Free-flow electrophoresis on an anodic bonded glass microchip

A micro-free-flow electrophoresis chip has been fabricated into a glass wafer etched with 20-microm-deep channels. Wafers were bonded anodically using an intermediate amorphous silicon film. Electric fields as high as 283 V/cm were applied across the separation channel to obtain baseline resolution of fluorescent standards in 4.8 s. The effect of electric fields ranging from 0 to 283 V/cm on the separations and resulting resolutions were examined. Resolution was shown to increase linearly with the applied electric field. Joule heating was not significant under the conditions tested. Instead, the generation of electrolysis products at higher currents proved to be the limiting factor preventing higher separation potentials from being used.     

Fast electrophoretic separation optimization using gradient micro free-flow electrophoresis

The continuous nature of micro free-flow electrophoresis (mu-FFE) was used to monitor the effect of a gradient of buffer conditions on the separation. This unique application has great potential for fast optimization of separation conditions and estimation of equilibrium constants. COMSOL was used to model pressure profiles in the development of a new mu-FFE design that allowed even application of a buffer gradient across the separation channel. The new design was fabricated in an all glass device using our previously published multiple-depth etch method (Fonslow, B. R.; Barocas, V. H.; Bowser, M. T. Anal. Chem. 2006, 78, 5369-5374, ref 1). Fluorescein solutions were used to characterize the applied gradients in the separation channel. Linear gradients were observed when buffer conditions were varied over a period of 5-10 min. The effect of a gradient of 0-50 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the separation of a group of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) labeled primary amines was monitored as a proof of concept experiment. Direct comparisons to capillary electrophoresis (CE) separations performed under the same conditions were made. Gradient mu-FFE recorded 60 separations during a 5 min gradient allowing nearly complete coverage across a range of HP-beta-CD concentrations. In comparison, 4 h were required to assess 15 sets of conditions across the same range of HP-beta-CD concentrations using CE. Qualitatively, mu-FFE separations were predictive of the migration order and spacing of peaks in CE electropherograms measured under the same conditions. Data were fit to equations describing 1:1 analyte-additive binding to allow a more quantitative comparison between gradient mu-FFE and CE.     

Palladium film decoupler for amperometric detection in electrophoresis chips

Demonstrated in this article is that a palladium metal film can be applied to decouple the electric circuitry of electrochemical detection from that of the electrophoretic separation in an electrophoresis chip. The Pd solid-state field decoupler, as well as the working electrodes, is thermally evaporated onto the plastic chip and oriented vertically across the separation channel. After the sample zones flow over the Pd decoupler, their electrochemical response is measured at working electrodes in the downstream pathway. Because the electrodes are on the separation channel, the electrode channel alignment is no longer a problem. For a separation channel of roughly 200 microm in width and 75 microm in depth in 10 mM phosphate (pH 5.1), the noise level at the working electrode is < 15 pA at an electric field of 570 V/cm.     

Electroosmotically induced hydraulic pumping with integrated electrodes on microfluidic devices

Electroosmotic manipulation of fluids was demonstrated using thin metal electrodes integrated within microfluidic channels at the substrate and cover plate interface. Devices were fabricated by photolithographically patterning electrodes on glass cover plates that were then bonded to polymeric substrates into which the channels were cast. Polymeric substrates were used to provide a permeable membrane for the transport and removal of gaseous electrolysis products generated at the electrodes. Electroosmotic flow between interdigitated electrodes was demonstrated and provided electric field-free pumping of fluids in sections of the channel outside of the electrode pairs. The resultant pumping velocities were shown to be dependent on the applied voltage, not on the applied field strength, and independent of the length of the electroosmotically pumped region.     

Concentration and separation of proteins in microfluidic channels on the basis of transverse IEF

The use of microfluidic channels formed by two electrodes made of gold or palladium to perform transverse isoelectric focusing (IEF) is presented as a means for continuous concentration and fractionation of proteins. The microchannels were 40 mm long with an electrode gap of 1.27 mm and a depth of 0.354 mm. The properties of pH gradients formed as a result of the electrolysis of water were influenced by variation of parameters such as the initial pH, ionic strength, and flow rate. Transverse IEF in pressure-driven flow is demonstrated using bovine serum albumin in a single ampholyte buffer as well as in multiple-component buffers. Experimental results of protein focusing compare well to predictions of a mathematical model. Optimal conditions for efficient continuous fractionation of a protein mixture are summarized and discussed.     

