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.     

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.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

Rapid and variable-volume sample loading in sieving electrophoresis microchips using negative pressure combined with electrokinetic force

A rapid and variable-volume sample loading scheme for chip-based sieving electrophoresis was developed by negative pressure combined with electrokinetic force. This was achieved by using a low-cost microvacuum pump and a single potential supply at a constant voltage. Both 12% linear polyacrylamide (LPA) with a high viscosity of 15000 cP and 2% hydroxyethylcellulose (HEC) with a low viscosity of 102 cP were chosen as the sieving materials to study the behavior and the versatility of the proposed method. To reduce the hydrodynamic resistance in the sampling channel, sieving material was only filled in the separation channel between the buffer waste reservoir (BW) to the edge of the crossed intersection. By applying a subambient pressure to the headspace of sample waste reservoir (SW), sample and buffer solution were drawn immediately from sample reservoir (S) and buffer reservoir (B) across the intersection to SW. At the same time, the charged sample in the sample flow was driven across the interface between the sample flow and the sieving matrix into the sieving material filled separation channel by the applied electric field. The injected sample plug length is in proportion with the loading time. Once the vacuum in SW reservoir was released to activate electrophoretic separation, flows from S and B to SW were immediately terminated by the back flow induced by the difference of the liquid levels in the reservoirs to prevent sample leakage during the separation stage. The sample consumption was about 1.7 x 10(2) nL at a loading time of 1 s for each cycle. Only 0.024 s was required to transport bias-free analyte to the injection point. It is easy to freely choose the sample plug volume in this method by simply changing the loading time and to inject high quality sample plug with non-distorted shape into the separation channel. The system has been proved to possess an exciting potential for improving throughput, repeatability, sensitivity and separation performance of chip-based sieving electrophoresis.     

Electrophoretic injection bias in a microchip valving scheme

The pinched injection strategy, implemented on microfabricated fluidic devices (microchips), was investigated for an electrophoretic injection bias. Both the sample loading and dispensing steps were found to contribute to the injection bias whereby neutral species were injected preferentially to anionic species. In the sample loading step, neutral species filled a larger volume in the cross intersection than anionic species. Similarly, in the dispensing step, a larger volume of neutral analyte was injected than anionic analyte. Up to a 27% difference in injected volumes was observed. Fluorescently labeled amino acids were used as model analytes.     

Study of injection bias in a simple hydrodynamic injection in microchip CE

The electrokinetically pinched method is the most commonly used mode for sample injection in microchip capillary electrophoresis (microCE) due to its simplicity and well-defined sample volume. However, the limited injection volume and the electrophoretic bias of the pinched injection may limit its universal usage to specific applications. Several hydrodynamic injection methods in microCE have been reported; however, almost all claimed that their methods are bias-free without considering the dispensing bias. To investigate the dispensing bias, a simple hydrodynamic injection was developed in single-T and double-T glass microchips. The sample flow was produced by hydrostatic pressure generated by the liquid level difference between the sample reservoir and the other reservoirs. The reproducibility of peak area and peak area ratio was improved to a significant extent using large-surface reservoirs for the buffer reservoir and the sample waste reservoir to reduce the Laplace pressure effect. Without a voltage applied on the sample solution, the voltage-related sample bias was eliminated. The dispensing bias was analyzed theoretically and studied experimentally. It was demonstrated that the dispensing bias existed and could be reduced significantly by appropriately setting up the voltage configuration and by controlling the appropriate liquid level difference.     

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。     

Rapid and efficient isotachophoretic preconcentration in free solution coupled with gel electrophoresis separation on a microchip using a negative pressure sampling technique

We present a novel isotachophoresis–gel electrophoresis (ITP–GE) microchip system designed for rapid and efficient isotachophoretic preconcentration coupled with gel electrophoresis separation by using a negative pressure sampling technique. The overall ITP–GE procedure involves only three steps: sample loading, ITP preconcentration and GE separation and was controlled by a simple and compact negative pressure sampling device, which is composed of a vacuum vessel, a three-way electromagnetic valve and a single high voltage power supply. During the sample loading stage, a negative pressure was applied via a three-way electromagnetic valve in headspace of the two sealed sample waste reservoirs (SWs). A sandwiched sample zone between a leading and a terminating electrolyte zone was formed in the channel intersection in less than 1 s. Once the three-way electromagnetic valve was switched to connect SWs to ambient atmosphere to release vacuum in SWs, ITP preconcentration in free solution and GE separation in the 4% hydroxyethylcellulose (HEC) sieving material were consequently activated under the electric potentials applied. The performance of present approach was evaluated by using DNA fragments as model analytes. Compared to conventional cross microchip GE using electrokinetic pinched injection, an average signal enhancement of 185-fold was obtained with satisfactory resolution. The results demonstrated the ITP–GE approach possessing an exciting potential of high sensitivity and short sampling time with significant simplification in operation and instrumentation.     

