Microchannel electrophoretic separation of biogenic amines by micellar electrokinetic chromatography

Fast, efficient separation of most common biogenic amines was successfully performed on a glass microchip capillary electrophoresis device. The amines putrescine, histamine, tyramine, cadaverine, phenethylamine, tryptamine, spermidine and spermine were derivatized prior to fluorescence detection with fluorescein isothiocyanate. Separation was carried out using a channel length of 28 mm, a cross section of 50 x 8 microm, and a field strength of 600 V/cm. After optimization of buffer electrolyte conditions (120 mM boric acid, pH 9.4, modified with 40 mM SDS), fluorescein thiocarbamyl amine derivatives were successfully resolved. Analysis time was as short as 75 s. Determination of the biogenic amines was achieved in soy sauce samples.     

Fluorescence detection in short capillary and chip using a variable wavelength epi-fluorescence microscope

Fast, efficient separation of five free acid forms of porphyrins was achieved in a short capillary and a chip, respectively. The capillary was 6 cm long from injection end to detector with an electric field strength of 214 V cm(-1). Separations were performed within 5 min. A glass microchip device was fabricated using standard photolithographic procedures and chemical wet etching. The channels were sealed using a direct bonding technique. For a separation length of 2.8 cm with electric field strength of 500 V cm(-1), electrophoretic separations with baseline resolution were achieved in less than 2 min. A variable wavelength epi-fluorescence microscope was used as an on-column detector.     

聚甲基丙烯酸甲酯电泳芯片电化学检测氨基酸

采用集成铜微电极的聚甲基丙烯酸甲酯(PMMA)电泳芯片,实现对氨基酸的分离与检测。考察了制备的铜微电极对氨基酸的催化活性以及检测电位和分离场强对分离效果的影响。在优化条件(30mmol／L NaOH缓冲溶液中,检测电位为0．7V(vs.Ag／AgCl),进样与分离电场强度均为50V／cm)下,用3cm长的通道实现了精氨酸(Arg)与亮氨酸(Leu)的分离。     

Application of plasma-polymerized films for isoelectric focusing of proteins in a capillary electrophoresis chip

The first use of plasma polymerization technique to modify the surface of a glass chip for capillary isoelectric focusing (cIEF) of different proteins is reported. The electrophoresis separation channel was machined in Tempax glass chips with length 70 mm, 300 microm width and 100 microm depth. Acetonitrile and hexamethyldisiloxane monomers were used for plasma polymerization. In each case 100 nm plasma polymer films were coated onto the chip surface to reduce protein wall adsorption and minimize the electroosmotic flow. Applied voltages of 1000 V, 2000 V and 3000 V were used to separate mixtures of cytochrome c (pI 9.6), hemoglobin (pI 7.0) and phycocyanin (pI 4.65). Reproducible isoelectric focusing of each pI marker protein was observed in different coated capillaries at increasing concentration 2.22-5 microg microL(-1). Modification of the glass capillary with hydrophobic HMDS plasma polymerized films enabled rapid cIEF within 3 min. The separation efficiency of cytochrome c and phycocyanin in both acrylamide and HMDS coated capillaries corresponded to a plate number of 19600 which compares favourably with capillary electrophoresis of neurotransmitters with amperometric detection.     

Characterization of SU-8 for electrokinetic microfluidic applications

The characterization of SU-8 microchannels for electrokinetic microfluidic applications is reported. The electroosmotic (EO) mobility in SU-8 microchannels was determined with respect to pH and ionic strength by the current monitoring method. Extensive electroosmotic flow (EOF), equal to that for glass microchannels, was observed at pH > or =4. The highest EO mobility was detected at pH > or =7 and was of the order of 5.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer. At pH < or =3 the electroosmotic flow was shown to reverse towards the anode and to reach a magnitude of 1.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer (pH 2). Also the zeta-potential on the SU-8 surface was determined, employing lithographically defined SU-8 microparticles for which a similar pH dependence was observed. SU-8 microchannels were shown to perform repeateably from day to day and no aging effects were observed in long-term use.     

