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.     

Chemiluminescence detection for a microchip capillary electrophoresis system fabricated in poly(dimethylsiloxane)

Chemiluminescence (CL) detection integrated with a microchip capillary electrophoresis (MCE) system that was fabricated in poly(dimethylsiloxane) was demonstrated for chemical and biochemical analyses. Two model CL systems were involved here: metal ion-catalyzed luminol-peroxide reaction and dansyl species conjugated peroxalate-peroxide reaction. Different strategies based on three chip patterns (cross, cross combining with Y, and cross combining with V) to perform on-line CL detection for MCE were evaluated and compared in terms of sensitivity, reproducibility, and peak symmetry. The chip pattern of cross combining with Y proved to be promising for the luminol-peroxide CL system, while the chip pattern of cross combining with V was preferred for the peroxalate-peroxide system where CL reagent could not be effectively transported by electroosmotic flow. A detection limit down to submicromolar concentrations (midattomole) was achieved with good reproducibility and symmetric peak shape. Successful separation of three metal cations such as Cr(III), Co(II), and Cu(II) and chiral recognition of dansyl phenylalanine enantiomers within 1 min revealed distinct advantages of combining MCE with CL detection for rapid and sensitive analyses.     

A prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane)

A method for carrying out 2D gel electrophoresis in a capillary format is presented. In this method, separation in the first dimension is carried out in a 1D capillary, with this system physically isolated from the capillaries that provide the separation in the second dimension. After completion of the first separation, the 1D channel is physically connected to the 2D capillaries, and a second separation is carried out in an orthogonal set of parallel capillaries. The ability of poly(dimethylsiloxane) (PDMS) to support the fabrication of 3D microfluidic systems makes it possible to produce membranes that both enclose the gel used in the first separation in a capillary and provide passages for the proteins to migrate into the array of orthogonal capillaries. The elastomeric nature of PDMS makes it possible to make reversible connections between pieces of PDMS. The feasibility of this system is demonstrated using a protein mixture containing fluorescein-conjugated carbonic anhydrase, fluorescein-conjugated BSA, and Texas Red-conjugated ovalbumin. This work suggests one type of design that might form the basis for a microfabricated device for 2D capillary electrophoresis.     

Miniaturized tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection cell for capillary electrophoresis and flow injection analysis

The design and performance of a miniaturized chip-type tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] electrochemiluminescence (ECL) detection cell suitable for both capillary electrophoresis (CE) and flow injection (FI) analysis are described. The cell was fabricated from two pieces of glass (20 x 15 x 1.7 mm), and the 0.5-mm-diameter platinum disk was used as working electrode held at +1.15 V (vs silver wire quasi-reference), the stainless steel guide tubing as counter electrode, and the silver wire as quasi-reference electrode. The performance traits of the cell in both CE and FI modes were evaluated using tripropylamine, proline, and oxalate and compared favorably to those reported for CE and FI detection cells. The advantages of versatility, sensitivity, and accuracy make the device attractive for the routine analysis of amine-containing species or oxalate by CE and FI with Ru(bpy)3(2+) ECL detection.     

Design and characterization of poly(dimethylsiloxane)-based valves for interfacing continuous-flow sampling to microchip electrophoresis

This work describes the fabrication and evaluation of a poly(dimethyl)siloxane (PDMS)-based device that enables the discrete injection of a sample plug from a continuous-flow stream into a microchannel for subsequent analysis by electrophoresis. Devices were fabricated by aligning valving and flow channel layers followed by plasma sealing the combined layers onto a glass plate that contained fittings for the introduction of liquid sample and nitrogen gas. The design incorporates a reduced-volume pneumatic valve that actuates (on the order of hundreds of milliseconds) to allow analyte from a continuously flowing sampling channel to be injected into a separation channel for electrophoresis. The injector design was optimized to include a pushback channel to flush away stagnant sample associated with the injector dead volume. The effect of the valve actuation time, the pushback voltage, and the sampling stream flow rate on the performance of the device was characterized. Using the optimized design and an injection frequency of 0.64 Hz showed that the injection process is reproducible (RSD of 1.77%, n = 15). Concentration change experiments using fluorescein as the analyte showed that the device could achieve a lag time as small as 14 s. Finally, to demonstrate the potential uses of this device, the microchip was coupled to a microdialysis probe to monitor a concentration change and sample a fluorescein dye mixture.     

Potentiometric titrations in a poly(dimethylsiloxane)-based microfluidic device

This paper describes a microfluidic device, fabricated in poly(dimethylsiloxane), that is used for potentiometric titrations. This system generates step gradients of redox potentials in a series of microchannels. These potentials are probed by microelectrodes that are integrated into the chip; the measured potentials were used to produce a titration curve from which the end point of a reaction was measured.     

Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices

This paper describes the compatibility of poly(dimethylsiloxane) (PDMS) with organic solvents; this compatibility is important in considering the potential of PDMS-based microfluidic devices in a number of applications, including that of microreactors for organic reactions. We considered three aspects of compatibility: the swelling of PDMS in a solvent, the partitioning of solutes between a solvent and PDMS, and the dissolution of PDMS oligomers in a solvent. Of these three parameters that determine the compatibility of PDMS with a solvent, the swelling of PDMS had the greatest influence. Experimental measurements of swelling were correlated with the solubility parameter, delta (cal(1/2) cm(-3/2)), which is based on the cohesive energy densities, c (cal/cm(3)), of the materials. Solvents that swelled PDMS the least included water, nitromethane, dimethyl sulfoxide, ethylene glycol, perfluorotributylamine, perfluorodecalin, acetonitrile, and propylene carbonate; solvents that swelled PDMS the most were diisopropylamine, triethylamine, pentane, and xylenes. Highly swelling solvents were useful for extracting contaminants from bulk PDMS and for changing the surface properties of PDMS. The feasibility of performing organic reactions in PDMS was demonstrated by performing a Diels-Alder reaction in a microchannel.     

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.     

Polyamine deactivation of integrated poly(dimethylsiloxane) structures investigated by radionuclide imaging and capillary electrophoresis experiments

The poly(dimethylsiloxane) (PDMS) material provides a number of advantageous features, such as flexibility, elasticity, and transparency, making it useful in integrated analytical systems. Hard fused-silica capillary structures and soft PDMS channels can easily be combined by a tight fit, which offers many alternatives for structure combinations. PDMS and fused silica are in different ways prone to adsorption of low levels of organic compounds. The need for modification of the inner wall surface of PDMS channels may often be necessary, and in this paper, we describe an easy and effective method using the amine-containing polymer PolyE-323 to deactivate both fused-silica and PDMS surfaces. The adsorption of selected peptides to untreated surfaces was compared to PolyE-323-modified surfaces, using both radionuclide imaging and capillary electrophoresis experiments. The polyamine modification displayed a substantially reduced adsorption of three hydrophobic test peptides compared to the native PDMS surface. Filling and storage of aqueous solution were also possible in PolyE-323-modified PDMS channels. In addition, hybrid microstructures of fused silica and PDMS could simultaneously be deactivated in one simple coating procedure.     

Development of a microfabricated palladium decoupler/electrochemical detector for microchip capillary electrophoresis using a hybrid glass/poly(dimethylsiloxane) device

The fabrication and evaluation of a palladium decoupler and working electrode for microchip capillary electrophoresis (CE) with electrochemical detection is described. The use of the Pd decoupler allows the working electrode to be placed directly in the separation channel and eliminates the band-broadening characteristic of the end-channel configuration. The method used for fabrication of the decoupler and working electrode was based on thin-layer deposition of titanium followed by palladium onto a glass substrate. When employed as the cathode in CE, palladium absorbs the hydrogen gas that is generated by the hydrolysis of water. The effect of the decoupler size on the ability to remove hydrogen was evaluated with regard to reproducibility and longevity. Using boric acid and TES buffer systems, 500 microm was determined to be the optimum decoupler size, with effective voltage isolation lasting for approximately 6 h at a constant field strength of 600 V/cm. The effect of distance between the decoupler and working electrode on noise and resolution for the separation of dopamine and epinephrine was also investigated. It was found that 250 microm was the optimum spacing between the decoupler and working electrode. At this spacing, laser-induced fluorescence detection at various points around the decoupler established that the band broadening due to pressure-induced flow that occurs after the decoupler did not significantly affect the separation efficiency of fluorescein. Limits of detection, sensitivity, and linearity for dopamine (500 nM, 3.5 pA/microM, r(2) = 0.9996) and epinephrine (2.1 microM, 2.6 pA/microM, r(2) = 0.9996) were obtained using the palladium decoupler in combination with a Pd working electrode.     

Development of a poly(dimethylsiloxane) interface for on-line capillary column liquid chromatography-capillary electrophoresis coupled to sheathless electrospray ionization time-of-flight mass spectrometry

An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI-MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC-CE-TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 microL LC injection of 25 microg/mL peptide standard.     

Poly(dimethylsiloxane)-based microchip for two-dimensional solid-phase extraction-capillary electrophoresis with an integrated electrospray emitter tip

A microchip in poly(dimethylsiloxane) (PDMS) for in-line solid-phase extraction-capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry (SPE-CE-ESI-TOF-MS) has been developed and evaluated. The chip was fabricated in a novel one-step procedure where mixed PDMS was cast over steel wires in a mold. The removed wires defined 50-microm cylindrical channels. Fused-silica capillaries were inserted into the structure in a tight fit connection. The inner walls of the inserted fused-silica capillaries and the PDMS microchip channels were modified with a positively charged polymer, PolyE-323. The chip was fabricated in a two-level cross design. The channel at the lower level was packed with 5-microm hyper-cross-linked polystyrene beads acting as a SPE medium used for desalting. The upper level channel acted as a CE channel and ended in an integrated emitter tip coated with conducting graphite powder to facilitate the electrical contact for sheathless ESI. An overpressure continuously provided fresh CE electrolyte independently of the flows in the different levels. Further studies were carried out in order to investigate the electrophoretic and flow rate properties of the chip. Finally, six-peptide mixtures, in different concentrations, dissolved in physiological salt solution was injected, desalted, separated, and sprayed into the mass spectrometer for analysis with a limit of detection in femtomole levels.     

An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications

This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.     

High-throughput detection of unknown mutations by using multiplexed capillary electrophoresis with poly(vinylpyrrolidone) solution

Single-nucleotide polymorphism detection has been the focus of much attention recently. Although many methods have been reported, low-cost, high-throughput, and high-detection-rate methods are still in demand. We present a fast and reliable mutation detection scheme based on temperature-gradient capillary electrophoresis. A large temperature gradient (10 degrees C) was applied with a precision of 0.02 degrees C and a temperature ramp of 0.7 degrees C/min. Multiple unlabeled samples from PCR were injected and analyzed. Ethidium bromide was used as the intercalating dye for laser-induced fluorescence detection. Mutations can be recognized by comparing the electrophoretic patterns of the heteroduplex with that of a homoduplex reference without prior knowledge of the exact type of mutation present. Mutations in all five test samples were successfully detected with high confidence. This scheme is demonstrated in 96-capillary array electrophoresis for screening single-point polymorphism in large numbers of samples prior to full sequencing of only the positive samples to identify the nature of the mutation.     

Chemical oxidation of p-hydroxyphenylpyruvic acid in aqueous solution by capillary electrophoresis with an electrochemiluminescence detection system

A system of capillary electrophoresis with electrochemiluminescence detection (CE-ECL) together with UV spectroscopic and electrochemical methods were used to study the chemical oxidation of p-hydroxyphenylpyruvic acid (pHPP) by dissolved oxygen in aqueous solution. The pHPP was observed to be readily oxidized by dissolved oxygen in alkaline solution and yielded a compound that strongly enhanced the electrochemiluminescence of Ru(bpy)23+. This compound was separated and detected by a new CE-ECL system and revealed to be oxalate by being compared with an authentic sample of oxalate. The chemical oxidation mechanism of pHPP by dissolved oxygen was discussed in this paper.     

Contactless conductivity detection for capillary electrophoresis

A contactless capacitively coupled conductivity detector for capillary electrophoresis is introduced. The detector consists of two electrodes which are placed cylindrically around the outer polyimide coating of the fused-silica capillary with a detection gap of 2 mm. The electrodes form a cylindrical capacitor, and the electric conductivity of the solution in the gap between the electrodes is measured. A high audio or low ultrasonic frequency for coupling of the ac voltage is used in order to minimize the influence of reactance of the liquid. For an improved version of the detector, two syringe cannulas are used as the electrodes and the capillary is simply assembled into the tubing. This allows an easy placement of the detector on various positions along the capillary. The limit of detection of inorganic cations and anions is 200 ppb, as determined for sodium and chloride, respectively.     

Passive conductivity detection for capillary electrophoresis

A passive electrochemical detection principle that can be applied to capillary electrophoresis is presented. The separation electrical field is used to generate a potential difference between two electrodes located along the channel. For constant-current electrophoresis, the generated signal is proportional to the resistance of the solution passing between the two electrodes. Contrary to conductivity detectors that are ac driven and need to be decoupled from the separation field, the passive detection directly takes advantage of the separation field. The signal is simply measured by a high-impedance voltmeter. The detection concept has been validated by numerical simulations showing how the magnitude of the signal is related to the ratio between the electrode distance and the length of the sample plug. As a proof of the principle, this detection concept has been demonstrated by the electrophoretic separation of three alkali ions on a polymer microchip. Based on preliminary results, a detection limit of 20 microM and a dynamic range of up to 3 orders of magnitude have been achieved.     

Batch-type chemiluminescence detection cell for sensitization and simplification of capillary electrophoresis

A simple and convenient chemiluminescence detection cell was designed for capillary electrophoresis. The detection cell easily combined with capillary electrophoresis equipment. Luminol chemiluminescence was adapted for use with the detection cell. Detailed analysis and testing of the system revealed that luminol could be determined over a range of 2.5 x 10(-10)-6.5 x 10(-7) M (correlation coefficient, 0.999), with a detection limit (S/N = 3) of 2.5 x 10(-10) M (7 amol). Furthermore, each component in a mixture of glycine, glycylglycine, and glycylglycylgycine, which were labeled with isoluminol isothiocyanate, was baseline separated and sensitively detected. Moreover, the stacking procedure was applied to postcolumn detection in capillary electrophoresis. When acetonitrile stacking was used under certain conditions in the present system, chemiluminescence intensities of luminol and labeled compounds were about 1 order of magnitude higher than those obtained without stacking. The detection limit for luminol was 1.5 x 10(-11) M (S/N = 3), representing the highest sensitivity of luminol yet reported. Finally, the effect of p-iodophenol as an enhancer of luminol chemiluminescence was examined under weak alkaline conditions. The chemiluminescence intensity of luminol was approximately 2 orders of magnitude higher than that in the unenhanced reaction. A preliminary immunoassay using horseradish peroxidase-labeled anti-mouse IgG was also developed.     

Surface-modified poly(methyl methacrylate) capillary electrophoresis microchips for protein and peptide analysis

Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (muCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were 1 order of magnitude better with derivatized compared to untreated PMMA muCE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA muCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 x10(4) plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses.     

Reinforcement of elastomeric poly(dimethylsiloxane) by glassy poly(diphenylsiloxane)

Some novel approaches were taken to provide the improvements in mechanical properties that are almost always necessary to prepare a commercially useful elastomer from poly(dimethylsiloxane) (PDMS) [-Si(CH₃)₂O-]. The reinforcement was provided by poly(diphenylsiloxane) (PDPS) [-Si(C₆H₅)₂O-], a hard glassy polymer, which was introduced into the PDMS by two rather different techniques. In the first, the PDPS was prepared separately by condensation polymerization of diphenylsilanediol and then solution-blended into the PDMS. In the second, it was generated by in situ polymerization of the same monomer absorbed into the PDMS network. The resulting materials were characterized by scanning electron microscopy and by stress-strain isotherms in elongation. At least under some conditions both techniques were found to be successful, leading to increases in ultimate strength by a factor of two or more.