微流控分析芯片中分光光度法吸收光谱分析的研究
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摘要
微流控芯片是微型全分析系统(μTAS)中当前最为活跃的领域和发展前沿，它集中地体现了将分析实验室的功能转移到芯片上的思想。而微流控芯片上的探测器的总体性能将影响整个芯片分析系统的检出限、检测速度、适用范围、体积等指标，是微流控芯片分析系统的一个关键部分。本课题针对微流控芯片探测过程中需要样品剂量少、与微流体芯片集成等要求和目前微流控芯片中吸收光度检测器存在的缺点，设计了两种在芯片内用分光光度法对混合液体的吸收光谱进行探测的方法。
采用直接吸收探测时：透镜使得液体中的光束为平行光束，样品对光有一定的吸收，比较进入样品前后的光谱来确定样品中某种成分的浓度。这种传统的生化分析方法已经广泛地应用于医院。微流控芯片中探测池通常较小，探测池在传输方向的长度限制最大光程，如果在其他方向的可以制作得足够小的话，所需要的样品量就可以大大减小。基于这种思想，本课题首次采用了微型自聚焦透镜作为探测光输入输出器件，使得在光程不减小的情况下，需要探测的样品量由传统的1～2mL减少到50μL。
采用消逝波间接探测时：利用光纤传感器中消逝波的产生和吸收原理[补1]，制作具有高灵敏度的消逝波光纤传感器，消逝波光纤传感器使得光纤芯与被探测样品直接接触，当可见光在光纤中传播时，光纤芯外的消逝场与吸收样品发生作用，光强会有减弱，通过比较发生作用前后的光谱的变化确定样品的浓度。
利用卤钨灯可见光光源、自行研发的微型光纤光谱仪，利用直接探测法和消逝波间接探测法对生化样品(主要是血清与溴钾酚绿混合液)进行了探测。实验通过探测吸收光谱，比较液体的吸收峰来确定血清中白蛋白的含量。与λ—9双光路光谱仪探测相比较，直接探测法吸光度误差为5.5％；消逝波探测与直接探测法相比，消逝波探测的吸光度灵敏度为直接探测的52.3倍。
结果表明，两种方法均适用于微流控芯片，直接探测法具有原理简单，消耗样品量少，结构容易实现等优点；消逝波探测法具有需要样品量极少、灵敏度高、易于集成等的优点。
Micro-fluidic chip is the most flourish and develop fastest in the field of miniaturized total analysis system( u TAS). It demonstrates the excellent idea that focuses the total analyzing system into a small chip. On the Micro-fluidic chip, the detector is an important part; it will determine the limitation, speed, applying area and volume of the chip. For the need of biochemical chip, which consumes fewer specimens and is easy to integrate with micro-fluid chip, two kinds of spectrophotometric analysis methods, are described in this paper.
The first method is based on schemes of direct absorption in which two gradual refraction index lens are hybrid integrated in the system. Its minimum optical path length limits it length in this direction. However, if we use two gradual refraction index lens which is very small, the length of the cuvette in other two directions can be small enough, base on this idea we can reduce the sample consumption a lot.
The second one is a fiber evanescent sensor. It is obtained by removing a small length of the cladding of a commercial single mode fiber or multimode fiber and replacing by an absorbing medium. The evanescent field in the cladding interacts with the absorption medium surrounding the core and power transmitted by the fiber is attenuated. Comparing the variety of the spectrum before and after absorption, we can affirm the concentration of the sample.
Both direct detection method and evanescent wave detection method are used to detect the spectral absorption of mixture of blood serum and BCG reagent. The light source is visible light(460~800nm). Comparing the results with standard instrument λ -9 photometer, it proved that the spectral analysis in micro-fluidic chips meets the needs of optical detection.
The experimental results proved that direct detection is simple and evident; on the other hands evanescent wave detection method consumes reagent and easy to integrated on micro micro-fluidic chips.
