芯片电泳分离—电化学发光检测联用研究
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摘要
本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效分离和电化学发光检测高灵敏度为目标,进行了一些探索性工作:
第一部分,芯片电泳是近年来发展快速且具有广泛应用前景的新技术,其中PDMS电泳芯片的表面改性是目前研究的热点之一。聚苯乙烯微球作为色谱介质或被修饰到电泳管道内已引起科学家们的关注,但将纳米级聚苯乙烯微球固定到PDMS管道内尚未见报道。本文合成了纳米级表面荷正电的聚苯乙烯微球,并采用气-液界面自组装的方法将其制备成二维聚苯乙烯微球阵列,然后将该阵列转移至PDMS电泳管道内壁上,随后将聚阴离子-聚苯乙烯磺酸钠引入管道内,通过静电吸附使管道表面带有丰富的负电荷,以改进管道内壁荷电情况。修饰后的电泳管道电渗流稳定,被分析物吸附减少,分离效率显著提高。将修饰好的电泳芯片与柱端碳盘电极电化学检测联用,用于多巴胺、5-羟色胺、肾上腺素这3种神经递质以及儿茶酚的分离,这4种物质在修饰后的管道上达到了基线分离,其中多巴胺和5-羟色胺的分离度达到了12.5,显著优于已有文献报道。
第二部分,电化学发光分析法(ECL)由于其可控性好、灵敏度高、仪器简单等优点已成功应用于环境科学、生命科学和材料科学等领域。鲁米诺是最常见的发光试剂之一,它具备很好的发光性能,尤其是有活性氧存在时强烈增敏其发光,提供了优异的检测平台。鲁米诺的电化学发光作为酶催化反应的信号输出,研制ECL生物传感器是现代分析化学的前沿领域之一。本文在碳纳米管/铁氰化钾修饰的铂电极上吸附胆碱氧化酶(ChOx),基于鲁米诺的电化学发光构建了一种对胆碱有灵敏和选择性响应的ECL生物传感器,实验优化了传感器研制和检测的最佳条件。酶反应中产生的过氧化氢增强鲁米诺的电化学发光强度,从而可得到发光强度与溶液中底物胆碱浓度的线性关系。对所制备传感器分析性能进行了表征,并应用于大鼠血清中胆碱的检测。该传感器显示了对胆碱的快速响应并具有良好的稳定性和重复性,较宽的线性范围和更低的检出限,为胆碱检测在医疗领域的应用提供了一种高性能的新方法。
第三部分,电化学发光修饰电极提高和改善了电化学发光的分析性能,ITO玻璃作为一种常见修饰电极基底,具有成本低、表面电阻小、光透性好等优点。实验中发现,在碱性溶液中没有发光试剂存在时,向ITO电极施加脉冲电压依然可监测到发光信号,本文对这一发光现象及其机理进行了探索。实验证明:发光实体为溶液中的活性氧,而ITO玻璃对发光有增强作用,向ITO施加脉冲电压时其对发光的增强作用更为明显。量子点作为一种新兴的电化学发光纳米材料,具有高灵敏度、高选择性、快速和成本低等优点。且研究表明,纳米金或碳纳米管对量子点电化学发光有增强作用。本文合成了水溶性CdTe量子点,并将其与碳纳米管和壳聚糖纳米复合材料混合修饰到导电玻璃(ITO)电极上,制备了性能稳定的电化学发光电极并将其应用到分析检测中。由于不同的研究工作对电化学发光仪器的要求不尽相同,所以至今电化学发光研究所用的仪器多由研究者自己自行设计、搭建,尤其是电化学发光池的设计与使用更是如此。本文设计了一种基于CdTe量子点纳米复合材料修饰ITO电化学发光电极的纳升级微型电化学发光检测池。其特征在于:检测池体积小、几无死体积;测试溶液输入流路正对工作电极、灵敏度高;ITO玻璃电极兼具发光池光窗的作用,正对PMT光窗,光信号采集效率高。将流动注射装置与该发光池联用,结合了电化学发光的高灵敏度和流动注射的实用性,单流路,简单高效,并以此为基础,优化了电化学发光脉冲电压、助反应剂三乙胺(TEA)的浓度和流动注射流速等因素,研究了对电化学发光有猝灭作用的多巴胺的检测,检测限为3.6pM,质量检测限达1.08amol,是目前可检索文献中灵敏度最高的方法之一。
第四部分,微型电化学发光检测池研制的终极目标就是使之与芯片电泳分离联用,以实现高效分离和高效检测的结合。本文对芯片电泳分离-电化学发光检测联用技术进行了初步研究。考察了电泳高压场对电化学发光过程的影响,工作电极和芯片电泳管道出口端的距离等因素,并在此联用装置上实现了对多巴胺的检测。初步研究结果为下一步研究指明了方向,包括:1、结合本研究中已经采用的聚苯乙烯纳米小球分离管道修饰技术以提高分离效率,改善溶质峰峰形并同时得到更高检测灵敏度;2、对分离管道尺寸和检测池的匹配进行优化,以充分发挥多巴胺等物质对CdTe量子点电化学发光的猝灭效率而提高检测灵敏度;3、针对芯片电泳进样量较小限制检测灵敏度这一重要因素,采用进样富集等技术进一步提高检测灵敏度。
Including in the fast progress in development of microchip analytic technique in the past decade, studies on modification of microchennal and miniaturization of detector appear to be the hot research topics. Electrochemiluminescence (ECL), also known as electrogenerated chemiluminescence, is an excellent analytic method that possessed the advantages of both electrochemical and chemiluminescent methods. It has the benefits such as simplicity, inexpensive instrumentation, low background noise, high sensitivity, good controllability and wide dynamic range. The present paper focuses on the modification of microchannel, fabrication of ECL detection cell and their hyphenated technique.
