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.     

Numerical simulation of electrokinetic injection techniques in capillary electrophoresis microchips

The effective design and control of a capillary electrophoresis (CE) microchip requires a thorough understanding of the electrokinetic transport phenomena associated with its microfluidic injection system. The present study utilizes a numerical simulation approach to investigate these electrokinetic transport processes and to study the control parameters of the injection process. Injection systems with a variety of different configurations are designed and tested, including the cross-form, T-form, double-T-form, variable-volume focused flow cross-form, and variable-volume triple-T-form configuration. Each injection system cycles through a predetermined series of steps in which the magnitudes and distributions of the applied electric field are precisely manipulated in order to effectuate a virtual valve. This study investigates the sample leakage effect associated with each of the injection configurations and applies the double-L, pullback, and focusing injection techniques to minimize the sample leakage effect. The injection methods presented in this paper have the exciting potential for use in high-quality, high-throughput chemical analysis applications and throughout the micro-total-analysis systems field.     

Microautosamplers for discrete sample injection and dispensation

Microfluidic systems show considerable potential for use in the continuous reaction and analysis of biosamples for various applications, such as drug screening and chemical synthesis. Typically, microfluidic chips are externally connected with large-scale autosamplers to inject specific volumes of discrete samples in the continuous monitoring and analysis of multiple samples. This paper presents a novel microelectromechanical system (MEMS)-based autosampler capable of performing the discrete injection and dispensation of variable-volume samples. This microdevice can be integrated with other microfluidic devices to facilitate the continuous monitoring and analysis of multiple biosamples. By means of electroosmotic focusing and switching controlled by the direct application of electric sources on specific fluid reservoirs, a precise sample volume can be injected into the specified outlet port. Fluorescence dye images verify the performance of the developed device. An injection-and-washing scheme is developed to prevent cross-contamination during the continuous injection of different samples. This approach renders feasible the injection of several discrete samples using a single microchip. Compared to its large-scale counterparts, the developed microautosampler is compact in size, has low fabrication costs, is straightforward to control, and most importantly, is readily integrated with other microfluidic devices (e.g., microcapillary electrophoresis chips) to form a microfluidic system capable of the continuous monitoring and analysis of bioreactions. The proposed microautosampler could be promising towards realizing the micrototal analysis system (mu-TAS) concept.     

Design of an interface to allow microfluidic electrophoresis chips to drink from the fire hose of the external environment

An interface design is presented that facilitates automated sample introduction into an electrokinetic microchip, without perturbing the liquids within the microfluidic device. The design utilizes an interface flow channel with a volume flow resistance that is 0.54-4.1 x 10(6) times lower than the volume flow resistance of the electrokinetic fluid manifold used for mixing, reaction, separation, and analysis. A channel, 300 microm deep, 1 mm wide and 15-20 mm long, was etched in glass substrates to create the sample introduction channel (SIC) for a manifold of electrokinetic flow channels in the range of 10-13 microm depth and 36-275 microm width. Volume flow rates of up to 1 mL/min were pumped through the SIC without perturbing the solutions within the electrokinetic channel manifold. Calculations support this observation, suggesting a leakage flow to electroosmotic flow ratio of 0.1:1% in the electrokinetic channels, arising from 66-700 microL/min pressure-driven flow rates in the SIC. Peak heights for capillary electrophoresis separations in the electrokinetic flow manifold showed no dependence on whether the SIC pump was on or off. On-chip mixing, reaction and separation of anti-ovalbumin and ovalbumin could be performed with good quantitative results, independent of the SIC pump operation. Reproducibility of injection performance, estimated from peak height variations, ranged from 1.5-4%, depending upon the device design and the sample composition.     

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10-100 fold in comparison with conventional IEF.     

Optimal configuration of capillary electrophoresis microchip with expansion chamber in separation channel

This study develops a novel capillary electrophoresis (CE) microfluidic device featuring a conventional cross-form injection system and an expansion chamber located at the inlet of the separation channel. The combined injection system/expansion chamber arrangement is designed to deliver a high-quality sample band into the separation channel such that the detection performance of the device is enhanced. Numerical simulations are performed to investigate the electrokinetic transport processes in the microfluidic device and to establish the optimal configuration of the expansion chamber. The results indicate that an expansion chamber with an expansion ratio of 2.5 and an expansion length of 500 microm delivers a sample plug with the correct shape and orientation. With this particular configuration, the peak intensities of the sample are sharp and clearly distinguishable in the detection region of the separation channel. Therefore, this configuration is well suited for capillary electrophoresis applications which require a highly sensitive resolution of the sample plug. The novel CE microfluidic device developed in this study has an exciting potential for use in high-performance, high-throughput chemical analysis applications and in many other applications throughout the field of micro-total-analysis-systems.     

