基于微全分析系统PCR基因芯片的光谱检测技术研究
|
摘要
微型全分析系统(Miniaturized Total Analysis System,μTAS)又称芯片实验室(lab on a chip, LOC)是将生化分析的许多过程与步骤,即生化分析实验室的“功能集成结构缩微”在100毫米见方左右(或更小)的芯片上,具有检测速度快、试样用量少、通量高等显著的特点。目前常见的生物芯片分为两大类:微阵列芯片与微流控芯片。
检测系统是芯片的关键之一。光谱检测由于其具有良好的选择性、较宽的线性范围、微量定性定量分析和非破坏性检测等特点,已逐渐成为该领域研究中应用最广泛,灵敏度最高的检测技术之一。因此符合“功能集成和结构缩微”理念的光谱检测系统研究,也已成为近年来世界各国研究并急求解决的热点课题。从目前的国内外相关研究发展现状看,都处在初级探索研究阶段。
本论文针对在上述两大类生物芯片中具有代表性的,并基于聚合酶链(PCR)反应技术基因芯片的光谱检测技术,展开了相关研究,为进一步的“功能集成和结构缩微”研究建立了基础。
针对微流控PCR基因芯片的荧光光谱检测技术,展开了下列研究:建立了一套微流控微通道动态检测系统,其意义在于:在生物微流控PCR荧光芯片使用时,对生物分析结果进行检测;在光谱检测微型化与集成化研究中,提供实验基础。同时在制备芯片时,为了优化最佳制作工艺参数,研究了激光制备微流控芯片工艺研究的生物PCR荧光分析方法。为了保证该荧光分析方法在芯片生产工艺和质量检验的量值统一,研究了生物PCR荧光分析标准比对模板。为了使微流控荧光PCR扩增循环的实际流速与理论设计流速吻合,保证芯片分析检验全过程的准确完成,研究了荧光测控流速系统。“荧光实时测控速系统”的实时测控速功能,是目前国内外有关文献中描述的几种微流体流速测量方法做不到的。
针对微阵列纳米探针PCR基因芯片的可见光度法光谱检测技术,展开了下列研究:
根据微阵列纳米探针PCR基因芯片光谱检测特点,研究了提高生物芯片上纳米探针“双光放大法”沉淀物的可见光度法光谱检测分辨率的工艺,获得检测高分辨率和研发使用的系统方案。使用半导体发光材料和光学导光系统以及包括准分子激光在内的激光微细处理加工技术,设计研发了薄板高均匀性散射平面光源系统。
Miniaturized Total Analysis System,μTAS (or lab on a chip , LOC) is the process and step of biochemistry analyses, the biochemistry analyses laboratory of the " integrated function micro structure " on 100 millimeters of square retinue (or smaller) chip, having notable characteristics :detecting speed quickly, sample dosages small, exchanging amounts advanced. Common bio-chip is divided into two categories: micro-array chip and micro-fluidic chip.
Detecting system is one of the key that the chip system studies. In a lot of detecting method, spectrum detecting as nicer selectivity, broader linearity range, the micro quantitative analysis and no destroyed detecting, becomes one of maximal detecting of sensitiveness technology in the detecting field. So with "functional integration and structural microfilm" the idea of spectral detection system has become to study and solve the hot topic in recent years in the world. Judging from the current domestic and foreign research and development related to the status quo, are at the initial stage of exploration and research.
This paper for the above two categories of chip representative, and based on polymerase chain (PCR) reaction of the spectrum detection technology of gene chip, launched a related study, to further the "functional and structural integration of microfilm," study the establishment of a foundation.
