双波长共聚焦生物芯片扫描仪的研究与开发
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摘要
生物芯片技术是近十年来影响最深远的重大科技进展之一，具有重大战略意义，又蕴藏着无限商机。为避免因没有自己的专利技术而受制于人的被动局面，提高生物芯片技术在我国的研究和应用水平，研究开发具有自主知识产权的生物芯片扫描仪，具有非常重要的意义。
本课题是国家自然科学基金的资助项目(30227002)，并同深圳益生堂生物企业有限公司进行相关合作，从项目开展以来，已取得阶段性的成果，达到了生物芯片扫描仪的样机生产水平，并有望在近期投入临床应用。
论文首先对生物芯片技术作了总体介绍，比较分析了当前国内外研究中的各检测系统的特点，提出了一种新颖的基于共聚焦扫描技术和光机二维扫描技术的双波长生物芯片扫描仪，并对其扫描原理作了分析和阐述。重点研究了根据扫描方案设计的实验系统电路原理及功能实现，实现了生物芯片的扫描检测、数据采集和图像重建等功能，并在此基础上着重研究了实验系统的优化，实现了为改善扫描性能，对系统f-θ物镜非线性扫描误差的校正及补偿等技术。完成了双波长共聚焦生物芯片扫描仪样机的设计、调试和性能测试，实现了系统的高速扫描、全自动的数据分析，532nm绿光扫描时，基本达到了5μm的扫描分辨率，1个荧光分子／μm~2(cy3)的灵敏度等性能指标，论文最后提出了研究中存在的问题及对研究工作的总结和展望。
Biochip technique is one of most important development of science and technology since the last ten years, which has momentous strategic meaning and immense commercial opportunities. To avoid the passive status resulted from having no technical monopoly, improve the level of research and application of biochip technique in our country, research and development of the biochip scanner that holding the intellectual copyright has great significance.
This project (30227002) is supported by NSFC (the national natural science foundation of China), and cooperated with Shenzhen Yishengtang Biological Products Co. Ltd. Then the phased fruits have been achieved since the project was unfolded, and approached to the level of producing the model machine of biochip scanner. It will be used to clinical application in the coming days.
In this paper, biochip technique is firstly introduced as a whole and kinds of detective systems' characters in domestic and international research are compared and analyzed respectively. Thus a novel type of double-wavelength biochip scanner is put forward, which based on confocal-scanning technique and optical-mechanical two-dimensional scanning technique. And its scanning principle is also discussed and analyzed. Secondly, the experimental system-circuits are designed according to the scanning conceptual design, the circuitous philosophy and implementation of its function are focused emphatically. And scanning biochip, data acquisition and imaging are implemented by the system-circuits in experiments. On the base of the experiments, the system' s optimization is studied emphatically, and the techniques are achieved, which can be used to improve the scanning performance such as nonlinear scanning error' s calibration of f-theta lens and so on. The model machine' s design, debug and performance testing are
finished, moreover the high speed scanning and data automated analysis are realized, and the performance index of 5Mm resolution and 1fluo/um2 (Cy3) etc are achieved basically when scanning by 532nm green laser. At last, the open questions are brought forward, and research are summarized and expected in this paper.
本课题是国家自然科学基金的资助项目(30227002)，并同深圳益生堂生物企业有限公司进行相关合作，从项目开展以来，已取得阶段性的成果，达到了生物芯片扫描仪的样机生产水平，并有望在近期投入临床应用。
论文首先对生物芯片技术作了总体介绍，比较分析了当前国内外研究中的各检测系统的特点，提出了一种新颖的基于共聚焦扫描技术和光机二维扫描技术的双波长生物芯片扫描仪，并对其扫描原理作了分析和阐述。重点研究了根据扫描方案设计的实验系统电路原理及功能实现，实现了生物芯片的扫描检测、数据采集和图像重建等功能，并在此基础上着重研究了实验系统的优化，实现了为改善扫描性能，对系统f-θ物镜非线性扫描误差的校正及补偿等技术。完成了双波长共聚焦生物芯片扫描仪样机的设计、调试和性能测试，实现了系统的高速扫描、全自动的数据分析，532nm绿光扫描时，基本达到了5μm的扫描分辨率，1个荧光分子／μm~2(cy3)的灵敏度等性能指标，论文最后提出了研究中存在的问题及对研究工作的总结和展望。
Biochip technique is one of most important development of science and technology since the last ten years, which has momentous strategic meaning and immense commercial opportunities. To avoid the passive status resulted from having no technical monopoly, improve the level of research and application of biochip technique in our country, research and development of the biochip scanner that holding the intellectual copyright has great significance.
This project (30227002) is supported by NSFC (the national natural science foundation of China), and cooperated with Shenzhen Yishengtang Biological Products Co. Ltd. Then the phased fruits have been achieved since the project was unfolded, and approached to the level of producing the model machine of biochip scanner. It will be used to clinical application in the coming days.
In this paper, biochip technique is firstly introduced as a whole and kinds of detective systems' characters in domestic and international research are compared and analyzed respectively. Thus a novel type of double-wavelength biochip scanner is put forward, which based on confocal-scanning technique and optical-mechanical two-dimensional scanning technique. And its scanning principle is also discussed and analyzed. Secondly, the experimental system-circuits are designed according to the scanning conceptual design, the circuitous philosophy and implementation of its function are focused emphatically. And scanning biochip, data acquisition and imaging are implemented by the system-circuits in experiments. On the base of the experiments, the system' s optimization is studied emphatically, and the techniques are achieved, which can be used to improve the scanning performance such as nonlinear scanning error' s calibration of f-theta lens and so on. The model machine' s design, debug and performance testing are
finished, moreover the high speed scanning and data automated analysis are realized, and the performance index of 5Mm resolution and 1fluo/um2 (Cy3) etc are achieved basically when scanning by 532nm green laser. At last, the open questions are brought forward, and research are summarized and expected in this paper.
