蛋白质/多肽的微流控二维芯片电泳*

在微流控芯片上构建多维分离系统,为蛋白质组学研究提供了一个有发展前景的高效分离分析技术平台。本文介绍了二维芯片电泳系统耦联模式选取及正交性评价的方法;综述了针对蛋白质/多肽分离分析的各种耦联模式微流控二维芯片电泳分析系统,如胶束电动力学色谱（MEKC）与毛细管区带电泳（CZE）,开管电色谱（OECE）与CZE,等电聚焦（IEF）与CZE, IEF与SDS毛细管凝胶电泳（CGE）, SDS-CGE与MEKC等。特别对二维电泳芯片切换接口的类型进行了分类,探讨了用于微流控二维芯片电泳系统的检测技术,并展望了微流控二维电泳芯片在蛋白质组学研究中的应用前景和发展方向。     

二维毛细管区带电泳/胶束电动毛细管色谱分离尿样中的药物及其对映体

建立了毛细管区带电泳(CZE)/胶束电动毛细管色谱(MEKC)二维毛细管电泳分离平台,CZE毛细管和MEKC毛细管通过一段带微孔的聚四氟乙烯(polytetrafluoroethylene, PTFE)套管固定。样品在CZE毛细管中分离后进入MEKC毛细管进一步分离,在二维转换过程中采用动态pH连接-胶束扫集法避免第一维分离区带在接口处扩散。将该方法成功用于鼠尿样品中4种药物及其对映体的分离,各组分的理论塔板数为(2.8～4.3)×104/m,检出限为0.015～0.052 mg/L,实际样品中峰面积和迁移时间的相对标准偏差(n=7)分别为1.7%～3.8%和1.3%～4.6%。方法重现性好、灵敏度和分离度高、峰容量大,适用于尿样中多种药物组分及其对映体的同时分离检测。     

在线富集二维毛细管电泳分析新体系检测尿样中药物及对映体

将在线富集技术同二维(2D)毛细管电泳(CE)分离相结合同时提高复杂样品中痕量组分的分离度和检测灵敏度.毛细管区带电泳(CZE)作为第一维,分析物根据淌度不同进行分离,第一维流出组分进入第二维毛细管,根据分配系数不同进行胶束电动毛细管色谱(MEKC)分离.采用阳离子选择性耗尽进样(CSEI)在柱预富集,延长进样时间,增大进样量;同时在二维毛细管接口处采用动态pH联接/胶束扫集在线富集技术不仅避免第一维分离组分在接口处扩散,还可进一步压缩样品区带.同常规电动进样CE分离相比,该在线富集二维分离技术的分离能力远远高于一维CZE或MEKC分离,富集倍数达到(0.5～1.2)×104.该法成功应用于人体尿样中四种药物及对映体的分析测定,浓度检出限为0.1～0.3μg/L.进一步研究了人体尿样中四种药物24h内的药代动力学规律.     

微流控芯片二维电泳研究进展

综述了微流控芯片二维电泳技术及其在生命科学中的应用,包括胶束电动力学毛细管色谱(MEKC)与毛细管区带电泳(CZE)、等电聚焦(IEF)与CZE、开管电色谱(OCEC)与CZE耦联等模式的二维微流控芯片。展望了二维微流控芯片的应用前景。     

二维毛细管电泳电化学检测尿样中的β-阻断剂

设计并制作了在柱电化学(EC)检测池,用于在同一根毛细管中进行中心切割二维毛细管电泳(2D-CE)在线纯化分离检测尿样中的6种β-阻断剂.尿样先在15 mmol/L NaAc缓冲液中进行毛细管区带电泳(CZE)分离,带正电荷的β-阻断剂与中性和带负电荷的干扰物质分成不同区带,然后在检测端施加13.8 kPa压力将干扰成分从毛细管入口端排出,同时将目标组分驱送到毛细管入口端,最后在90 mmol/L NaAc-30 mmol/L SDS缓冲液中进行胶束电动毛细管色谱(MEKC)分离.场放大样品堆积(FASS)/胶束推扫在柱双重富集技术不仅有效抵消压力驱送过程中产生的区带扩散,还可进一步压缩样品区带,提高检测灵敏度.本方法成功用于服药后鼠尿样品中6种β-阻断剂的分离测定,经第一维CZE分离排除干扰后,在未涂层毛细管柱(60 cm ×50 μm i.d.)、90 mmol/L NaAc/HAc-30 mmol/L SDS运行缓冲液、检测电位0.8 V、运行电压10 kV条件下,对6种β-阻断剂进行在线富集分离,峰高、峰面积和迁移时间的相对标准偏差(RSD)分别为2.0%～4.1%, 1.4%～3.7%和0.9%～2.7%(n=6).本研究为毛细管电泳在复杂样品在线纯化分析等方面的应用提供了新方法.     