For micro-PCR gene-a-chip fluorescence spectrometry detection technology, launched on the following:
We have set up a micro-flow control micro-channel dynamic detection system, its significance lies: in micro-biological fluorescence-a-chip using PCR, the analysis of the results of biological testing in the spectrum of micro-detection and integrated research, provide experimental basis. At the same time to prepare the chip, in order to optimize the best production process parameters, a study of laser-on-a-chip technology of biological fluorescence PCR analysis. In order to ensure the reunification of the fluorescence analysis in chip production technology and quality inspection, we study the standard template of the biological fluorescence PCR analysis. In order to make micro-controlled fluorescent PCR amplification cycle of actual flow velocity anastomosis with the theoretical design to ensure that the entire process of chip testing of the exact completion of the study fluorescence flow monitoring and control system. "Fluorescent real-time monitoring and control speed system”is the first described in the literature of several micro-fluid flow measurement methods at home and abroad.
For micro-array gene chip PCR method visible spectrum of detection technologies, launched on the following:
According to nano-probe micro-array PCR gene chip testing spectrum characteristics, carried the research on nano-bio-chip probe the "double optical amplification method" of the sediments and enhanced resolving power of spectral detection method, get resolving power and System solutions. LED semiconductor materials and the use of optical-guided excimer laser system and including micro-processing, laser processing technology, design research and development of the high uniformity of thin flat light scattering system.
检测系统是芯片的关键之一。光谱检测由于其具有良好的选择性、较宽的线性范围、微量定性定量分析和非破坏性检测等特点,已逐渐成为该领域研究中应用最广泛,灵敏度最高的检测技术之一。因此符合“功能集成和结构缩微”理念的光谱检测系统研究,也已成为近年来世界各国研究并急求解决的热点课题。从目前的国内外相关研究发展现状看,都处在初级探索研究阶段。
本论文针对在上述两大类生物芯片中具有代表性的,并基于聚合酶链(PCR)反应技术基因芯片的光谱检测技术,展开了相关研究,为进一步的“功能集成和结构缩微”研究建立了基础。
针对微流控PCR基因芯片的荧光光谱检测技术,展开了下列研究:建立了一套微流控微通道动态检测系统,其意义在于:在生物微流控PCR荧光芯片使用时,对生物分析结果进行检测;在光谱检测微型化与集成化研究中,提供实验基础。同时在制备芯片时,为了优化最佳制作工艺参数,研究了激光制备微流控芯片工艺研究的生物PCR荧光分析方法。为了保证该荧光分析方法在芯片生产工艺和质量检验的量值统一,研究了生物PCR荧光分析标准比对模板。为了使微流控荧光PCR扩增循环的实际流速与理论设计流速吻合,保证芯片分析检验全过程的准确完成,研究了荧光测控流速系统。“荧光实时测控速系统”的实时测控速功能,是目前国内外有关文献中描述的几种微流体流速测量方法做不到的。
针对微阵列纳米探针PCR基因芯片的可见光度法光谱检测技术,展开了下列研究:
根据微阵列纳米探针PCR基因芯片光谱检测特点,研究了提高生物芯片上纳米探针“双光放大法”沉淀物的可见光度法光谱检测分辨率的工艺,获得检测高分辨率和研发使用的系统方案。使用半导体发光材料和光学导光系统以及包括准分子激光在内的激光微细处理加工技术,设计研发了薄板高均匀性散射平面光源系统。
Miniaturized Total Analysis System,μTAS (or lab on a chip , LOC) is the process and step of biochemistry analyses, the biochemistry analyses laboratory of the " integrated function micro structure " on 100 millimeters of square retinue (or smaller) chip, having notable characteristics :detecting speed quickly, sample dosages small, exchanging amounts advanced. Common bio-chip is divided into two categories: micro-array chip and micro-fluidic chip.
Detecting system is one of the key that the chip system studies. In a lot of detecting method, spectrum detecting as nicer selectivity, broader linearity range, the micro quantitative analysis and no destroyed detecting, becomes one of maximal detecting of sensitiveness technology in the detecting field. So with "functional integration and structural microfilm" the idea of spectral detection system has become to study and solve the hot topic in recent years in the world. Judging from the current domestic and foreign research and development related to the status quo, are at the initial stage of exploration and research.
This paper for the above two categories of chip representative, and based on polymerase chain (PCR) reaction of the spectrum detection technology of gene chip, launched a related study, to further the "functional and structural integration of microfilm," study the establishment of a foundation.
