微流控芯片荧光检测系统的研制
摘要
微流控芯片技术被用于进行生化分析，与传统的毛细管电泳相比具有高效、快速、试样用量少、结果可靠的特点。它已成功地用于DNA序列测定，蛋白质、单细胞等的分离检测。本课题研制了一种采用488nm的氩离子激光器和FITC荧光染料激光诱导荧光检测系统。
了解系统信噪比特性、进行系统优化设计是把微流控芯片荧光检测系统推向实用化、商品化的关键。为此，我们根据已有资料，分析了影响系统整体性能的因素，并使用有关器件，在光学平台上搭建了简易的透射式和反射式微流控芯片荧光检测系统，包括光学、机械和电路部分的设计和实现。系统通过主物镜收集微流控芯片微通道中的荧光信号，然后被光电倍增管接收并转换成电信号，最后通过电路处理、采样，在计算机上显示出电泳的曲线。实验结果与理论分析基本吻合，进一步明确了激光器、针孔尺寸和显微物镜等因素与系统整体性能(信噪比)的关系，并对仪器的整体结构、器件参数和电路部分提出了改进和具体设计结果。
本论文共分为六章：第一章介绍了微流控芯片的概念、简要发展史、发展趋势、主要应用领域和主要检测方式；第二章简单介绍了毛细管电泳的相关概念和荧光的特性，以及共焦技术提高光学系统性能的理论基础；第三章详细介绍了我们所搭建的透射式和反射式荧光检测实验系统；第四章给出了使用实验系统对样品进行检测的结果以及对结果的分析；第五章从整体结构、主物镜、针孔、滤色片组合和电路等几个方面阐述了现有系统的不足，并提出了相应的改进方法；第六章是对全文的总结和对本课题的展望。
Microfluidic chip, which is also called integrated capillary electrophoresis (ICE), planar, capillary electrophoresis (CE) chip, is used in the biochemical analyses such as DNA sequencing and the separation of the protein or monoplast. As compared to the conventional capillary eletrophoresis, it shows many advantages: less reagent, minitype, high efficiency and sensitivity. We develop a laser induced fluorescence (LIF) detection system for use with the ICE chip, which is based on a 488nm argon ion laser and FITC fluorescent dye.
Optimization of the LIF detection system is the key of commercialization and utilization. Therefore, we analyze the effect of various factors on signal-noise ratio in LIF detection system. Then we set up adjustable mini LIF detection system of transmission style and reflection style, which are consist of optical module, mechanical module and electronic module. In this detection system, the excitation laser beam from the argon ion laser illuminates some point that lies in the separation micro channel. Then the fluorescent signal is emitted when the fluid
that contains FITC dye flows through the point. After that, an objective lens collects the optical signal and transmits it to the Photoelectric Multiplier (PMT). Finally, the signal is sampled and the electropherogram is displayed on the computer screen. The results of the experiments match the theoretical conclusions. Furthermore, they let us know the relationships among the laser, the size of pinhole, the objective and the signal-noise ratio more clearly. So we improve our detection system by changing its structure, the parameter of components and the circuit.
This paper includes six chapters. Chapter one gives a brief introduction of microfluidic chip and its detection methods. Chapter two explains the mutuality theory about the detection of microfluidic chip, including confocal theory. Chapter three introduces each module in our experimentation system, including optical module, photoelectric conversion module, and electronic module. Chapter four is about the results of these experiments. Chapter five shows the shortage of our system and gives the corresponding advices.to improve it thrbugh the aspects such as objective,
pinhole, filters, circuits and so on. Chapter six sums up this paper.
了解系统信噪比特性、进行系统优化设计是把微流控芯片荧光检测系统推向实用化、商品化的关键。为此，我们根据已有资料，分析了影响系统整体性能的因素，并使用有关器件，在光学平台上搭建了简易的透射式和反射式微流控芯片荧光检测系统，包括光学、机械和电路部分的设计和实现。系统通过主物镜收集微流控芯片微通道中的荧光信号，然后被光电倍增管接收并转换成电信号，最后通过电路处理、采样，在计算机上显示出电泳的曲线。实验结果与理论分析基本吻合，进一步明确了激光器、针孔尺寸和显微物镜等因素与系统整体性能(信噪比)的关系，并对仪器的整体结构、器件参数和电路部分提出了改进和具体设计结果。
本论文共分为六章：第一章介绍了微流控芯片的概念、简要发展史、发展趋势、主要应用领域和主要检测方式；第二章简单介绍了毛细管电泳的相关概念和荧光的特性，以及共焦技术提高光学系统性能的理论基础；第三章详细介绍了我们所搭建的透射式和反射式荧光检测实验系统；第四章给出了使用实验系统对样品进行检测的结果以及对结果的分析；第五章从整体结构、主物镜、针孔、滤色片组合和电路等几个方面阐述了现有系统的不足，并提出了相应的改进方法；第六章是对全文的总结和对本课题的展望。
Microfluidic chip, which is also called integrated capillary electrophoresis (ICE), planar, capillary electrophoresis (CE) chip, is used in the biochemical analyses such as DNA sequencing and the separation of the protein or monoplast. As compared to the conventional capillary eletrophoresis, it shows many advantages: less reagent, minitype, high efficiency and sensitivity. We develop a laser induced fluorescence (LIF) detection system for use with the ICE chip, which is based on a 488nm argon ion laser and FITC fluorescent dye.