引文
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[3] J. Xu, X. He, Y. Zhou, et al. Research and applications of biochip technologies. Chinese Sci. Bull., 2000, 45: 101～107.
[4] E. S. Lander. Array of hope. Nat. Genet., 1999, 21(1 Suppl): 3～4.
[5] N. Dhiman, R. Bonilla, D. J. O'Kane, et al. Gene expression microarrays: a 21st century tool for directed vaccine design. Vaccine, 2001, 20: 22～30.
[6] 徐炳森，项黎新，邵健忠．生物芯片及其研究进展[J]，浙江大学学报(理学版)，2001，28(4)：443～449．
[7] 尚春庆，胡耀铭，邓春晖，胡克季．生物芯片技术应用进展[J]，中国基础科学，2001，(5)：20～25．
[8] 杨军，李卓荣，曾志雄等．生物芯片技术的现状和发展前景[J]，生命科学仪器，2003，1(1)：19～22．
[9] 张天浩，张春平，张光寅．DNA芯片制作原理及其杂交信号检测方法[J]，生物工程进展，2000，20(2)：64～68．
[10] 许俊泉，贺学忠，周玉祥，刘理天，程京．生物芯片技术的发展与应用[J]，科学通报，1999，44(24)：2600～2606．
[11] 马立人，蒋中华．生物芯片(第二版)．北京：化学工业出版社，2002，p170～210．
[12] S. K. Moore. Making chips to probe genes. IEEE Spectrum, 2001, 38: 54～60.
[13] A. C. Pease, D. solas, E. J. Sullivan, et al. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. USA, 1994, 91: 5022～5026.
[14] G. McGall. Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists. Proc. Natl. Acad. Sci. USA, 1996, 93: 13555～13560.
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[16] M. Schena, R. A. Heller, T. P. Theriault, et al. Microarrays: biotechnology's discovery platform for functional genomics. Trends Biotechnol., 1998, 16: 301～306.
[17] M. Schena, D. Shalon, R. W. Davis, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995, 270: 467～470.
[18] M. Schena, D. Shalon, R. Heller, et al. Parrel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. USA, 1996, 93: 10614～10619.
[19] D. Shalon, S. J. Smith and P. O. Brown. A DNA microarray system for
analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res., 1996, 6: 639～645.
[20] T. M. Brennan. Method and apparatus for conducting an array of chemical reactions on a support surface. US patent, 1995, #5, 474, 796.
[21] G. Van den Engh and C. Farmer. Photo-Bleaching and Photon Saturation in Flow Cytometry. Cytometry, 1992, 13: 669～677.
[22] R. A. Mathies and K. Peck. Optimization of high-sensitivity fluorescence detection. Anal. Chem., 1990, 62: 1786～1791.
[23] S. Pickett. Understanding and evaluating fluorescent microarray imaging instruments. IVD technology, 2003.
[24] 陆祖宏．生物芯片的研究、发展和应用[J]，电子科技导报，1998，(11)：5～9．
[25] 石岩，倪旭翔，陆祖康．生物芯片测试方法进展[J]，光学仪器，2003，25(3)：52～55．
[26] 王立强，陆祖康，林斌．生物芯片及其荧光信号检测[J]．光学仪器，2002，24(4-5)：7～13．
[27] D. J. Rachlin. Optimized approach for microarray scanning. SPIE, 2002, 4623: 13～26.
[28] 齐华，程光伟．基于荧光标记的生物芯片信号检测技术[J]，现代电子技术，2003，5：41～46．
[29] 马平，唐小萍，罗正全．生物芯片扫描仪弱信号检测方法[J]，微纳电子技术，2002，(7)：35～38．
[30] Wenkui Wang, Min Liu, Qiquan Hu. A novel microarray scanner[J], SPIE, 2002, 4916: 339～341.
[31] J. Montagu, P. Honkanen. Fluorescence ArrayScanner[J], SPIE, 1999, 3779: 284～292.
[32] K. Mossberg and M. Ericsson, Detection of doubly stained fluorescent specimens using confocal microscopy. J. Microsc., 1990, 158: 215～224.
[33] K. Carlsson, N. slund, K. Mossberg, et al. Simultaneous confocal recording of multiple fluorescent labels with improved channel separation. J. Microsc., 1994, 176: 287～299.
[34] MINSKY M. Memoir on inventing me confocal scanning microscope[J]. Scanning, 1988, 128～138.
[35] Davidovits P, Egger M D. Scanning laser microscope for biological investigations[J]. APPl. Opt, 1971, 10(7): 1615～1619
[36] Sheppard C J R, Choudhury A. Image formation in the scanning microscope[J]. Optia Acta, 1977, 24(10): 1051～1073.
[37] Wilson T, Sheppard C J R. Theory ond Practice of Scanning Optical Microscopy[M], London: Academic Press, 1984, 1～9.
[38] C. J. R. Sheppard. Stray light and noise in confocal microscopy. Micron Microsc. Acta, 1991, 22: 239～243.
[39] C. J. R. Sheppard, C. J. Cogswell and M. Gu. Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture. Scanning, 1991, 13: 233～240.
[40] J. B. Pawley. Sources of noise in three-dimensional microscopical data
sets. Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, Academic Press, San Diego, 1994, p47～94.
[41] 上海博联生物信息有限公司．ScanArray常见问题．
[42] 张建民．机电一体化系统设计[M]，北京：北京理工大学出版社，1996：15～20．
[43] 李仁定．电机的微机控制[M]，北京：机械工业出版社，1999：55～71．
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