A low-leakage sample plug injection scheme for crossform microfluidic capillary electrophoresis devices incorporating a restricted cross-channel intersection

This study develops a crossform CE microfluidic device in which a single-circular barrier or a double-circular barrier is introduced at the cross-channel intersection. Utilizing a conventional crossform injection scheme, it is shown that these barriers reduce sample leakage and deliver a compact sample band into the separation channel, thereby ensuring an enhanced detection performance. A series of numerical and experimental investigations are performed to investigate the effects of the barrier type and the barrier ratio on the flow streamlines within the microchannel and to clarify their respective effects on the sample leakage ratio and sample plug variance during the injection process. The results indicate that a single-circular barrier injector with a barrier ratio greater than 20% and a double-circular barrier injector with a barrier ratio greater than 40% minimize the sample leakage ratio and produce a compact sample plug. As a result, both injectors have an excellent potential for use in high-quality, high-throughput chemical analysis procedures and in many other applications throughout the micro-total analysis systems field.     

Numerical analysis of an electrokinetic double-focusing injection technique for microchip CE

The injection techniques in electrophoresis microchips play an important role in the sample-handling process, whose characteristics determine the separation performance achieved, and the shape of a sample plug delivered into the separation channel has a great impact on the high-quality separation performance as well. This paper describes a numerical investigation of different electrokinetic injection techniques to deliver a sample plug within electrophoresis microchips. A novel double-focusing injection system is designed and fabricated, which involves four accessory arm channels in which symmetrical focusing potentials are loaded to form a unique parallel electric field distribution in the intersection of injection channel and separation channel. The parallel electric field effectuates virtual walls to confine the spreading of a sample plug at the intersection and prevents sample leakage into separation channel during the dispensing step. The key features of this technique over other injection techniques are the abilities to generate regular and nondistorted shape of sample plugs and deliver the variable-volume sample plugs by electrokinetic focusing. The detection peak in the proposed injection system is uniform regardless of the position of the detection probe in the separation channel, and the peak resolution is greatly enhanced. Finally, the double-focusing injection technique shows the flexibility in detection position and ensures improved signal sensitivity with good peak resolution due to the delivered high-quality sample plug.     

小型可连续进样微流控芯片分析仪的研制

报道了一种结构简单、可连续进样的小型微流控芯片分析仪的研制。顺序注射分析系统通过芯片上制作的接口将试样连续引入芯片 ,并采用自行设计的紧凑型光纤式激光诱导荧光检测器进行检测。该仪器用于芯片毛细管电泳分离实验室合成Cy5荧光染料 ,实现了连续进样和换样。峰高RSD为 1 .9% (n=1 1 ) ,试样通量 3 5 h ;相邻试样携出 <4%。     

Electrochemical Detection Using an Engraved Microchip – Capillary Electrophoresis Platform

The present study describes a simple strategy to integrate electrochemical detection with an assembled microchip‐capillary electrophoresis platform. The electrochemical cell was integrated with a microfluidic device consisting of five plastic squares interconnected with fused silica capillaries, forming a four‐way injection cross between the separation channel and three side‐arms (each of 15 mm in length) acting as buffer/sample reservoirs. The performance of the system was evaluated using electrodes made with either carbon ink, carbon nanotubes, or gold and under different experimental conditions of pH, capillary length, and injection time. Using this system it was possible to separate the neurotransmitters dopamine and cathecol and to quantify phenol from a real sample using a linear calibration curve with a calculated LOD of 0.7 µM. A similar concept was applied to determine glucose, by including a pre‐reactor filled with beads modified with glucose oxidase (GOx). The latter system was used to determine glucose in a commercial sample, with a recovery of 95.2 %. Overall, the presented approach represents a simple, inexpensive, and versatile approach to integrate electrochemical detection with CE separations without requiring access to microfabrication facilities.     

pH-mediated acid stacking with reverse pressure for the analysis of cationic pharmaceuticals in capillary electrophoresis