Isoelectric focusing in a microfluidically defined electrophoresis channel

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.     

On-chip microfluidic transport and mixing using electrowetting and incorporation of sensing functions

An integrated system was developed that performs microfluidic transport, mixing, and sensing on a single chip. The operation principle for the microfluidic transport was based on electrowetting. A solution to be transported was confined in a space between a row of gold working electrodes and a protruding poly(dimethylsiloxane) (PDMS) structure. When a negative potential was applied to one of the gold working electrodes, it became hydrophilic, and the solution was transported through the flow channel. The solution could be transported in any desired direction in a network of flow channels by switching on necessary electrodes one by one. Furthermore, two solutions transported through two flow channels could be mixed using a mixing electrode based on the same principle. To demonstrate the applicability of a lab-on-a-chip, an air gap ammonia electrode was integrated by taking advantage of the open structure of the flow channel. Gaseous ammonia that was produced after pH adjustment and diffused through an air gap caused a pH change in the electrolyte layer, which was measured with an iridium oxide pH indicator electrode. The 90% response time was less than 1 min for the millimolar order of ammonia. The calibration curve was linear down to 10 microM. The ammonia-sensing system was also applied to construct biosensing systems for urea and creatinine. A linear relationship was observed between the potential and the logarithm of the concentration of the analytes down to 50 microM for both urea and creatinine. The developed microfluidic system can be a basic building block for future systems.     

Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bpy)3(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 microM (S/N = 3) and a linear range from 5 to 600 microM.     

Mechanistic investigation of nanoparticle motion in pulsed voltage miniaturized electrical field flow fractionation device by in situ fluorescence imaging

In our previous study, we reported a miniaturized electrical field flow fractionation device (micro-EFFF) that used a pulsed voltage (PV) to increase the effective electric field and, hence, improved the separation performance. In this work, we developed two micro-EFFFs with planar or segmented electrode design and investigated the particle movement in the flow channels under a PV. Numerical simulation was used to understand the electric field distribution in the micro-EFFFs. When the calculations for the micro-EFFF with a segmented electrode (segmented micro-EFFF) and the micro-EFFF with planar electrodes (planar micro-EFFF) are compared, a stronger electric field at the top electrode segments is found in the segmented micro-EFFF, with the strongest field at the edges of the electrode segments. Nanoparticle motion in both devices was in situ visualized by using a fluorescence microscope equipped with a CCD-camera. Results reveal that electrophoresis governs the nanoparticle movement in the planar micro-EFFF and dielectrophoresis dominates the movement in the segmented micro-EFFF. Two models are postulated to explain the experimental observations of the nanoparticle movement. The mechanistic understanding of controlling nanoparticle motion in a miniaturized environment will help the design and application of micro-EFFF for the separation of charged biomolecules (proteins and DNAs).     

Electrochemical reduction of nitrobenzene at carbon nanotube electrode

The electrochemical behaviors of nitrobenzene at a pyrolytic graphite electrode modified with carbon nanotubes (CNTs) were studied using cyclic voltammetry and constant-potential electrolysis technique, and the CNT-modified electrode was characterized with Fourier transform infrared spectroscopy (FTIR), high resolution transmission electron microscopy (HRTEM) and scanning electron microscopy (SEM) measurements. A CNT-modified packed-bed flow reactor was also constructed for electrocatalytic reduction of nitrobenzene. The results showed that CNTs exhibited high activity for nitrobenzene reduction to aniline and the electrochemical reduction of nitrobenzene at CNT-modified electrode followed the pathway of nitrobenzene-->phenylhydroxylamine-->aniline. CNTs had been functionalized with profuse carboxylic group and other oxygen-containing groups, became open with some lacuna on the wall, and were distributed symmetrically on the electrode with forming a three-dimensional layer, resulting in the high catalytic-activity for nitrobenzene reduction to aniline. The removal of nitrobenzene was over 95% with electrolysis for 50 min at -1.20 V in pH 5 solution using the CNT-modified packed-bed flow reactor, and no other product was obtained except aniline. The removal of nitrobenzene was over 95% with electrolysis for 80 min at -1.20 V in pH 7 solution and was 87% with electrolysis for 120 min in pH 9 solution. A little phenylhydroxylamine besides aniline was obtained during the initial electrolysis stage, and then all reduced to aniline. The average current efficiency at pH 5, 7 and 9 was 46, 51 and 63%, respectively. The electrolysis products were mineralized easily through aerobiotic biodegradation.     

