提高环烯烃共聚物微芯片电泳分离性能的途径及应用
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摘要
微流控芯片电泳具有试剂和样品量消耗少、分析速度快等优势,但玻璃和石英芯片的制作成本较高,限制了其广泛应用。聚合物芯片易制作、成本低,可替代玻璃和石英芯片,但大多数塑料表面疏水性强,会非特异性吸附生物大分子,不仅污染通道表面,而且改变电渗流,影响分离效率和分析的重现性。另外,电泳芯片的分离通道一般比较短,储液池中细微的液面差异或液面形状变化都会在通道中引起额外的压力流动,影响分离柱效和重现性。
为了解决上述问题,本学位论文在前人工作的基础上,致力于探索消除分析物或样品基体非特异性吸附,提高分离效率和分析重现性的途径。本论文主要的创新工作包括：
(1)以羟丙基纤维素(HPC)为缓冲添加剂,建立了一种快速检测农产品中草甘膦和草铵膦残留的COC芯片电泳方法。
(2)以两种罗丹明染料为模型分析物考察了聚丙烯酸作为COC芯片电泳多功能缓冲添加剂的性能,并将其应用于食品中罗丹明染料的检测。
(3)以丙酮和环己烷的混合溶液作为通道修复溶剂,实现了几种具有缺陷的COC芯片微通道的原位修复。
(4)通过在简单的十字芯片上增加额外的样品废液池和缓冲废液池,设计了一种适合夹切进样的电泳芯片,提高了分析的重现性。
论文共分为五章：
第一章：对微流控技术做了系统的介绍,内容包括微芯片的制作、微芯片电泳样品的引入方式、微通道表面修饰和微芯片电泳在样品分析中的应用。
第二章：建立了一种激光诱导荧光-芯片电泳快速高效无干扰的检测草甘膦和草铵膦的新方法。系统优化了分离条件,在含有10mmol/L硼砂和2.0%羟丙基纤维素、pH为9.0的分离缓冲液中,草甘膦和草铵膦能从样品基质中成功分离。这两种分析物的理论塔板数高达1.0×106/m,草甘膦和草铵膦的检测限分别是0.34和0.18μg/L。在不经任何样品富集条件下,该方法被成功用于检测黄河水样、西兰花、大豆中这两种农残的含量,这些样品中草甘膦和草铵膦的加标回收率分别在84.0-101.0%和90.0-103.0%之间。
第三章：以聚丙烯酸为多功能缓冲添加剂动态修饰COC通道内表面,减少了罗丹明类染料在通道表面的吸附,增加了缓冲液的粘度减小了压差流动对分离柱效的影响,实现了罗丹明B和罗丹明6G在COC芯片中的高效分离。考察了电泳条件对分离的影响,在含有20,mmol/L磷酸盐、0.36%(m/v)聚丙烯酸、70%甲醇,pH为5.0的缓冲液中,分离的理论塔板数可达到7.0×105/m。在选定的分离条件下,罗丹明B和罗丹明6G迁移时间的RSD不大于0.72%,峰面积的RSD不大于2.6%,两种分析物的检测限分别为0.17和0.34μg/L。RITC标记的氨基酸也能达到很好的分离。实验还发现聚丙烯酸的存在会显著影响表观电渗流,在聚丙烯酸浓度较高时甚至能反转表观电渗流的方向。该法被用于检测番茄酱、火腿肠、辣椒面中这两种罗丹明染料的含量,加标回收率为84.6-106.7%。
第四章：利用对COC有一定溶解性能的混合溶剂冲洗COC芯片微通道,实现了微通道的表面的清洗更新和缺陷修复。本工作考察了体积比为8：2的丙酮和环己烷溶液流过COC芯片微通道对各种性能不佳的芯片的修复效果,实验结果证实对于外观这种方法对微通道表面性质不均匀、芯片通道封接不严、微通道被蛋白污染等情况都有明显的修复效果,原子力显微镜测试显示该溶剂修复可以降低微通道表面的粗糙程度。经过该法修复的芯片可以实现多种氨基酸的高效分离,但该法不适合通道堵塞的芯片修复。同时,实验结果还证明对于性能良好的芯片,经过该法处理分析柱效基本不变,不会对无缺陷芯片带来不良影响。
第五章：设计了一种新的芯片结构,在简单的十字芯片基础上增加了额外的样品废液池和缓冲废液池,并且在样品池和额外的样品废液池、缓冲废液池和额外的缓冲废液池之间维持合适的液面差,电极置于这两个额外的储液池中。进样时,由于液面差的存在,在样品池和额外的样品废液池之间的通道里样品持续流动,避免了样品被分离时回退的溶液稀释,提高分析的重现性。还可以通过优化样品引入的时间,保证目标分析物进入分离通道,剔除迁移速度较慢的基体和其它组分。额外缓冲废液池的使用则避免了前一次分析的样品在样品引入阶段返回分离通道而引起的基线抬高,有利于提高分析重现性。实验结果证实采用这种结构的芯片两种FITC标记的有机磷除草剂可以连续分析120以上。
Microchip based capillary electrophoresis (MCE) exhibits the advantages of extremely low consumption of reagents/samples and fast analysis speed, but the relative high fabrication cost of glass or fused silica chips limits its wide application in the real analysis. Polymer microchips can be easily made and are much cheaper, have been widely used instead of glass and fused silica chips. However, most of plastics are hydrophobic and can unspecifically adsorb biomacromolecules, which cause surface contamination and alter the electroosmotic flow, degrade the separation efficiency and repeatability. Also, due to the relative short separation channel of microchips, tiny difference of liquid levels or surface curvature in different reservoirs may cause unexpected pressurized flow, negatively affect the separation efficiency and the repeatability.
