基于光学检测的电泳微芯片关键技术研究
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摘要
电泳微芯片技术作为微流控芯片中重要的组成部分之一,在近二十年来的发展过程中,经历了材料选取、工艺实现、功能完善以及分析仪器系统的集成化、微型化和便携化等几个发展阶段。本文基于毛细管电渗流基本理论,以光学探测方法,针对电泳微芯片现有技术中存在的芯片制作工艺复杂、注液清洗过程繁复以及光学测试方法中微米量级光程影响检测灵敏度等问题进行研究。
电渗性能作为衡量电泳微芯片性能的指标之一,其大小直接影响分离情况和分析结果的精密度和准确度。以电场中稀溶液流动的Navier-Stokes方程为基础,借鉴低雷诺数流理论忽略斯托克斯方程中的惯性力项,建立电渗驱动方程模型。利用有限差分法和多步打靶法分别求解PMMA基材电泳微沟道内电渗流形、电场和微流体流速的关系。计算结果表明PMMA基电泳芯片电渗流形为扁平塞状流,流体流速与电场强度保持线性关系,μeo=2.3×10-4cm2/Vs。设计电流监测法电渗流测速实验,以硼砂为缓冲溶液,PMMA基电泳微芯片测试的电渗迁移率为μeo=3.02×10-4cm2/Vs。利用XPS谱进行加工表面成分和价态分析,表明铣削方式加工制作的PMMA电泳微芯片微沟道表面存在不饱和价键结构,导致定域电荷面密度较大,而造成电渗强度较大。
以毛细管电渗理论为基础,进行电泳微芯片设计、制作以及实验过程研究。以CFDRC软件进行微沟道构型、几何尺寸以及电场施加策略等优化设计,将微沟道设计为宽度大于50μm,可施加场强200V/cm~250V/cm。
针对PDMS和PMMA材料分别进行芯片加工实现,提出二次模板法加工PDMS微器件以及氨水浸泡法实现PDMS芯片不可逆键合法,解决了PDMS芯片加工过程中的脱模问题和键合问题;
采用超精密数控铣削加工模式进行PMMA电泳微芯片加工,实现加工即成形目的。设计加工PMMA基压电驱动平面无阀微泵,最大流量可达350μl/min,在1min内完成沟道清洗次数最高可达487次,可完成电泳微芯片微沟道内溶液的灌注、清洗以及进样功能。PMMA微泵设计工艺与制作电泳微芯片工艺兼容,一体化的设计加工模式提高了芯片的可使用性。
搭建了以TW30SX光敏二极管为光电转换器件的紫外-可见吸收检测系统,可检测10-11A数量级的光电流;总延迟时间在150~250ms之间;输出噪声±5mV。总重量2kg左右,体积为280×200×150mm3。以该系统测试单层PMMA芯片对紫外光源的吸收率为0.035,键合后芯片对光强的衰减为16.87%。
同时针对无机阴离子(Cl-和SO42-)的电泳实验,以间接紫外吸收检测方式,设计背景缓冲溶液为10mmol/L Na2CrO4+0.5mmol/LCTAB的混合液,其电渗性能测试结果表明在此浓度溶液组合下,电渗流方向成功实现反转,反向电渗迁移率为μeo=9.15×10-4cm2/Vs,可满足无机阴离子毛细管电泳实验测试需求。
最后利用电迁移进样方式在单一微沟道布局芯片上,实现无机阴离子Cl-和SO42-电泳间接紫外吸收检测,单峰重复性实验结果效果良好,双峰可初步实现基线分离,检测限可达10-9mol。PMMA电泳微芯片以及光检测系统性能稳定,为PMMA基材电泳微芯片在紫外吸收检测方面的进一步应用奠定基础。
As one of important parts of microfluidics, microchip electrophoresis (MCE) technology has gone through several seedtimes during the last 20 years,including fabrication processing and selection of materials, function improvement, as well as system integrated, miniaturized and portable. There are some problems still impeding the development of MCE, such as the complexity of fabrication technique, the operation process complexity of solution introducing into the microchannel. Also the micron-scale optical-path length is so short that the sensitivity of optical detection system is affected seriously. In this paper,based on the basic theory of capillary electro-osmosis flow (EOF), with optical detection methods, all attention are focused on finding solutions to the problems that exist in the MCE technology.