When using capillary electrophoresis (CE) for the analysis of biological samples, it is often necessary to employ techniques to overcome peak-broadening that results from having a high-conductivity sample matrix. To improve the concentration detection limits and separation efficiency of cationic pharmaceuticals in CE, pH-mediated acid stacking was performed to electrofocus the sample, improving separation sensitivity for the analyzed cations by 60-fold. However, this method introduces a large titrated acid plug into the capillary. To overcome the limitations this low-conductivity plug poses to stacking, the plug was removed prior to the separation step by applying reverse pressure to force it out of the anode of the capillary. Employing this technique allows for roughly twice the volume of sample to be injected. A maximum sample injection time of 240 s was attainable with baseline peak resolution compared to a maximum sample injection time of 120 s without reverse pressure, leading to a twofold decrease in the limits of detection of the analytes used. Separation efficiency overall is also improved when utilizing the reverse pressure step. For example, a 60 s sample injection time results in 94,000 theoretical plates as compared to 60,500 theoretical plates without reverse pressure. This reverse-pressure method was used for detection and quantitation of several cationic pharmaceuticals that were prepared in Ringer's solution to simulate microdialysis sampling conditions.     

Negative pressure pinched sample injection for microchip-based electrophoresis

A simple method for injecting well-defined non-biased sample plugs into the separation channel of a microfluidic chip-based capillary electrophoresis system was developed by a combination of flows generated by negative pressure, electrokinetic and hydrostatic forces. This was achieved by using only a single syringe pump and a single voltage supply at constant voltage. In the loading step, a partial vacuum in the headspace of a sealed sample waste reservoir was produced using a syringe pump equipped with a 3-way valve. Almost instantaneously, sample was drawn from the sample reservoir across the injection intersection to the sample waste reservoir by negative pressure. Simultaneously, buffer flow from the remaining two buffer reservoirs pinched the sample flow to form a well-defined sample plug at the channel intersection. In the subsequent separation stage, the vacuum in headspace of the sample waste reservoir was released to terminate all flows generated by negative pressure, and the sample plug at the channel intersection was electrokinetically injected into the separation channel under the potential applied along the separation channel. The liquid levels of the four reservoirs were optimized to prevent sample leakage during the separation stage. The approach considerably simplified the operations and equipment for pinched injection in chip-based CE, and improved the throughput. Migration time precisions of 3.3 and 1.5% RSD for rhodamine123 (Rh123) and fluorescein sodium (Flu) in the separation of a mixture of Flu and Rh123 were obtained for 56 consecutive determinations with peak height precisions of 6.2% and 4.4% RSD for Rh123 and Flu, respectively.     

Parallel analysis of biomolecules on a microfabricated capillary array chip

This paper focused on a self-developed microfluidic array system with microfabricated capillary array electrophoresis (mu-CAE) chip for parallel chip electrophoresis of biomolecules. The microfluidic array layout consists of two common reservoirs coupled to four separation channels connected to sample injection channel on the soda-lime glass substrate. The excitation scheme for distributing a 20 mW laser beam to separation channels in an array is achieved. Under the control of program, the sample injection and separation in multichannel can be achieved through six high-voltage modules' output. A CCD camera was used to monitor electrophoretic separations simultaneously in four channels with LIF detection, and the electropherograms can be plotted directly without reconstruction by additional software. Parallel multichannel electrophoresis of series biomolecules including amino acids, proteins, and nucleic acids was performed on this system and the results showed fine reproducibility.     

Flow manipulation for sweeping with a cationic surfactant in microchip capillary electrophoresis

Flow manipulation in sweeping microchip capillary electrophoresis (CE) is complicated by the free liquid communication between channels at the intersection, especially when the electroosmotic flows are mismatched in the main channel. Sweeping in traditional CE with cationic micelles is an effective way to concentrate anionic analytes. However, it is a challenge to transfer this method onto microchip CE because the dynamic coating process on capillary walls by cationic surfactants is interrupted when the sample solution free of surfactants is introduced into the microchip channels. This situation presents a difficulty in the sample loading, injection and dispensing processes. By adding surfactant at a concentration around the critical micelle concentration and by properly designing the voltage configuration, the flows in a microchip were effectively manipulated and this sweeping method was successfully moved to microchip CE using tetradecyltrimethylammonium bromide (TTAB). The sweeping effect of cationic surfactant in the sample solution was discussed theoretically and studied experimentally in traditional CE. The flows in a microchip were monitored with fluorescence imaging, and the injection and sweeping processes were studied by locating the detection point along the separation channel. A detection enhancement of up to 500-fold was achieved for 5-carboxyfluorescein.     