Therefore, this dissertation focuses on the research of approaches to eliminate the non-specific adsorption of analytes or sample matrices, and improve the separation efficiency and reproducibility. Originalities include:
(1) Hydroxypropyl cellulose (HPC) was used as buffer additive to establish a method for rapid separation and detection of glyphosate and glufosinate residues in agricultural products.
(2) Polyacrylic acid was used as a multifunctional additive in COC microchip electrophoresis for the separation of rhodamine B (RhB) and rhodamine6G (Rh6G). The method was used for the detection of these two compounds in rea food samples.
(3) A mixed solution of acetone and cyclohexane as a solvent for channel restoration, defective channels were successfully repaired.
(4) By implementation of an extra sample waste reservoir and an extra buffer waste reservoir on the cross chip, a design for effective pinched injection was proposed and the performance of the chip electrophoresis was improved.
The thesis contains five chapters:
Chapter1:Microfluidics was shortly reviewed, manily on the fabrication process of the microchips, injection methods for microchip electrophoresis, surface modification of microfluidic channels and the applications of microchip electrophoresis in real analysis.
Chapter2:A microchip electrophoresis system with laser induced fluorescence (LIF) detection for rapid and sensitive analysis of glyphosate (GLYP) and glufosinate (GLUF) residues was described. Systematic optimization of experimental conditions was performed to achieve highly efficient analysis. Under the selected condition, GLYP and GLUF were efficiently resolved from sample matrices with a buffer containing10mmol/L borax and2.0%(m/v) hydroxypropyl cellulose at pH9.0. The number of theoretical plates of1.0×106/m was attained for both analytes. Derivatization at lower concentrations (＜10μg/L) was also examined, successful detection of0.34μg/L GLYP and0.18μg/L GLUF was confirmed. The system was applied for the determination of both analytes in real samples without any preconcentration involved. Recoveries of GLYP and GLUF spiked in these samples were84.0-101.0%and90.0-103.0%, respectively.
