基于表面等离子共振的葡萄糖浓度测量技术研究
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摘要
传统的人体葡萄糖浓度的测量主要是通过从人体取血后使用大型的生化分析仪或小型的血糖仪来完成。由于测量过程中会给病人带来一定的痛苦,因而不适用于长期连续的测量,同时由于无法实时地对人体葡萄糖浓度变化做出快速的响应,限制了这些方法在人体葡萄糖检测上的进一步应用,使得检测方法与检测需求之间的矛盾日益地突出。另外,如何提高葡萄糖测量的专一性以及灵敏度也是测量方法中亟待解决的问题。
本文探讨了一种新颖的葡萄糖检测的方法及其系统,用于解决人体葡萄糖检测中遇到测量精度不高以及选择性测量效果不好等问题。在研究中充分利用了表面等离子共振技术测量精度高,能够进行选择性测量的特点,使用微型表面等离子共振传感器构建了一套自动化的葡萄糖浓度检测系统。并为这套系统设计了机械结构,控制软件,以及算法软件等。围绕该系统进行了葡萄糖溶液以及人体组织液的直接和间接的测量,制订了实验的流程,分析了测量系统的性能,研究了影响实验结果的一些因素并提出了改进的办法。在直接测量中,系统成功地分辨出了浓度为0.1g/L的葡萄糖溶液。随着葡萄糖溶液浓度的不同,测量所获得的折射率呈现很好的线性关系。同时,我们表达出了一种与葡萄糖有亲合力的D-半乳糖/D-葡萄糖结合蛋白(GGBP),并且通过在GGBP的两个位点E149,G74进行的定点突变,获得了GGBP的两种突变蛋白,通过在表面等离子共振传感器表面固定这两种蛋白质,间接地进行葡萄糖浓度测量。测量系统的分辨率进一步提高,达到了1mg/L(G74C)和6.25mg/L(E149C),同时对组织液中的葡萄糖有很好的选择性测量效果,显示出了这种蛋白质以及表面等离子共振测量方法在人体葡萄糖连续检测领域的良好应用前景。我们还构建了一套表面等离子共振的光学实验系统,为今后开展进一步的表面等离子共振测量研究奠定了基础。为了使表面等离子共振葡萄糖测量系统能够适应今后便携性、方便性的需要,文中还提出了将葡萄糖测量系统小型化的方案。
The traditional ways for glucose monitoring are realized by extracting blood from people and measuring the blood by large biochemical analyzing instrument or small blood glucose watch. Due to the pain brought by these methods, they do not adapt to continuous and frequent measurement. In addition, owing to lack of the capacity to quickly reflect the change of glucose level, the traditional way of glucose detection fails to meet the demand of application in continuous measurement. The confliction between measuring way and measuring demand becomes increasingly austere. What is more, the finding of approaches to improve the specificity and sensitivity in glucose detection is also an urgent problem that needs to be solved.
In this thesis, a novel way and system are proposed to handle the problems met in glucose detection, such as low precision and poor performance in selective measurement. Basing on a miniature surface plasmon resonance sensor, we constructed an automatic measuring system. Together with this system, mechanical structure, control software, and arithmetic were also developed. Basing on this system, a large number of researches on direct and indirect measurement of glucose solution and interstitial fluid were carried through, as well as the research on experiment procedure, system performance, influence, and method to improve the system. Direct way that measures the refractive index in the surface of SPR sensor directly has succeeded in distinguish a glucose solution with the concentration of 0.1g/L, the measured refractive index of glucose solution rises linearly with the increase of concentration. By immobilizing GGBP which has specific affinity to glucose on the surface of SPR sensor, we indirectly measure the concentration of glucose solution and interstitial fluid. The detecting resolution increases to 1mg/L (G74C GGBP) and 6.25mg/L (E149C GGBP), and the SPR measuring system shows good effect in selective measurement of glucose in interstitial fluid. The experimental result reveals the bright future in applying this technique into diabetes monitoring. Besides of the automatic system, an experimental SPR system and a blue print to minimize the system are also researched.