For micro-PCR gene-a-chip fluorescence spectrometry detection technology, launched on the following:
We have set up a micro-flow control micro-channel dynamic detection system, its significance lies: in micro-biological fluorescence-a-chip using PCR, the analysis of the results of biological testing in the spectrum of micro-detection and integrated research, provide experimental basis. At the same time to prepare the chip, in order to optimize the best production process parameters, a study of laser-on-a-chip technology of biological fluorescence PCR analysis. In order to ensure the reunification of the fluorescence analysis in chip production technology and quality inspection, we study the standard template of the biological fluorescence PCR analysis. In order to make micro-controlled fluorescent PCR amplification cycle of actual flow velocity anastomosis with the theoretical design to ensure that the entire process of chip testing of the exact completion of the study fluorescence flow monitoring and control system. "Fluorescent real-time monitoring and control speed system”is the first described in the literature of several micro-fluid flow measurement methods at home and abroad.
For micro-array gene chip PCR method visible spectrum of detection technologies, launched on the following:
According to nano-probe micro-array PCR gene chip testing spectrum characteristics, carried the research on nano-bio-chip probe the "double optical amplification method" of the sediments and enhanced resolving power of spectral detection method, get resolving power and System solutions. LED semiconductor materials and the use of optical-guided excimer laser system and including micro-processing, laser processing technology, design research and development of the high uniformity of thin flat light scattering system.
引文
[1] 方肇伦. 微流控分析芯片的制作及应用[M].北京:化学工业出版社,2005:1-3.
[2] 范树国.生物芯片在医学上的应用,生物芯片在医学和食品安检中的应用大会.2008 年 4 月:228
[3] Manz A, Graber N, Widmer H M. Miniaturized total chemical analysis system: a novel concept for chemical sensing. Sensors and Actuators B, 1990, 1:224-248
[4] Manz A, Harrison D J, Verpoorte E et al. Planar Chip Technology of Separation System: A Developing Perspective in Chemical Monitoring. Advances in Chromatography, 1993, 33: 1-66
[5] Harrison D.J., Fluri K., Seiler K., Fan Z.,Effenhauser C.S., Manz A., Science, 1993, 261:895
[6] Ramsey J.M.,Jacobson S.C. ,Knapp M.R. ,Nature Med., 1995 ,1: 1093
[7] 陈忠斌.生物芯片技术[M]. 北京:化学工业出版社,2005.5.
[8] Woolley,A.T., Mathies, R.A. ,Anal. Chem., 1995, 67:3676
[9] Woolley,A.T., Hadley D., Landre P., de Mello A.J., Mathies, R.A., Northrup M.A., Anal. Chem., 1996, 68:4081
[10] Kopp M.U. ,de Mello A.J., Manz A., Science, 1998,280:1046
[11] Woolley AT, Landre P. Analytical Chemistry [J].1996, 68:4081-4086
[12] 朱睿,肖松山,范世福.微流控芯片检测技术进展.纳米技术与精密工程[J],2005.3.
[13] 梁 创 , 廖 静 , 梁 冰 , 等 . 硅 雪 崩 光 电 二 级 管 单 光 子 探 测 器 [J]. 光 子 学报,2002,29(12):1142-1147.