Chapter3:This chapter describes the utilization of polyacrylic acid as multifunctional buffer additive for dynamic modification of COC to reduce adsorption of rhodamine dyes onto the microchannel, and suppress the effect of pressurized flow for the separation efficieny through increase of the buffer viscosity. Rhodamine B (RhB) and Rhodamine6G (Rh6G) were well resolved. The parameters were systematically investigated and by using20mmol/L phosphate buffer with0.36%polyacrylic acid in70%methanol at pH5.0, the number of theoretical plates of7.0×105/m was achieved. Under the selected condition, the relative standard deviations of RhB and Rh6G detection (n=5) were not more than0.72%for migration time, and not more than2.6%for peak area, respectively. Limits of detection (S/N=3) were0.17and0.34μg/L repectively. RITC-labeled amino acids were well resolved with the selected system too. It was also found that polyacrylic acid might significantly affect the observed electroosmotic flow, and it was even reversed at higher polyacrylic acid concentrations. The method was applied for the determination both RhB and Rh6G in ham, tomato sauce, chili and satisfactory standard addition recoveries of84.6-106.7%were attained.
Chapter4:Remedy and surface recovery of COC microchannels were realized through rinsing with a solvent mixture that with proper solubility of COC. In this work, the effectiveness of rinsing of microchannel with a8:2(v/v) mixture of acetone and cyclohexane for recovering the microchips with poor performance was examined. The results indicated that the method could recover the mcirochips with non-uniform surface status, improperly sealed microchannels and contaminated microcahnnels by proteins. Atomic force microscopic measurement showed that the solvent rinsing could reduce the roughness of the microchannel surfaces. Efficient separation of several FITC labeled amino acids was achieved on microchips remedied by the proposed method. But the clogged microchannels could not be remedied by this method. Meanwhile, the experiments also showed that for microchip with good performance, treatment with the solvent mixture had no adverse effects.
Chapter5:A novel chip structure is used, with an extra sample waste reservoir and an extra buffer waste reservoir. Proper level differences between sample reservoir and the extra sample waste reservoir, buffer waste reservoir and the extra buffer waste reservoir were maintained. The electrodes were placed in the extra sample and buffer waste reservoirs. In the sample injection stage, because of the continuous flow in the channel between the sample reservoir and the extra sample waste reservoir, the retreated sample was conducted into the extra sample waste reservoir, preventing the dilution of the sample by the retreated buffer and improving the reproducibility. By optimizing the sample injection time, it may be possible to ensure analytes entering the separation channel while removing the slower migrating sample matrices and other components. The use of extra buffer waster reservoir may avoid the waste sample of the previous separation entering the separation channel and leading to baseline elevation, which is also benefical to the reproducibility. Experimental results showed that FITC labeled organophosphorus herbicides could be repeatedly analyzed more than120times on the proposed microchips.