Miniaturized electrochemical flow cells

Several novel types of miniaturized electrochemical flow cells are described. The flow cells are fabricated in fluorinated ethylene propylene using a novel technique where channels with inner diameters down to 13 microm are integrated with electrodes. The channel is formed by shrinking and simultaneous melting of a heat shrink/melt tubing around a channel template (a tungsten wire) and electrodes followed by removal of the channel template. The technique allows incorporation of different electrode materials of different sizes. The electrode configuration consists of one or two working electrodes inside the channel and a counter electrode located in the channel outlet reservoir. Electrode configurations with different channel and working electrode sizes, different electrode materials including carbon fibers, glassy carbon rods, poly(tetrafluoroethylene)/carbon composite material, and platinum wires, and different arrangements have been assembled. Hydrodynamic voltammograms in dual-electrode (generator-collector) experiments indicate good potential control for cells with 25-microm channels, while there is some iR drop in cells with 13-microm channels. Cells prepared with a cylindrical working electrode tangent and perpendicular to a flow channel show a flow rate dependence consistent with thin-layer cell behavior. Electrode areas can be made in the range of 10(-10)-10(-8) m2.     

Microfluidic characterization and continuous separation of cells and particles using conducting poly(dimethyl siloxane) electrode induced alternating current-dielectrophoresis

This paper presents a poly(dimethyl siloxane) (PDMS) polymer microfluidic device using alternating current (ac) dielectrophoresis (DEP) for separating live cells from interfering particles of similar sizes by their polarizabilities under continuous flow and for characterizing DEP behaviors of cells in stagnant flow. The ac-DEP force is generated by three-dimensional (3D) conducting PDMS composite electrodes fabricated on a sidewall of the device main channel. Such 3D PDMS composite electrodes are made by dispersing microsized silver (Ag) fillers into PDMS gel. The sidewall AgPDMS electrodes can generate a 3D electric field that uniformly distributes throughout the channel height and varies along the channel lateral direction, thereby producing stronger lateral DEP effects over the entire channel. This allows not only easy observation of cell/particle lateral motion but also using the lateral DEP force for manipulation of cells/particles. The former feature is used to characterize the frequency-dependent DEP behaviors of Saccharomyces cerevisiae (yeast) and Escherichia coli (bacteria). The latter is utilized for continuous separation of live yeast and bacterial cells from similar-size latex particles as well as live yeast cells from dead yeast cells. The separation efficiency of 97% is achieved in all cases. The demonstration of these functions shows promising applications of the microfluidic device.     

Direct electrochemical oxidation of disulfides at anodically pretreated boron-doped diamond electrodes

