A thin cover glass chip for contactless conductivity detection in microchip capillary electrophoresis

A microfabricated thin glass chip for contactless conductivity detection in chip capillary electrophoresis is presented in this contribution. Injection and separation channels were photolithographed and chemically etched on the surface of substrate glass, which was bonded with a thin cover glass (100 μm) to construct a new microchip. The chip was placed over an independent contactless electrode plate. Owing to the thinness between channel and electrodes, comparatively low excitation voltage (20–110 V in V_p–p) and frequency (40–65 kHz) were suitable, and favorable signal could be obtained. This microchip capillary electrophoresis device was used in separation and detection of inorganic ions, amino acids and alkaloids in amoorcorn tree bark and golden thread in different buffer solutions. The detection limit of potassium ion was down to 10 μmol/L. The advantages of this microchip system exist in the relative independence between the microchip and the detection electrodes. It is convenient to the replacement of chip and other operations. Detection in different position of the channel would also be available.     

Impact of reservoir potentials on the analyte behavior in microchip electrophoresis: computer simulation and experimental validation for DNA fragments

Fundamental understanding of the impact of reservoir potentials on the analyte behavior on the microfluidic chips is an important issue in microchip electrophoresis (MCE) for suitable injection and separation of analytes, since the applied potentials may significantly affect the shape of sample plug, sample leakage from the injection channel to the separation channel, injected sample amount, and separation efficiency. This study addressed this issue for the case of a conventional cross-geometry microchip with four reservoirs using computer simulations, the results of which were verified by the analysis of DNA fragments. For the microchip with a definite structure and migration distance, the injected sample amount was shown to be the vital parameter for improving the limit of detection and resolution. During injection, the shape of the sample plug could be adjusted by varying the reservoir potentials. It was demonstrated that a "magnified injection" (applying high voltage on the three reservoirs to the sample reservoir) is useful to enhance the detection sensitivity depending on the analyte composition, although such injection was previously avoided because of introducing too large amounts of the analyte in comparison with two established modes, floating and pinched injection. Optimal magnified injection was proved to improve the sensitivity for about 4 times over that of pinched injection for the analysis of DNA step ladders using microchip gel electrophoresis (MCGE). Sample leakage of DNA fragments could be suppressed by applying a high positive voltage on injection channel during separation, but the voltage degraded the injected amount and resolution.     

基于微芯片电泳的脱氧核糖核酸片段的浓缩和分离

采用超负荷电动供给(electrokinetic supercharging, EKS)预浓缩技术,在微芯片电泳(MCE)上对脱氧核糖核酸（DNA）片段进行浓缩和分离。EKS是集合样品电动进样(EKI)和过渡等速电泳(tITP)的一种在线浓缩方法。研究表明：采用该方法后,在40.5 mm长的单通道芯片上能够实现对低浓度样品的大量进样、浓缩和基线分离。在普通的紫外检测条件(检测波长为260 nm)下,对DNA片段的平均检出限(S/N=3)约为0.07 mg/L,仅为十字芯片上的微芯片电泳检出限的1/40。本文还对浓缩过程中的一些关键因素和定性分析进行了探讨。     

Integration of a flow-type chemiluminescence detector on a glass electrophoresis chip

A glass electrophoresis microchip integrated a flow-type chemiluminescence (CL) detection cell has been developed and evaluated. The chip pattern is a double-T-type electrophoretic sample injection and separation combining with a Y-type chemiluminecent detector. The double-T geometry allows for high-efficiency sample injection and geometric definition of sample plug size. The branch of Y was used as CL reagent channel, and the CL reagent was delivered by a lab-made micropump. Bis[(2,4,6-trichlorophenyl)]oxalate-H₂O₂ CL system was employed to detect dansyl amino acids. On this microchip, dansyl-phenylalanine and -sarcosine were successfully separated by electrophoresis and detected within 250 s. The detection limits (S/N=3) of dansyl-phenylalanine and -sarcosine could reach to 2.8 and 3.2 μM, respectively, due to the vigorous dilution of sample with CL reagent and timely removal of the waste solution from reaction area.     

Rapid separation of antimicrobial metabolites by microchip electrophoresis with UV linear imaging detection

This research examines microchip electrophoresis with linear imaging UV detection for the analysis of antimicrobial metabolites, monoacetylphloroglucinol (MAPG) and 2,4-diacetylphloroglucinol (2,4-DAPG) from Pseudomonas fluorescens F113. Initial results show the separation of MAPG, 2,4-DAPG and resorcinol in less than 20 s. This was achieved using a quartz microchip with a separation channel length of 25 mm. In order to quantitate the amount of MAPG and 2,4-DAPG in a microbial cultured supernatant sample, on-chip sample introduction in a methanol/buffer matrix was investigated. Sample introduction/injection parameters were optimized to improve sensitivity and thus decrease the limit of detection (LOD). The amount of antimicrobial metabolites present was quantitated with a separation time of 15 s. A previously developed capillary electrophoretic method was compared to the microchip method in relation to speed, efficiency, precision, linear range and limit of detection. This investigation shows the fastest separation so far of these antimicrobial metabolites with high efficiency.     

Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis

A laser-induced fluorescence detection system coupled with a highly sensitive silicon-intensified target (SIT) camera is successfully applied to the imaging of a band for DNA fragment labeling by fluorescence dye in a microchannel, and to the visualizing of the separation process on a microfabricated chip. We demonstrated that an only 6 mm separation channel is sufficient for the separation of triplet repeat DNA fragment and DNA molecular marker within only 12 s. The separation using the microfabricated capillary electrophoresis device is confirmed to be at least 18 times faster than the same separation carried out by conventional capillary electrophoresis with 24.5 cm effective length. The use of a short capillary with 8.5 cm effective length is also efficient for fast separation of DNA; however, the microchip technology is even faster than capillary electrophoresis using a short capillary.     

Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase,TniA

This work deals with zone electrophoresis (ZE) separations of proteins on a poly(methyl methacrylate) chip with integrated conductivity detection. Experiments were performed in the cationic mode of the separation (pH 2.9) with a hydrodynamically closed separation compartment and suppressed electroosmotic flow. The test proteins reached the detector in less than 10 min under these working conditions and their migration times characterized excellent repeatabilities (0.1–0.6% RSD values). The chip-to-chip agreements of the migration times, evaluated from the ZE runs performed on three chips, were within 1.5%. The conductivity detection provided for protein, loaded on the chip at 10–1000 μg/ml concentrations, detection responses were characterized by 1–5% RSD values of their peak areas. Such migration and detection performances made a frame for reproducible baseline separations of a five-constituent mixture (cytochrome c, avidin, conalbumin, human hemoglobin and trypsin inhibitor). On the other hand, a high sample injection channel/separation compartment volume ratio of the chip (500 nl/8500 nl) restricted the resolution of proteins of very close effective mobilities in spite of the fact that in the initial phase of the separation an electric field stacking was applied. A maximum macroconstituent/trace constituent ratio attainable for proteins on the chip was assessed for cytochrome c (quantifiable when its concentration in the loaded sample was 10 μg/ml) and apo-transferrin (containing a trace constituent migrating in the position of cytochrome c detectable when the load of apo-transferrin was 2000 μg/ml). This assessment indicated that a ratio of 1000:1 is attainable with the aid of conductivity detection on the present chip.     

Analysis of multiplex PCR fragments with PMMA microchip

Microchip electrophoresis is a promising technique for analysis of bio-molecules. It has the advantages of fast analysis, high sensitivity, high resolution and low-cost of samples. Plastic chip has the potential of mass production for clinical use for its advantages in biocompatibility and low cost. In this work, the method for fabrication of poly(methyl methacrylate) (PMMA) chip was described, and conditions for DNA separation were investigated with the chip. The PMMA microchip was used for detection of multiplex PCR products of 18 and 36 cases with SARS and hepatitis B virus infection under optimized separation conditions. Microchip electrophoresis showed higher sensitivity, higher resolution and less time consumption when compared with gel electrophoresis. The microchip electrophoresis with PMMA chip provided a rapid, sensitive and reliable method for analysis of multiplex PCR products.     

Novel multi-depth microfluidic chip for single cell analysis

A novel multi-depth microfluidic chip was fabricated on glass substrate by use of conventional lithography and three-step etching technology. The sampling channel on the microchip was 37 microm deep, while the separation channel was 12 microm deep. A 1mm long weir was constructed in the separation channel, 300 microm down the channel crossing. The channel at the weir section was 6 microm deep. By using the multi-depth microfluidic chip, human carcinoma cells, which easily aggregate, settle and adhere to the surface of the channel, can be driven from the sample reservoir to the sample waste reservoir by hydrostatic pressure generated by the difference of liquid level between sample and sample waste reservoirs. Single cell loading into the separation channel was achieved by applying a set of pinching potentials at the four reservoirs. The loaded cell was stopped by the weir and precisely positioned within the separation channel. The trapped cell was lysed by sodium dodecyl sulfate (SDS) containing buffer solution in 20s. This approach reduced the lysing time and improved the reproducibility of chip-based electrophoresis separations. Reduced glutathione (GSH) and reactive oxygen species (ROS) were used as model intracellular components in single human carcinoma cells, and the constituents were separated by chip-based electrophoresis and detected by laser-induced fluorescence (LIF). A throughput of 15 samples/h, a migration time precision of 3.1% RSD for ROS and 4.9% RSD for GSH were obtained for 10 consecutively injected cells.     

Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis

A microchip structure for field amplification stacking (FAS) was developed, which allowed the formation of comparatively long, volumetrically defined sample plugs with a minimal electrophoretic bias. Up to 20-fold signal gains were achieved by injection and separation of 400 microm long plugs in a 7.5 cm long channel. We studied fluidic effects arising when solutions with mismatched ionic strengths are electrokinetically handled on microchips. In particular, the generation of pressure-driven Poiseuille flow effects in the capillary system due to different electroosmotic flow velocities in adjacent solution zones could clearly be observed by video imaging. The formation of a sample plug, stacking of the analyte and subsequent release into the separation column showed that careful control of electric fields in the side channels of the injection element is essential. To further improve the signal gain, a new chip layout was developed for full-column stacking with subsequent sample matrix removal by polarity switching. The design features a coupled-column structure with separate stacking and capillary electrophoresis (CE) channels, showing signal enhancements of up to 65-fold for a 69 mm long stacking channel.     

Membrane-mediated ultrafast restriction digestion and subsequent rapid gel microchip electrophoresis of DNA

Ultrafast, membrane-mediated restriction digestion of DNA molecules followed by rapid gel microchip electrophoresis of the resulting fragments is described. Combination of restriction endonuclease digestion on small pore-size microfibrous membranes with sample loading and electrophoresis analysis in a multilane (up to 96) format resulted in very fast restriction digest based microscale DNA analysis. Complete digestion of several nanogram target DNA was accomplished on the microporous membrane at room temperature just in a few minutes with a single or a combination of various restriction enzymes, using only submicroliter quantities of samples and reagents. The reaction mixture containing membrane also served as sample loading device for the subsequent gel microchip electrophoresis based analysis. This work establishes methods for high-speed, high-throughput DNA analysis, featuring extremely low sample and reagent consumption, and fast restriction digestion in combination with sample loading and rapid gel microchip analysis of the resulting fragments. The entire restriction digestion, sample loading and electrophoresis analysis process required less than 20 min.     

电泳芯片结构和芯片电泳分离操作参数的模拟、优化和实验验证

选择了L-精氨酸和L-苯丙氨酸为分离样品体系,根据电泳实验提出样品基本参数,通过模拟计算考察了进样管道宽度和进样时间对进样方差的贡献;根据分离度与分离长度拟合曲线确定电泳芯片的有效分离长度;对化学发光柱后衍生管道施加的夹流电压进行了模拟优化,得出氨基酸体系分离分析的电泳芯片设计方案和操作参数为:进样管道宽度为分离管道宽度的1/2,简单进样充样时间应大于5 s,分离管道有效分离长度为30 mm,衍生夹流比1.0~1.6。根据模拟优化结果提出了电泳芯片设计方案,采用整体浇注法制作带有柱后衍生反应器的PDMS电泳芯片,按照模拟计算提出的电压操作参数实现了精氨酸和苯丙氨酸样品体系的准确进样、芯片电泳分离和柱后衍生化学发光检测。电泳过程模拟结果和实验结果相结合,考察了柱后衍生对样品谱带展宽的影响,简单进样过程样品泄露引起的谱峰拖尾现象,并讨论了夹流进样法对减小进样方差和抑制样品泄露的贡献。     

Fabrication of quartz microchips with optical slit and development of a linear imaging UV detector for microchip electrophoresis systems

We have developed quartz microchips for electrophoresis and a linear imaging UV detector along with the microchip. The microchips have an optical slit, which cut off the stray light in order to improve the sensitivity of UV absorption detection on the chip, at the bonding interface. They have been successfully fabricated on synthesized quartz glass substrates using the hydrofluoric acid (HF) solution bonding method. The signal level of UV absorption detection was effectively improved by applying microchips with the "on-chip" optical slit. It is also possible to improve the signal-to-noise ratio by repetitive scanning of linear photodiode array located along the separation channel, and signal averaging during elimination of the potential. Furthermore, the analysis may be performed until the separation of the target component is complete, because the real-time migration pattern of each component in the sample can be seen just as in a slab-gel electrophoresis, thus enabling a shorter analysis time.     

Fast parallel detection of feline panleukopenia virus DNA by multi‐channel microchip electrophoresis with programmed step electric field strength

A multi‐channel microchip electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge‐coupled device camera were used to simultaneously detect the separations in three parallel channels using laser‐induced fluorescence detection. The parallel separations of a 100‐bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M_r = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single‐run and one‐step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel electrophoresis. The proposed multi‐channel microchip electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease‐related DNA fragments in parallel with high speed, throughput, and accuracy.     

