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.     

Dual-channel method for interference-free in-channel amperometric detection in microchip capillary electrophoresis

A novel in-channel amperometric detection method for microchip capillary electrophoresis (CE) has been developed to avoid the interference from applied potential used in the CE separation. Instead of a single separation channel as in conventional CE microchips, we use a dual-channel configuration consisting of two different parallel separation and reference channels. A working electrode (WE) and a reference electrode (RE) are placed equally at a distance 200 microm from its outlet on each channel. Running buffer flows through the reference channel. Our dual-channel CE microchips consist of a poly(dimethylsiloxane) (PDMS) upper plate and a glass lower plate to form a PDMS/glass hybrid chip. Amperometric signals are measured without any potential shift and interference from the applied CE potential, and CE separation maintains its high resolution because this in-channel configuration does not allow additional band broadening that is notorious in end-channel and off-channel configurations. The high performance of this new in-channel electrochemical detection methodology for CE has been demonstrated by analyzing a mixture of electrochemically active biomolecules: dopamine (DA), norepinephrine, and catechol. We have achieved a 0.1 pA detectability from the analysis of DA, which corresponds to a 1.8 nM concentration.     

A polymeric microfluidic chip for CE/MS determination of small molecules

A polymeric microfluidic chip made of Zeonor 1020 was fabricated using conventional embossing techniques to perform capillary electrophoresis for selected ion monitoring and selected reaction monitoring mass spectrometric detection of small molecules. A silicon master was microfabricated using photolithographic and dry etching processes. The microfluidic channel was embossed in the plastic from a silicon master. The embossed chip was thermally bonded with a Zeonor 1020 cover to form an enclosed channel. This channel (60-microm width, 20-microm depth, 2.0- and 3.5-cm length) provided capillary electrophoresis (CE) separation of polar small molecules without surface treatment of the polymer. A microsprayer coupled via a microliquid junction provided direct electrospray mass spectrometric detection of CE-separated components. An electric field of 0.5-2 kV/cm applied between the microsprayer and a separation buffer reservoir produced a separation of carnitine, acylcarnitine, and butylcarnitine with separation efficiencies ranging from 1,650 to 18,000 plates. Injection quantities of 0.2 nmol of these compounds produced a separation of the targeted polar small molecules without surface treatment of the polymer-abundant ion current signals and baseline separation of these compounds in less than 10 s. These results suggest the feasibility of polymeric chip-based devices for ion spray CE/MS applications.     

Fabrication and characterization of a fritless microfabricated electroosmotic pump with reduced pH dependence

A fritless electroosmotic pump with reduced pH dependence has been fabricated on a glass microchip and its performance characterized. The chip design consists of two 500-microm channels, one packed with anion exchange beads and the other packed with cation exchange beads, which produce convergent electroosmotic flow streams. The electroosmotically pumped solution flows away from the intersection of the two pumping channels through a field-free channel. This simple design allows for the production of a fritless electroosmotic pump and easy replacement of the ion exchange beads whose charged surfaces generate the flow. The pump was found to produce volumetric flow rates of up to 2 microL/min for an applied voltage of 3 kV at a pH of 6.8. Moreover, the electroosmotic pump can generate high flow rates over an extended pH range of at least 2-12, a significant advantage over previously fabricated electroosmotic pumps, which typically have a more limited range in which they can achieve high flow rates.     

Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.     

Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.     

Formation of Single Micro‐ and Nanowires with Extreme Aspect Ratios in Microfluidic Channels

Shown here is the site‐specific formation of single extraordinarily long metal–organic micro‐ and nanowires using a microfluidic device made of poly(dimethylsiloxane) (PDMS). This approach exploits two concepts, i) the diffusion of organic precursor molecules through PDMS and ii) the use of microfluidic channels as a growth template. To initiate wire formation, metal and organic precursor solutions are filled into different supply channels that are separated by PDMS. As the precursor diffuses through PDMS, and thereby infiltrates the adjacent channel, the growth of micro‐ and nanowires starts at the side walls of this adjacent channel. The formation yields single wires with sizes ranging from several hundreds of micrometers to millimeters at diameters of 0.5–2 µm. The principles of this formation pathway are demonstrated with the reaction of tetrathiafulvalene (TTF) and gold(III) ions that yields Au‐TTF wires. The influence of various reaction parameters including the choice of solvents and the chip fabrication protocol on the reaction are evaluated. Based on these findings, a further microfluidic device design with orthogonally arranged channels is developed, and the formation of single wires in a channel‐defined pattern is demonstrated. Moreover, the possibility of pulsed precursor supply allows for advanced control over the growth of the wires.     

