LSPR传感与微流体系统集成化的生物芯片研究
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摘要
随着生命科学的不断发展，生物医学检测和生物化学分析得到越来越多的重视，微纳米结构的微流体系统已经逐渐成为研究的热点。本文的主要工作是在研究金纳米粒子的局域表面等离子共振（LSPR）消光特性的基础上，将行波压电微泵与LSPR传感器件集成制作在玻璃基片上，形成一体化的高灵敏度生物芯片系统，实现整体系统的小型化和低成本。在行波压电微泵的制作中，设计了新型的微泵管道结构，提高了微泵输送液体的效率。为了给LSPR传感区域的金纳米粒子消光性质提供理论依据，采用离散偶极子近似（DDA）等仿真算法分析了不同间距的金纳米粒子阵列的消光性质，得出其分布密度对整体灵敏度的影响。并利用金纳米粒子在可见光区的LSPR消光特性，分析纳米粒子表面的分子反应过程。本文的研究目的，是为了开发用于生物医学领域的小型、廉价、高灵敏度、集成化的LSPR生物芯片检测系统。主要研究工作包括：
1．行波压电微泵的理论研究与仿真分析。选用压电双晶片作为行波压电微泵的执行器，详细分析了压电双晶片振幅模型的理论推导和行波在弹性管道上的形成机理；利用有限元分析软件ANSYS对压电双晶片进行建模仿真，分析了几个重要因素对压电双晶片振幅的影响，对设计参数进行优化改进；同时还分析了与弹性管道接触的压电双晶片输出不同位移时，管道内部产生的位移响应。
2．行波压电微泵的设计和制作。以PMMA为材料，用微机械加工方法制作行波压电微泵基底和微泵管道模具；将液体高分子材料PDMS注入到PMMA模具中，经过固化处理，制作出弹性微泵管道；在微泵基底上制作了PDMS薄膜，利用热键合工艺将微泵管道与基底粘合在一起，并将压电双晶片阵列安装在泵区管道上方，完成行波压电微泵的制作；将行波压电微泵的泵区管道设计为多级扩散结构的锯齿形管道，在较低的驱动电压下，提高了微泵的最大平均流速和背压，分别达到33.36μL/min和1.13kPa；在相同的测试条件下，与平直结构管道的行波压电微泵的测试数值（24.88μL/min和0.64kPa）相比，新型结构的微泵性能有较大的提高。
3．对不同纳米粒子及其阵列消光特性的仿真分析。采用离散偶极子近似（DDA）方法和时域有限差分（FDTD）方法，分析了金属纳米粒子的材料、形状、大小以及阵列结构等参数对其LSPR消光特性的影响；利用DDA方法仿真分析了不同间距的球形纳米粒子阵列的LSPR消光特性，分析结果说明消光峰值波长和强度随纳米粒子间距的减小而增大，但在间距较大的情况下，纳米粒子阵列的折射率灵敏度基本不变。因此，当纳米粒子阵列的间距较大时，其局部分布的不均匀不会影响LSPR传感芯片的整体性能。
4． LSPR生物传感芯片的测试。用柠檬酸钠还原法水相合成球形金纳米粒子，并将金纳米粒子自组装在硅烷化的玻璃基片表面，形成LSPR敏感膜；测试比较不同大小、不同分布密度的金纳米粒子阵列在不同介质中的消光特性，研究不同参数的敏感膜的LSPR消光光谱，从实验结果上说明金纳米粒子间距对折射率灵敏度的影响。
5．集成化LSPR生物传感系统的制作。以LSPR生物传感芯片为基底，利用紫外线键合工艺将行波压电微泵制作在芯片基底上，用来向敏感膜输送样品；并将检测光纤引入到LSPR敏感膜处，通过光纤将传感区域与片外分光光度计连接在一起，检测LSPR消光光谱，完成集成化LSPR生物传感系统的制作。
6．生物免疫测量的应用。用行波压电微泵将巯基烷酸、交联剂（EDC/NHS）和羊抗人IgG先后输送到固定有金纳米粒子阵列的LSPR敏感膜处，完成探针分子的修饰，测定各个反应步骤的LSPR消光光谱和折射率灵敏度，制备出高灵敏度的生物传感芯片。
Along with the continuous development of life sciences, biomedical detectionand biochemical analysis have getting more and more attention of the researchers,and the microfluidic systems have also gradually become a research hotspot. Themain work of this paper is fabricating a high sensitivity biochip system, whichintegrated the traveling-wave piezoelectric micropump and Localized SurfacePlasmon Resonance (LSPR) sensing device onto the glass substrate, to achieve theminiaturized and inexpensive test, on the basis of the study of LSPR extinctionproperties of the gold nanoparticles. The novel microchannel structure of thetraveling-wave piezoelectric micropump was designed to improve the micropumpefficiency in the fabrication of the micropump. In order to provide the theoreticalanalysis of the gold nanoparticles at the LSPR sensing area, the extinction propertiesof the gold nanoparticles array was simulated by Discrete Dipole Approximation(DDA) method to obtain the sensitivity influence of the array density. Furthermore,the molecular reaction procedure on the nanoparticles' surface was tested by theextinction properties in the visible region. The research of this paper developed anintegrated LSPR biochip system applied to the biomedical test with the advantages ofminiaturezation, low cost and high sensitivity. The main work includes:
1. Theoretical simulation of the traveling-wave piezoelectric micropumps. Theamplitude theoretical model of piezoelectric bimorphs and the formationmechanism of microchannels were analyzed in detail, while the piezoelectricbimorphs were selected as the micropump actuators; the piezoelectric bimorphwas modeled by Finite Element Analysis software ANSYS, and the influence ofthe important factors toward the amplitude of the piezoelectric bimorph weresimulated to optimize the design parameters; meanwhile, the inside displacementresponse of the PDMS microchannel was simulated when the piezoelectricbimorphs generated different displacement on the microchannel.
2. Design and fabrication of the traveling-wave piezoelectric micropump. The micropump substrate and microchannel mold were fabricated with PMMA bymicromachining technology, and the microchannel was formed by liquid polymermaterial PDMS using injection molding process; the traveling-wave piezoelectricmicropump would be done while the microchannel was bonded onto the substateby thermal bonding process, and the piezoelectric bimorphs array was fixed onthe pump area of the microchannel; the pump area configuration of themicrochannel was designed as a saw-tooth stucture of multi-stage diffuser, whichwas improved the maximum flow rate and the maximum back pressure of themicropump at the lower voltage, the average flow rate and back pressurerespectively reached33.36μL/min and1.13kPa; with the same testing conditions,these two parameters of straight microchannel were respectively24.88μL/minand0.64kPa, which are much smaller than those of saw-tooth microchannel.
3. The simulation of the extinction properties of different nanoparticles andnanoparticles arrays. The LSPR extinction of nanoparticles with differentmaterial, shape, size and array structure were simulated by Discrete DipoleApproximation (DDA) and Finite-Difference Time-Domain (FDTD) method; theextinction properties of spherical nanoparticles arrays with different spacing weresimulated by DDA method, and the simulation results indicate that the extinctionwavelength and intensity were increasing with the decreasing nanoparticlesspacing, but the refractive index (RI) sensitivity remained a constant essentiallywhen the nanoparticles spacing was large enough. Therefore, the local unevendistribution of the nanoparticles array would not change its RI sensitivity as thespacing between nanoparticles is large enough.
4. The testing of the LSPR biosensor chip. The spherical gold nanoparticles wassynthetized by sodium citrate reduction method, and fixed on the silanized glasssubstrate by self-assembly technology, forming a monolayer of gold nanoparticlesarray; the extinction properties of gold nanoparticles arrays in different mediumwas tested, and the influence to the RI sensitivity of the biosensor chip withdifferent array spacing was researched.
5. The fabrication of integrated LSPR biosensing system. The traveling-wavepiezoelectric micropump, which applied to deliver the sample to the sensing area, was integrated on the LSPR biosensor chip by ultraviolet bonding process, andthe optical fiber was introduced into the LSPR sensing area; thus, the LSPRsensor and the off-chip optical detection equipment were connected together toforming the integrated LSPR biosensing system.