Anodically oxidized diamond electrodes have been used to oxidize disulfides, thiols, and methionine in aqueous acidic media and tested for amperometric detection of these compounds after chromatographic separation. Cyclic voltammetric signals for 1 mM glutathione disulfide (GSSG) were observed at 1.39 and 1.84 V vs SCE, the values being less positive than those of its as-deposited counterpart as well as glassy carbon electrode. The voltammetric and chronocoulometric results have indicated the high stability of the electrode with negligible adsorption. A positive shift in the peak potential with increasing pH indicated the attractive electrostatic interaction between the anodically oxidized diamond surface and the positively charged GSSG in acidic media that promoted its analytical performance. The results of the electrolysis experiments of disulfides and thiols showed that the oxidation reaction mechanism of glutathione (GSH) and GSSG involves oxygen transfer. Following separation by liquid chromatography (LC), the determination of both GSH and GSSG in rat whole blood was achieved at a constant potential (1.50 V vs Ag/AgCl), and the limits of detection for GSH and GSSG were found to be 1.4 nM (0.028 pmol) and 1.9 nM (0.037 pmol) with a linear calibration range up to 0.25 mM. These detection limits were much lower than those reported for the amperometry using Bi-PbO2 electrodes and LC-mass spectrometry, and the LC method using diamond electrodes were comparable with enzymatic assay in real sample analysis. The high response stability and reproducibility together with the possibility of regeneration of the electrode surface by on-line anodic treatment at 3 V for 30 min further support the applicability of anodically pretreated diamond for amperometric detection of disulfides.     

In-channel electrochemical detection in the middle of microchannel under high electric field

We propose a new method for performing in-channel electrochemical detection under a high electric field using a polyelectrolytic gel salt bridge (PGSB) integrated in the middle of the electrophoretic separation channel. The finely tuned placement of a gold working electrode and the PGSB on an equipotential surface in the microchannel provided highly sensitive electrochemical detection without any deterioration in the separation efficiency or interference of the applied electric field. To assess the working principle, the open circuit potentials between gold working electrodes and the reference electrode at varying distances were measured in the microchannel under electrophoretic fields using an electrically isolated potentiostat. In addition, "in-channel" cyclic voltammetry confirmed the feasibility of electrochemical detection under various strengths of electric fields (～400 V/cm). Effective separation on a microchip equipped with a PGSB under high electric fields was demonstrated for the electrochemical detection of biological compounds such as dopamine and catechol. The proposed "in-channel" electrochemical detection under a high electric field enables wider electrochemical detection applications in microchip electrophoresis.     

High-throughput microfabricated CE/ESI-MS: automated sampling from a microwell plate

A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is presented. The approach combines a sample-loading port, a separation channel, and a liquid junction, the latter for coupling the device to the MS with a miniaturized subatmospheric electrospray interface. The microdevice was attached to a polycarbonate manifold with external electrode reservoirs equipped for electrokinetic and pressure-fluid control. A computer-activated electropneumatic distributor was used for both sample loading from the microwell plate and washing of channels after each run. Removal of the electrodes and sample reservoirs from the microdevice structure significantly simplified the chip design and eliminated the need both for drilling access holes and for sample/buffer reservoirs. The external manifold also allowed the use of relatively large reservoirs that are necessary for extended time operation of the system. Initial results using this microfabricated system for the automated CE/ESI-MS analysis of peptides and protein digests are presented.     

Silicon microfabricated column with microfabricated differential mobility spectrometer for GC analysis of volatile organic compounds

A 3.0-m-long, 150-microm-wide, 240-microm-deep channel etched in a 3.2-cm-square silicon chip, covered with a Pyrex wafer, and coated with a dimethyl polysiloxane stationary phase is used for the GC separation of volatile organic compounds. The column, which generates approximately 5500 theoretical plates, is temperature-programmed in a conventional convection oven. The column is connected through a heated transfer line to a microfabricated differential mobility spectrometer. The spectrometer incorporates a 63Ni source for atmospheric-pressure chemical ionization of the analytes. Nitrogen or air transport gas (flow 300 cm(3)/min) drives the analyte ions through the cell. The spectrometer operates with an asymmetric radio frequency (RF) electric field between a pair of electrodes in the detector cell. During each radio frequency cycle, the ion mobility alternates between a high-field and a low-field value (differential mobility). Ions oscillate between the electrodes, and only ions with an appropriate differential mobility reach a pair of biased collectors at the downstream end of the cell. A compensation voltage applied to one of the RF electrodes is scanned to allow ions with different differential mobilities to pass through the cell without being annihilated at the RF electrodes. A unique feature of the device is that both positive and negative ions are detected from a single experiment. The combined microfabricated column and detector is evaluated for the analysis of volatile organic compounds with a variety of functionalities.     

Dielectrophoretic manipulation of particles and cells using insulating ridges in faceted prism microchannels

This paper presents a novel device for the dielectrophoretic manipulation of particles and cells. A two-level isotropic etch of a glass substrate was used to create three-dimensional ridge-like structures in micrometer-sized channels. Due to the insulating properties of glass, locally patterned regions of nonuniform electric field form near the ridges when a dc field is applied along the channel. The ridges are designed using the method of faceted prisms, such that substantially uniform fields are produced on each side of the faceted interfaces that form each ridge. The dielectrophoretic force that results from the electric field gradient near the ridges is used to affect particle motion parallel to the ridges in the absence of a bulk pressure-driven flow. Trapping and deflection of particles and continuous concentration and separation of Bacillus subtilis from a two-component sample mixture are demonstrated. The flow of B. subtilis is restricted to a selected channel of a planar, multichannel device as a result of negative dielectrophoresis arising from the presence of the insulating ridges when the applied electric field exceeds a threshold of 30 V/mm. Dielectrophoresis has a negligible impact on 200-nm-diameter polystyrene particles under the same conditions.     

Bubble-free operation of a microfluidic free-flow electrophoresis chip with integrated Pt electrodes

In order to ensure a stable and efficient separation in microfluidic free-flow electrophoresis (FFE) devices, various methods and chips have been presented until now. A major concern hereby is the generation of gas bubbles caused by electrolysis and the resulting disturbances in the position of the separated analyte lanes. Instable lane positions would lead to a decreased resolution in sample collection over time which certainly would be problematic when incorporating a stationary detector system. In contrast to our previous publications, in which we implemented laborious semipermeable membranes to keep bubbles outside the separation region, here we describe an electrochemical approach to suppress the electrolysis of water molecules and therefore bubble formation. This approach allowed a simpler and additionally a closed chip device with integrated platinum electrodes. With the use of this chip, the successful separation of three fluorescent compounds was demonstrated. Quinhydrone, which is a complex of hydroquinone and p-benzoquinone, was added only to the local flow streams along the electrodes, preventing mixing with the separation media and sample. The electrical current was generated via the oxidization and reduction of hydroquinone and p-benzoquinone up to a certain limit of the electrical current without gas formation. The separation stability was investigated for the chip with and without quinhydrone, and the results clearly indicated the improvement. In contrast to the device operating without quinhydrone, a 2.5-fold increase in resolution was achieved. Furthermore, separation was demonstrated within tens of milliseconds. This chemical approach with its high miniaturization possibilities offers an interesting alternative, in particular for low-current miniaturized FFE systems, in which large and open electrode reservoirs are not tolerable.     

Electroosmotically induced hydraulic pumping on microchips: differential ion transport

The theory behind and operation of an electroosmotically induced hydraulic pump for microfluidic devices is reported. This microchip functional element consists of a tee intersection with one inlet channel and two outlet channels. The inlet channel is maintained at high voltage while one outlet channel is kept at ground and the other channel has no electric potential applied. A pressure-induced flow of buffer is created in both outlet channels of the tee by reducing electroosmosis in the ground channel relative to that of the inlet channel. Spatially selective reduction of electroosmosis is accomplished by coating the walls of the ground channel with a viscous polymer. The pump is shown to differentially transport ions down the two outlet channels. This ion discrimination ability of the pump is examined as a function of an analyte's electrophoretic velocity. In addition, we demonstrate that an anion can be rejected from the ground channel and made to flow only into the field-free channel if the electrophoretic velocity of the anion is greater than the pressure-generated flow in the ground channel. The velocity threshold at which anion rejection occurs can be selectively tuned by changing the flow resistance in the field-free channel relative to the ground channel.     

Thermal fluxes in a generator of low temperature plasma with a divergent channel of the outlet electrode

A short review is presented of experimental results concerning distribution of heat fluxes over the length of a generator of low temperature plasma (plasmatron). Comparative analysis is given of the operation of plasma generators with a divergent channel of the outlet electrode against plasma generators with a cylindrical channel of constant cross section. Experimental studies were performed on a specially created facility consisting of four plasma generators with sectioned and continuous outlet electrodes and automated measuring system.