EOF,as one of the most important performance indicators of MCE, will influence the precision and veracity of the analysis result. Based on Navier-Stokes equation of the dilute solution under the electric field, neglected the inertial force item in Stokes equations as the theory of low Reynolds number, the EOF mathematics model in MCE micro-channel is set up. Then with finite difference method and multi-step shooting method, the EOF flow shape in the micro-channel on PMMA MCE and the relationship between E and veo are solved respectively. The calculation results indicate that the EOF flow shape in the micro-channel is flat plug-like, and the E ? veo curve is kept the lineariy relation,μeo=2.3×1 0-4cm2/Vs. Current-monitoring method for measuring the EOF rate is adopted. The buffer is borax. The electroosmosis mobility isμeo=3.02×10-4cm2/Vs. The microchannel surface component and valent-state are analysed with XPS spectrum, the results show that the unsaturated bond are existing on the PMMA surface with the milling process, leading to increase the surface charge density, then resulting in a greater intensity of electro-osmotic. According to EOF basic theory,MCE structure design is carried out, at the same time, the study for the fabrication process and the electophoresis experimental is followed along. With CFDRC software, the configuration and geometry parameter of the MCE micro-channel, also the strategy of electric field apllied, are simulated and optimized designing. The optimization parameter of the microchannel width is 50μm, and the electric field strength E is 200V/cm~ 250V/cm.
The fabrication techniques about the PDMS and PMMA material are described detailed in this paper. The technique that fabricated the PDMS device with twice mold is presented firstly.With this method the PDMS devices could be released from the PMMA mold easily and perfectly. Then an irreversible bonding process of PDMS chip with ammonia immersion method is developed. These two methods completely solved the fabrication problems of PDMS chip.
The ultra-precision computer numerical control (CNC) milling manufacture method is presented in fabrication the PMMA substrate MCE with an ultra-precision CNC milling. As for the PMMA materials chip fabrication, this method is easier to realize the purpose of processing that is forming. A PMMA substrate plane valveless micro-pump driven by the PZT piezoelectric thick film is designed and fabricated with this method too. Then the chip’s function and practicability could be increased. The maxium flow rate is 350μl/min, and could complete rinsing microchannel up to 487 times in 1 min. The function of solution introducing, rinsing and sample injection could be completed. Especially, the fabrication process of the PMMA micropump is compatibile with MCE chip’s. Integrated design and processing mode improve the availability of the chip.
With the TW30SX photodiode as the photo-electric converter device, a UV detection system is set up. With this detection system, 10-11A magnitude of photocurrent can be detected. The total delay time is between 150 ~ 250ms, the output noise is±5mV, the weight of the whole detect system is about 2kg, and the volume is 280×200×150mm3. The absorption rate of single-layer PMMA chip is 0.035at UV light source with the system, while attenuation of the light strength is 16.87% with the bonding PMMA chip.
The whole system electrophoresis experiment are developed with indirect UV detection methods. The inorganic anions(Cl-, SO42-)is used as the separation ions. The background buffer solution is 10mmol/L Na2CrO4+0.5mmol/LCTAB, which electroosmotic test results show that the direction of EOF flow is from the cathode to the anode, reversal with the borax buffer. And the mobility of this buffer isμeo=9.15×10-4cm2/Vs. This condition is suitable for the test demand of inorganic anion electrophoresis experiment
Finally, with the electromigration sample injection mode in a single micro- channel MCE chip, inorganic anion Cl-and SO42- electrophoresis peak are detected with indirect UV detection method. The single-peak repeatability results are better, also the mixed two ions could achieve the baseline separation, the detection limit is 10-9mol. The performance and stability are better during the experiment of PMMA MCE with the portable detection system. All these work lay a stable foundation for the PMMA MCE further application in the UV absorbance detection.
