激光诱导荧光DNA分析系统的研究
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摘要
DNA分析技术是从生物的分子—基因水平揭示生物的特性,它为生命科学研究提供了最有效、最便捷的技术和手段。因此,DNA分析仪器的研究十分重要,它是获取信息的源头和基础。本论文开展了激光诱导荧光DNA分析系统的相关理论和研制工作,先后完成了两套检测系统的设计与构建。它们是基于微通道毛细管电泳技术的现代光学检测系统,具有结构简单、性能良好、成本较低的优点,分辨率达到1bp,也就是单碱基分辨。灵敏度高,对标准DNA样品pGEM-3Zf(+)/HaeⅢMarkers的检测限是2.4×10~(-11)mol/L。满足测序和短串联重复序列(short tandem repeat,STR)分型对分辨率和灵敏度的要求。主要工作包括两大部分:微通道毛细管电泳部分和激光诱导荧光检测部分。
电泳部分主要包括:对微通道内壁进行改性,以控制电渗流和抑制吸附;制备性能良好的筛分介质能够对样品电泳分离;对涂层、灌胶、进样系统以及荧光染料的选择进行优化,选用非交联的线性聚丙烯酰胺(Linear Polyacrylamide,LPA)作为涂层材料和可替换的筛分介质;测量得到的自制涂层管的电渗流淌度为2.5165×10~(-8)m~2V~(-1)s~(-1),对电渗流的控制满足DNA样品分离的要求;研究了电场强度与迁移率的关系,得到电场强度与电泳电流的关系曲线;对于本筛分系统,提出了一种新的Ogston修正模型,从而能够简单、良好地解释实验结果;考察了DNA样品中不同长度的片段在不同浓度筛分介质中,随电场强度条件变化的信号强度、展宽和分辨率的变化情况,优化筛分质浓度为4%LPA。
检测部分设计为共焦光路,建立了分别以488nmAr+激光器和532nm固体激光器为光源的两套系统,由激光器激发电泳到检测窗口的DNA片段(标记有荧光染料),发射的荧光信号由光电倍增管收集检测,激发光路和收集光路三维可调。通过理论分析和实验优化本系统的设计:优化聚焦于微通道中的激发光斑大小,增大聚焦到毛细管内径中的激发光斑尺寸,使光斑尺寸由最小的10μm增大到50μm(充满了整个毛细管内径),光电倍增管接收到的荧光信号提高了2-3倍,从而提高系统的信噪比和灵敏度;提出在聚焦物镜处加折射率匹配液的方法,加匹配液后的荧光信号得到了增强,因而提高了荧光收集效率和灵敏度。通过理论和实验分析阵列毛细管电泳技术中杂散光的强度与分布,激发光束直接照射到毛细管的内径中心时,毛细管产生的杂散光强度是无毛细管的情况下的2.7025倍;对不同内径的微通道毛细管进行比较,优化内径为50μm;分析了不同间距的阵列毛细管杂散光情况;所得结果对提高检测系统荧光收集效率、提高信噪比、采取最佳扫描方式十分有意义。
DNA analysis is the technology to reveal the biological characteristic in molecular and genetic level; it is the most effective and convenient way for research in life sciences. So, the study of DNA analyzer is very important; it is the source of the information. The laser induced fluorescence DNA analysis system was researched and developed. Two detection systems were designed and setup, which were the modern optical detection systems based on microchannel and capillary electrophoresis. They were simple, cost effective, high sensitivity and good performance. Single basepair could be resolved and the limit of detection (LOD) of DNA sample pGEM-3Zf(+)/Hae III Markers was 2.4×10~(-11)mol/L. which meet the requirement of DNA sequencing and short tandem repeat (STR) typing. The system could be divided in two parts: microchannel capillary electrophoresis and laser induced fluorescence detection.
The electrophoresis section included the work as follows. The inner wall of the microchannel was modified to control the electroosmotic flow and restrain adsorption. The good performance sieving gel medium was prepared to separate the samples. The processes of coating, gel injection, sample injection and the flouorescece selection were optimized. The replacebale noncross-link linear polyacrylamide (LPA) was selected to be coating material and sieving medium. The electroosmotic flow mobility (EOF) of coated microchannel was 2.5165×10~(-8) m~2 V~(-1) s~(1) which suited DNA separation. The characteristics of mobility were investigated and a new modified Ogston model was applied. The model was simple and suitable for the results of experiments. The electric field strength versus current curve was obtained. The signal intensity, band broading and resolution were analysed using different sizes of DNA fragment in different concentrations of sieving medium. The optimal concentration was 4%LPA.
The detection section was designed of confocal optical path. Two systems were setup using 488nmAr+ laser and 532nm solid laser respectively. The laser excited the DNA fragments labeled dyes when they electrophoresed to the detection window. F fluorescence signals were collected by photomultiplier (PMT); the exciting and collecting optical system could be 3D adjusted. The design was optimized for maximum signal intensity by simulation and experiments. The size of focal spot in the micro-channel capillary was optimized. Fluorescence signal was enhanced 2～3 times when increasing the diameter of focal spot from 10μm to 50μm (full of the inner wall of capillary), through which the sensitivity and signal-noise ratio (SNR) were also improved. A technique of using refractive matching liquid was presented. The fluorescence signals were boosted up and the signal collection efficiency was also meliorated when using this method. The intensity and distribution of stray light in capillary any system were analysed by simulation and experiments. When the exciting beams illuminated at the center of capillaries, the stray light was strongest and was 2.7025 times of that without capillary. Stray light was compared for capillaries with different inner diameters; the optimum value was 50μm. The stray light of array with different spaces between capillaries was also researched. The results all above were useful for improving the signal collection efficiency, SNR and the mode selection of scanning.