6. The application of Biological immune measurement. The mercapto acid,crosslinking agent (EDC/NHS) and goat anti-human IgG were successivelytransported to the LSPR sensing membrane of gold nanoparticles array bytraveling-wave piezoelectric micropump to modify the probe molecules, and theLSPR extinction spectrum of each step of the modifying reaction procedure wastest by spectrophotometer to obtain the biosensor chip with higher sensitivity.
1．行波压电微泵的理论研究与仿真分析。选用压电双晶片作为行波压电微泵的执行器，详细分析了压电双晶片振幅模型的理论推导和行波在弹性管道上的形成机理；利用有限元分析软件ANSYS对压电双晶片进行建模仿真，分析了几个重要因素对压电双晶片振幅的影响，对设计参数进行优化改进；同时还分析了与弹性管道接触的压电双晶片输出不同位移时，管道内部产生的位移响应。
2．行波压电微泵的设计和制作。以PMMA为材料，用微机械加工方法制作行波压电微泵基底和微泵管道模具；将液体高分子材料PDMS注入到PMMA模具中，经过固化处理，制作出弹性微泵管道；在微泵基底上制作了PDMS薄膜，利用热键合工艺将微泵管道与基底粘合在一起，并将压电双晶片阵列安装在泵区管道上方，完成行波压电微泵的制作；将行波压电微泵的泵区管道设计为多级扩散结构的锯齿形管道，在较低的驱动电压下，提高了微泵的最大平均流速和背压，分别达到33.36μL/min和1.13kPa；在相同的测试条件下，与平直结构管道的行波压电微泵的测试数值（24.88μL/min和0.64kPa）相比，新型结构的微泵性能有较大的提高。
3．对不同纳米粒子及其阵列消光特性的仿真分析。采用离散偶极子近似（DDA）方法和时域有限差分（FDTD）方法，分析了金属纳米粒子的材料、形状、大小以及阵列结构等参数对其LSPR消光特性的影响；利用DDA方法仿真分析了不同间距的球形纳米粒子阵列的LSPR消光特性，分析结果说明消光峰值波长和强度随纳米粒子间距的减小而增大，但在间距较大的情况下，纳米粒子阵列的折射率灵敏度基本不变。因此，当纳米粒子阵列的间距较大时，其局部分布的不均匀不会影响LSPR传感芯片的整体性能。
4． LSPR生物传感芯片的测试。用柠檬酸钠还原法水相合成球形金纳米粒子，并将金纳米粒子自组装在硅烷化的玻璃基片表面，形成LSPR敏感膜；测试比较不同大小、不同分布密度的金纳米粒子阵列在不同介质中的消光特性，研究不同参数的敏感膜的LSPR消光光谱，从实验结果上说明金纳米粒子间距对折射率灵敏度的影响。
5．集成化LSPR生物传感系统的制作。以LSPR生物传感芯片为基底，利用紫外线键合工艺将行波压电微泵制作在芯片基底上，用来向敏感膜输送样品；并将检测光纤引入到LSPR敏感膜处，通过光纤将传感区域与片外分光光度计连接在一起，检测LSPR消光光谱，完成集成化LSPR生物传感系统的制作。
6．生物免疫测量的应用。用行波压电微泵将巯基烷酸、交联剂（EDC/NHS）和羊抗人IgG先后输送到固定有金纳米粒子阵列的LSPR敏感膜处，完成探针分子的修饰，测定各个反应步骤的LSPR消光光谱和折射率灵敏度，制备出高灵敏度的生物传感芯片。
Along with the continuous development of life sciences, biomedical detectionand biochemical analysis have getting more and more attention of the researchers,and the microfluidic systems have also gradually become a research hotspot. Themain work of this paper is fabricating a high sensitivity biochip system, whichintegrated the traveling-wave piezoelectric micropump and Localized SurfacePlasmon Resonance (LSPR) sensing device onto the glass substrate, to achieve theminiaturized and inexpensive test, on the basis of the study of LSPR extinctionproperties of the gold nanoparticles. The novel microchannel structure of thetraveling-wave piezoelectric micropump was designed to improve the micropumpefficiency in the fabrication of the micropump. In order to provide the theoreticalanalysis of the gold nanoparticles at the LSPR sensing area, the extinction propertiesof the gold nanoparticles array was simulated by Discrete Dipole Approximation(DDA) method to obtain the sensitivity influence of the array density. Furthermore,the molecular reaction procedure on the nanoparticles' surface was tested by theextinction properties in the visible region. The research of this paper developed anintegrated LSPR biochip system applied to the biomedical test with the advantages ofminiaturezation, low cost and high sensitivity. The main work includes:
1. Theoretical simulation of the traveling-wave piezoelectric micropumps. Theamplitude theoretical model of piezoelectric bimorphs and the formationmechanism of microchannels were analyzed in detail, while the piezoelectricbimorphs were selected as the micropump actuators; the piezoelectric bimorphwas modeled by Finite Element Analysis software ANSYS, and the influence ofthe important factors toward the amplitude of the piezoelectric bimorph weresimulated to optimize the design parameters; meanwhile, the inside displacementresponse of the PDMS microchannel was simulated when the piezoelectricbimorphs generated different displacement on the microchannel.