毛细管电泳法在手性分离中的应用及进展

近年来,毛细管电泳(CE) 手性分离方法的研究主要集中在各种手性添加剂与对映体药物的匹配及实验条件的最优化选择上.目前,较为成熟的CE分离模式有:区带电泳(CZE)、凝胶电泳(CGE)、等速电泳(CITP)、胶束电动色谱(MEKC)和非水电泳(NACE)等,并已成功地用于手性化合物对映体的分离.CE手性分离研究正朝着新型手性选择剂的研制和实现与其他各种定性分析仪器及其他色谱分离模式的联用方向发展.     

不同缓冲体系对毛细管电泳法分离15种核苷类化合物效果的比较

对毛细管电泳法分离15种核苷类化合物所用的不同缓冲液体系进行了系统比较,确定不同模式毛细管电泳法分析多种核苷类化合物的最适合背景缓冲液体系(BGE)。分别以四硼酸钠、磷酸氢二钠、乙酸钠、碳酸氢钠、乙酸铵和乙二胺(DEA)为背景电解质,对毛细管区带电泳(CZE)、毛细管电泳-电喷雾飞行时间质谱(CE-ESI-TOF/MS)以及胶束电动毛细管电泳(MEKC)3种模式进行比较,并对其中几种优势缓冲体系进行了优化。结果表明,CZE模式下使用四硼酸钠和磷酸氢二钠缓冲体系无法同时分离15种核苷类化合物,因此只适用于分析核苷类化合物数量较少的样品。而使用含有2%丙酮的300 mmol/L DEA能完全分开15种核苷类化合物,且分辨率和峰形良好。MEKC模式下,以25 mmol/L磷酸氢二钠(添加70 mmol/L十二烷基磺酸钠(SDS))为缓冲盐的分离结果最佳,并且此方法能成功应用于海洋生物海葵中核苷类化合物的分离。CE-ESI-TOF/MS分析中,以20 mmol/L乙酸铵(pH 10.0)为背景电解质,正离子模式检测,15种核苷类化合物的质谱信号均良好,检测灵敏度明显优于文献中报道的使用DEA缓冲体系的结果。本研究阐明了不同缓冲体系对15种核苷类化合物分离的适用性,为毛细管电泳技术在复杂基质中多种核苷类化合物的分离方法中的应用奠定了基础。     

黄酮类化合物的高效毛细管电泳分析

综述了近10年来高效毛细管电泳在黄酮类分析中的应用．着重介绍了黄酮类化合物毛细管电泳的CZE与MEKC两种模式的分离情况,同时简要介绍了黄酮类的结构与其电泳迁移行为的关系．     

毛细管等电聚焦/毛细管无胶筛分电泳二维蛋白质分离平台的构建

设计并制作了一种新型的中空纤维接口,以此为核心构建了毛细管等电聚焦(CLEF)／毛细管无胶筛分(CNGE)电泳二维蛋白质分离技术平台,实现了将二维凝胶电泳从平板转移到毛细管中。利用该二维分离平台,对血红蛋白样品进行了高效、快速分离分析,验证了该二维分离平台的可行性及分离效能。实验结果表明：二维分离系统总的峰个数和分离效能都比各自一维分离系统有较大提高。     

微量金属元素的毛细管电泳分析方法及应用

综述了毛细管电泳分析微量金属元素的基本原理、分离模式(CZE、MKEC、非水电泳、芯片分离等)、检测方法(紫外、荧光、化学发光、安培、电导、质谱联用技术)等的进展和该技术在环境、生物医学领域的研究与应用。引用文献94篇。     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

On-chip chiral separation based on bovine serum albumin-conjugated carbon nanotubes as stationary phase in a microchannel

A novel method of chiral separation based on protein-stationary phase immobilized in a poly(methyl methacrylate) microfluidic chip was developed. BSA conjugated with the shortened carboxylic single-walled carbon nanotubes (SWNTs) was employed as the chiral selector. Successful separation of tryptophan enantiomers was achieved in less than 70 s with a resolution factor of 1.35 utilizing a separation length of 32 mm. This is the first example of chiral separation based on SWNTs-BSA conjugates as stationary phase immobilized in microchip channel. The stability of the stationary phase in the channel was examined by microchip electrophoresis with laser-induced fluorescence detection. Factors that influenced the chiral separation resolution were examined. Under the optimized conditions, the proposed modified chip revealed adequate repeatability concerning run-to-run. These results show that the use of SWNTs-BSA conjugates within microfluidic channels hold great promise for a variety of analytical schemes.     