[14] Holgate C S. J. Histochem. Cytochem, 1978, 26: 1074—1081
[15] Mirkin C A, Letsinger R L , Mucic P C, et al. Nature , 1996 ,382: 607 —609
[16] 张阳德(Zhang YD) . 纳米生物材料(Nano biology Materials) .北京: 化学工业出版社(Beijing: Chemical Industry Press) ,2005. 61—62
[17] 姚李英,陈涛,王升启,左铁钏,高聚物基PCR微流控芯片技术,中国生物工程杂志,(3),2004,90-97
[18] 姚李英,刘保安,陈涛等,采用KrF准分子激光制备聚合酶链式反应微流控芯片,中国激光,32(8),2005,1137-1142
[19] 吴坚, 曹文祺 聚合酶链式反应(PCR) 荧光检测研究. 计量学报 2002年4月 第23卷第2期
[20] 马立人,蒋中华,生物芯片,化学工业出版社,2000,2
[21] 姚李英,张瑜,刘保安等,PMMA基于微流控芯片的准分子激光制备方法研究, 高等学校化学学报, 25(Supple),2004,37-38
[22] Schrum K F, Lancaster J M, Johnston S E, et al. Anal Chem, 2000, 72: 4317
[23] Pittman J L, Henry C S, Gilman S D. Anal Chem, 2003, 75: 361
[24] Paul P H, Garguilo M G, Rakestraw D J. Anal Chem, 1998, 70: 2459
[25] Ross D, Johnson T J, Locascio L E. Anal Chem, 2001, 73: 2509
[26] StClaire J C, Hayes M A. Anal Chem, 2000, 72: 4726
[27] Santiago J G, Wereley S T, Meinhart C D, et al. Exp Fluids, 1998, 25: 316
[28] Meinhart C D, Wereley ST, Santiago J G. Exp Fluids, 1999, 27: 414
[29]由长福, 祁海鹰,徐旭常。流体力学实验与测量,2003,17:84
[30] Singh A K, Cummings E B, Throckmorton D J. Anal Chem, 2001, 73: 1057
[31]Gosch M, Blom H, Holm J, et al. Anal Chem, 2000, 72: 3260
[32] Edel J B, Hill E K, de Mello A J. Analyst, 2001, 126: 1953
[33] Wu S, Lin Q, Yuen Y, et al. Sens Actuators A, 2001, 89: 152
[34] 林炳承.毛细管电泳导论.科学出版社,1996年第一版:78-80
[35] Espinoza LA, Smulson ME. Macroarray analysis of the effects of JP-8 jet fuel on gene expression in Jurkat cells [J]. Toxicology, 2003,189(3): 181-190.
[36] Gabriel MN, Calloway CD, Reynolds RL, et al. Identification of human remains by immobilized sequence-specific oligonucleotide probe analysis-is of mtDNA hyper variable regions I and II [J7 .Croat Med J,2003,44(3):293-298.
[37] Enserink M. Biodefense hampered inadequate tests [J]. Science, 2001, 294(5545):1266-1267.
[38] Park SJ, Taton TA, Mirkin CA. Array-based electrical detection of DNA with Nan particle probes [J].Science, 2002,295 (5559):1503-1506.
[39] Service RF.Analytical chemistry. New test could speed bio-weapon detection [J]. Science, 2002, 295 (5559):1447.
[40] 逢键涛,文思远,王升启. 金纳米颗粒聚集以及金纳米探针一微阵列技术研究进展. 分析化学 (FENXI HUAXUE) 评述与进展[J]. 2006年第34卷第6期 884—888
[2] 范树国.生物芯片在医学上的应用,生物芯片在医学和食品安检中的应用大会.2008 年 4 月:228
[3] Manz A, Graber N, Widmer H M. Miniaturized total chemical analysis system: a novel concept for chemical sensing. Sensors and Actuators B, 1990, 1:224-248
[4] Manz A, Harrison D J, Verpoorte E et al. Planar Chip Technology of Separation System: A Developing Perspective in Chemical Monitoring. Advances in Chromatography, 1993, 33: 1-66
[5] Harrison D.J., Fluri K., Seiler K., Fan Z.,Effenhauser C.S., Manz A., Science, 1993, 261:895
[6] Ramsey J.M.,Jacobson S.C. ,Knapp M.R. ,Nature Med., 1995 ,1: 1093
[7] 陈忠斌.生物芯片技术[M]. 北京:化学工业出版社,2005.5.
[8] Woolley,A.T., Mathies, R.A. ,Anal. Chem., 1995, 67:3676
[9] Woolley,A.T., Hadley D., Landre P., de Mello A.J., Mathies, R.A., Northrup M.A., Anal. Chem., 1996, 68:4081
[10] Kopp M.U. ,de Mello A.J., Manz A., Science, 1998,280:1046
[11] Woolley AT, Landre P. Analytical Chemistry [J].1996, 68:4081-4086
[12] 朱睿,肖松山,范世福.微流控芯片检测技术进展.纳米技术与精密工程[J],2005.3.