为了解决上述问题,本学位论文在前人工作的基础上,致力于探索消除分析物或样品基体非特异性吸附,提高分离效率和分析重现性的途径。本论文主要的创新工作包括：
(1)以羟丙基纤维素(HPC)为缓冲添加剂,建立了一种快速检测农产品中草甘膦和草铵膦残留的COC芯片电泳方法。
(2)以两种罗丹明染料为模型分析物考察了聚丙烯酸作为COC芯片电泳多功能缓冲添加剂的性能,并将其应用于食品中罗丹明染料的检测。
(3)以丙酮和环己烷的混合溶液作为通道修复溶剂,实现了几种具有缺陷的COC芯片微通道的原位修复。
(4)通过在简单的十字芯片上增加额外的样品废液池和缓冲废液池,设计了一种适合夹切进样的电泳芯片,提高了分析的重现性。
论文共分为五章：
第一章：对微流控技术做了系统的介绍,内容包括微芯片的制作、微芯片电泳样品的引入方式、微通道表面修饰和微芯片电泳在样品分析中的应用。
第二章：建立了一种激光诱导荧光-芯片电泳快速高效无干扰的检测草甘膦和草铵膦的新方法。系统优化了分离条件,在含有10mmol/L硼砂和2.0%羟丙基纤维素、pH为9.0的分离缓冲液中,草甘膦和草铵膦能从样品基质中成功分离。这两种分析物的理论塔板数高达1.0×106/m,草甘膦和草铵膦的检测限分别是0.34和0.18μg/L。在不经任何样品富集条件下,该方法被成功用于检测黄河水样、西兰花、大豆中这两种农残的含量,这些样品中草甘膦和草铵膦的加标回收率分别在84.0-101.0%和90.0-103.0%之间。
第三章：以聚丙烯酸为多功能缓冲添加剂动态修饰COC通道内表面,减少了罗丹明类染料在通道表面的吸附,增加了缓冲液的粘度减小了压差流动对分离柱效的影响,实现了罗丹明B和罗丹明6G在COC芯片中的高效分离。考察了电泳条件对分离的影响,在含有20,mmol/L磷酸盐、0.36%(m/v)聚丙烯酸、70%甲醇,pH为5.0的缓冲液中,分离的理论塔板数可达到7.0×105/m。在选定的分离条件下,罗丹明B和罗丹明6G迁移时间的RSD不大于0.72%,峰面积的RSD不大于2.6%,两种分析物的检测限分别为0.17和0.34μg/L。RITC标记的氨基酸也能达到很好的分离。实验还发现聚丙烯酸的存在会显著影响表观电渗流,在聚丙烯酸浓度较高时甚至能反转表观电渗流的方向。该法被用于检测番茄酱、火腿肠、辣椒面中这两种罗丹明染料的含量,加标回收率为84.6-106.7%。
第四章：利用对COC有一定溶解性能的混合溶剂冲洗COC芯片微通道,实现了微通道的表面的清洗更新和缺陷修复。本工作考察了体积比为8：2的丙酮和环己烷溶液流过COC芯片微通道对各种性能不佳的芯片的修复效果,实验结果证实对于外观这种方法对微通道表面性质不均匀、芯片通道封接不严、微通道被蛋白污染等情况都有明显的修复效果,原子力显微镜测试显示该溶剂修复可以降低微通道表面的粗糙程度。经过该法修复的芯片可以实现多种氨基酸的高效分离,但该法不适合通道堵塞的芯片修复。同时,实验结果还证明对于性能良好的芯片,经过该法处理分析柱效基本不变,不会对无缺陷芯片带来不良影响。
第五章：设计了一种新的芯片结构,在简单的十字芯片基础上增加了额外的样品废液池和缓冲废液池,并且在样品池和额外的样品废液池、缓冲废液池和额外的缓冲废液池之间维持合适的液面差,电极置于这两个额外的储液池中。进样时,由于液面差的存在,在样品池和额外的样品废液池之间的通道里样品持续流动,避免了样品被分离时回退的溶液稀释,提高分析的重现性。还可以通过优化样品引入的时间,保证目标分析物进入分离通道,剔除迁移速度较慢的基体和其它组分。额外缓冲废液池的使用则避免了前一次分析的样品在样品引入阶段返回分离通道而引起的基线抬高,有利于提高分析重现性。实验结果证实采用这种结构的芯片两种FITC标记的有机磷除草剂可以连续分析120以上。
Microchip based capillary electrophoresis (MCE) exhibits the advantages of extremely low consumption of reagents/samples and fast analysis speed, but the relative high fabrication cost of glass or fused silica chips limits its wide application in the real analysis. Polymer microchips can be easily made and are much cheaper, have been widely used instead of glass and fused silica chips. However, most of plastics are hydrophobic and can unspecifically adsorb biomacromolecules, which cause surface contamination and alter the electroosmotic flow, degrade the separation efficiency and repeatability. Also, due to the relative short separation channel of microchips, tiny difference of liquid levels or surface curvature in different reservoirs may cause unexpected pressurized flow, negatively affect the separation efficiency and the repeatability.
Therefore, this dissertation focuses on the research of approaches to eliminate the non-specific adsorption of analytes or sample matrices, and improve the separation efficiency and reproducibility. Originalities include:
(1) Hydroxypropyl cellulose (HPC) was used as buffer additive to establish a method for rapid separation and detection of glyphosate and glufosinate residues in agricultural products.
(2) Polyacrylic acid was used as a multifunctional additive in COC microchip electrophoresis for the separation of rhodamine B (RhB) and rhodamine6G (Rh6G). The method was used for the detection of these two compounds in rea food samples.
(3) A mixed solution of acetone and cyclohexane as a solvent for channel restoration, defective channels were successfully repaired.
