Continuous monitoring with microfabricated capillary electrophoresis chip devices

We report on the direct coupling of hydrodynamically flowing stream to a microchip capillary electrophoresis (CE) for continuous assays of liquid samples. The new interface relies on mounting the sample tubing onto a sharp inlet tip and allows rapid, convenient and reproducible electrokinetic loading from a continuously flowing stream directly into the narrow separation microchannel. The sharp inlet interface is characterized by its efficiency, stability and simplicity. The effect of the sample flow rate, applied voltages and other relevant variables, is described. It was found that the peak intensity is independent of the flow rate. The performance of the new interface is illustrated for on-line CE-electrochemical monitoring of phenolic and explosive compounds. Conditions simulating continuous long-term monitoring, led to a highly stable response for a 15 ppm 1,3,5-trinitrobenzene solution (RSD = 3.7%, n= 40). Such ability to continuously introduce flowing samples into micrometer channels makes 'lab-on-a-chip' devices highly compatible with real-life monitoring applications.     

Electrophoresis microchips with sharp inlet tips, for contactless conductivity detection, fabricated by in-situ surface polymerization

A novel method based on in-situ surface polymerization of methyl methacrylate (MMA) has been developed for rapid fabrication of poly(methyl methacrylate) (PMMA) electrophoresis microchips with sharp inlet tips. Prepolymerized MMA containing an ultraviolet (UV) initiator was directly sandwiched between a nickel template and a PMMA plate. The image of the relief on the nickel template was precisely replicated in the synthesized PMMA layer on the surface of the commercially available PMMA plate during UV-initiated polymerization at room temperature. The chips were subsequently assembled by thermal bonding of channel plates and cover sheets. The sample was directly introduced into the separation channel through a sharp inlet tip, which was placed in the sample vial, without use of an injection cross. The attractive performance of the novel PMMA microchips has been demonstrated by using contactless conductivity detection for determination of several inorganic ions. Such rapid and simple sample introduction leads to highly reproducible signals with relative standard deviations of less than 5% for peak responses. These new approaches significantly simplify the process of fabricating PMMA devices and show great promise for high-speed microchip analysis.      

Poly（dimethylsiloxane） Microchips with Two Sharpened Stretching Tips and Its Application to Protein Separation Using Dynamic Coating

An integrated poly（dirnethylsiloxane） （PDMS） microchip with two sharpened stretching tips for convenient sample injecting, running buffer refreshing and channel cleaning has been presented. The sample was directly introduced into the separation channel through the stretching inlet tip without complicated power switching supplies and injection cross channel. The operation of running buffer refreshing or channel cleaning was simplified by vacuuming one end of the tip and placing the other tip into the solution vial. Therefore, this fabrication method can be easily applied to most analytical laboratories economically without soft lithography and plasma bonding equipments. The attractive performance of the novel PDMS microchips has been demonstrated by using laser-induced fluorescence detection for separation of proteins. The addition of 0.04% Brij 35 in 0.04 mol/L phosphate buffer （pH 7.0） can reduce the adhesion of proteins in multienzyme tablet and make separation more easily. The electroosmotic flow （EOF） exhibits pH-independence in the range of 3-1 1 in dynamic modified microchannel.     

Fabrication of a novel poly(dimethylsiloxane) microchips with two sharpened stretching tips

An integrated poly(dimethylsiloxane) (PDMS) microchip with two sharpened stretching has been presented. The sample was directly introduced into the separation channel through the stretching inlet tip without complicated power switching supplies and without injection cross-channel. Operations of running buffer refreshing or channel cleaning also becomes simple by vacuumed in one end and placed another tip into solution vial. The fabrication method can be easily applied in most analytical laboratories at low cost in the absence of soft lithography and plasma bonding equipments. Characteristics of the chips were tested and it can be used to separate fluorescence labeled molecules.     

Recent developments in electrochemical detection for microchip capillary electrophoresis

Significant progress in the development of miniaturized microfluidic systems has occurred since their inception over a decade ago. This is primarily due to the numerous advantages of microchip analysis, including the ability to analyze minute samples, speed of analysis, reduced cost and waste, and portability. This review focuses on recent developments in integrating electrochemical (EC) detection with microchip capillary electrophoresis (CE). These detection modes include amperometry, conductimetry, and potentiometry. EC detection is ideal for use with microchip CE systems because it can be easily miniaturized with no diminution in analytical performance. Advances in microchip format, electrode material and design, decoupling of the detector from the separation field, and integration of sample preparation, separation, and detection on-chip are discussed. Microchip CEEC applications for enzyme/immunoassays, clinical and environmental assays, as well as the detection of neurotransmitters are also described.     