电渗性能作为衡量电泳微芯片性能的指标之一,其大小直接影响分离情况和分析结果的精密度和准确度。以电场中稀溶液流动的Navier-Stokes方程为基础,借鉴低雷诺数流理论忽略斯托克斯方程中的惯性力项,建立电渗驱动方程模型。利用有限差分法和多步打靶法分别求解PMMA基材电泳微沟道内电渗流形、电场和微流体流速的关系。计算结果表明PMMA基电泳芯片电渗流形为扁平塞状流,流体流速与电场强度保持线性关系,μeo=2.3×10-4cm2/Vs。设计电流监测法电渗流测速实验,以硼砂为缓冲溶液,PMMA基电泳微芯片测试的电渗迁移率为μeo=3.02×10-4cm2/Vs。利用XPS谱进行加工表面成分和价态分析,表明铣削方式加工制作的PMMA电泳微芯片微沟道表面存在不饱和价键结构,导致定域电荷面密度较大,而造成电渗强度较大。
以毛细管电渗理论为基础,进行电泳微芯片设计、制作以及实验过程研究。以CFDRC软件进行微沟道构型、几何尺寸以及电场施加策略等优化设计,将微沟道设计为宽度大于50μm,可施加场强200V/cm~250V/cm。
针对PDMS和PMMA材料分别进行芯片加工实现,提出二次模板法加工PDMS微器件以及氨水浸泡法实现PDMS芯片不可逆键合法,解决了PDMS芯片加工过程中的脱模问题和键合问题;
采用超精密数控铣削加工模式进行PMMA电泳微芯片加工,实现加工即成形目的。设计加工PMMA基压电驱动平面无阀微泵,最大流量可达350μl/min,在1min内完成沟道清洗次数最高可达487次,可完成电泳微芯片微沟道内溶液的灌注、清洗以及进样功能。PMMA微泵设计工艺与制作电泳微芯片工艺兼容,一体化的设计加工模式提高了芯片的可使用性。
搭建了以TW30SX光敏二极管为光电转换器件的紫外-可见吸收检测系统,可检测10-11A数量级的光电流;总延迟时间在150~250ms之间;输出噪声±5mV。总重量2kg左右,体积为280×200×150mm3。以该系统测试单层PMMA芯片对紫外光源的吸收率为0.035,键合后芯片对光强的衰减为16.87%。
同时针对无机阴离子(Cl-和SO42-)的电泳实验,以间接紫外吸收检测方式,设计背景缓冲溶液为10mmol/L Na2CrO4+0.5mmol/LCTAB的混合液,其电渗性能测试结果表明在此浓度溶液组合下,电渗流方向成功实现反转,反向电渗迁移率为μeo=9.15×10-4cm2/Vs,可满足无机阴离子毛细管电泳实验测试需求。
最后利用电迁移进样方式在单一微沟道布局芯片上,实现无机阴离子Cl-和SO42-电泳间接紫外吸收检测,单峰重复性实验结果效果良好,双峰可初步实现基线分离,检测限可达10-9mol。PMMA电泳微芯片以及光检测系统性能稳定,为PMMA基材电泳微芯片在紫外吸收检测方面的进一步应用奠定基础。
As one of important parts of microfluidics, microchip electrophoresis (MCE) technology has gone through several seedtimes during the last 20 years,including fabrication processing and selection of materials, function improvement, as well as system integrated, miniaturized and portable. There are some problems still impeding the development of MCE, such as the complexity of fabrication technique, the operation process complexity of solution introducing into the microchannel. Also the micron-scale optical-path length is so short that the sensitivity of optical detection system is affected seriously. In this paper,based on the basic theory of capillary electro-osmosis flow (EOF), with optical detection methods, all attention are focused on finding solutions to the problems that exist in the MCE technology.