本文探讨了一种新颖的葡萄糖检测的方法及其系统,用于解决人体葡萄糖检测中遇到测量精度不高以及选择性测量效果不好等问题。在研究中充分利用了表面等离子共振技术测量精度高,能够进行选择性测量的特点,使用微型表面等离子共振传感器构建了一套自动化的葡萄糖浓度检测系统。并为这套系统设计了机械结构,控制软件,以及算法软件等。围绕该系统进行了葡萄糖溶液以及人体组织液的直接和间接的测量,制订了实验的流程,分析了测量系统的性能,研究了影响实验结果的一些因素并提出了改进的办法。在直接测量中,系统成功地分辨出了浓度为0.1g/L的葡萄糖溶液。随着葡萄糖溶液浓度的不同,测量所获得的折射率呈现很好的线性关系。同时,我们表达出了一种与葡萄糖有亲合力的D-半乳糖/D-葡萄糖结合蛋白(GGBP),并且通过在GGBP的两个位点E149,G74进行的定点突变,获得了GGBP的两种突变蛋白,通过在表面等离子共振传感器表面固定这两种蛋白质,间接地进行葡萄糖浓度测量。测量系统的分辨率进一步提高,达到了1mg/L(G74C)和6.25mg/L(E149C),同时对组织液中的葡萄糖有很好的选择性测量效果,显示出了这种蛋白质以及表面等离子共振测量方法在人体葡萄糖连续检测领域的良好应用前景。我们还构建了一套表面等离子共振的光学实验系统,为今后开展进一步的表面等离子共振测量研究奠定了基础。为了使表面等离子共振葡萄糖测量系统能够适应今后便携性、方便性的需要,文中还提出了将葡萄糖测量系统小型化的方案。
The traditional ways for glucose monitoring are realized by extracting blood from people and measuring the blood by large biochemical analyzing instrument or small blood glucose watch. Due to the pain brought by these methods, they do not adapt to continuous and frequent measurement. In addition, owing to lack of the capacity to quickly reflect the change of glucose level, the traditional way of glucose detection fails to meet the demand of application in continuous measurement. The confliction between measuring way and measuring demand becomes increasingly austere. What is more, the finding of approaches to improve the specificity and sensitivity in glucose detection is also an urgent problem that needs to be solved.
In this thesis, a novel way and system are proposed to handle the problems met in glucose detection, such as low precision and poor performance in selective measurement. Basing on a miniature surface plasmon resonance sensor, we constructed an automatic measuring system. Together with this system, mechanical structure, control software, and arithmetic were also developed. Basing on this system, a large number of researches on direct and indirect measurement of glucose solution and interstitial fluid were carried through, as well as the research on experiment procedure, system performance, influence, and method to improve the system. Direct way that measures the refractive index in the surface of SPR sensor directly has succeeded in distinguish a glucose solution with the concentration of 0.1g/L, the measured refractive index of glucose solution rises linearly with the increase of concentration. By immobilizing GGBP which has specific affinity to glucose on the surface of SPR sensor, we indirectly measure the concentration of glucose solution and interstitial fluid. The detecting resolution increases to 1mg/L (G74C GGBP) and 6.25mg/L (E149C GGBP), and the SPR measuring system shows good effect in selective measurement of glucose in interstitial fluid. The experimental result reveals the bright future in applying this technique into diabetes monitoring. Besides of the automatic system, an experimental SPR system and a blue print to minimize the system are also researched.
引文
[1] Continuous Glucose Monitoring: Innovation in the Management of Diabetes, Report of New England Healthcare Institute, 2005
[2] Chuji W, Susan T S, Delwar H. Measurements of cavity ringdown spectroscopy of acetone in the ultraviolet and near-Infrared spectral regions: potential for development of a breath analyze. Applied spectroscopy, 2004, 58(7):784~791
[3] The Scout: A Noninvasive Screening for Type 2 Diabetes, www.medgadget.com
[4] Someya D, Nikawa Y, Yamamoto M. Measurement of blood sugar level using millimeter wave. Korea-Japan Microwave workshop Proceeding, Korea, 2000, 32~35
[5] Cameron B D, Gorde H, Cote G L. Development of an optical polarimeter for in vivo glucose monitoring. Proceedings of SPIE, 1999, 3599:43~49.
[6] Zhao Z M, Myllyla R. The effect of optical scattering on pulsed photoacoustic measurement in weakly absorbing liquid, Measurement science and technology, 2001, 12: 2172~2177
[7] Pan S T, Chung H, Mark A A. Near-infrared spectroscopic measurement of physiological glucose levels in variable matrics of protein and triglycerudes. Analytical Chemistry, 1996, 68(7): 1124~1134
[8] The diabetes research in children network study group. Accuracy of the GlucoWatch G2 biographer and the continuous glucose monitoring system during hypoglycemia, Diabetes Care, 2004, 27(3).