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 polymeric microchip with integrated tips and in situ polymerized monolith for electrospray mass spectrometry

We describe the integration of a cyclo-olefin polymer based microchip with a sheathless capillary tip for electrospray ionization-mass spectrometry (ESI-MS). The microchip was fabricated by hot embossing and thermal bonding. Its design includes a side channel for adjusting the composition of the electrospray solution so that analytes in 100% water can be analyzed. The fused silica capillaries, used for sample introduction, and the electrospray tips for MS coupling were directly inserted into the microchannel before thermal bonding of the device. A microfabricated on-chip gold microelectrode was used to apply the electrospray voltage. Annealing the device after thermal bonding increased the pressure resistance of the microchip. The cross section of the microchannel was imaged by scanning electron microscopy to estimate the effects of the annealing step. The relationship between the applied electrospray voltages and MS signal was measured at different flow rates by coupling the device to an ion trap mass spectrometer. The performance of the microchip was evaluated by MS analysis of imipramine in ammonium acetate buffer solution by direct infusion. An alkylacrylate based monolith polymer bed for on-chip sample pretreatment and separation was polymerized in the microchannel and tested for ESI-MS applications.     

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.     

An automated electrokinetic continuous sample introduction system for microfluidic chip-based capillary electrophoresis

An automated and continuous sample introduction system for microfluidic chip-based capillary electrophoresis (CE) was developed in this work. An efficient world-to-chip interface for chip-based CE separation was produced by horizontally connecting a Z-shaped fused silica capillary sampling probe to the sample loading channel of a crossed-channel chip. The sample presentation system was composed of an array of bottom-slotted sample vials filled alternately with samples and working electrolyte, horizontally positioned on a programmable linearly moving platform. On moving the array from one vial to the next, and scanning the probe, which was fixed with a platinum electrode on its tip, through the slots of the vials, a series of samples, each followed by a flow of working electrolyte was continuously introduced electrokinetically from the off-chip vials into the sample loading channel of the chip. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine and phenylalanine with LIF detection, by continuously introducing a train of different samples. Employing 4.5 kV sampling voltage (1000 V cm(-1) field strength) for 30 s and 1.8 kV separation voltage (400 V cm(-1) field strength) for 70 s, throughputs of 36 h(-1) were achieved with <1.0% carryover and 4.6, 3.2 and 4.0% RSD for arginine, FITC and phenylalanine, respectively (n = 11). Net sample consumption was only 240 nL for each sample.     

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance.     

High-throughput characterization for organic pollutants in environmental waters using a capillary electrophoresis chip.

To evaluate organic pollution in water, we did preliminarily studies on high-throughput characterization of organic pollution in water using microchip-based capillary electrophoresis (CE) with laseer-induced fluorescence (LIF) detection. The applied voltage was investigated to control the gated valve injection and CE separation for conventional cross type microchips using a self-made personal computer (PC)-based controller as the voltage supply. We obtained high-throughput data for the reproducible separation of fluorescein isothiocyanate (FITC)-labeled river-water samples using a zwitter-ion based buffer solution to avoid adsorption of the labeled sample onto the channel of a microchip made from quartz glass. We used real samples from the Hino River that flows into Lake Biwa, from ten sampling points and obtained several reproducible peaks in different separation patterns for each sample within 2 min. We successfully demonstrated high-throughput characterization of dissolved organic carbon (DOC) in environmental water using the microchip.     

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.     

Fabrication of a monolithic sampling probe system for automated and continuous sample introduction in microchip-based CE

A fabrication process for producing monolithic sampling probes on glass chips, with tip diameters of a few hundred micrometers was developed, using simple tools including a glass cutter and a bench drill. Microfluidic chips with probes fabricated by this approach were coupled to a linearly moving slotted-vial array sample presentation system for performing continuous sample introduction in the chip-based CE system. On-chip horizontal tubular reservoirs containing working electrolyte and waste were used to maintain a stable hydrostatic pressure in the chip channels during prolonged working periods. The performance of the system was demonstrated in the separation of FITC-labeled amino acids with LIF detection, by continuously introducing a train of different samples without interruption. Throughputs of 30-60/h were achieved with <1.0% carry-over and reproducibilities in peak height of 3.6, 3.3, and 3.5% RSD for arginine, FITC, and phenylalanine, respectively (n = 11). Continuous analysis of a mixture of FITC-labeled amino acids for 2 h, involving 60 analytical cycles, yielded an RSD of 7.5 and 6.8% for arginine and FITC (n = 60), respectively. An extremely low sample consumption of 30 nL for each analysis was obtained. Separation efficiencies in plate numbers were in the range of 0.8-2x10(5)/m. In addition to the application in sample introduction, the sample/reagent introduction system was also used to produce working electrolyte gradients during a CE separation to improve the separation efficiency. Comparing with isocratic electrophoresis separation, gradient CE demonstrated better separation efficiencies for a mixture of FITC-labeled amino acids.     

Simultaneous microchip enzymatic measurements of blood lactate and glucose

A miniaturized capillary electrophoretic (CE) microchip device for the simultaneous measurements of lactate and glucose is described. The new microchip bioassay protocol integrates an electrophoretic separation of lactate and glucose, post-column enzymatic reactions of these metabolites with their respective oxidase enzymes, and an amperometric (anodic) detection of enzymatically-liberated hydrogen peroxide at a gold-coated thick-film carbon detector. Factors influencing the response have been examined and optimized, and the analytical performance has been characterized. Applicability of the microchip assay to clinical samples, such as serum and blood, is demonstrated. The microchip protocol obviates cross enzymatic reactions and interferences from major oxidizable constituents common to dual glucose-lactate enzyme electrodes. Such ability to rapidly separate and quantitate lactate and glucose on a small microchip platform should find important clinical and biotechnological applications.     