(4) By implementation of an extra sample waste reservoir and an extra buffer waste reservoir on the cross chip, a design for effective pinched injection was proposed and the performance of the chip electrophoresis was improved.
The thesis contains five chapters:
Chapter1:Microfluidics was shortly reviewed, manily on the fabrication process of the microchips, injection methods for microchip electrophoresis, surface modification of microfluidic channels and the applications of microchip electrophoresis in real analysis.
Chapter2:A microchip electrophoresis system with laser induced fluorescence (LIF) detection for rapid and sensitive analysis of glyphosate (GLYP) and glufosinate (GLUF) residues was described. Systematic optimization of experimental conditions was performed to achieve highly efficient analysis. Under the selected condition, GLYP and GLUF were efficiently resolved from sample matrices with a buffer containing10mmol/L borax and2.0%(m/v) hydroxypropyl cellulose at pH9.0. The number of theoretical plates of1.0×106/m was attained for both analytes. Derivatization at lower concentrations (＜10μg/L) was also examined, successful detection of0.34μg/L GLYP and0.18μg/L GLUF was confirmed. The system was applied for the determination of both analytes in real samples without any preconcentration involved. Recoveries of GLYP and GLUF spiked in these samples were84.0-101.0%and90.0-103.0%, respectively.
Chapter3:This chapter describes the utilization of polyacrylic acid as multifunctional buffer additive for dynamic modification of COC to reduce adsorption of rhodamine dyes onto the microchannel, and suppress the effect of pressurized flow for the separation efficieny through increase of the buffer viscosity. Rhodamine B (RhB) and Rhodamine6G (Rh6G) were well resolved. The parameters were systematically investigated and by using20mmol/L phosphate buffer with0.36%polyacrylic acid in70%methanol at pH5.0, the number of theoretical plates of7.0×105/m was achieved. Under the selected condition, the relative standard deviations of RhB and Rh6G detection (n=5) were not more than0.72%for migration time, and not more than2.6%for peak area, respectively. Limits of detection (S/N=3) were0.17and0.34μg/L repectively. RITC-labeled amino acids were well resolved with the selected system too. It was also found that polyacrylic acid might significantly affect the observed electroosmotic flow, and it was even reversed at higher polyacrylic acid concentrations. The method was applied for the determination both RhB and Rh6G in ham, tomato sauce, chili and satisfactory standard addition recoveries of84.6-106.7%were attained.
Chapter4:Remedy and surface recovery of COC microchannels were realized through rinsing with a solvent mixture that with proper solubility of COC. In this work, the effectiveness of rinsing of microchannel with a8:2(v/v) mixture of acetone and cyclohexane for recovering the microchips with poor performance was examined. The results indicated that the method could recover the mcirochips with non-uniform surface status, improperly sealed microchannels and contaminated microcahnnels by proteins. Atomic force microscopic measurement showed that the solvent rinsing could reduce the roughness of the microchannel surfaces. Efficient separation of several FITC labeled amino acids was achieved on microchips remedied by the proposed method. But the clogged microchannels could not be remedied by this method. Meanwhile, the experiments also showed that for microchip with good performance, treatment with the solvent mixture had no adverse effects.
Chapter5:A novel chip structure is used, with an extra sample waste reservoir and an extra buffer waste reservoir. Proper level differences between sample reservoir and the extra sample waste reservoir, buffer waste reservoir and the extra buffer waste reservoir were maintained. The electrodes were placed in the extra sample and buffer waste reservoirs. In the sample injection stage, because of the continuous flow in the channel between the sample reservoir and the extra sample waste reservoir, the retreated sample was conducted into the extra sample waste reservoir, preventing the dilution of the sample by the retreated buffer and improving the reproducibility. By optimizing the sample injection time, it may be possible to ensure analytes entering the separation channel while removing the slower migrating sample matrices and other components. The use of extra buffer waster reservoir may avoid the waste sample of the previous separation entering the separation channel and leading to baseline elevation, which is also benefical to the reproducibility. Experimental results showed that FITC labeled organophosphorus herbicides could be repeatedly analyzed more than120times on the proposed microchips.
引文
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