电泳部分主要包括:对微通道内壁进行改性,以控制电渗流和抑制吸附;制备性能良好的筛分介质能够对样品电泳分离;对涂层、灌胶、进样系统以及荧光染料的选择进行优化,选用非交联的线性聚丙烯酰胺(Linear Polyacrylamide,LPA)作为涂层材料和可替换的筛分介质;测量得到的自制涂层管的电渗流淌度为2.5165×10~(-8)m~2V~(-1)s~(-1),对电渗流的控制满足DNA样品分离的要求;研究了电场强度与迁移率的关系,得到电场强度与电泳电流的关系曲线;对于本筛分系统,提出了一种新的Ogston修正模型,从而能够简单、良好地解释实验结果;考察了DNA样品中不同长度的片段在不同浓度筛分介质中,随电场强度条件变化的信号强度、展宽和分辨率的变化情况,优化筛分质浓度为4%LPA。
检测部分设计为共焦光路,建立了分别以488nmAr+激光器和532nm固体激光器为光源的两套系统,由激光器激发电泳到检测窗口的DNA片段(标记有荧光染料),发射的荧光信号由光电倍增管收集检测,激发光路和收集光路三维可调。通过理论分析和实验优化本系统的设计:优化聚焦于微通道中的激发光斑大小,增大聚焦到毛细管内径中的激发光斑尺寸,使光斑尺寸由最小的10μm增大到50μm(充满了整个毛细管内径),光电倍增管接收到的荧光信号提高了2-3倍,从而提高系统的信噪比和灵敏度;提出在聚焦物镜处加折射率匹配液的方法,加匹配液后的荧光信号得到了增强,因而提高了荧光收集效率和灵敏度。通过理论和实验分析阵列毛细管电泳技术中杂散光的强度与分布,激发光束直接照射到毛细管的内径中心时,毛细管产生的杂散光强度是无毛细管的情况下的2.7025倍;对不同内径的微通道毛细管进行比较,优化内径为50μm;分析了不同间距的阵列毛细管杂散光情况;所得结果对提高检测系统荧光收集效率、提高信噪比、采取最佳扫描方式十分有意义。
DNA analysis is the technology to reveal the biological characteristic in molecular and genetic level; it is the most effective and convenient way for research in life sciences. So, the study of DNA analyzer is very important; it is the source of the information. The laser induced fluorescence DNA analysis system was researched and developed. Two detection systems were designed and setup, which were the modern optical detection systems based on microchannel and capillary electrophoresis. They were simple, cost effective, high sensitivity and good performance. Single basepair could be resolved and the limit of detection (LOD) of DNA sample pGEM-3Zf(+)/Hae III Markers was 2.4×10~(-11)mol/L. which meet the requirement of DNA sequencing and short tandem repeat (STR) typing. The system could be divided in two parts: microchannel capillary electrophoresis and laser induced fluorescence detection.
The electrophoresis section included the work as follows. The inner wall of the microchannel was modified to control the electroosmotic flow and restrain adsorption. The good performance sieving gel medium was prepared to separate the samples. The processes of coating, gel injection, sample injection and the flouorescece selection were optimized. The replacebale noncross-link linear polyacrylamide (LPA) was selected to be coating material and sieving medium. The electroosmotic flow mobility (EOF) of coated microchannel was 2.5165×10~(-8) m~2 V~(-1) s~(1) which suited DNA separation. The characteristics of mobility were investigated and a new modified Ogston model was applied. The model was simple and suitable for the results of experiments. The electric field strength versus current curve was obtained. The signal intensity, band broading and resolution were analysed using different sizes of DNA fragment in different concentrations of sieving medium. The optimal concentration was 4%LPA.
The detection section was designed of confocal optical path. Two systems were setup using 488nmAr+ laser and 532nm solid laser respectively. The laser excited the DNA fragments labeled dyes when they electrophoresed to the detection window. F fluorescence signals were collected by photomultiplier (PMT); the exciting and collecting optical system could be 3D adjusted. The design was optimized for maximum signal intensity by simulation and experiments. The size of focal spot in the micro-channel capillary was optimized. Fluorescence signal was enhanced 2～3 times when increasing the diameter of focal spot from 10μm to 50μm (full of the inner wall of capillary), through which the sensitivity and signal-noise ratio (SNR) were also improved. A technique of using refractive matching liquid was presented. The fluorescence signals were boosted up and the signal collection efficiency was also meliorated when using this method. The intensity and distribution of stray light in capillary any system were analysed by simulation and experiments. When the exciting beams illuminated at the center of capillaries, the stray light was strongest and was 2.7025 times of that without capillary. Stray light was compared for capillaries with different inner diameters; the optimum value was 50μm. The stray light of array with different spaces between capillaries was also researched. The results all above were useful for improving the signal collection efficiency, SNR and the mode selection of scanning.
引文
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