基于微流控芯片的小型流式细胞仪研究
|
摘要
随着微流控芯片加工技术逐渐普及和微流控分析方法研究的不断深入,微型全分析系统(Miniaturized Total Analysis Systems,μTAS)已将分析实验室的部分功能转移到便携化的分析设备中,为化学分析设备的微型化与集成化提供了基础,最终可以实现分析实验室的“个性化”、“家用化”,使分析仪器从化学实验室解放出来,进入千家万户。本研究组在2005年提出了应用于微流控芯片的正交型激光诱导荧光检测光路新模式,具有光路结构简单,易于搭建的特点。该检测模式曾被用于构建微流控芯片流式细胞分析装置,该实验中,在荧光微球前向小角度(6°)方向上检测前向光强度的变化,实现了芯片上荧光微球的计数功能;利用芯片侧壁作为采集面,实现荧光微球荧光的同时采集。在上述工作基础上,本文进一步进行了正交光路激光诱导荧光检测-芯片流式细胞分析仪的小型化研究。该仪器具有仪器结构简单、体积小、试样消耗量少、价格低廉等优点。
第一章,对流式细胞仪原理和基于微流控芯片的流式细胞仪的发展进行了综述。
第二章,基于芯片正交光路检测模式,搭建了一套小型的微流控芯片流式细胞仪。以532nm小型半导体激光器作为激发光源,激光束被透镜聚焦于芯片通道中央,采用光电二极管检测芯片通道内流动细胞的前向散射光信号,采用小型光电倍增管检测荧光信号,整套仪器体积为19cm×15cm×25cm(长度×宽度×高度),具有结构简单、体积小、价格低廉等特点。荧光检测系统对四甲基罗丹明异硫氰酸的检测限为4.4×10~(-8)mol/L,采用6μm荧光微球作为模型样品考察了仪器的分析性能,同时初步实现了细胞样品的分析。
In 2005, a simple laser-induced fluorescence (LIF) detection system based on an orthogonal optical arrangement for microfluidic chips was developed by our group. Later, the orthogonal LIF detection systems were employed in microfluidic flow cytometry. In this work, a compact microfluidic chip-based flow cytometer has been developed based on the orthogonal optical arrangement. The present system has many advantages, including simple structure, small size and low cost.
In the first chapter, the concept of traditional flow cytometry and the development of microfluidic chip based on flow cytometer were reviewed.
In the second chapter, a compact microfluidic chip-based flow cytometer has been developed based on the orthogonal optical arrangement. A 532 nm laser diode was used as the light source. Both photodiode and integrated photomultiplier tube were used as detectors for the forward scattered light and fluorescence, respectively. The size of the flow cytometer was 19 cm×15 cm×25 cm (L×H×W). The detection limit of the system for tetramethylrhodamine isothiocyanate (TRITC) was 4.4×10~(-8) mol/L. The performance of the system was demonstrated in the analysis of fluorescent beads (6μm) and cell samples. The advantages of the present system included simple structure, small size and low cost.
第一章,对流式细胞仪原理和基于微流控芯片的流式细胞仪的发展进行了综述。
第二章,基于芯片正交光路检测模式,搭建了一套小型的微流控芯片流式细胞仪。以532nm小型半导体激光器作为激发光源,激光束被透镜聚焦于芯片通道中央,采用光电二极管检测芯片通道内流动细胞的前向散射光信号,采用小型光电倍增管检测荧光信号,整套仪器体积为19cm×15cm×25cm(长度×宽度×高度),具有结构简单、体积小、价格低廉等特点。荧光检测系统对四甲基罗丹明异硫氰酸的检测限为4.4×10~(-8)mol/L,采用6μm荧光微球作为模型样品考察了仪器的分析性能,同时初步实现了细胞样品的分析。
In 2005, a simple laser-induced fluorescence (LIF) detection system based on an orthogonal optical arrangement for microfluidic chips was developed by our group. Later, the orthogonal LIF detection systems were employed in microfluidic flow cytometry. In this work, a compact microfluidic chip-based flow cytometer has been developed based on the orthogonal optical arrangement. The present system has many advantages, including simple structure, small size and low cost.
In the first chapter, the concept of traditional flow cytometry and the development of microfluidic chip based on flow cytometer were reviewed.
In the second chapter, a compact microfluidic chip-based flow cytometer has been developed based on the orthogonal optical arrangement. A 532 nm laser diode was used as the light source. Both photodiode and integrated photomultiplier tube were used as detectors for the forward scattered light and fluorescence, respectively. The size of the flow cytometer was 19 cm×15 cm×25 cm (L×H×W). The detection limit of the system for tetramethylrhodamine isothiocyanate (TRITC) was 4.4×10~(-8) mol/L. The performance of the system was demonstrated in the analysis of fluorescent beads (6μm) and cell samples. The advantages of the present system included simple structure, small size and low cost.