Capillary liquid chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.     

芯片毛细管电泳-安培检测系统

由于安培检测具有的高灵敏度、低成本、低能耗、易集成化便携化、与微加工技术匹配等特点,芯片毛细管电泳-安培检测系统（μCE-AD）的研究近年来得到人们广泛的关注。本文结合本课题组的研究工作,对近年来μCE-AD的研究进展进行评述;重点讨论了近年来在芯片的设计、集成化电极的制备、消除分离电压的干扰等方面的进展;同时介绍了利用分离电场拓展检测范围、阵列电极和阵列通道、化学修饰电极的应用、新型进样技术和试样预处理等方面的新成就;最后展望了未来μCE-AD的发展趋势。     

Use of a heterogeneous buffer combination in microchip electrophoresis for high-resolution separation by on-line concentration of DNA samples

We have developed a novel high-resolution separation technique of DNA fragments in a heterogeneous combination of a sample buffer and a separation buffer. The use of a heterogeneous buffer combination is a simple method for on-line concentration of DNA fragments, in which a sample buffer is simply exchanged with one including taurine anions. The mobility of taurine anions, co-ions for DNA, is lower than the that of acetate anions in a separation buffer. The difference in the mobility invokes transient isotachophoresis. The current technique allows DNA fragments to be effectively concentrated and the separation length of microchips to be shorter than that of conventional ones by a factor of three without deterioration in separation resolution and any modification of a chip design. Fragments of 100-bp DNA ladders (100-1000 bp) were separated with high resolution (0.72-10.7) within 60 s with a 10 mm separation length on a polymethyl methacrylate chip. Furthermore, fragments of 10-bp DNA ladders (10-330 bp) were separated with high resolution (0.69-2.00) with a 10 mm separation length within 50 s without band broadening. The current achievements will make it possible to fabricate compact devices for microchip electrophoresis.     

Frontal analysis on a microchip

Conventional microchip applications involving capillary electrophoresis (CE) typically inject a sample along one channel and use an intersection of two channels to define the sample plug--the portion of sample to be analysed along a second channel. In contrast to this method of zone separation, frontal analysis proceeds by injecting sample continuously into a single channel or column. Frontal analysis is more common in macroscopic procedures but there are benefits in sensitivity and device density to its application to electrophoresis on microchips. This work compares conventional microchip zone analysis with frontal analysis in the separation of PCR products. Although we detect on the order of 5000 fluorophores with a compact instrument using the zone separation CE method, we found a several-fold increase in the effective signal-to-noise ratio by using a frontal analysis method. By removing the need for additional channels and reservoirs the frontal method would allow device densities to be significantly increased, potentially improving the cost-effectiveness of microchip analyses in applications such as medical diagnostics.     

Further improvement of hydrostatic pressure sample injection for microchip electrophoresis

Hydrostatic pressure sample injection method is able to minimize the number of electrodes needed for a microchip electrophoresis process; however, it neither can be applied for electrophoretic DNA sizing, nor can be implemented on the widely used single-cross microchip. This paper presents an injector design that makes the hydrostatic pressure sample injection method suitable for DNA sizing. By introducing an assistant channel into the normal double-cross injector, a rugged DNA sample plug suitable for sizing can be successfully formed within the cross area during the sample loading. This paper also demonstrates that the hydrostatic pressure sample injection can be performed in the single-cross microchip by controlling the radial position of the detection point in the separation channel. Rhodamine 123 and its derivative as model sample were successfully separated.     

Sensitivity enhancing injection from a sample reservoir and channel interface in microchip electrophoresis

The stacking of a cationic analyte (i.e., rhodamine B) at the interface between a sample reservoir and channel in a microchip electrophoresis device is described for the first time. Stacking at negative polarity was by micelle to solvent stacking where the dye was prepared in a micellar solution (5 mM sodium dodecyl sulfate in 25 mM phosphoric acid, pH 2.5) and the channel was filled with high methanol content background solution (70% methanol in 50 mM phosphoric acid, pH 2.5). The injection of the stacked dye into the channel was by simple reversal of the voltage polarity with the sample solution and background solution at the anodic and cathodic reservoirs of the straight channel, respectively. The enrichment of rhodamine B at the interface and injection of the stacked dye into the channel was clearly visualized using an inverted fluorescence microscope. A notable sensitivity enhancement factor of up to 150 was achieved after 2 min at 1 kV of micelle to solvent stacking. The proposed technique will be useful as a concentration step for analyte mixtures in simple and classical cross‐channel microchip electrophoresis devices or for the controlled delivery of enriched reagents or analytes as narrow plugs in advanced microchip electrophoresis devices.