集成毛细管电泳芯片系统的制作、测试及应用

使用标准光刻和化学湿法腐蚀技术,在玻璃板材上制作了由样样管道和分离管道内构成的集成毛细管网路系统,对影响芯片质量的一些因素进行了讨论,并进行了性能测试和评价。芯片上毛细管道散热良好。使用激光诱导荧光和CCD成像检测系统,以电渗作用为驱动力,对混合样品进行了进样、快速分离（20s以内）和监测,证明了自制集成毛细管电泳芯片及检测系统的可行性。比较了两种注样方式（float和pinched)的不同;证明了在分离时可以优化加电策略,防止拖尾,改善峰形。     

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.     

Influence of channel position on sample confinement in two-dimensional planar microfluidic devices

We report enhanced sample confinement on microfluidic devices using a combination of electrokinetic flow from adjacent control channels and electric field shaping with an array of channels perpendicular to the sample stream. The basic device design consisted of a single first dimension (1D) channel, intersecting an array of 32 or 96 parallel second dimension (2D) channels. To minimize sample dispersion and leakage into the parallel channels as the sample traversed the sample transfer region, control channels were placed to the left and right of the 1D and waste channels. The electrokinetic flow from the control channels confined the sample stream and acted as a buffer between the sample stream and the 2D channels. To further enhance sample confinement, the electric field was shaped parallel to the sample stream by placing the channel array in close proximity to the sample transfer region. Using COMSOL Multiphysics, initial work focused on simulating the electric fields and fluid flows in various device geometries, and the results guided device design. Following the design phase, we fabricated devices with 40, 80, and 120 microm wide control channels and evaluated the sample stream width as a function of the electric field strength ratio in the control and 1D channels (E(C)/E(1D)). For the 32 channel design, the 40 and 80 microm wide control channels produced the most effective sample confinement with stream widths as narrow as 75 microm, and for the 96 channel design, all three control channel widths generated comparable sample stream widths. Comparison of the 32 and 96 channel designs showed sample confinement scaled easily with the length of the sample transfer region.     

Effects of injector geometry and sample matrix on injection and sample loading in integrated capillary electrophoresis devices

The double-T injector design employed in many microchip capillary electrophoresis devices allows for the formation of very small (50-500 pL) sample plugs for subsequent analysis on-chip. In this study, we show that sample plugs formed at the channel junction can be geometrically defined. The channel width and injector symmetry prove to be of great importance to good performance. A unique pushback of solvent into the side channels can be induced when the side channels have a very low resistance to flow, and this helps to better define the injected sample plug. Samples and running buffers of differing ionic strength (e.g., 10 mM KCl buffer and 20 mM KCl sample) can yield widely variable results in terms of plug shape and amount injected (variations of 1.5 to 10x). Applying bias voltages to all the intersecting channels aids in controlling the plug shape. However, when the ionic strengths of buffer and sample are not matched, the actual amount injected (up to 10x variations) can be inconsistent with the appearance of the plug formed in the injector (up to only 30 % variations). Operating at constant pH and ionic strength produced the most consistent results. This report examines the effects of altering the injector geometry and solution ionic strengths, and presents the results of using bias voltages to control plug formation. The observed results should provide a benchmark for modeling of the fluid dynamics in channel intersections.     

Hydrodynamic injection with pneumatic valving for microchip electrophoresis with total analyte utilization

A novel hydrodynamic injector that is directly controlled by a pneumatic valve has been developed for reproducible microchip CE separations. The PDMS devices used for the evaluation comprise a separation channel, a side channel for sample introduction, and a pneumatic valve aligned at the intersection of the channels. A low pressure (≤ 3?psi) applied to the sample reservoir is sufficient to drive sample into the separation channel. The rapidly actuated pneumatic valve enables injection of discrete sample plugs as small as ~ 100?pL for CE separation. The injection volume can be easily controlled by adjusting the intersection geometry, the solution back pressure, and the valve actuation time. Sample injection could be reliably operated at different frequencies (< 0.1?Hz to > 2?Hz) with good reproducibility (peak height relative standard deviation ≤ 3.6%) and no sampling biases associated with the conventional electrokinetic injections. The separation channel was dynamically coated with a cationic polymer, and FITC-labeled amino acids were employed to evaluate the CE separation. Highly efficient (≥ 7.0 × 103 theoretical plates for the ~2.4-cm-long channel) and reproducible CE separations were obtained. The demonstrated method has numerous advantages compared with the conventional techniques, including repeatable and unbiased injections, little sample waste, high duty cycle, controllable injected sample volume, and fewer electrodes with no need for voltage switching. The prospects of implementing this injection method for coupling multidimensional separations for multiplexing CE separations and for sample-limited bioanalyses are discussed.     