A poly(dimethylsiloxane) (PDMS) microchip with an amperometric detector was developed for the electrophoretic separation and determination of neurotransmitters. Although the polystyrene (PS) microsphere is widely used as the modifier for chromatography, there is no report on electrophoretic separation on self-assembled nano-PS modified PDMS microchannel. To enhance the separation efficiency, the positive charged PS nano-sphere (PSNS) has been solvothermal synthesized and was self-assembly modified onto a PDMS microchannel to obtain a quasi-ordered PSNS monolayer. Followed by driving through the PSS solution, the final PSNS/PSS modified layer was built on the channel surface. Thus a stable electroosmotic ?ow (EOF) and high separation efficiency are obtained in resulted modified microchannel. Under optimized conditions, dopamine, epinephrine, catechol, and serotonin are acceptably baseline separated in this 3.5 cm length separation channel with the theoretical plate number from 4.6×104 to 2.1×105 per meter and resolution from 1.29 to 12.5, is obviously higher than some reported papers. The practicability of this microchip is validated by the recovery test with cerebrospinal ?uid as real sample which resulted from 91.7% to 106.5%.
ECL is a simple and sensitive method for analytical detection. It provides a real-time analytical approach. An ECL choline biosensor is developed by drop- coating of choline oxidase (ChOx) onto a carbon nanotubes (CNTs) / potassium ferricyanide modified platinum electrode with ECL of luminol as readout signal. Due to the improvement of biocompatibility and electron transfer of electrode surface from CNTs, meanwhile the activation for enzyme and the ECL emission from K3Fe(CN)6, the developed biosensor possesses excellent analytical properties. It gives optimal results while the Pt basal electrode was modified with 4μL of 0.33g/L CNTs dispersoid, 2μL of 0.1mol/L K3Fe(CN)6 and 1.5U of ChOx. In the PBS buffer (pH 7.4) containing 8×10-6mol/L luminol, the ECL signal linearly responded the concentration of choline from 1×10-7mol/L to 4×10-3mol/L (r=0.994) with detection limit of 1.21×10-8mol/L under 30°C of detection temperature. The developed biosensor was applied to assay the concentration of choline in rat blood sample. The result of 0.268 mg/100mL was obtained with average recovery of 101.1%. It shows a fast response to choline with good reproducibility.
Indium tin oxide (ITO) glass is generally used as the substrate of modified electrode, but the authors have once found a very significant phenomenon when using the indium tin oxide (ITO) glass as the anode in electrochemical studies. There were notable luminescent signals had been observed when a pulse potential was applied on the ITO electrode in alkaline solution without any luminescent reagent.The mechanism of this luminescence was discussed in details. After all of the studies, it was revealed that the yielded reactive oxygen species (ROSs) as O2??, OH˙ and H2O2 during the electrolysis and whereafter the produced singlet oxygen (1O2) acted as the indispensable right-emitting entity and the ITO was a critical determinant intensifier.
Since the first work on the ECL of silicon quantum dots (QDs) was reported, the ECL phenomena of CdS, CdSe and CdTe in the presence of co-reactants have been studied, and further applied for the development of ECL sensors. There the TGA-capped CdTe QDs were synthesized in our lab by an improved method, which had a high quantum yield of over 50%, and an ECL electrode has been developed which was constructed based on the immobilization of TGA-capped CdTe QDs by nanocomposite of CNTs and chitosan (Chit) on ITO glass. The developed ECL electrode displayed high ECL intensity, excellent stability and good biocompatibility. It provides an excellent platform for further applications.
To miniaturize the analytical detector is one of the trends of instrumental analysis. A nano-liter sized ?ow-cell is developed for constructing a ?ow injection analytic (FIA) system with ECL detection. The CdTe QDs composite modified ECL electrode is applied as the working electrode in this ?ow-cell. It has been demonstrated to have the efficient anodic ECL performance with the triethylamine (TEA) as the co-reactant. The ?ow-cell gives the stable ECL background under optimized conditions for parameters such as electrolytic pulse, concentration of TEA and ?ow rate, etc. The sensitive ECL quenching response of dopamine (DA) is realized on this FIA system within the linear range from 10 pM to 4 nM and a detection limit as low as 3.6 pM. It is practically used to determine the neurotransmitters in cerebro-spinal ?uid (CSF) with DA as the index and with an average recovery of 94%.
And then the nano-liter sized ECL detection cell was hyphenated with PDMS microchip to construct a microchip-ECL electrophoretic system. The preliminary study of this microchip-ECL system reveals the practicability after the investigation on pivotal factors such as the effect of high voltage on luminescence detection and the distance between microchannel and detector. There is a confirmable electrophoretic peak of dopamine to be recorded in this microchip-ECL electrophoretic system. The study provides the foundation of experimental technique, and suggests the direction for the further researches. 1, Apply the channel modification technique with PSNS to improve the separation efficiency, outline of electrophoretic peak and therefore the detection sensitivity; 2, Optimize the matching degree between separation channel and the detection cell to improve the detection performance; 3, Adopt the technique of sampling-enrichment to conquer the limitation of little sampling quantity to enhance the detection sensitivity.
第一部分,芯片电泳是近年来发展快速且具有广泛应用前景的新技术,其中PDMS电泳芯片的表面改性是目前研究的热点之一。聚苯乙烯微球作为色谱介质或被修饰到电泳管道内已引起科学家们的关注,但将纳米级聚苯乙烯微球固定到PDMS管道内尚未见报道。本文合成了纳米级表面荷正电的聚苯乙烯微球,并采用气-液界面自组装的方法将其制备成二维聚苯乙烯微球阵列,然后将该阵列转移至PDMS电泳管道内壁上,随后将聚阴离子-聚苯乙烯磺酸钠引入管道内,通过静电吸附使管道表面带有丰富的负电荷,以改进管道内壁荷电情况。修饰后的电泳管道电渗流稳定,被分析物吸附减少,分离效率显著提高。将修饰好的电泳芯片与柱端碳盘电极电化学检测联用,用于多巴胺、5-羟色胺、肾上腺素这3种神经递质以及儿茶酚的分离,这4种物质在修饰后的管道上达到了基线分离,其中多巴胺和5-羟色胺的分离度达到了12.5,显著优于已有文献报道。
第二部分,电化学发光分析法(ECL)由于其可控性好、灵敏度高、仪器简单等优点已成功应用于环境科学、生命科学和材料科学等领域。鲁米诺是最常见的发光试剂之一,它具备很好的发光性能,尤其是有活性氧存在时强烈增敏其发光,提供了优异的检测平台。鲁米诺的电化学发光作为酶催化反应的信号输出,研制ECL生物传感器是现代分析化学的前沿领域之一。本文在碳纳米管/铁氰化钾修饰的铂电极上吸附胆碱氧化酶(ChOx),基于鲁米诺的电化学发光构建了一种对胆碱有灵敏和选择性响应的ECL生物传感器,实验优化了传感器研制和检测的最佳条件。酶反应中产生的过氧化氢增强鲁米诺的电化学发光强度,从而可得到发光强度与溶液中底物胆碱浓度的线性关系。对所制备传感器分析性能进行了表征,并应用于大鼠血清中胆碱的检测。该传感器显示了对胆碱的快速响应并具有良好的稳定性和重复性,较宽的线性范围和更低的检出限,为胆碱检测在医疗领域的应用提供了一种高性能的新方法。
第三部分,电化学发光修饰电极提高和改善了电化学发光的分析性能,ITO玻璃作为一种常见修饰电极基底,具有成本低、表面电阻小、光透性好等优点。实验中发现,在碱性溶液中没有发光试剂存在时,向ITO电极施加脉冲电压依然可监测到发光信号,本文对这一发光现象及其机理进行了探索。实验证明:发光实体为溶液中的活性氧,而ITO玻璃对发光有增强作用,向ITO施加脉冲电压时其对发光的增强作用更为明显。量子点作为一种新兴的电化学发光纳米材料,具有高灵敏度、高选择性、快速和成本低等优点。且研究表明,纳米金或碳纳米管对量子点电化学发光有增强作用。本文合成了水溶性CdTe量子点,并将其与碳纳米管和壳聚糖纳米复合材料混合修饰到导电玻璃(ITO)电极上,制备了性能稳定的电化学发光电极并将其应用到分析检测中。由于不同的研究工作对电化学发光仪器的要求不尽相同,所以至今电化学发光研究所用的仪器多由研究者自己自行设计、搭建,尤其是电化学发光池的设计与使用更是如此。本文设计了一种基于CdTe量子点纳米复合材料修饰ITO电化学发光电极的纳升级微型电化学发光检测池。其特征在于:检测池体积小、几无死体积;测试溶液输入流路正对工作电极、灵敏度高;ITO玻璃电极兼具发光池光窗的作用,正对PMT光窗,光信号采集效率高。将流动注射装置与该发光池联用,结合了电化学发光的高灵敏度和流动注射的实用性,单流路,简单高效,并以此为基础,优化了电化学发光脉冲电压、助反应剂三乙胺(TEA)的浓度和流动注射流速等因素,研究了对电化学发光有猝灭作用的多巴胺的检测,检测限为3.6pM,质量检测限达1.08amol,是目前可检索文献中灵敏度最高的方法之一。
第四部分,微型电化学发光检测池研制的终极目标就是使之与芯片电泳分离联用,以实现高效分离和高效检测的结合。本文对芯片电泳分离-电化学发光检测联用技术进行了初步研究。考察了电泳高压场对电化学发光过程的影响,工作电极和芯片电泳管道出口端的距离等因素,并在此联用装置上实现了对多巴胺的检测。初步研究结果为下一步研究指明了方向,包括:1、结合本研究中已经采用的聚苯乙烯纳米小球分离管道修饰技术以提高分离效率,改善溶质峰峰形并同时得到更高检测灵敏度;2、对分离管道尺寸和检测池的匹配进行优化,以充分发挥多巴胺等物质对CdTe量子点电化学发光的猝灭效率而提高检测灵敏度;3、针对芯片电泳进样量较小限制检测灵敏度这一重要因素,采用进样富集等技术进一步提高检测灵敏度。
Including in the fast progress in development of microchip analytic technique in the past decade, studies on modification of microchennal and miniaturization of detector appear to be the hot research topics. Electrochemiluminescence (ECL), also known as electrogenerated chemiluminescence, is an excellent analytic method that possessed the advantages of both electrochemical and chemiluminescent methods. It has the benefits such as simplicity, inexpensive instrumentation, low background noise, high sensitivity, good controllability and wide dynamic range. The present paper focuses on the modification of microchannel, fabrication of ECL detection cell and their hyphenated technique.
A poly(dimethylsiloxane) (PDMS) microchip with an amperometric detector was developed for the electrophoretic separation and determination of neurotransmitters. Although the polystyrene (PS) microsphere is widely used as the modifier for chromatography, there is no report on electrophoretic separation on self-assembled nano-PS modified PDMS microchannel. To enhance the separation efficiency, the positive charged PS nano-sphere (PSNS) has been solvothermal synthesized and was self-assembly modified onto a PDMS microchannel to obtain a quasi-ordered PSNS monolayer. Followed by driving through the PSS solution, the final PSNS/PSS modified layer was built on the channel surface. Thus a stable electroosmotic ?ow (EOF) and high separation efficiency are obtained in resulted modified microchannel. Under optimized conditions, dopamine, epinephrine, catechol, and serotonin are acceptably baseline separated in this 3.5 cm length separation channel with the theoretical plate number from 4.6×104 to 2.1×105 per meter and resolution from 1.29 to 12.5, is obviously higher than some reported papers. The practicability of this microchip is validated by the recovery test with cerebrospinal ?uid as real sample which resulted from 91.7% to 106.5%.