引文
[1]Gucker F T,O'KonscH B,Pitts J N.J.Am.Chem.Soc.1947,68:2422-243.
[2]Moldavan A.Science.1934,180:188-189.
[3]Mandy F F,Bergeron M,Minkus T.Transfus Sci.1995,16:303-314.
[4]Grogan W M.Guide to flow cytometry methods.New York:Marcel Dekker Inc.1990:1-20.
[5]Wang Z,Ei-Ali J,Engelund M,Gotsaed T,Perch-Nielsen I R,Mogensen K B,Snakenborg D,Kutter J P,WolfA.Lab Chip.2004,4:372-377.
[6]Yang S Y,Hsiung S K,Hung Y C,Chang C M,Liao T L,Lee G B.Meas.Sci.Technol.2006,17:2001-2009.
[7]Sobek D,Senturia S,Gray M.Solid State Sensor and Actuator Workshop,Hilton Head,S C:1994,120.
[8]Larsen U,Blankenstein G,Branebjerg J.Solid State Sensors and Actuators,TRANSDUCERS'97 Chicago.1997,2:1319-1322.
[9]Gawad S,Schild L,Renaud.Lab Chip.2001,1:76-82.
[10]Chun H G,Chung T D,Kim H C.Anal.Chem.2005,77:2490-2495.
[11]Schrum D P,Culbertson C T,Jacobson S C,Ramsey J M.Anal.Chem.1999,71:4173-4177.
[12]McClain M A,Culbertson C T,Jacobson S C,Ramsey J M.Anal.Chem.2001,73:5334-5338.
[13]Agronskaia A,Schins J M,Grooth B G,Greve J.Anal.Chem.1999,71:4684-4689.
[14]Pamme N,Koyama R,Manz A.Lab Chip,2003,3:187-192.
[15]Lin C H,Lee G B.J.Micromech.Microeng.2003,13:447-453.
[16]Lee G B,Lin C H,Chang S C.J.Micromech.Microeng.2005,15:447-454.
[17]Tung Y C,Torisawa Y S,Futaia N,Takayama S.Lab Chip.2007,7:1497-503.
[18]Cheng X h,Irimia D,Dixon M,Sekine K,Demirci U,Zamir L,Tompkins R G,Rodriguez W,Tone M.Lab Chip.2007,7:170-178.
[19]Wang Y N,Kang YJ,Xu D Y,Chon C H,Barnett L,Kalams S A,Lib D,Li D Q.Lab Chip.2008,8:309-315.
[20]Wolff A,Perch-Nielsen I R,Larsen U D,Friis P,Goranovic G,Poulsen C R,Kutter J P,Telleman P.Lab Chip.2003,3:22-27.
[21]Yao B,Luo G A,Feng X,Wang W,Chena L X,Wang Y M.Lab Chip.2004,4:603-607.
[22]Wang M M,Tu E,Raymond D E,Yang J M,Zhang H C,Hagen N,Dees B,Mercer E M,Marchand P J,Butler W F.Nat Biotechnol.2005,23:83-87.
[23]Fu AY,Chou H P,Spence C,Arnold F H,Quake S R.Anal.Chem.2002,74:2451-2457.
[24]Fuhr G.Zimmermann U,Shirley S G.Principles and Potential in Electromanipulation ofCells(eds.)Zimmerman U,Neu GA.Boca Raton:CRC Press.1996:259-328
[25]高健,殷学锋,方肇伦.化学进展.2004,16:975-983.
[26]Pohl H A.Dielectrophoresis.Cambridge:Cambridge University Press.1978
[27]Tai C H,Hsiung S K,Chen C Y,Tsai M L,G B Lee.Biomed.Microdevices.2007,9:533-543.
[28]Wang L,Flanagan LA,Jeon N L,Monukib E,Leeac A P.Lab Chip.2007,7:1114-1120.
[29]Kruger J,Singh K,O'Neill A,Jackson C,Morrison A,P O'Brien.J.Micromech.Microeng.2002,12:486-494.
[30]Singh K,Su X T,Liu C,Capjack C,Rozmus W,Backhouse C J.International Society for Analytical Cytology.2006,69:307-315.
[31]Li Y L,Dalton C,Crabtree H J,Nilsson G,Kaler KV I S.Lab Chip.2007,7:239-248.