A dynamic loading method for controlling on-chip microfluidic sample injection

A new technique for controlling discrete sample injection in straight-cross microfluidic chips is presented here. This technique involves a three-part process with a dynamic loading step in between the steady-state loading step and the dispensing step. During the intermediate step, sample is pumped into the intersection and into the three connecting channels. The key features of this technique are the ability to dynamically control the sample size and the ability to inject well-defined samples at the original sample concentration. Injections of these samples with lengths varying from 2 channel widths (100 microm) to 20 channel widths (millimeter-sized) are demonstrated. The sample concentration profiles obtained are compared with those of focused and less-focused pinched-valve injections. In applications such as high-speed capillary zone electrophoresis, this technique can provide an increase in signal with a small increase in sample length. This technique is especially applicable to many large-sample applications in which the offset twin-T microchip has been previously employed.     

Pressure pinched injection of nanolitre volumes in planar micro-analytical devices

A new method for injecting and driving fluids by means of a multi-port injection valve and syringe pumps in a micro-channel network is described. A structure composed of two micro-channels arranged as a cross is connected with capillary tubes to an external multi-port injection valve. The fluid flows are driven by pressure and the multi-port valve controls the direction of the flow within the different sections of the structure. The first position of the multi-port valve allows the preparation of the loading of the sample, which is pinched in the cross section of the two micro-channels. The second position allows the precise injection of nL volumes. No dead volume exists between injection and separation modes. The system can be used to prepare a sample plug by pressure in order to perform chromatography with a broad range of buffered or non-buffered solutions. Thanks to the insensitivity to the ionic strength of the sample, this injection method is useful for the injection of complex biological samples in microchip analysis. In order to demonstrate the feasibility of the method, different solutions of ionic or fluorescent molecules were injected and detected in a photoablated planar polymer device.     

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.     

Pressure-driven sample injection with quantitative liquid dispensing for on-chip electrophoresis.

A novel pressure-driven sample injection method was developed as an alternative to electrokinetic injection, and electrophoretic separation was carried out on a microfabricated device employing this method. This method enables a defined volume of liquid dispensing, followed by instantaneous injection driven by pneumatic pressure, greatly simplifying the injection procedure. A particular microstructure, called a "metering chamber", has been designed for the quantitative dispensing of an ultra-low volume of sample liquid; a "hydrophobic passive valve" equipped with an air vent channel is employed for injecting a dispensed sample into the separation channel. The reproducibility of dispensing was 3.3% (n = 15), expressed by the variation of dispensed volumes. The electrophoretic separation of DNA fragments was performed using this injection method, varying the injection volumes from 0.45 to 4.0 nL, and the separation efficiencies were compared. This precise injection method, easily variable in injection volumes, is highly suitable for quantitative as well as qualitative electrophoretic analyses.     

Numerical calculation of the electroosmotic flow at the cross region in microfluidic chips.

A numerical study is presented for the electroosmotic flow (EOF) at the cross region in microfluidic chips. The distributions of the electric potential due to the electric double layer (EDL) and the external electric field are discussed and the calculation of the latter can give rough speculations on the flow tendencies in the channels during various operation modes. Simplification of the two-dimensional Navier-Stokes (N-S) equations is obtained by focusing on the solution of interior flows, and the numerical calculation results show good agreement with the experimental images. The sample leakage to the separation channel during the "float" sampling proved to be caused not only by the sample diffusion, but also by the weak extension of the sampling electric field. It is also verified that with suitable voltage configuration, the "pinch" sampling mode is better than the "float" mode in sample plug control.     

Control-free air vent system for ultra-low volume sample injection on a microfabricated device.

An improved method of sample injection was demonstrated for introducing ultra-low volume liquid on a microfabricated device. In our previous study, a pressure-driven injection method has been introduced and was applied to on-chip electrophoresis. In this study, the need for control of the air vent, which was indispensable for sample injection in the previous study, was completely eliminated, facilitating sample injection with great simplicity and high accuracy. This was realized by altering the topology of the air vent channel, which is connected to a hydrophobic and narrow channel (called a passive valve). Several types of air vent channels were designed and their injection performances were tested. In addition, by modifying the shape and the position of air vent channel and passive valve, the residual liquid volume inside the passive valve after sample injection was decreased to approximately 0.5% of the injected volume, a value which showed high reproducibility.     

A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis

A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 microm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes.