微流控芯片及有机发光二极管荧光检测系统的研究
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摘要
本论文从分析方法和仪器搭建两方面入手,以体现微流控芯片系统快速、高通量、微型化和集成化几个特点为目标,进行了一些探索性工作:
针对电场力驱动细胞进样效率低的问题,利用储液池液面差引起的静压力驱动细胞向微通道中进样,然后在静压力和电场力共同作用下实现细胞的聚焦和迁移,在十字型微流控芯片上实现了流式细胞检测技术,并用于紫外线照射诱导HeLa细胞凋亡和坏死情况的半定量检测。
将传统流式细胞仪的各组成部分集成在一块微流控芯片上,发展了以细胞自身的重力为动力,驱动细胞在竖直放置的芯片微管道中运动的模式,避免了电场力驱动时对细胞的损伤作用引起的细胞凋亡,保持了细胞的活性。还发展了根据细胞表面带电荷情况不同,在重力和电场力共同作用下进行细胞分选的模式,并采用流体力学方法对细胞在重力场和电场中的受力和运动情况进行了分析。该系统不必外加泵阀等设备,进一步减小了体积,简化了结构。
上述微流控芯片流式细胞检测系统虽然通过微加工技术实现功能单元的集成,使得体积大为降低,但该系统采用的激光诱导荧光检测器体积庞大,成为芯片系统微型化的关键问题。针对这一问题,以有机发光二极管(OLED)为激发光源,搭建了用于微流控芯片的微型化荧光检测系统。针对OLED发射光谱范围宽的弱点,设计加工了厚度仅为300μm的激发光滤光片,将干扰荧光检测的激发光滤除～95%。对于Alexa532染料,在进样体积为0.7 nL时,得到的检测限为14 fmol,系统的灵敏度与文献报道的结果相比大幅提高。
根据OLED器件具有便于加工和可集成的特点,进一步以OLED作为二维光源,建立了微流控芯片等电聚焦(IEF)电泳荧光柱成像检测系统。与文献报道的柱成像检测系统相比体积大为降低,结构也大大简化。该系统可以对IEF过程进行实时动态监测,并实现了快速分离分析,15 s内完成整个聚焦过程和检测。荧光蛋白R-phycoerythrin的检出限约为2.5×10~(-9) mol/L,即进样体积75 nL的绝对检出量约45 pg。此外,还发展了三通道IEF柱成像检测,提高了系统的样品分析通量。
Several novel microfluidic systems including integrated flow cytometors and miniaturized fluorescence detectors using organic light emitting diode as single point or two dimensional light sources were developed for cellular manipulation, apoptotic analysis and whole column imaging detection, respectively.
A simple cross-channel microchip was employed for flow cytometry and single cell fluorescence detection, where cells were introduced into micro-channels and driven passing through the detector by combined effect of hydrodynamic and electric power. This miniaturized flow cytometor was applied to estimate the damage degree of HeLa cells exposed to ultraviolet (UV) radiation for 10, 20 and 40 min respectively by the number of cells determined and their fluorescence intensity.
As cells were found damaged in electric field when electric force was employed for driving cells in flow cytometor, a novel microfluidic device based on gravity driving was developed for flow cytometry and cell sorting. In the experiments, cells flowed spontaneously under their own gravity in an upright-put microchip, passed through the detection point and then entered into the sorting electric field one by one at average velocity of 0.55 mm/s for cell sorting. In order to study the dynamical and kinematical characteristics of single cell in gravity and electric field, physical and numerical module based on Newton’s Law of motion was established and optimized. This system was also applied to estimate necrotic and apoptotic effect of UV radiation on HeLa cells and results shown that UV radiation induced membrane damage contributed to apoptosis and necrosis of HeLa cells.
However, a bulky laser induced fluorescence detection system was still an obstacle for further miniaturization of microfluidic systems. Therefore, a simply fabricated microfluidic device using a green organic light emitting diode (OLED) and thin film interference filter as integrated excitation source was then presented and applied to fluorescence detection of proteins. A layer-by-layer compact systemconsisting of glass/PDMS microchip, pinhole, excitation filter and OLED were designed and equipped with a coaxial optical fiber for fluorescence detection. A 300μm thick excitation filter was employed for eliminating nearly 95% of the unwanted light emitted by OLED which had overlaped with fluorescence spectrum of the dyes. The distance between OLED illuminant and microchannels was limited to ~1mm for sensitive detection. This system was used for fluorescence detection of Rhodamine6G, Alexa532 and BSA conjugates in 4% linear polyacrymide (LPA) buffer (in 1×TBE, pH8.3) and 1.4 fmol of mass detection limit at 0.7nL injection volume for Alexa532 was obtained.
Although sensitivity achieved by the above OLED induced fluorescence detection system was roughly six orders of magnitude poorer than good laser-induced fluorescence detection in capillary electrophoresis because of its low irradiance and purity, OLEDs have the potential to be used as a two dimensional light source for whole column imaging detection in isoelectric focusing (IEF) systems, which is problematic for lasers and high performance LEDs. So an integrated and simplified microfluidic device using OLED array as the two dimensional light source for single- and multi-channel whole column imaging detection was developed and used for IEF of R-phycoerythrin. The IEF conditions were optimized and the total analysis time was extremely reduced to 15s for 1cm long microchannels at 700V/cm of electric field strength without the presence of electroosmotic flow (EOF). The compression of pH gradient caused by electrolytes drawning into the microchannels was efficiently restrained when 1% of hydroxylpropylmethyl cellulose (HPMC) in 2% ampholyte was used as the carrier for IEF. Under optimized IEF conditions, the detection limit of this system was～2.5×10~(-9)9 mol/Lor 45 pg at 75nL per column injection of R-phycoerythrin.