[1]Jaroszeski M J,Radcliff G.Mol Biotechnol.1999,11:37-53.
[2]Keating P,Cambrosio A.J Hist Biol.1994,27:449-479.
[3]Manz A,Harrison D J,Verpoorte E M J,Fettinger JC.J.Chromatogr.1992,593:253-260.
[4]Harrison D J,Fluri K,Seiler K,Fan Z,Effenhauser C S,Manz A.Science.1993,261:895-903.
[5]Manz A,Becker H.Microsystem Technology in Chemistry and Life Sciences.New York:Springer-Verlag,1999.
[6]Sobek D,Sentuffa S,Gray M.Solid-State Sensor and Actuator Workshop,Hilton Head,S C.1994,120-121.
[7]Woolley A T,Mathies R A.Anal.Chem.1995,67:3676-3680.
[8]Haab B B,Mathies R A.Anal.Chem.1999,71:5137-5148.
[9]Liang Z H,Chime N,Ocvirk G,Tang T,Fluff K,Harrison D J.Anal.Chem.1999,68:1040-1046.
[10]Chabinyc M L,Chiu D T,McDonald J C,Stroock A D,Christian J F,Karger A M,Whitesides G M.Anal.Chem.2001,73:4491-4410.
[11]Jacobson S C,Ramsey J M.Anal.Chem.1996,68:720-723.
[12]Fister J C,Jacobson S C,Ramsey J M.Anal.Chem.1999,71:4460-4475.
[13]McClain M A,Culbertson C T,Jacobson S C,Ramsey J M.Anal.Chem.2001,73:5334-5338.
[14]Pamme N,Koyama R,Manz A.Lab Chip.2003,3:187-192.
[15]Dittffch P S,Schwille P.Anal.Chem.2003,75:5767-5774.
[16]Fang Qun(方群),Fu Jinglin(富景林),Huang yanzhen(黄艳贞).Chinese Patent(中国发明专利),Nov.No.:ZL03141572.5,2006.
[17]Fu J L,F Q,Z T,Jin X H,Fang Z L.Anal.Chem.2006,78:3827-3834.
[18]Kellar K L,Iannone M A.Experimental Hematology.2002,30:1227-1237.
[19]MeClain M A,Culbertson C T,Jaeobson S C.Anal.Chem.2001,73:5334-5338.
[20]杨蕊,牟颖,邹明强,吴站,金钦汉.高等学校化学学报.2004,25:1816-1819.
[21]姚波,冯雪,罗国安,王义明.高等学校化学学报.2005,26:43-45.
[2]Moldavan A.Science.1934,180:188-189.
[3]Mandy F F,Bergeron M,Minkus T.Transfus Sci.1995,16:303-314.
[4]Grogan W M.Guide to flow cytometry methods.New York:Marcel Dekker Inc.1990:1-20.
[5]Wang Z,Ei-Ali J,Engelund M,Gotsaed T,Perch-Nielsen I R,Mogensen K B,Snakenborg D,Kutter J P,WolfA.Lab Chip.2004,4:372-377.
[6]Yang S Y,Hsiung S K,Hung Y C,Chang C M,Liao T L,Lee G B.Meas.Sci.Technol.2006,17:2001-2009.
[7]Sobek D,Senturia S,Gray M.Solid State Sensor and Actuator Workshop,Hilton Head,S C:1994,120.
[8]Larsen U,Blankenstein G,Branebjerg J.Solid State Sensors and Actuators,TRANSDUCERS'97 Chicago.1997,2:1319-1322.
[9]Gawad S,Schild L,Renaud.Lab Chip.2001,1:76-82.
[10]Chun H G,Chung T D,Kim H C.Anal.Chem.2005,77:2490-2495.
[11]Schrum D P,Culbertson C T,Jacobson S C,Ramsey J M.Anal.Chem.1999,71:4173-4177.
[12]McClain M A,Culbertson C T,Jacobson S C,Ramsey J M.Anal.Chem.2001,73:5334-5338.
[13]Agronskaia A,Schins J M,Grooth B G,Greve J.Anal.Chem.1999,71:4684-4689.
[14]Pamme N,Koyama R,Manz A.Lab Chip,2003,3:187-192.
[15]Lin C H,Lee G B.J.Micromech.Microeng.2003,13:447-453.
[16]Lee G B,Lin C H,Chang S C.J.Micromech.Microeng.2005,15:447-454.
[17]Tung Y C,Torisawa Y S,Futaia N,Takayama S.Lab Chip.2007,7:1497-503.
[18]Cheng X h,Irimia D,Dixon M,Sekine K,Demirci U,Zamir L,Tompkins R G,Rodriguez W,Tone M.Lab Chip.2007,7:170-178.