Integrated isotachophoretic stacking and gel electrophoresis on a plastic substrate and variations in detection dynamic range

In this study, we demonstrated an integrated ITP-gel electrophoresis (GE) device on a plastic substrate, in which 50 nL of samples could be hydrodynamically or electrokinetically injected and enriched by ITP into narrow bands and then subsequently introduced into a homogeneous GE channel for separation and detection. This microchip design rendered a simple introduction scheme for creating sandwiched stacking buffer system and flexibilities in choosing separation and stacking buffers independently. We used gel sieving buffers which compositions were different from those for stacking buffers to separate DNA and protein molecules based on sizing mechanism. Compared to conventional microchip GE, the sensitivity of microchip ITP-GE was estimated to increase by one to two orders of magnitude based on the dilution factor of the injected sample and the S/N ratio detected from the electropherogram. Moreover, it is interesting to note that ITP stacking leads to a preferential enhancement for analytes with lower concentrations compared to those with higher concentrations. Therefore, a reduction in the detection dynamic range for ITP-GE was gained. We demonstrated that ITP-GE could lead to 2-4-folds of reduction in the signal dynamic range for two PCR products in a mixture. Such advantage is demonstrated to be useful for the detection of two products amplified from a multiplex PCR in which one product is poorly amplified compared to the other.     

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.     

Automated sampling system for the analysis of amino acids using microfluidic capillary electrophoresis

An improved automated continuous sample introduction system for microfluidic capillary electrophoresis (CE) is described. A sample plate was designed into gear-shaped and was fixed onto the shaft of a step motor. Twenty slotted reservoirs for containing samples and working electrolytes were fabricated on the “gear tooth” of the plate. A single 7.5-cm long Teflon AF-coated silica capillary serves as separation channel, sampling probe, as well as liquid-core waveguide (LCW) for light transmission. Platinum layer deposited on the capillary tip serves as the electrode. Automated continuous sample introduction was achieved by scanning the capillary tip through the slots of reservoirs. The sample was introduced into capillary and separated immediately in the capillary with only about 2-nL gross sample consumption. The laser-induced fluorescence (LIF) method with LCW technique was used for detecting fluorescein isothiocyanate (FITC)-labeled amino acids. With electric-field strength of 320 V/cm for injection and separation, and 1.0-s sample injection time, a mixture of FITC-labeled arginine and leucine was separated with a throughput of 60/h and a carryover of 2.7%.     

Poly(methylmethacrylate) and Topas capillary electrophoresis microchip performance with electrochemical detection

A capillary electrophoresis (CE) microchip made of a new and promising polymeric material: Topas (thermoplastic olefin polymer of amorphous structure), a cyclic olefin copolymer with high chemical resistance, has been tested for the first time with analytical purposes, employing an electrochemical detection. A simple end-channel platinum amperometric detector has been designed, checked, and optimized in a poly-(methylmethacrylate) (PMMA) CE microchip. The end-channel design is based on a platinum wire manually aligned at the exit of the separation channel. This is a simple and durable detection in which the working electrode is not pretreated. H(2)O(2) was employed as model analyte to study the performance of the PMMA microchip and the detector. Factors influencing migration and detection processes were examined and optimized. Separation of H(2)O(2) and L-ascorbic acid (AsA) was developed in order to evaluate the efficiency of microchips using different buffer systems. This detection has been checked for the first time with a microchip made of Topas, obtaining a good linear relationship for mixtures of H(2)O(2) and AsA in different buffers.     

A simple and compact blue diode laser powered excitation source for fluorescence detection in capillary electrochromatographic microchip separation

A simple and compact fluorescence excitation source was prepared using a 405 nm blue laser diode module and characterized in capillary electrochromatographic or capillary electrophoretic microchip separation. An inexpensive blue laser diode module with a tiny focusing lens was simply mounted at the center of an aluminum block on a miniature linear motion guide for heat dissipation and position control. A slit unit has a series of fifteen laser-machined slits with 1 mm space along the direction of the separation channel of the microchip above this unit. The laser beam was focused through a slit with 50 μm width to the separation channel at the position of a desired length. Although the excitation source unit was connected to a simple current controlled power supply, it was stable with 0.1% drift per hour and 1.3% (1σ) fluctuation in intensity. This simple excitation source can be prepared easily with inexpensive minimum optical components and mounted with a microchip on the stage of an ordinary fluorescence microscope for daily separation studies using a CE or CEC microchip. The applicability of the excitation source was evaluated with FITC-amino acid derivative mixtures using a polymer based CEC microchip packed fully with submicron silica beads in its microchannel.     

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.