[13] 梁 创 , 廖 静 , 梁 冰 , 等 . 硅 雪 崩 光 电 二 级 管 单 光 子 探 测 器 [J]. 光 子 学报,2002,29(12):1142-1147.
[14] Holgate C S. J. Histochem. Cytochem, 1978, 26: 1074—1081
[15] Mirkin C A, Letsinger R L , Mucic P C, et al. Nature , 1996 ,382: 607 —609
[16] 张阳德(Zhang YD) . 纳米生物材料(Nano biology Materials) .北京: 化学工业出版社(Beijing: Chemical Industry Press) ,2005. 61—62
[17] 姚李英,陈涛,王升启,左铁钏,高聚物基PCR微流控芯片技术,中国生物工程杂志,(3),2004,90-97
[18] 姚李英,刘保安,陈涛等,采用KrF准分子激光制备聚合酶链式反应微流控芯片,中国激光,32(8),2005,1137-1142
[19] 吴坚, 曹文祺 聚合酶链式反应(PCR) 荧光检测研究. 计量学报 2002年4月 第23卷第2期
[20] 马立人,蒋中华,生物芯片,化学工业出版社,2000,2
[21] 姚李英,张瑜,刘保安等,PMMA基于微流控芯片的准分子激光制备方法研究, 高等学校化学学报, 25(Supple),2004,37-38
[22] Schrum K F, Lancaster J M, Johnston S E, et al. Anal Chem, 2000, 72: 4317
[23] Pittman J L, Henry C S, Gilman S D. Anal Chem, 2003, 75: 361
[24] Paul P H, Garguilo M G, Rakestraw D J. Anal Chem, 1998, 70: 2459
[25] Ross D, Johnson T J, Locascio L E. Anal Chem, 2001, 73: 2509
[26] StClaire J C, Hayes M A. Anal Chem, 2000, 72: 4726
[27] Santiago J G, Wereley S T, Meinhart C D, et al. Exp Fluids, 1998, 25: 316
[28] Meinhart C D, Wereley ST, Santiago J G. Exp Fluids, 1999, 27: 414
[29]由长福, 祁海鹰,徐旭常。流体力学实验与测量,2003,17:84
[30] Singh A K, Cummings E B, Throckmorton D J. Anal Chem, 2001, 73: 1057
[31]Gosch M, Blom H, Holm J, et al. Anal Chem, 2000, 72: 3260
[32] Edel J B, Hill E K, de Mello A J. Analyst, 2001, 126: 1953
[33] Wu S, Lin Q, Yuen Y, et al. Sens Actuators A, 2001, 89: 152
[34] 林炳承.毛细管电泳导论.科学出版社,1996年第一版:78-80
[35] Espinoza LA, Smulson ME. Macroarray analysis of the effects of JP-8 jet fuel on gene expression in Jurkat cells [J]. Toxicology, 2003,189(3): 181-190.
[36] Gabriel MN, Calloway CD, Reynolds RL, et al. Identification of human remains by immobilized sequence-specific oligonucleotide probe analysis-is of mtDNA hyper variable regions I and II [J7 .Croat Med J,2003,44(3):293-298.
[37] Enserink M. Biodefense hampered inadequate tests [J]. Science, 2001, 294(5545):1266-1267.
[38] Park SJ, Taton TA, Mirkin CA. Array-based electrical detection of DNA with Nan particle probes [J].Science, 2002,295 (5559):1503-1506.
[39] Service RF.Analytical chemistry. New test could speed bio-weapon detection [J]. Science, 2002, 295 (5559):1447.
[40] 逢键涛,文思远,王升启. 金纳米颗粒聚集以及金纳米探针一微阵列技术研究进展. 分析化学 (FENXI HUAXUE) 评述与进展[J]. 2006年第34卷第6期 884—888