采用直接吸收探测时：透镜使得液体中的光束为平行光束，样品对光有一定的吸收，比较进入样品前后的光谱来确定样品中某种成分的浓度。这种传统的生化分析方法已经广泛地应用于医院。微流控芯片中探测池通常较小，探测池在传输方向的长度限制最大光程，如果在其他方向的可以制作得足够小的话，所需要的样品量就可以大大减小。基于这种思想，本课题首次采用了微型自聚焦透镜作为探测光输入输出器件，使得在光程不减小的情况下，需要探测的样品量由传统的1～2mL减少到50μL。
采用消逝波间接探测时：利用光纤传感器中消逝波的产生和吸收原理[补1]，制作具有高灵敏度的消逝波光纤传感器，消逝波光纤传感器使得光纤芯与被探测样品直接接触，当可见光在光纤中传播时，光纤芯外的消逝场与吸收样品发生作用，光强会有减弱，通过比较发生作用前后的光谱的变化确定样品的浓度。
利用卤钨灯可见光光源、自行研发的微型光纤光谱仪，利用直接探测法和消逝波间接探测法对生化样品(主要是血清与溴钾酚绿混合液)进行了探测。实验通过探测吸收光谱，比较液体的吸收峰来确定血清中白蛋白的含量。与λ—9双光路光谱仪探测相比较，直接探测法吸光度误差为5.5％；消逝波探测与直接探测法相比，消逝波探测的吸光度灵敏度为直接探测的52.3倍。
结果表明，两种方法均适用于微流控芯片，直接探测法具有原理简单，消耗样品量少，结构容易实现等优点；消逝波探测法具有需要样品量极少、灵敏度高、易于集成等的优点。
Micro-fluidic chip is the most flourish and develop fastest in the field of miniaturized total analysis system( u TAS). It demonstrates the excellent idea that focuses the total analyzing system into a small chip. On the Micro-fluidic chip, the detector is an important part; it will determine the limitation, speed, applying area and volume of the chip. For the need of biochemical chip, which consumes fewer specimens and is easy to integrate with micro-fluid chip, two kinds of spectrophotometric analysis methods, are described in this paper.
The first method is based on schemes of direct absorption in which two gradual refraction index lens are hybrid integrated in the system. Its minimum optical path length limits it length in this direction. However, if we use two gradual refraction index lens which is very small, the length of the cuvette in other two directions can be small enough, base on this idea we can reduce the sample consumption a lot.
The second one is a fiber evanescent sensor. It is obtained by removing a small length of the cladding of a commercial single mode fiber or multimode fiber and replacing by an absorbing medium. The evanescent field in the cladding interacts with the absorption medium surrounding the core and power transmitted by the fiber is attenuated. Comparing the variety of the spectrum before and after absorption, we can affirm the concentration of the sample.
Both direct detection method and evanescent wave detection method are used to detect the spectral absorption of mixture of blood serum and BCG reagent. The light source is visible light(460~800nm). Comparing the results with standard instrument λ -9 photometer, it proved that the spectral analysis in micro-fluidic chips meets the needs of optical detection.
The experimental results proved that direct detection is simple and evident; on the other hands evanescent wave detection method consumes reagent and easy to integrated on micro micro-fluidic chips.
引文
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[8] Sergio B Mendes, Lifeng Li. Broad-Band Attenuated Total Reflection Spectroscopy of a Hydrated Protein Film on a single Mode Planar Waveguide. Langmuir, 996.12,12,3374-3376.
[9] F.A.Muhanmmad, G. Stewart, W Jin. Sensitivity enhancement of D-fibre methane gas sensor using high-index overlay. IEE PROCEEDINGS-J, 1993 APRIL,Vol. 140, No.2.
[10] Hong S. Haddock, P.M. Shankar. Fabrication of biconical tapered optical fibers using hydrofluoric acid. Material Science and Engineering. B97(2003): 87-93.
[11] Hong S. Haddock, P.M. Shankar. Evanescent sensing of biomolecules and cells. Sensors and Actuators B88 (2003):67-74.
[12] 鞠挥．用于生化分析的光谱仪微小型化的研究．中国科学院研究生院博士学位论文．
[13] 邓勃，宁永成．仪器分析．清华大学出版社．
[14] 乔亚天．剃度折射率光学．科学出版社，1991．
[15] 郝寅雷．梯度折射率棒透镜制备技术的研究．中国科学院研究生院博士后研究工作报告．
[16] 尹真．电动力学．南京大学出版社．1999．
[17] 郭硕鸿．电动力学．高等教育出版社 1995：132～133．
[18] Gordon L Mitchell, senior member IEEE. Absorption Spectroscopy in Scattering
Samples Using Integrated Optics.
[19] 国分太雄．光波工程．科学出版社．
[20] 李玉权，崔敏，光波导理论与技术．人民邮电出版社，2002，97～99．
[21] (澳) A．W．斯奈德．J．D．洛夫．光波导理论．人民邮电出版社，1991．
[22] G.STEWART, B. CULSHAW Optical waveguide modeling and design for evanescent field chemical sensors. Optical and Quantum Electronics 26 (1994) S249-S259.
[23] Y. Zaatar. D. Zaouk. J. Bechara Fa.brication and characterization of an evanescent wave fiber optic sensor for air pollution control. Materials science& Engineering B74(2000): 296～298.
[24] Oscar Esteban, M Cruz Navarrete. Simple model of compound waveguide structures used as fiber-optic sensors. Optics and Lasers in Engineering 33 (2000): 219～230.