Improvements on the electrokinetic injection technique for microfluidic chips

This paper presents a T-form electrokinetic injection system for the discrete time-based loading and dispensing of samples of variable-volume in a microfluidic chip. A novel push-pull effect is produced during the loading and dispensing processes by the application of an appropriate control voltage distribution. The experimental and numerical results show that this push-pull loading technique produces compact sample plugs and hence improves the detection resolution of the microfluidic device. The injection system is integrated with a microflow switch, and a suitable voltage control scheme is proposed to guide the sample to the desired outlet port such that the microfluidic device can function as a microdispenser. The time-based variable-volume T-form injection method presented in this study is performed using a compact geometry and a simple control scheme and can be readily integrated with other microfluidic devices to form a microfluidic system capable of continuous monitoring and analysis of bioreactions in the life science and biochemistry fields.     

Flexible particle flow‐focusing in microchannel driven by droplet‐directed induced‐charge electroosmosis

We report herein a novel microfluidic particle concentrator that utilizes constriction microchannels to enhance the flow‐focusing performance of induced‐charge electroosmosis (ICEO), where viscous hemi‐spherical oil droplets are embedded within the mainchannel to form deformable converging‐diverging constriction structures. The constriction region between symmetric oil droplets partially coated on the electrode strips can improve the focusing performance by inducing a granular wake flow area at the diverging channel, which makes almost all of the scattered sample particles trapped within a narrow stream on the floating electrode. Another asymmetric droplet pair arranged near the outlets can further direct the trajectory of focused particle stream to one specified outlet port depending on the symmetry breaking in the shape of opposing phase interfaces. By fully exploiting rectification properties of induced‐charge electrokinetic phenomena at immiscible water/oil interfaces of tunable geometry, the expected function of continuous and switchable flow‐focusing is demonstrated by preconcentrating both inorganic silica particles and biological yeast cells. Physical mechanisms responsible for particle focusing and locus deflection in the droplet‐assisted concentrentor are analyzed in detail, and simulation results are in good accordance with experimental observations. Our work provides new routes to construct flexible electrokinetic framework for preprocessing on‐chip biological samples before performing subsequent analysis.     

Characterization of electrokinetic gating valve in microfluidic channels

Electrokinetic gating, functioning as a micro-valve, has been widely employed in microfluidic chips for sample injection and flow switch. Investigating its valving performance is fundamentally vital for microfluidics and microfluidics-based chemical analysis. In this paper, electrokinetic gating valve in microchannels was evaluated using optical imaging technique. Microflow profiles at channels junction were examined, revealing that molecular diffusion played a significant role in the valving disable; which could cause analyte leakage in sample injection. Due to diffusion, the analyte crossed the interface of the analyte flow and gating flow, and then formed a cometic tail-like diffusion area at channels junction. From theoretical calculation and some experimental evidences, the size of the area was related to the diffusion coefficient and the velocity of analytes. Additionally, molecular diffusion was also believed to be another reason of sampling bias in gated injection.     

微流控芯片二维电泳研究进展

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

Split injection: A simple introduction of subnanoliter sample volumes for chip electrophoresis

The ability to accurately inject small volumes of sample into microfluidic channels is of great importance in electrophoretic separations. While electrokinetic injection of nanoliter scale volumes is commonly utilized in microchip capillary electrophoresis (MCE), mobility and matrix bias makes quantitation difficult. Herein, we describe a new injection method based on the simple patterning of the crossing of channels that does not require sophisticated instrumentation. The sample volume injected into the separation channel is dependent on the ratio of the widths of the crossing channels. This injection method is capable of introducing, into a separation channel, multiple plugs of sample on a large scale. This injection technique is tested for zone electrophoresis in native and surface modified poly(dimethylsiloxane) (PDMS) chips.     

Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing

Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic flow cytometer developed in this paper provides a practical platform that can be used for routine analysis in biological laboratories. Additionally, the 3D hydrodynamic focusing channel design can also be applied to various applications that can advance the lab on a chip research.     