注塑型聚甲基丙烯酸甲酯多通道微流控芯片的研制及其性能考察

微流控芯片技术因具有微量、快速、高效和高通量等特点,已成为分析化学领域中的研究热点之一．在微流控芯片中,最常见的可用作芯片的材料为玻璃、石英和各种塑料．玻璃和石英有很好的电渗性和光学性质,可采用标准的刻蚀工艺加工和用化学方法进行表面改性,但加工成本较高,封接难度较大．     

电场耦合法芯片电泳柱上电导检测特性研究

通过电场耦合作用对毛细管电泳柱上直流电导检测原理进行了分析, 利用带有双T结构检测池的玻璃和聚合物芯片对其特性进行了研究. 在电泳分离过程中, 电泳高电场通过检测通道耦合到出口的检测电极上并产生可被检测的电势差. 当导电性与支持电解质不同的组分谱带通过分离通道上的检测池时造成该电位差的变化, 该变化与溶液电导率、检测池长度和电场强度直接相关. 检测信号与基线相除得到的信号只和检测池中溶液的物理特征参数(电导率及电阻)有关. 用自制高阻抗信号转换系统可使芯片电泳电场强度提高到450 V/cm以上, 以1 mmol/L Tris-HCl为支持电解质, 在12 s内实现了K＋和Na＋的高重现性分离检测, 检出限达到5 μmol/L, 线性范围达两个数量级(10 μmol/L~2 mmol/L).     

电泳芯片的制作及其进样与分离

利用微细加工技术研究在玻璃上制作电泳芯片的方法,测试了微管道的伏安特性曲线。在该电泳芯片上进行了注样和分离实验,采用激光诱导荧光法进行检测,利用CCD拍摄了进样和分离的全过程。分析了电泳芯片上施加不同的电压对样品注样的影响,给出了FITC－OH和FITC－Arg分离谱图。     

Temperature gradient focusing in a PDMS/glass hybrid microfluidic chip

This paper reports the application of temperature gradient focusing (TGF) in a PDMS/glass hybrid microfluidic chip. With TGF, by the combination of a temperature gradient along a microchannel, an applied electric field, and a buffer with a temperature-dependent ionic strength, analytes are focused by balancing their electrophoretic velocities against the bulk velocity of the buffer containing the analytes. In this work, Oregon Green 488 carboxylic acid was concentrated approximately 30 times as high as the initial concentration in 45 s at moderate electric strength of 70 V/cm and a temperature gradient of 55 degrees C across the PDMS/glass hybrid microfluidic chip with a 1 cm long capillary.     

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips

MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.     

Continuous capillary electrophoresis with flow injection and its application for determination of Ephedrine and Pseudo-ephedrine in Chinese medicinal preparations

This article presents a new, simple and rapid continuous separation method by combination of flow injection with capillary electrophoresis designed for the analysis of basic traditional Chinese medicines. The device was produced using commercial capillary and components readily available in analytical laboratory. In double-T configuration, the designed horizontal separation channel was 25 microm i.d. x 146 mm length (an effective separation length of 93 mm) quartz capillary, with two vertical elicitation arms produced from 0.5 mm i.d. pump tubing. The capillary was embedded in a 40 x 20 x 3 mm organic glass base. Using the double-T configuration, continuous introduction of a series of samples was achieved. More than 3.00 resolution for ephedrine and pseudo-ephedrine were obtained using 100 mm borate buffer (pH 9.80) within 8 min in 25 microm separation channel with an electrical field strength of 137 V/cm (UV detection at 215 nm). The linear calibration range was 50-1500 microg/mL (ephedrine, r = 0.9982; pseudo-ephedrine, r = 0.9990) for both analytes. The limits of detection were 2.65 micro g/mL for ephedrine and 2.92 microg/mL for pseudo-ephedrine. In this device, the contents of ephedrine and pseudo-ephedrine in five Chinese medicinal preparations were determined with RSDs (n = 5) in range 1.16-4.51% and recoveries in range 90.4-114.6%.     