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.     

High-resolution DNA separation in microcapillary electrophoresis chips utilizing double-L injection techniques

An experimental and numerical investigation into the use of high-resolution injection techniques to separate DNA fragments within electrophoresis microchips is presented. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered, and several variable-volume injection methods are discussed. A detailed analysis is provided of a double-L injection technique, which employs appropriate electrokinetic manipulations to reduce sample leakage within the microchip. The leakage effect in electroosmotic flow (EOF) is investigated using a sample composed of rhodamine B and Cy3 dye. Meanwhile, the effects of sample leakage in capillary electrophoresis (CE) separation are studied by considering the separation of 100-base pairs (bp) DNA ladders and HaeIII-digested PhiX-174 DNA samples. The present experimental and simulation results indicate that the unique injection system employed in the current microfluidic chip has the ability to replicate the functions of both the conventional cross-channel and the shift-channel injection systems. Furthermore, applying the double-L injection method to these two injection systems is shown to reduce sample leakage significantly. The proposed microfluidic chip and double-L injection technique developed in this study have an exciting potential for use in high-resolution, high-throughput biochemical analysis applications and in many other applications throughout the micrototal analysis systems field.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Multiplexed detection of nitrate and nitrite for capillary electrophoresis with an automated device for high injection efficiency

A simple automated nanoliter scale injection device which allows for reproducible 5 nL sample injections from samples with a volume of <1 microL is successfully used for conventional capillary electrophoresis (CE) and Hadamard transform (HT) CE detection. Two standard fused silica capillaries are assembled axially through the device to function as an injection and a separation capillary. Sample solution is supplied to the injection capillary using pressure controlled with a solenoid valve. Buffer solution flows gravimetrically by the junction of the injection and separation capillaries and is also gated with a solenoid valve. Plugs of sample are pushed into the space between the injection and separation capillaries for electrokinectic injection. To evaluate the performance of the injection device, several optimizations are performed including the influence of flow rates, the injected sample volume and the control of the buffer transverse flow on the overall sensitivity. The system was then applied to HT-CE-UV detection for the signal-to-noise ratio (S/N) improvement of the nitric oxide (NO) metabolites, nitrite and nitrate. In addition, signal averaging was performed to explore the possibility of greater sensitivity enhancements compared to single injections.     

Electrophoretic separations on microfluidic chips

This review presents a brief outline and novel developments of electrophoretic separation in microfluidic chips. Distinct characteristics of microchip electrophoresis (MCE) are discussed first, in which sample injection plug, joule heat, channel turn, surface adsorption and modification are introduced, and some successful strategies and recognized conclusions are also included. Important achievements of microfluidic electrophoresis separation in small molecules, DNA and protein are then summarized. This review is aimed at researchers, who are interested in MCE and want to adopt MCE as a functional unit in their integrated microsystems.     

超高速平板通道毛细管电泳

超高速平板通道毛细管电泳是９０年代发展的一种秒级分离的新颖技术。应用现代微电子光刻技术将化学反应。进样、分离和检测等组合在数厘米玻片上。实现分离分析的小型化、集成化、一体化和自动化。     

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.     

Design and simulation of sample pinching utilizing microelectrodes in capillary electrophoresis microchips

The paper proposed novel designs to pinch the transverse diffusion of the sample in the injection mode using microelectrodes to generate the potential difference at the channel intersection in the capillary electrophoresis (CE) microchip. A pair of microelectrodes was used to conduct the injection channel and the separation channel, which directly provided the potential to pinch the sample without using a power supply. These new designs of the CE microchip simplify the electric circuitry and improve performance. Simulations were performed using the CFD-ACE[trade mark sign] software. The mechanisms of diffusion and electrophoresis were employed in the numerical simulation. The injection and separation processes of the sample were simulated and the parameters of the present design were investigated numerically.     