[9] Stefan Z, Doerte F, Boris S. A microneedle-based glucose monitor:Fabricated on a wafer-level using in-device enzyme immobilization, the 12th International conference on Solid State Sensor, Actuators and Microsystems, June 2003, 8(12): 99~102
[10] Wang Z, Chen Y, Analysis of mono- and oligosaccharides by multiwavelength surface plasmon resonance (SPR) Spectroscopy. Carbohydrate Research, 2001, (332):209~213
[11] Lam W W, Chu L H, Wong C L, Zhang Y T, A surface plasmon resonance system for the measurement of glucose in aqueous solution. Sensors and Actuators B, 2005, (105):138~143
[12] Hsieh H V, Pfeiffer Z A, Amiss T J, Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein, Biosensors and Bioelectronics, 2004, 19(7):653~660
[13] Eiji F, Kaori N, Application of the Absorption-Based Surface Plasmon Resonance Principle to the Determination of Glucose Using an Enzyme Reaction, Instrumentation Science & Technology, 2003,31(4):343~356
[14] Ballerstadt R, Schultz J S, Kinetics of dissolution of concanvalin A dextra sols in response to glucose measured by surface plasmon resonance, Sensors and Actuators B, 1998,(46):50~55
[15] Wood R W. On a remarkable case of uneven distribution of light in a diffraction grating spectrum, Phil. Magm,1902,4: 396~402
[16] Otto A. Excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection. A. Physik, 1968, 216: 398~410
[17] Kretschmann E, Raether H, Radiative decay of non-radiative surface plasmons excited by light. Z. Naturforsch, 1968,23A: 2135~2136.
[18] Chien F C, Chen S J, A sensitivity comparison of optical biosensor based on four different surface plasmon resonance modes, Biosensors and Bioelectronics,2004 20: 633~642.
[19] Jorgenson R C, Yee S S. A fiber-optic chemical sensor based on surface plasmon resonance. Sensors and Actuators B, 1993, 12: 213~220
[20] Tiefenthaler K, Lukosz W.Sensitivity of grating couplers as integrated-optical chemical sensors[J].Opt.Soc.Am.B,1989,6:209~212.
[21] Homola J, Koudela I, Yee S. Surface plasmon resonance sensor based on diffraction gratings and prism couplers: sensitivity comparison, Sensors and Actuators B, 1999, 54: 16~24.
[22] Nikitin P I, Eloglazov A A. A multi-purpose sensor based on surface plasmon polariton resonance in a Schottky structure, Sensors and Actuators, 1994, A(41-42) :547~552.
[23] 蒋稼欢,生物医学微系统技术及医用,北京,化学工业出版社,2006,25~29
[24] Cooper J M, Greenough K R, McNeil C J. Direct electron transfer reactions between immobilized cytochrome and modified gold electrode, J. Electro. Chem, 1993, 347~267
[25] Biacore knowledge and training products, Amine Coupling.
[26] Melendez J, Carr R, Bartholomew D U, A commercial solution for surface plasmon sensing, Sens. and Actuators, B: Chemical, 1996, 35 (1-3), 212~216
[27] Kukanskis K, Elkind J, Melendez J. Detection of DNA hybridization using the TISPR-1 surface plasmon resonance biosensor Anal. Biochem, 1997, 274: 7~17
[28] Nenninger G., Piliarik M, Homola J. Data analysis for optical sensors based on spectroscopy of surface plasmons, Meas. Sci. Technol, 2002, 13: 2038~2046
[29] Stenberg E, Persson B, Roos H. Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins J. Colloid Interf. Sci, 1991,143:513~26
[30] Kurihara K, Nakamura K, Suzuki K. Asymmetric SPR sensor response curve-fitting. equation for the accurate determination of SPR resonance angle. Sens. and Actuators B, 2002,86:49~57
[31] Johnston K S, Yee S S, Booksh K S. Calibration of Surface Plasmon Resonance Refractometers Using Locally Weighted Parametric Regression. Anal Chem, 1997, 69(10):1851~1884.
[32] Thirstrup C, Zong W C, Data Analysis for Surface Plasmon Resonance Sensors Using Dynamic Baseline Algorithm, Sens. and Actuators B. 2005, 106(2):796~802
[33] Karlsson, R. and Falt, A. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors, Journal of Immunological Methods 1997, 200:121~133
[2] Chuji W, Susan T S, Delwar H. Measurements of cavity ringdown spectroscopy of acetone in the ultraviolet and near-Infrared spectral regions: potential for development of a breath analyze. Applied spectroscopy, 2004, 58(7):784~791
[3] The Scout: A Noninvasive Screening for Type 2 Diabetes, www.medgadget.com
[4] Someya D, Nikawa Y, Yamamoto M. Measurement of blood sugar level using millimeter wave. Korea-Japan Microwave workshop Proceeding, Korea, 2000, 32~35
[5] Cameron B D, Gorde H, Cote G L. Development of an optical polarimeter for in vivo glucose monitoring. Proceedings of SPIE, 1999, 3599:43~49.