ECL is a simple and sensitive method for analytical detection. It provides a real-time analytical approach. An ECL choline biosensor is developed by drop- coating of choline oxidase (ChOx) onto a carbon nanotubes (CNTs) / potassium ferricyanide modified platinum electrode with ECL of luminol as readout signal. Due to the improvement of biocompatibility and electron transfer of electrode surface from CNTs, meanwhile the activation for enzyme and the ECL emission from K3Fe(CN)6, the developed biosensor possesses excellent analytical properties. It gives optimal results while the Pt basal electrode was modified with 4μL of 0.33g/L CNTs dispersoid, 2μL of 0.1mol/L K3Fe(CN)6 and 1.5U of ChOx. In the PBS buffer (pH 7.4) containing 8×10-6mol/L luminol, the ECL signal linearly responded the concentration of choline from 1×10-7mol/L to 4×10-3mol/L (r=0.994) with detection limit of 1.21×10-8mol/L under 30°C of detection temperature. The developed biosensor was applied to assay the concentration of choline in rat blood sample. The result of 0.268 mg/100mL was obtained with average recovery of 101.1%. It shows a fast response to choline with good reproducibility.
Indium tin oxide (ITO) glass is generally used as the substrate of modified electrode, but the authors have once found a very significant phenomenon when using the indium tin oxide (ITO) glass as the anode in electrochemical studies. There were notable luminescent signals had been observed when a pulse potential was applied on the ITO electrode in alkaline solution without any luminescent reagent.The mechanism of this luminescence was discussed in details. After all of the studies, it was revealed that the yielded reactive oxygen species (ROSs) as O2??, OH˙ and H2O2 during the electrolysis and whereafter the produced singlet oxygen (1O2) acted as the indispensable right-emitting entity and the ITO was a critical determinant intensifier.
Since the first work on the ECL of silicon quantum dots (QDs) was reported, the ECL phenomena of CdS, CdSe and CdTe in the presence of co-reactants have been studied, and further applied for the development of ECL sensors. There the TGA-capped CdTe QDs were synthesized in our lab by an improved method, which had a high quantum yield of over 50%, and an ECL electrode has been developed which was constructed based on the immobilization of TGA-capped CdTe QDs by nanocomposite of CNTs and chitosan (Chit) on ITO glass. The developed ECL electrode displayed high ECL intensity, excellent stability and good biocompatibility. It provides an excellent platform for further applications.
To miniaturize the analytical detector is one of the trends of instrumental analysis. A nano-liter sized ?ow-cell is developed for constructing a ?ow injection analytic (FIA) system with ECL detection. The CdTe QDs composite modified ECL electrode is applied as the working electrode in this ?ow-cell. It has been demonstrated to have the efficient anodic ECL performance with the triethylamine (TEA) as the co-reactant. The ?ow-cell gives the stable ECL background under optimized conditions for parameters such as electrolytic pulse, concentration of TEA and ?ow rate, etc. The sensitive ECL quenching response of dopamine (DA) is realized on this FIA system within the linear range from 10 pM to 4 nM and a detection limit as low as 3.6 pM. It is practically used to determine the neurotransmitters in cerebro-spinal ?uid (CSF) with DA as the index and with an average recovery of 94%.
And then the nano-liter sized ECL detection cell was hyphenated with PDMS microchip to construct a microchip-ECL electrophoretic system. The preliminary study of this microchip-ECL system reveals the practicability after the investigation on pivotal factors such as the effect of high voltage on luminescence detection and the distance between microchannel and detector. There is a confirmable electrophoretic peak of dopamine to be recorded in this microchip-ECL electrophoretic system. The study provides the foundation of experimental technique, and suggests the direction for the further researches. 1, Apply the channel modification technique with PSNS to improve the separation efficiency, outline of electrophoretic peak and therefore the detection sensitivity; 2, Optimize the matching degree between separation channel and the detection cell to improve the detection performance; 3, Adopt the technique of sampling-enrichment to conquer the limitation of little sampling quantity to enhance the detection sensitivity.
引文
1. A. Manz, N. Graber, H. M. Widmer, Sens.Actuators, 1990, B1, 244
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