基于PMMA毛细管电泳芯片的多位点突变检测研究

利用基于激光诱导荧光(LIF)检测的芯片毛细管电泳平台,批量制作了低成本聚甲基丙烯酸甲酯(PMMA)芯片,通过修饰管道,优化有效分离距离、分离介质等条件,可在90s内完成DNA片段的分离检测,实现单碱基分离,并在此平台上成功地对遗传性耳聋三个常见突变位点实现分型检测,为这种低成本的PMMA芯片应用于分型相关的临床诊断领域奠定了基础。     

基于复合型微流控芯片的紫外检测毛细管电泳系统

提出了一种基于芯片-毛细管复合装置的紫外检测-微流控芯片毛细管电泳分析系统.采用小死体积的耦合技术实现了石英毛细管与“十”字通道型微流控玻璃芯片的耦合.本系统的紫外检测灵敏度与商品化毛细管电泳仪相当.采用夹流进样方式,达到较高的进样重现性,2mmol/L苯甲酸的峰高相对标准偏差(RSD)为1.5%(n=11).可用于复方磺胺甲唑片剂的两种有效成分的快速分离.     

Capacitance‐to‐digital: The upgrades of single chip detector

The capacitance‐to‐digital single chip detector was upgraded. The paper discusses hardware issues and benefits of the designed/upgraded detector. The device can be operated from rechargeable lithium‐ion battery as stand‐alone, portable system and is capable of transmitting real‐time data wirelessly. The detector and additional modules (battery, battery holder, microcontroller board, wireless module) weight is less than 85 g. Electrophoretic separation in low conductivity 20 mM MES/L‐His buffer, pH 6.1, was performed in order to evaluate detection parameters. The system is capable of quantification of potassium ions down to 0.31 μM. Investigation of differential signal acquisition configuration showed improved performance regarding external noise and temperature fluctuations. The system can be a solution for stand‐alone, field‐portable capillary format separation detector.     

微流体芯片电泳技术对人血清蛋白的快速分离

通过微流体芯片电泳技术分离人血清蛋白,探讨了常见十字形微流体芯片上样品的电动进样与分离过程,分析了在十字芯片上的进样时间和电压设置对后续样品检测和定量的影响。采用的缓冲体系为:100mmol/L H3BO3,50mmol/L NaCl,5%Dextran(以NaOH调至pH 8.3),该缓冲液能够有效分离人血清蛋白中的白蛋白(Albumin)和4种球蛋白(α1-,α2-,β-,和γ-globulin),并且给出了它们在该缓冲体系中的淌度估算范围为5.15×10-5～47.2×10-5 cm2/(V.s)。在芯片上2min之内可以完成进样和分离,相比于常用的毛细管区带电泳,提高了分析速度。     

Determination of oxalate in beer by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel combined with a 500 nL sample injection channel) and a pair of on‐chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in beer was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (3.8), implemented by aspartic acid and bis‐tris propane, provided an adequate selectivity in the separation of oxalate from anionic beer constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 0.5 μmol/L concentration also in samples containing chloride (a major anionic constituent of beer) at a 1800 higher concentration. Such a favorable analyte/matrix concentration ratio made possible accurate and reproducible [typically, 2–5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample] determination of oxalate in 500 nL volumes of 20–50‐fold diluted beer samples. Short analysis times (about 200 s), minimum sample preparation, and reproducible migration times of this analyte (0.5–1.0% RSD values) were characteristic for ZE on the chip.     

Development and optimization of an integrated PDMS based‐microdialysis microchip electrophoresis device with on‐chip derivatization for continuous monitoring of primary amines

An all‐PDMS on‐line microdialysis‐microchip electrophoresis with on‐chip derivatization and electrophoretic separation for near real‐time monitoring of primary amine‐containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene‐2,3,‐dicarboxyaldehyde/cyanide (NDA/CN^?), and the separation was performed using a 15‐cm serpentine channel. The analytes were detected using LIF detection.     

Quantitative determination of dopamine in single rat pheochromocytoma cells by microchip electrophoresis with only one high‐voltage power supply

We developed a method for the direct identification of dopamine in single cultured rat pheochromocytoma cells by capillary electrophoresis using an end‐channel carbon fiber nanoelectrode amperometric detector. The operation mode was designed to achieve single‐cell injection and lysis in microfluidic chip electrophoresis with only one high‐voltage power supply. The separation and detection conditions were optimized. Four catecholamines were baseline‐separated and determined with this system, and the cell density and liquid height of the reservoirs were accommodated for single cell loading, docking and analysis. The microchip capillary electrophoresis system was successfully applied to determine dopamine in single cultured rat pheochromocytoma cells.     

Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis

In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%.