Application of a capillary‐assembled microfluidic system for separation of cephalosporins

This paper demonstrates a simple and easy setting up of a fused‐silica capillary‐assembled microfluidic system (μCE). This system incorporates a split‐flow pressure injection of the sample into a microfluidic system made from PDMS and a short (～20 cm) length of fused‐silica capillary as a separation unit. The on‐capillary detection was carried out by fiber optic spectrometry. A mixture of six cephalosporin antibiotics was separated in the μCE system and the obtained results were compared to those achievable by conventional CE. The six components could be separated within 8.5 min with the number of theoretical plates around 10 000.     

Micro sequential injection: automated insulin derivatization and separation using a lab-on-valve capillary electrophoresis system

Automated sampling and fluorogenic derivatization of islet proteins (insulin, proinsulin, c-peptide) are separated and analyzed by a novel lab-on-valve capillary electrophoresis (LOV-CE) system. This fully integrated device is based on a micro sequential injection instrument that uses a lab-on-valve manifold to integrate capillary electrophoresis. The lab-on-valve manifold is used to perform all microfluidic tasks such as sampling, fluorogenic labeling, and CE capillary rejuvenation providing a very reliable system for reproducible CE separations. Fluorescence detection was coupled to an epiluminescence fluorescence microscope using a customized capillary positioning plate. This customized plate incorporated two fused-silica fiber optic probes that allow for simultaneous absorbance and fluorescence detection, extending the utility of this device. Derivatization conditions with respect to the sequence of addition, timing, injection position, and volumes were optimized through iterative series of experiments that are executed automatically by software control. Reproducibility in fluorogenic labeling was tested with repetitive injections of 3.45 mM insulin, yielding 1.3% RSD for peak area, 0.5% RSD for electromigration time, and 2.8% RSD for peak height. Fluorescence detection demonstrated a linear dynamic range of 3.43 to 6.87 microM for insulin (r2 = 0.99999), 0.39 to 1.96 pM for proinsulin (r2 = 0.99195) and 260 to 781 nM for c-peptide (r2 = 0.99983). By including hydrodynamic flushing immediately after the detection of the last analyte, the sampling frequency for islet protein analysis was increased. Finally, an in vitro insulin assay using rat pancreatic islet excretions was tested using this lab-on-valve capillary electrophoresis system.     

Continuous on-line concentration based on dynamic pH junction for trimethoprim and sulfamethoxazole by microfluidic capillary electrophoresis combined with flow injection analysis system

A novel, rapid and continuous on-line concentration approach based on dynamic pH junction for the analysis of trimethoprim (TMP) and sulfamethoxazole (SMZ) by microfluidic capillary electrophoresis (CE) combined with flow injection analysis is developed in this paper. Stacking is due to decreases in the velocity of analytes when migrating from the low-pH sample zone (sample was dissolved in 50 mM HCl) to a relatively high-pH buffer (30 mM phosphate buffer, pH 8.5) filled in the capillary. This results in 2.9-4.7-fold improvement in concentration sensitivity relative to conventional capillary electrophoresis methods. The separation could be achieved within 2 min and sample throughput rate can reach up to 38 h(-1).     

A three-dimensionally adjustable amperometric detector for microchip electrophoretic measurement of nitroaromatic pollutants

A microchip capillary electrophoresis (CE)–amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an amperometric detection cell containing a one-dimensionally adjustable disc detection electrode in a Plexiglas holder. It facilitates the precise three-dimensional alignment between the channel outlet and the detection electrode without a complicated three-dimensional manipulator. The performance of this unique system was demonstrated by separating four nitroaromatic pollutants (nitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and p-nitrobenzene). Factors influencing their separation and detection processes were examined and optimised. The four analytes have been well-separated within 120 s in a 75 cm long separation channel at a separation voltage of +2000 V using an electrophoretic separation medium containing 15 mM borax and 15 mM sodium dodecyl sulfate (pH 9.2). Highly linear response is obtained for the four analytes over the range of 0–5 ppm with the detection limits ranging from 12 to 52 ppb. The present system demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n = 9). The new approach for the microchannel–electrode alignment should find a wide range of applications in other microfluidic analysis systems.