, we used two fixed detection wavelength channels to carry out the fluorescence intensity of the donor and the receptor respectively. At the same time, the fluorescence resonance energy transfer efficiency (38.56-69.58%) of the donor and the receptor was measured accurately, while the fluorescence resonance energy transfer system was successfully separated from other "excess" fluorescence by capillary electrophoresis. The fluorescence resonance energy transfer efficiency (12.77-52.37%) obtained by the fluorescence intensity measurement method is improved to a certain extent, and the observation of the fluorescence resonance energy transfer is more intuitive, the sensitivity is higher, and the sample consumption is less. This work provides a new analytical method for the study of fluorescence resonance energy transfer.
(3) using the avidin biotin system and the direct covalent coupling method, the CdTe quantum dots with the fluorescence emission wavelength of 585 and 650 nm were connected with the two molecular beacons with different base sequences, and two quantum dot molecular beacons were constructed and the capillaries of the quantum dot sub beacon probe hybridized with the different targets were obtained. The results of tube electrophoresis show that these two quantum dots - molecular beacon probes are only specific to the targets that are completely complementary to their ring part sequences, and all have the ability to recognize single base mutations. Using these two quantum dots - molecular beacon probes, we have accomplished two loci of specific nucleic acid sequences by capillary electrophoresis. The simultaneous detection of single base mutation has not only provided an important means for the detection of single nucleotide mutation in multiple sites in the future, but also has a wide application prospect in the field of high sensitivity detection and single nucleotide polymorphisms.
(4) the CdSe/ZnS quantum dots and gold nanoparticles were coupled with the complementary DNA single strand of different base lengths, and the quantum dots were connected to gold nanoparticles through the complementary hybridization of the DNA chain. A model was constructed to study the fluorescence of the metal enhanced quantum dots in the solution. Then the gold nanoparticles were investigated by capillary electrophoresis. The effect of the distance between the particles and the quantum dots on the fluorescence enhancement of the quantum dots. The experimental results show that the fluorescence of the gold nanoparticles in the solution has a strong distance dependence. Only when the gold nanoparticles and the quantum dots are apart from 6.8-18.7nm, the quantum dots appear to be enhanced by the fluorescence enhancement, and the 11.9 nm is a gold nanoparticle enhanced quantum dot fluorescein. At the best distance of the light, the fluorescence intensity of the quantum dots is enhanced to 2.3 times that of the original. Then, we add the same base sequence of the DNA chain to the quantum dots in the system of quantum dots and the nano gold particles 11.9 nm apart, and use the specific competition between the target DNA and the quantum dot -DNA to achieve the DNA of the 19.6 PG (15 nM). This work not only provides an important reference for the study of metal enhanced quantum dot fluorescence in the future, but also has a wide range of applications in the fields such as DNA hybridization analysis and high sensitivity DNA detection.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

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