Silicate glass coated microchannels through a phase conversion process for glass-like electrokinetic performance

The surface modified polydimethylsiloxane (PDMS) microchannels show a much more inferior performance to the durable and reproducible glass chip. In this paper, a facile approach to preparing a silicate glass modified PDMS microchannel for glass-like performance is presented. This glass-like performance is made possible by a phase conversion of a preceramic polymer--allylhydridopolycarbosilane (AHPCS). The, several hundred nanometer thick, polymer that coats the PDMS channel is hydrolyzed to form hydrophilic silicate glass via phase conversion under an aqueous alkali condition. It is characterized by XPS, FTIR-ATR, AFM, and contact angle measurements. The silicate glass coated PDMS channel from AHPCS has an excellent solvent resistance, delivers a high electroosmotic flow (EOF) that is stable in the long-term (4.9±0.1×10(-4) cm(2) V(-1) s(-1)) and a reliable capillary electrophoresis (CE), which are comparable to those of native glass channels. Moreover, the silicate glass PDMS channel allows easy regeneration of the electrokinetic behavior, just as in a glass channel, by a simple treatment with alkali solution. This coating approach can be applied to other polymer substrates such as polyimide (PI).     

Separation of double-stranded DNA fragments in plastic capillary electrophoresis chips by using E99P69E99 as separation medium.

The separation of double-stranded DNA (dsDNA) fragments in polymethylmethacrylate (PMMA) capillary electrophoresis (CE) chips by using E99P69E99 as a separation medium has been demonstrated. The PMMA CE chips were simply manufactured by micromachining and adhesive tape sealing. To make the separation channel compatible with the separation medium, a dynamic nonionic surfactant coating procedure was developed, which made the plastic separation channel sufficiently hydrophilic to allow the separation medium to fill the channel by capillary action. Subsequent separation of DNA fragments was successful with a separation efficiency of the order of 10(4) theoretical plates over an effective separation distance of 1.5 cm. By using an applied electric field strength of 200 V/cm, the separation of low DNA mass ladder was completed within 5 min. The simple coating procedure, together with the self-assembled viscosity-adjustable separation medium, should be useful to meet some of the essential requirements for developing single-use disposable CE chips. Coating the channels with polymer blends of PMMA and the separation medium also showed promise.     

Continuous analysis of dye-loaded, single cells on a microfluidic chip

Continuous analysis of two dyes loaded into single mammalian cells using laser-based lysis combined with electrophoretic separation was developed and characterized on microfluidic chips. The devices employed hydrodynamic flow to transport cells to a junction where they were mechanically lysed by a laser-generated cavitation bubble. An electric field then attracted the analyte into a separation channel while the membranous remnants passed through the intersection towards a waste reservoir. Phosphatidylcholine (PC)-supported bilayer membrane coatings (SBMs) provided a weakly negatively charged surface and prevented cell fouling from interfering with device performance. Cell lysis using a picosecond-pulsed laser on-chip did not interfere with concurrent electrophoretic separations. The effect of device parameters on performance was evaluated. A ratio of 2 : 1 was found to be optimal for the focusing-channel : flow-channel width and 3 : 1 for the flow-channel : separation-channel width. Migration times decreased with increased electric field strengths up to 333 V cm(-1), at which point the field strength was sufficient to move unlysed cells and cellular debris into the electrophoretic channel. The migration time and full width half-maximum (FWHM) of the peaks were independent of cell velocity for velocities between 0.03 and 0.3 mm s(-1). Separation performance was independent of the exact lysis location when lysis was performed near the outlet of the focusing channel. The migration time for cell-derived fluorescein and fluorescein carboxylate was reproducible with <10% RSD. Automated cell detection and lysis were required to reduce peak FWHM variability to 30% RSD. A maximum throughput of 30 cells min(-1) was achieved. Device stability was demonstrated by analyzing 600 single cells over a 2 h time span.     