[25] Hong S. Haddock. P.M. shankar. Evanescent sensing of biomolecules and cells. Sensors and Actuators B 88 (2003): 67～74.
[26] F.A. Muhammad. G. Stewart. Sensitivity enhancement of D-fibre methane gas sensor using high-index overlay. IE PROCEEDINGS-J Vol. 140, No.2 April 1993.
[27] 全国临床检验操作规程．东南大学出版社，1990．9．
[28] 赵华兵，鞠挥，李也凡，吴一辉．微型光谱仪的光谱数据分析及计算机实现．光学精密工程 2003 VOL．11 NO．4
[29] 王庆有．CCD应用技术．天津大学出版社，2000．
[30] Hong S. Haddock. P. M. Shankar. Evanescent sensing of biomolecules and cells. Sensors and Actuators.
[2] Ruzicka J, Hansen E J. Anal. Chim. Acta. 1975,78:145
[3] Fang Z-L. Flow Injection Separation and Preconcentration. VCH Publishers, Weinheim, 1993
[4] Woolley A T, Mathies R A. Anal. Chem. 1995, 67:3676
[5] Woolley A T, Hadley D, Landre P, demello A J, Mathies R A, Northrup M A. Anal. Chem. 1996, 68:4081
[6] 方肇伦．微流控分析芯片．科学出版社，2003．
[7] E. Verpoorte, A. Manz. A silicon flow cell for optical detection in miniaturized total chemical analysis systems. Sensors and Actuators B, 6(1992) 66-70.
[8] Sergio B Mendes, Lifeng Li. Broad-Band Attenuated Total Reflection Spectroscopy of a Hydrated Protein Film on a single Mode Planar Waveguide. Langmuir, 996.12,12,3374-3376.
[9] F.A.Muhanmmad, G. Stewart, W Jin. Sensitivity enhancement of D-fibre methane gas sensor using high-index overlay. IEE PROCEEDINGS-J, 1993 APRIL,Vol. 140, No.2.
[10] Hong S. Haddock, P.M. Shankar. Fabrication of biconical tapered optical fibers using hydrofluoric acid. Material Science and Engineering. B97(2003): 87-93.
[11] Hong S. Haddock, P.M. Shankar. Evanescent sensing of biomolecules and cells. Sensors and Actuators B88 (2003):67-74.
[12] 鞠挥．用于生化分析的光谱仪微小型化的研究．中国科学院研究生院博士学位论文．
[13] 邓勃，宁永成．仪器分析．清华大学出版社．
[14] 乔亚天．剃度折射率光学．科学出版社，1991．
[15] 郝寅雷．梯度折射率棒透镜制备技术的研究．中国科学院研究生院博士后研究工作报告．
[16] 尹真．电动力学．南京大学出版社．1999．
[17] 郭硕鸿．电动力学．高等教育出版社 1995：132～133．
[18] Gordon L Mitchell, senior member IEEE. Absorption Spectroscopy in Scattering
Samples Using Integrated Optics.
[19] 国分太雄．光波工程．科学出版社．
[20] 李玉权，崔敏，光波导理论与技术．人民邮电出版社，2002，97～99．
[21] (澳) A．W．斯奈德．J．D．洛夫．光波导理论．人民邮电出版社，1991．
[22] G.STEWART, B. CULSHAW Optical waveguide modeling and design for evanescent field chemical sensors. Optical and Quantum Electronics 26 (1994) S249-S259.
[23] Y. Zaatar. D. Zaouk. J. Bechara Fa.brication and characterization of an evanescent wave fiber optic sensor for air pollution control. Materials science& Engineering B74(2000): 296～298.
[24] Oscar Esteban, M Cruz Navarrete. Simple model of compound waveguide structures used as fiber-optic sensors. Optics and Lasers in Engineering 33 (2000): 219～230.
[25] Hong S. Haddock. P.M. shankar. Evanescent sensing of biomolecules and cells. Sensors and Actuators B 88 (2003): 67～74.
[26] F.A. Muhammad. G. Stewart. Sensitivity enhancement of D-fibre methane gas sensor using high-index overlay. IE PROCEEDINGS-J Vol. 140, No.2 April 1993.
[27] 全国临床检验操作规程．东南大学出版社，1990．9．
[28] 赵华兵，鞠挥，李也凡，吴一辉．微型光谱仪的光谱数据分析及计算机实现．光学精密工程 2003 VOL．11 NO．4
[29] 王庆有．CCD应用技术．天津大学出版社，2000．
[30] Hong S. Haddock. P. M. Shankar. Evanescent sensing of biomolecules and cells. Sensors and Actuators.