Optimization of the LIF detection system is the key of commercialization and utilization. Therefore, we analyze the effect of various factors on signal-noise ratio in LIF detection system. Then we set up adjustable mini LIF detection system of transmission style and reflection style, which are consist of optical module, mechanical module and electronic module. In this detection system, the excitation laser beam from the argon ion laser illuminates some point that lies in the separation micro channel. Then the fluorescent signal is emitted when the fluid
that contains FITC dye flows through the point. After that, an objective lens collects the optical signal and transmits it to the Photoelectric Multiplier (PMT). Finally, the signal is sampled and the electropherogram is displayed on the computer screen. The results of the experiments match the theoretical conclusions. Furthermore, they let us know the relationships among the laser, the size of pinhole, the objective and the signal-noise ratio more clearly. So we improve our detection system by changing its structure, the parameter of components and the circuit.
This paper includes six chapters. Chapter one gives a brief introduction of microfluidic chip and its detection methods. Chapter two explains the mutuality theory about the detection of microfluidic chip, including confocal theory. Chapter three introduces each module in our experimentation system, including optical module, photoelectric conversion module, and electronic module. Chapter four is about the results of these experiments. Chapter five shows the shortage of our system and gives the corresponding advices.to improve it thrbugh the aspects such as objective,
pinhole, filters, circuits and so on. Chapter six sums up this paper.
引文
1 王世立，方肇伦。光谱学与光谱分析，2000，20(2)：143～148
2 P C H Li, D J Harrison. Anal Chem., 1997, 69:1564
3 A Manz, Y Miyahara, J Miura et al. Sens. Actuators B, 1990, B1: 244
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8 http://www.calipertech.com/
9 方肇伦。大学化学，2001，16(2)：1
10 方肇伦，方群。现代科学仪器，2001，4：3
11 D J Harrison et al. Science, 1993, 261: 895
12 J M Ramsey et al. Nature Med, 1995, 1: 1093
13 A T Woolley, R A Mathies. Anal. Chem., 1995, 67:3676
14 A T Woolley et al. Anal. Chem., 1996, 68:4081
15 金亚，温涛等。现代科学仪器，2001，4：13
16 R A Mathies, X C Huang. Nature, 1992, 359:167
17 Y Shi, P C Simpson, R A Mathies et al. Anal Chem., 1999, 71:5354
18 Q F Xue, F Foret, B L Karger et al. Anal. Chem., 1997, 69:426
19 R S Ramsey, J M Ramsey. Anal. Chem., 1997, 69:1174
20 D Figeys, R Aebersold. Anal. Chem., 1998, 70:3721
21 N Chiem, D J Harrison. Anal. Chem., 1997, 69:373
22 S D Mangru, D J Harrison. Electrophoresis, 1998, 19:2301
23 A T Woolley, K Q Lao, R A Mathies. Anal. Chem., 1998, 70:684
2 P C H Li, D J Harrison. Anal Chem., 1997, 69:1564
3 A Manz, Y Miyahara, J Miura et al. Sens. Actuators B, 1990, B1: 244
4 M A Roberts, J S Rossier, P Bercier et al. Anal. Chem., 1997, 69:2035
5 A Manz, E Verpoorte, C S Effenhauser et al. Fresenius J. Anal. Chem., 1994, 348: 567
6 S C Jacobson, R Hergenroeder, L B Koutny et al. Anal. Chem., 1994, 66:1114
7 S C Jacobson, R Hergenroeder, A W Moore et al. Anal. Chem., 1994, 66:4127
8 http://www.calipertech.com/
9 方肇伦。大学化学，2001，16(2)：1
10 方肇伦，方群。现代科学仪器，2001，4：3
11 D J Harrison et al. Science, 1993, 261: 895
12 J M Ramsey et al. Nature Med, 1995, 1: 1093
13 A T Woolley, R A Mathies. Anal. Chem., 1995, 67:3676
14 A T Woolley et al. Anal. Chem., 1996, 68:4081
15 金亚，温涛等。现代科学仪器，2001，4：13
16 R A Mathies, X C Huang. Nature, 1992, 359:167
17 Y Shi, P C Simpson, R A Mathies et al. Anal Chem., 1999, 71:5354
18 Q F Xue, F Foret, B L Karger et al. Anal. Chem., 1997, 69:426
19 R S Ramsey, J M Ramsey. Anal. Chem., 1997, 69:1174
20 D Figeys, R Aebersold. Anal. Chem., 1998, 70:3721
21 N Chiem, D J Harrison. Anal. Chem., 1997, 69:373
22 S D Mangru, D J Harrison. Electrophoresis, 1998, 19:2301
23 A T Woolley, K Q Lao, R A Mathies. Anal. Chem., 1998, 70:684