[19]Wang Y N,Kang YJ,Xu D Y,Chon C H,Barnett L,Kalams S A,Lib D,Li D Q.Lab Chip.2008,8:309-315.
[20]Wolff A,Perch-Nielsen I R,Larsen U D,Friis P,Goranovic G,Poulsen C R,Kutter J P,Telleman P.Lab Chip.2003,3:22-27.
[21]Yao B,Luo G A,Feng X,Wang W,Chena L X,Wang Y M.Lab Chip.2004,4:603-607.
[22]Wang M M,Tu E,Raymond D E,Yang J M,Zhang H C,Hagen N,Dees B,Mercer E M,Marchand P J,Butler W F.Nat Biotechnol.2005,23:83-87.
[23]Fu AY,Chou H P,Spence C,Arnold F H,Quake S R.Anal.Chem.2002,74:2451-2457.
[24]Fuhr G.Zimmermann U,Shirley S G.Principles and Potential in Electromanipulation ofCells(eds.)Zimmerman U,Neu GA.Boca Raton:CRC Press.1996:259-328
[25]高健,殷学锋,方肇伦.化学进展.2004,16:975-983.
[26]Pohl H A.Dielectrophoresis.Cambridge:Cambridge University Press.1978
[27]Tai C H,Hsiung S K,Chen C Y,Tsai M L,G B Lee.Biomed.Microdevices.2007,9:533-543.
[28]Wang L,Flanagan LA,Jeon N L,Monukib E,Leeac A P.Lab Chip.2007,7:1114-1120.
[29]Kruger J,Singh K,O'Neill A,Jackson C,Morrison A,P O'Brien.J.Micromech.Microeng.2002,12:486-494.
[30]Singh K,Su X T,Liu C,Capjack C,Rozmus W,Backhouse C J.International Society for Analytical Cytology.2006,69:307-315.
[31]Li Y L,Dalton C,Crabtree H J,Nilsson G,Kaler KV I S.Lab Chip.2007,7:239-248.
[1]Jaroszeski M J,Radcliff G.Mol Biotechnol.1999,11:37-53.
[2]Keating P,Cambrosio A.J Hist Biol.1994,27:449-479.
[3]Manz A,Harrison D J,Verpoorte E M J,Fettinger JC.J.Chromatogr.1992,593:253-260.
[4]Harrison D J,Fluri K,Seiler K,Fan Z,Effenhauser C S,Manz A.Science.1993,261:895-903.
[5]Manz A,Becker H.Microsystem Technology in Chemistry and Life Sciences.New York:Springer-Verlag,1999.
[6]Sobek D,Sentuffa S,Gray M.Solid-State Sensor and Actuator Workshop,Hilton Head,S C.1994,120-121.
[7]Woolley A T,Mathies R A.Anal.Chem.1995,67:3676-3680.
[8]Haab B B,Mathies R A.Anal.Chem.1999,71:5137-5148.
[9]Liang Z H,Chime N,Ocvirk G,Tang T,Fluff K,Harrison D J.Anal.Chem.1999,68:1040-1046.
[10]Chabinyc M L,Chiu D T,McDonald J C,Stroock A D,Christian J F,Karger A M,Whitesides G M.Anal.Chem.2001,73:4491-4410.
[11]Jacobson S C,Ramsey J M.Anal.Chem.1996,68:720-723.
[12]Fister J C,Jacobson S C,Ramsey J M.Anal.Chem.1999,71:4460-4475.
[13]McClain M A,Culbertson C T,Jacobson S C,Ramsey J M.Anal.Chem.2001,73:5334-5338.
[14]Pamme N,Koyama R,Manz A.Lab Chip.2003,3:187-192.
[15]Dittffch P S,Schwille P.Anal.Chem.2003,75:5767-5774.
[16]Fang Qun(方群),Fu Jinglin(富景林),Huang yanzhen(黄艳贞).Chinese Patent(中国发明专利),Nov.No.:ZL03141572.5,2006.
[17]Fu J L,F Q,Z T,Jin X H,Fang Z L.Anal.Chem.2006,78:3827-3834.
[18]Kellar K L,Iannone M A.Experimental Hematology.2002,30:1227-1237.
[19]MeClain M A,Culbertson C T,Jaeobson S C.Anal.Chem.2001,73:5334-5338.
[20]杨蕊,牟颖,邹明强,吴站,金钦汉.高等学校化学学报.2004,25:1816-1819.
[21]姚波,冯雪,罗国安,王义明.高等学校化学学报.2005,26:43-45.