2. Design and fabrication of the traveling-wave piezoelectric micropump. The micropump substrate and microchannel mold were fabricated with PMMA bymicromachining technology, and the microchannel was formed by liquid polymermaterial PDMS using injection molding process; the traveling-wave piezoelectricmicropump would be done while the microchannel was bonded onto the substateby thermal bonding process, and the piezoelectric bimorphs array was fixed onthe pump area of the microchannel; the pump area configuration of themicrochannel was designed as a saw-tooth stucture of multi-stage diffuser, whichwas improved the maximum flow rate and the maximum back pressure of themicropump at the lower voltage, the average flow rate and back pressurerespectively reached33.36μL/min and1.13kPa; with the same testing conditions,these two parameters of straight microchannel were respectively24.88μL/minand0.64kPa, which are much smaller than those of saw-tooth microchannel.
3. The simulation of the extinction properties of different nanoparticles andnanoparticles arrays. The LSPR extinction of nanoparticles with differentmaterial, shape, size and array structure were simulated by Discrete DipoleApproximation (DDA) and Finite-Difference Time-Domain (FDTD) method; theextinction properties of spherical nanoparticles arrays with different spacing weresimulated by DDA method, and the simulation results indicate that the extinctionwavelength and intensity were increasing with the decreasing nanoparticlesspacing, but the refractive index (RI) sensitivity remained a constant essentiallywhen the nanoparticles spacing was large enough. Therefore, the local unevendistribution of the nanoparticles array would not change its RI sensitivity as thespacing between nanoparticles is large enough.
4. The testing of the LSPR biosensor chip. The spherical gold nanoparticles wassynthetized by sodium citrate reduction method, and fixed on the silanized glasssubstrate by self-assembly technology, forming a monolayer of gold nanoparticlesarray; the extinction properties of gold nanoparticles arrays in different mediumwas tested, and the influence to the RI sensitivity of the biosensor chip withdifferent array spacing was researched.
5. The fabrication of integrated LSPR biosensing system. The traveling-wavepiezoelectric micropump, which applied to deliver the sample to the sensing area, was integrated on the LSPR biosensor chip by ultraviolet bonding process, andthe optical fiber was introduced into the LSPR sensing area; thus, the LSPRsensor and the off-chip optical detection equipment were connected together toforming the integrated LSPR biosensing system.
6. The application of Biological immune measurement. The mercapto acid,crosslinking agent (EDC/NHS) and goat anti-human IgG were successivelytransported to the LSPR sensing membrane of gold nanoparticles array bytraveling-wave piezoelectric micropump to modify the probe molecules, and theLSPR extinction spectrum of each step of the modifying reaction procedure wastest by spectrophotometer to obtain the biosensor chip with higher sensitivity.
引文
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