Dual-electrode electrochemical detection for poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips

The development of a poly(dimethylsiloxane)-based (PDMS-based) microchip electrophoresis system employing dual-electrode electrochemical detection is described. This is the first report of dual-electrode electrochemical detection in a microchip format and of electrochemical detection on chips fabricated from PDMS. The device described in this paper consists of a top layer of PDMS containing the separation and injection channels and a bottom glass layer onto which gold detection electrodes have been deposited. The two layers form a tight reversible seal, eliminating the need for high-temperature bonding, which can be detrimental to electrode stability. The channels can also be temporarily removed for cleaning, significantly extending the lifetime of the chip. The performance of the chip was evaluated using catechol as a test compound. The response was linear from 10 to 500 microM with an LOD (S/N = 3) of 4 microM and a sensitivity of 45.9 pA/microM. Collection efficiencies for catechol ranged from 28.7 to 25.9% at field strengths between 200 and 400 V/cm. Dual-electrode detection in the series configuration was shown to be useful for the selective monitoring of species undergoing chemically reversible redox reactions and for peak identification in the electropherogram of an unresolved mixture.     

Fully packed capillary electrochromatographic microchip with self-assembly colloidal silica beads

A fully packed capillary electrochromatographic (CEC) microchip showing improved solution and chip handling was developed. Microchannels for the CEC microchip were patterned on a cyclic olefin copolymer substrate by injection molding and packed fully with 0.8-microm monodisperse colloidal silica beads utilizing a self-assembly packing technique. The silica packed chip substrate was covered and thermally press-bonded. After fabrication, the chip was filled with buffer solution by self-priming capillary action. The self-assembly packing at each channel served as a built-in nanofilter allowing quick loading of samples and running buffer solution without filtration. Because of a large surface area-to-volume ratio of the silica packing, reproducible control of electroosmotic flow was possible without leveling of the solutions in the reservoirs resulting 1.3% rsd in migration rate. The capillary electrophoretic separation characteristics of the chip were studied using fluorescein isothiocyanate (FITC)-derivatized amino acids as probe molecules. A mixture of FITC and four FITC-derivatized amino acids was successfully separated with 2-mm separation channel length.     

Fabrication of porous polymer monoliths in polymeric microfluidic chips as an electrospray emitter for direct coupling to mass spectrometry

Coupling of polymeric microfluidic devices to mass spectrometry is reported using porous polymer monoliths (PPM) as nanoelectrospray emitters. Lauryl acrylate-co-ethylene dimethacrylate porous polymer monolith was photopatterned for 5 mm at the end of the channel of microfluidic devices fabricated from three different polymeric substrate materials, including the following: poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and cyclic olefin copolymer (COC). Spraying directly from the end of the chip removes any dead volume associated with inserted emitters or transfer lines, and the presence of multiple pathways in the PPM prevents the clogging of the channels, which is a common problem in conventional nanospray emitters. Spraying from a microfluidic channel having a PPM emitter produced a substantial increase in TIC stability and increased sensitivity by as much as 70x compared to spraying from an open end chip with no PPM. The performance of PPM emitter in COC, PMMA, and PDMS chips was compared in terms of stability and reproducibility of the electrospray. COC chips showed the highest reproducibility in terms of chip-to-chip performance, which can be attributed to the ease and reproducibility of the PPM formation due to the favorable optical and chemical properties of COC. We have further tested the performance of the COC chips by constant infusion of poly(propylene glycol) solution at organic content ranging from 10 to 90% methanol and at flow rates ranging from 50 to 1000 nL/min, showing optimum spraying conditions (RSD < 5%) at 50-70% organic content and at flow rates from 100 to 500 nL/min. The PPM sprayer was also used for protein preconcentration and desalting prior to mass spectrometric detection, and results were comparable with a chip spraying from an electrospray tip.     

Selective modification for polydimethylsiloxane chip by micro-plasma

We report a method to selectively modify polydimethylsiloxane (PDMS) chip in a fast and facile way using micro-plasma approach in the atmospheric-pressure. Pure He and He/acrylic acid plasma were ignited directly in different channels of PDMS microchip. Our experiments results yielded strong hydrophilic property on the surface of PDMS by the plasma treatment.     

Fabrication of a microfluidic chip containing dam,weirs and gradient generator for studying cellular response to chemical modulation

A microfluidic chip was designed and fabricated for studying cellular response to chemical modulation. The microfluidic network comprised an up-stream gradient-generating module and a down-stream cell culture module. The microchip was composed of a piece of glass plate and a covered PDMS film. By using a two-step wet etching method, the dam structure was fabricated on the inlet of the cell chamber facilitating cell positioning, and a series of weir structures were fabricated on the bottom of cell culture reservoirs facilitating cell seeding. This microfluidics exploited the advantage of lab-on-a-chip technology by integrating the generation of chemical concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. Steady concentration gradients were generated by flowing two fluids in the network. Over time observation showed that the microchip was suitable for cell seeding and culture. The microchip described above was applied in studying the roles of As₂O₃ and buthionine sulfoximine (BSO) in mediating intracellular levels of reduced glutathione (GSH) and reactive oxygen species (ROS) in MCF-7 cells. MCF-7 cells showed dose dependent reaction to the chemical modulations. Upon the treatment with both As₂O₃ and BSO, GSH levels were down-regulated but ROS levels were up-regulated. As₂O₃ showed a stronger effect on ROS enhancement, while BSO was more effective on GSH depletion. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, thus holds great potential for extrapolation to the cell based high-content drug screening.     

Microchip capillary electrophoresis with solid-state electrochemiluminescence detector

We report microchip capillary electrophoresis (CE) coupling to a solid-state electrochemiluminescence (ECL) detector. The solid-state ECL detector was fabricated by immobilizing tris(2,2'-bipyridyl)ruthenium(II) (TBR) into an Eastman AQ55D-silica-carbon nanotube composite thin film on an indium tin oxide (ITO) electrode. After being made by a photolithographic method, the surface of the ITO electrode was coated with a thin composite film through a micromolding in capillary (MIMIC) technique using a poly(dimethylsiloxane) (PDMS) microchannel with the same pattern as an ITO electrode. Then the TBR was immobilized via ion exchange by immersing the ITO electrode containing the thin film in TBR aqueous solution. The whole system was built by reversibly sealing the TBR-modified ITO electrode plate with a PDMS layer containing electrophoresis microchannels. The results indicated that the present solid-state ECL detector displayed good durability and stability in the microchip CE-ECL system. Proline was selected to perform the microchip device with a limit of detection of 2 microM (S/N=3) and a linear range from 25 to 1000 microM. Compared with the CE-ECL of TBR in aqueous solution, while the CE microchip with solid-state ECL detector system gave the same sensitivity of analysis, a much lower TBR consumption and a high integration of the whole system were obtained. The present system was also used for medicine analysis.     

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.     

Three-electrode electrochemical detector and platinum film decoupler integrated with a capillary electrophoresis microchip for amperometric detection

This article demonstrates that a three-electrode electrochemical (EC) detector and an electric decoupler could be fabricated in the same glass chip and integrated with an O2-plasma-treated PDMS layer using microfabrication techniques to form the capillary electrophoresis (CE) microchip. The platinized decoupler could mostly decouple the electrochemical detection circuit from the interference of an separation electric field in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5) solution. The baseline offset of background current recorded from the working electrode with and without application of a separation electric field was maintained at less than 0.05 pA in 10 mM MES. In addition, the platinized pseudoreference electrode was demonstrated to offer a stable potential in electrochemical detection. As a consequence, the limit of detection of dopamine was 0.125 microM at a S/N = 4. The responses for dopamine to different concentrations were found to be linear between 0.25 and 50 microM with a correlation coefficient of 0.9974 and a sensitivity of 11.76 pA/microM. The totally integrated CE-EC microchip should be able to fulfill the ideal of miniaturization and commercialization.     

Gas chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry

An atmospheric pressure chemical ionization (APCI) microchip is presented for combining a gas chromatograph (GC) to a mass spectrometer (MS). The chip includes capillary insertion channel, stopper, vaporizer channel, nozzle and nebulizer gas inlet fabricated on the silicon wafer, and a platinum heater sputtered on a glass wafer. These two wafers are joined by anodic bonding creating a two-dimensional version of an APCI microchip. The sample from GC is directed via heated transfer line capillary to the vaporizer channel of the APCI chip. The etched nozzle forms narrow sample plume, which is ionized by an external corona discharge needle, and the ions are analyzed by a mass spectrometer. The GC-microchip APCI-MS combination provides an efficient method for qualitative and quantitative analysis. The spectra produced by microchip APCI show intensive protonated molecule and some fragmentation products as in classical chemical ionization for structure elucidation. In quantitative analysis the GC-microchip APCI-MS showed good linearity (r(2) = 0.9989) and repeatability (relative standard deviation 4.4%). The limits of detection with signal-to-noise ratio of three were between 0.5 and 2 micromol/L with MS mode using selected ion monitoring and 0.05 micromol/L with MS/MS using multiple reaction monitoring.     

Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics

A polymeric microfluidic device for solid-phase extraction (SPE)-based isolation of nucleic acids is demonstrated. The plastic chip can function as a disposable sample preparation system for different biological and diagnostic applications. The chip was fabricated in a cyclic polyolefin by hot-embossing with a master mold. The solid phase consisted of a porous monolithic polymer column impregnated with silica particles. The extraction was achieved due to the binding of nucleic acids to the silica particles in the monolith. The solid phase was formed within the channels of the device by in situ photoinitiated polymerization of a mixture of methacrylate and dimethacrylate monomers, UV-sensitive free-radical initiator, and porogenic solvents. The channel surfaces were pretreated via photografting to covalently attach the monolith to the channel walls. The solid phase prepared by this method allowed for successful extraction and elution of nucleic acids in the polymeric microchip.     

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.     

Generation of hydrophilic poly(dimethylsiloxane) for high-performance microchip electrophoresis

Poly(dimethylsiloxane) (PDMS) has become one of the most widely used materials for microchip capillary electrophoresis and microfluidics. The popularity of this material is the result of its low cost, simple fabrication, and rugged elastomeric properties. The hydrophobic nature of PDMS, however, limits its applicability for microchip CE, microfluidic patterning, and other nonelectrophoresis applications. The surface of PDMS can be made hydrophilic using a simple air plasma treatment; however, this property is quickly lost through hydrophobic recovery caused by diffusion of unreacted oligomer to the surface. Here, a simple approach for the generation of hydrophilic PDMS with long-term stability in air is presented. PDMS is rendered hydrophilic through a simple two-step extraction/oxidation process. First, PDMS is extracted in a series of solvents designed to remove unreacted oligomers from the bulk phase. Second, the oligomer-free PDMS is oxidized in a simple air plasma, generating a stable layer of hydrophilic SiO2. The conversion of surface-bound siloxane to SiO2 was followed with X-ray photoelectron spectroscopy. SiO2 on extracted-oxidized PDMS was stable for 7 days in air as compared to less than 3 h for native PDMS. Furthermore, the contact angle for modified PDMS was reduced to <40 degrees and remained low throughout the experiments. As a result of the decreased contact angle, capillary channels self-wet through capillary action, making the microchannels much easier to fill. Finally, the modification significantly improved the performance of the devices for microchip electrophoresis. The electroosmotic flow increased from 4.1 x 10(-4) to 6.8 x 10(-4) cm(2)/V.s for native compared to oxidized PDMS. Separation efficiencies for electrochemical detection also increased from 50 000 to 400 000 N/m for a 1.1-nL injection volume. The result of this modification is a significant improvement in the performance of PDMS for microchip electrophoresis and microfluidic applications.