[6] Zhao Z M, Myllyla R. The effect of optical scattering on pulsed photoacoustic measurement in weakly absorbing liquid, Measurement science and technology, 2001, 12: 2172~2177
[7] Pan S T, Chung H, Mark A A. Near-infrared spectroscopic measurement of physiological glucose levels in variable matrics of protein and triglycerudes. Analytical Chemistry, 1996, 68(7): 1124~1134
[8] The diabetes research in children network study group. Accuracy of the GlucoWatch G2 biographer and the continuous glucose monitoring system during hypoglycemia, Diabetes Care, 2004, 27(3).
[9] Stefan Z, Doerte F, Boris S. A microneedle-based glucose monitor:Fabricated on a wafer-level using in-device enzyme immobilization, the 12th International conference on Solid State Sensor, Actuators and Microsystems, June 2003, 8(12): 99~102
[10] Wang Z, Chen Y, Analysis of mono- and oligosaccharides by multiwavelength surface plasmon resonance (SPR) Spectroscopy. Carbohydrate Research, 2001, (332):209~213
[11] Lam W W, Chu L H, Wong C L, Zhang Y T, A surface plasmon resonance system for the measurement of glucose in aqueous solution. Sensors and Actuators B, 2005, (105):138~143
[12] Hsieh H V, Pfeiffer Z A, Amiss T J, Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein, Biosensors and Bioelectronics, 2004, 19(7):653~660
[13] Eiji F, Kaori N, Application of the Absorption-Based Surface Plasmon Resonance Principle to the Determination of Glucose Using an Enzyme Reaction, Instrumentation Science & Technology, 2003,31(4):343~356
[14] Ballerstadt R, Schultz J S, Kinetics of dissolution of concanvalin A dextra sols in response to glucose measured by surface plasmon resonance, Sensors and Actuators B, 1998,(46):50~55
[15] Wood R W. On a remarkable case of uneven distribution of light in a diffraction grating spectrum, Phil. Magm,1902,4: 396~402
[16] Otto A. Excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection. A. Physik, 1968, 216: 398~410
[17] Kretschmann E, Raether H, Radiative decay of non-radiative surface plasmons excited by light. Z. Naturforsch, 1968,23A: 2135~2136.
[18] Chien F C, Chen S J, A sensitivity comparison of optical biosensor based on four different surface plasmon resonance modes, Biosensors and Bioelectronics,2004 20: 633~642.
[19] Jorgenson R C, Yee S S. A fiber-optic chemical sensor based on surface plasmon resonance. Sensors and Actuators B, 1993, 12: 213~220
[20] Tiefenthaler K, Lukosz W.Sensitivity of grating couplers as integrated-optical chemical sensors[J].Opt.Soc.Am.B,1989,6:209~212.
[21] Homola J, Koudela I, Yee S. Surface plasmon resonance sensor based on diffraction gratings and prism couplers: sensitivity comparison, Sensors and Actuators B, 1999, 54: 16~24.
[22] Nikitin P I, Eloglazov A A. A multi-purpose sensor based on surface plasmon polariton resonance in a Schottky structure, Sensors and Actuators, 1994, A(41-42) :547~552.
[23] 蒋稼欢,生物医学微系统技术及医用,北京,化学工业出版社,2006,25~29
[24] Cooper J M, Greenough K R, McNeil C J. Direct electron transfer reactions between immobilized cytochrome and modified gold electrode, J. Electro. Chem, 1993, 347~267
[25] Biacore knowledge and training products, Amine Coupling.
[26] Melendez J, Carr R, Bartholomew D U, A commercial solution for surface plasmon sensing, Sens. and Actuators, B: Chemical, 1996, 35 (1-3), 212~216
[27] Kukanskis K, Elkind J, Melendez J. Detection of DNA hybridization using the TISPR-1 surface plasmon resonance biosensor Anal. Biochem, 1997, 274: 7~17
[28] Nenninger G., Piliarik M, Homola J. Data analysis for optical sensors based on spectroscopy of surface plasmons, Meas. Sci. Technol, 2002, 13: 2038~2046
[29] Stenberg E, Persson B, Roos H. Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins J. Colloid Interf. Sci, 1991,143:513~26
[30] Kurihara K, Nakamura K, Suzuki K. Asymmetric SPR sensor response curve-fitting. equation for the accurate determination of SPR resonance angle. Sens. and Actuators B, 2002,86:49~57
[31] Johnston K S, Yee S S, Booksh K S. Calibration of Surface Plasmon Resonance Refractometers Using Locally Weighted Parametric Regression. Anal Chem, 1997, 69(10):1851~1884.
[32] Thirstrup C, Zong W C, Data Analysis for Surface Plasmon Resonance Sensors Using Dynamic Baseline Algorithm, Sens. and Actuators B. 2005, 106(2):796~802
[33] Karlsson, R. and Falt, A. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors, Journal of Immunological Methods 1997, 200:121~133