EOF,as one of the most important performance indicators of MCE, will influence the precision and veracity of the analysis result. Based on Navier-Stokes equation of the dilute solution under the electric field, neglected the inertial force item in Stokes equations as the theory of low Reynolds number, the EOF mathematics model in MCE micro-channel is set up. Then with finite difference method and multi-step shooting method, the EOF flow shape in the micro-channel on PMMA MCE and the relationship between E and veo are solved respectively. The calculation results indicate that the EOF flow shape in the micro-channel is flat plug-like, and the E ? veo curve is kept the lineariy relation,μeo=2.3×1 0-4cm2/Vs. Current-monitoring method for measuring the EOF rate is adopted. The buffer is borax. The electroosmosis mobility isμeo=3.02×10-4cm2/Vs. The microchannel surface component and valent-state are analysed with XPS spectrum, the results show that the unsaturated bond are existing on the PMMA surface with the milling process, leading to increase the surface charge density, then resulting in a greater intensity of electro-osmotic. According to EOF basic theory,MCE structure design is carried out, at the same time, the study for the fabrication process and the electophoresis experimental is followed along. With CFDRC software, the configuration and geometry parameter of the MCE micro-channel, also the strategy of electric field apllied, are simulated and optimized designing. The optimization parameter of the microchannel width is 50μm, and the electric field strength E is 200V/cm~ 250V/cm.
The fabrication techniques about the PDMS and PMMA material are described detailed in this paper. The technique that fabricated the PDMS device with twice mold is presented firstly.With this method the PDMS devices could be released from the PMMA mold easily and perfectly. Then an irreversible bonding process of PDMS chip with ammonia immersion method is developed. These two methods completely solved the fabrication problems of PDMS chip.
The ultra-precision computer numerical control (CNC) milling manufacture method is presented in fabrication the PMMA substrate MCE with an ultra-precision CNC milling. As for the PMMA materials chip fabrication, this method is easier to realize the purpose of processing that is forming. A PMMA substrate plane valveless micro-pump driven by the PZT piezoelectric thick film is designed and fabricated with this method too. Then the chip’s function and practicability could be increased. The maxium flow rate is 350μl/min, and could complete rinsing microchannel up to 487 times in 1 min. The function of solution introducing, rinsing and sample injection could be completed. Especially, the fabrication process of the PMMA micropump is compatibile with MCE chip’s. Integrated design and processing mode improve the availability of the chip.
With the TW30SX photodiode as the photo-electric converter device, a UV detection system is set up. With this detection system, 10-11A magnitude of photocurrent can be detected. The total delay time is between 150 ~ 250ms, the output noise is±5mV, the weight of the whole detect system is about 2kg, and the volume is 280×200×150mm3. The absorption rate of single-layer PMMA chip is 0.035at UV light source with the system, while attenuation of the light strength is 16.87% with the bonding PMMA chip.
The whole system electrophoresis experiment are developed with indirect UV detection methods. The inorganic anions(Cl-, SO42-)is used as the separation ions. The background buffer solution is 10mmol/L Na2CrO4+0.5mmol/LCTAB, which electroosmotic test results show that the direction of EOF flow is from the cathode to the anode, reversal with the borax buffer. And the mobility of this buffer isμeo=9.15×10-4cm2/Vs. This condition is suitable for the test demand of inorganic anion electrophoresis experiment
Finally, with the electromigration sample injection mode in a single micro- channel MCE chip, inorganic anion Cl-and SO42- electrophoresis peak are detected with indirect UV detection method. The single-peak repeatability results are better, also the mixed two ions could achieve the baseline separation, the detection limit is 10-9mol. The performance and stability are better during the experiment of PMMA MCE with the portable detection system. All these work lay a stable foundation for the PMMA MCE further application in the UV absorbance detection.
引文
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