一种直接测定微流控芯片电渗流速度的新方法

随着微芯片技术的成熟,越来越迫切地需要有一个准确而简洁的电渗流速度的检测方法。根据荧光物质罗丹明123（Rh123）在不同pH缓冲溶液中迁移时间的变化,推导出Rh123在pH 9和10条件下分别有中性分子存在,而中性分子的移动速度等于电渗流速度,因此建立了直接以Rh123中性分子为标记物测定电渗流速度的方法。通过直接检测Rh123中性分子的迁移时间,计算得出所用玻璃微流控芯片在pH 9.3和pH 10.1的电渗流速度为3.9×10-4 cm2/(s·V)和4.1×10-4 cm2/(s·V),与经典方法对照无明显差异。     

Fabrication of plastic microchips by hot embossing

Plastic microchips with microchannels (100 microm wide, 40 microm deep) of varying designs have been fabricated in polymethylmethacrylate by a hot embossing process using an electroform tool produced starting with silicon chip masters. Hot-embossed chips were capped with a polymethylmethacrylate top using a proprietary solvent bonding process. Holes were drilled through the top of the chip to allow access to the channels. The chips were tested with fluid and shown to fill easily. The seal between the top of the chip and the hot embossed base was effective, and there was no leakage from the channels when fluid was pumped through the microchannels. The chips were also tested with a semen sample and the plastic chip performed identically to the previous silicon-glass and glass versions of the chip. This microfabrication technique offers a viable and potentially high-volume low cost production method for fabricating transparent microchips for analytical applications.     

Differential interference contrast microscopy for real-time dynamics and manipulation of single cells in microchannels

Nomarski differential interference contrast (DIC) microscopy was used for real-time dynamics of intact single cells in various microchannels for adaptation to microfluidic chip application. The cheek cell was chosen as a model, single cell and the dynamics was measured at the microchannels. The image resolution of single cell was shaper and more distinct in DIC than in conventional microscopy. The individual single living cells were also manipulated by both hydrodynamic and electrokinetic flow-driving forces at the microchannels. The DIC contrast was enhanced according to the order of round-, square-, and rectangle-type microchannels. The velocity of the single living cell was consistently increased with increasing electric field strength and pH. However, the velocity of cell was decreased with increasing run buffer concentration. The driving direction of the individual single cell was simply controlled by changing the polarity of the applied voltage and the electric field strength. The cells were consistently manipulated in the microchannel under the co-application of the low electric field of 2.44 V/cm, instead of the solo application of the hydrodynamic force.     

聚甲基丙烯酸甲酯微流控分析芯片的简易热压制作法

提出聚甲基丙烯酸甲酯(PMMA)微流控分析芯片的一种简易热压制作法,研究了镍基、单晶硅和玻璃3种阳模制备芯片及芯片的封合条件.采用扫描电镜(SEM)和电荷耦合检测器(CCD)对PMMA芯片的微通道及其横截面形貌进行了表征.SEM图和CCD图表明实现了热压封接.测定了PMMA芯片的伏安曲线和电渗流,其电渗流值与文献报道值基本一致.本法制作的PMMA芯片用于电泳分离Cy5荧光染料,峰高RSD为2.2%(n=11),理论塔板数7.4×10⁴m^-1.     

Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet

We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.     

Electrokinetic characterization of poly(dimethylsiloxane) microchannels

This paper characterizes the basic electrokinetic phenomena occurring within native poly(dimethylsiloxane) (PDMS) microchannels. Using simple buffers and current measurements, current density and electroosmosis data were determined in trapezoidal, reversibly sealed PDMS/PDMS and hybrid PDMS/glass channels with a cross-sectional area of 1035.5 microm(2) and about 6 cm length. This data was then compared to that obtained in an air-thermostated 50 microm inner diameter (1963.5 microm(2) cross-sectional area) fused-silica (FS) capillary of 70 cm length. Having a pH 7.8 buffer with an ionic strength (I) of 90 mM, Ohms's law was observed in the microchannels with electric field strengths of up to about 420 V/cm, which is about twice as high as for the FS capillary. The electroosmotic mobility (micro(EO)) in PDMS and FS is shown to exhibit the same general dependences on I and pH. For all configurations tested, the experimentally determined micro(EO) values were found to correlate well with the relationship micro(EO) = a + b log(I), where a and b are coefficients that are determined via nonlinear regression analysis. Electroosmotic fluid pumping in native PDMS also follows a pH dependence that can be estimated with a model based upon the ionization of silanol. Compared to FS, however, the magnitude of the electroosmotic flow in native PDMS is 50-70% smaller over the entire pH range and is difficult to maintain at acidic pH values. Thus, the origin of the negative charge at the inner wall of PDMS, glass, and FS appears to be similar but the density is lower for PDMS than for glass and FS.