Reduction in sample injection bias using pressure gradients generated on chip

Sample injection in microchip-based capillary zone electrophoresis (CZE) frequently rely on the use of electric fields which can introduce differences in the injected volume for the various analytes depending on their electrophoretic mobilities and molecular diffusivities. While such injection biases may be minimized by employing hydrodynamic flows during the injection process, this approach typically requires excellent dynamic control over the pressure gradients applied within a microfluidic network. The current article describes a microchip device that offers this needed control by generating pressure gradients on-chip via electrokinetic means to minimize the dead volume in the system. In order to realize the desired pressure-generation capability, an electric field was applied across two channel segments of different depths to produce a mismatch in the electroosmotic flow rate at their junction. The resulting pressure-driven flow was then utilized to introduce sample zones into a CZE channel with minimal injection bias. The reported injection strategy allowed the introduction of narrow sample plugs with spatial standard deviations down to about 45 μm. This injection technique was later integrated to a capillary zone electrophoresis process for analyzing amino acid samples yielding separation resolutions of about 4–6 for the analyte peaks in a 3 cm long analysis channel.     

Towards high concentration enhancement of microfluidic temperature gradient focusing of sample solutes using combined AC and DC field induced Joule heating

It is challenging to continuously concentrate sample solutes in microfluidic channels. We present an improved electrokinetic technique for enhancing microfluidic temperature gradient focusing (TGF) of sample solutes using combined AC and DC field induced Joule heating effects. The introduction of an AC electric field component services dual functions: one is to produce Joule heat for generating temperature gradient; the other is to suppress electroosmotic flow. Consequently the required DC voltages for achieving sample concentration by Joule heating induced TGF are reduced, thereby leading to smaller electroosmotic flow (EOF) and thus backpressure effects. As a demonstration, the proposed technique can lead to concentration enhancement of sample solutes of more than 2500-fold, which is much higher than the existing literature reported microfluidic concentration enhancement by utilizing the Joule heating induced TGF technique.     

Continuous concentration of bacteria in a microfluidic flow cell using electrokinetic techniques

A novel method for the concentration of bacterial solutions is presented that implements electrokinetic techniques, zone electrophoresis (ZE) and isoelectric focusing (IEF), in a microfluidic device. The method requires low power (< 3e-5 W) and can be performed continuously on a flowing stream. The device consists of two palladium electrodes held in a flow cell constructed from layers of polymeric film held together by a pressure-sensitive adhesive. Both ZE and IEF are performed with carrier-free solutions in devices in which the electrodes are in intimate contact with the sample fluid. IEF experiments were performed using natural pH gradients; no carrier ampholyte solution was required. Experiments performed in buffer alone resulted in significant electroosmotic flow. Pretreatment of the sample chamber with bleach followed by a concentrated solution of cationic detergent effectively suppressed electroosmotic flow.     

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

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

Effect of shell-side flows on the performance of hollow-fiber gas separation modules

A theoretical analysis of shell-side flow effects on the performance of hollow-fiber gas separation modules is presented. The theory uses Darcy’s law to relate fiber packing, pressure fields, and velocity fields within the shell. The resulting shell conservation equations are coupled to the lumen conservation equations through the permeation relationship. This two-dimensional (2-D) analysis quantifies the performance penalty associated with gas distribution across the fiber bundle at the shell inlet and outlet. Theoretical predictions for the production of nitrogen from air in a commercial shell-fed module are closer to experimental data than predictions obtained assuming one-dimensional (1-D) plug flow. Fluid flows primarily across fibers near the inlet and outlet ports, and along fibers between ports. Nitrogen composition increases along fluid streamlines, which leads to axial and radial concentration variations within the fiber bundle. Diffusional contributions to shell mass transfer are small for the modules considered here.     

High-efficiency electrokinetic micromixing through symmetric sequential injection and expansion

Rapid electric field switching is an established microfluidic mixing strategy for electrokinetic flows. Many such microfluidic mixers are variations on the T- or Y-form channel geometry. In these configurations, rapid switching of the electric field can greatly improve initial mixing over that achieved with static-field mixing. Due to a fundamental lack of symmetry, however, these strategies produce lingering cross-channel concentration gradients which delay complete mixing of the fluid stream. In this paper, a field switching microfluidic mixing strategy which utilizes a symmetric sequential injection geometry with an expansion chamber to achieve high efficiency microfluidic mixing is demonstrated experimentally. A three-inlet injector sequentially interlaces two dissimilar incoming solutions. Downstream of the injector, the sequence enters an expansion chamber resulting in a dramatic (two orders of magnitude) decrease in Peclet number and rapid axial diffusive mixing. The outlet concentration may be accurately varied over the full spectrum by tuning the duty cycle of the field switching waveform. The chips are designed with input from a previous numerical study, manufactured in poly(dimethylsiloxane) using soft-lithography based microfabrication, and tested using fluorescence microscopy. In the context of on-chip chemical processing for analytical operations, the demonstrated mixing strategy has several features: high mixing efficiency (99%), compact axial length (2.3 mm), steady outflow velocity, and readily variable outlet concentration (0.15 < c* < 0.95).     

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.