针对电场力驱动细胞进样效率低的问题,利用储液池液面差引起的静压力驱动细胞向微通道中进样,然后在静压力和电场力共同作用下实现细胞的聚焦和迁移,在十字型微流控芯片上实现了流式细胞检测技术,并用于紫外线照射诱导HeLa细胞凋亡和坏死情况的半定量检测。
将传统流式细胞仪的各组成部分集成在一块微流控芯片上,发展了以细胞自身的重力为动力,驱动细胞在竖直放置的芯片微管道中运动的模式,避免了电场力驱动时对细胞的损伤作用引起的细胞凋亡,保持了细胞的活性。还发展了根据细胞表面带电荷情况不同,在重力和电场力共同作用下进行细胞分选的模式,并采用流体力学方法对细胞在重力场和电场中的受力和运动情况进行了分析。该系统不必外加泵阀等设备,进一步减小了体积,简化了结构。
上述微流控芯片流式细胞检测系统虽然通过微加工技术实现功能单元的集成,使得体积大为降低,但该系统采用的激光诱导荧光检测器体积庞大,成为芯片系统微型化的关键问题。针对这一问题,以有机发光二极管(OLED)为激发光源,搭建了用于微流控芯片的微型化荧光检测系统。针对OLED发射光谱范围宽的弱点,设计加工了厚度仅为300μm的激发光滤光片,将干扰荧光检测的激发光滤除～95%。对于Alexa532染料,在进样体积为0.7 nL时,得到的检测限为14 fmol,系统的灵敏度与文献报道的结果相比大幅提高。
根据OLED器件具有便于加工和可集成的特点,进一步以OLED作为二维光源,建立了微流控芯片等电聚焦(IEF)电泳荧光柱成像检测系统。与文献报道的柱成像检测系统相比体积大为降低,结构也大大简化。该系统可以对IEF过程进行实时动态监测,并实现了快速分离分析,15 s内完成整个聚焦过程和检测。荧光蛋白R-phycoerythrin的检出限约为2.5×10~(-9) mol/L,即进样体积75 nL的绝对检出量约45 pg。此外,还发展了三通道IEF柱成像检测,提高了系统的样品分析通量。
Several novel microfluidic systems including integrated flow cytometors and miniaturized fluorescence detectors using organic light emitting diode as single point or two dimensional light sources were developed for cellular manipulation, apoptotic analysis and whole column imaging detection, respectively.
A simple cross-channel microchip was employed for flow cytometry and single cell fluorescence detection, where cells were introduced into micro-channels and driven passing through the detector by combined effect of hydrodynamic and electric power. This miniaturized flow cytometor was applied to estimate the damage degree of HeLa cells exposed to ultraviolet (UV) radiation for 10, 20 and 40 min respectively by the number of cells determined and their fluorescence intensity.
As cells were found damaged in electric field when electric force was employed for driving cells in flow cytometor, a novel microfluidic device based on gravity driving was developed for flow cytometry and cell sorting. In the experiments, cells flowed spontaneously under their own gravity in an upright-put microchip, passed through the detection point and then entered into the sorting electric field one by one at average velocity of 0.55 mm/s for cell sorting. In order to study the dynamical and kinematical characteristics of single cell in gravity and electric field, physical and numerical module based on Newton’s Law of motion was established and optimized. This system was also applied to estimate necrotic and apoptotic effect of UV radiation on HeLa cells and results shown that UV radiation induced membrane damage contributed to apoptosis and necrosis of HeLa cells.
However, a bulky laser induced fluorescence detection system was still an obstacle for further miniaturization of microfluidic systems. Therefore, a simply fabricated microfluidic device using a green organic light emitting diode (OLED) and thin film interference filter as integrated excitation source was then presented and applied to fluorescence detection of proteins. A layer-by-layer compact systemconsisting of glass/PDMS microchip, pinhole, excitation filter and OLED were designed and equipped with a coaxial optical fiber for fluorescence detection. A 300μm thick excitation filter was employed for eliminating nearly 95% of the unwanted light emitted by OLED which had overlaped with fluorescence spectrum of the dyes. The distance between OLED illuminant and microchannels was limited to ~1mm for sensitive detection. This system was used for fluorescence detection of Rhodamine6G, Alexa532 and BSA conjugates in 4% linear polyacrymide (LPA) buffer (in 1×TBE, pH8.3) and 1.4 fmol of mass detection limit at 0.7nL injection volume for Alexa532 was obtained.
Although sensitivity achieved by the above OLED induced fluorescence detection system was roughly six orders of magnitude poorer than good laser-induced fluorescence detection in capillary electrophoresis because of its low irradiance and purity, OLEDs have the potential to be used as a two dimensional light source for whole column imaging detection in isoelectric focusing (IEF) systems, which is problematic for lasers and high performance LEDs. So an integrated and simplified microfluidic device using OLED array as the two dimensional light source for single- and multi-channel whole column imaging detection was developed and used for IEF of R-phycoerythrin. The IEF conditions were optimized and the total analysis time was extremely reduced to 15s for 1cm long microchannels at 700V/cm of electric field strength without the presence of electroosmotic flow (EOF). The compression of pH gradient caused by electrolytes drawning into the microchannels was efficiently restrained when 1% of hydroxylpropylmethyl cellulose (HPMC) in 2% ampholyte was used as the carrier for IEF. Under optimized IEF conditions, the detection limit of this system was～2.5×10~(-9)9 mol/Lor 45 pg at 75nL per column injection of R-phycoerythrin.
引文
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