Micromachined separation chips with a precolumn reactor and end-column electrochemical detector

Glass microchips, integrating chemical derivatization reactions, electrophoretic separations, and amperometric detection, have been developed. The performance of the new integrated microfabricated devices is demonstrated for rapid on-chip measurements of amino acids utilizing precolumn reactions of amino acids with o-phthaldialdehyde/2-mercaptoethanol to generate electroactive derivatives that are separated electrophoretically and detected at the end-column electrochemical detector. The influence of the sample/reagent mixing ratio, reagent concentrations, driving voltage, detection potential, and other variables is explored. The integrated microsystem offers a rapid (6 min) simultaneous measurements of eight amino acids, down to approximately 2.5 x 10(-6) M (5 fmol) level, with linearity up to the 2 x 10(-4) M level examined, and good reproducibility (RSD = 2.2-2.7%). A step of the driving voltage is used for decreasing the migration time of late-eluting components and reducing the overall analysis time. The integrated microfabricated device expands the scope of on-chip electrochemical detection to nonelectroactive analytes and holds promise of being a powerful analytical tool.     

Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system

Microfabricated capillary electrophoresis chips containing an integrated sheath-flow electrochemical detector are developed with the goal of minimizing the influence of separation voltages on end-column detection while maintaining optimum performance. The microdevice consists of an upper glass wafer carrying the etched separation, injection, and sheath-flow channels and a lower glass wafer on which gold- and silver-plated electrodes have been fabricated. The sheath-flow channels join the end of the separation channel from each side, and gravity-driven flow carries the analytes to the electrochemical detector placed at working distances of 100, 150, 200, and 250 microm from the separation channel exit. The performance of this detector is evaluated using catechol and a detection limit of 4.1 microM obtained at a working distance of 250 microm. Detection of DNA restriction fragments and PCR product sizing is demonstrated using the electroactive intercalating dye, iron phenanthroline. Additionally, an allele-specific, PCR-based single-nucleotide polymorphism typing assay for the C282Y substitution diagnostic for hereditary hemochromatosis is developed and evaluated using ferrocene-labeled primers. This study advances the feasibility of high-speed, high-throughput chemical and genetic analysis using microchip electrochemical detection.     

Electrochemical detectors prepared by electroless deposition for microfabricated electrophoresis chips

Microfabricated capillary electrophoresis (CE) chips with integrated electrochemical detection have been developed on glass substrates. An electroless deposition procedure was used to deposit a gold film directly onto the capillary outlet to provide high-sensitivity electrochemical detection for catechol and several nitroaromatic explosives. Scanning electron microscopy revealed that the electroless gold film contains nanoscopic gold aggregates (100-150 nm) with an average thickness of 79 nm. The electroless deposition procedure can be easily and routinely performed in any wet-chemistry laboratory, and electroless gold can be deposited onto complex and internal surfaces. Intimate coupling of electrochemical detection and CE chips obviates the need for a coupling mechanism or tedious alignment procedures. With nitroaromatic compounds as a working model, microchip capillary electrophoresis equipped with electroless gold has proven to provide high sensitivity and fast response times for sensor applications. The CE microchip system was capable of separation and determination of explosive compounds including TNT in less than 130 s with detection limits ranging from 24 to 36 microg/L, i.e., 4-fold enhancements in detection efficiency in comparison to thick-film technology.     

Development of rotating electrochemical detectors for capillary electrophoresis

A rotating disk electrode (RDE) has been evaluated and optimized for the detection of electroactive species separated by capillary electrophoresis (CE). With catechol as a working model, the limit of detection was estimated to be 0.3 microM, i.e., approximately 2.5-fold better than that of the stationary disk electrode (0.7 microM). Separation efficiency was significantly improved as exemplified by an increase of theoretical plates from 26,000 plates/m at 0 rpm to 67,000 plates/m at 500 rpm. Of particular importance was the capability of RDE to alleviate electrode passivation and electrical interference associated with high separation potential fields. Therefore, rotation amperometry was especially useful for analytes such as phenolic compounds that tended to rapidly foul the electrode surface. The RDE/ CE system was capable of separation and determination of pentachlorophenol in contaminated soils, and the result obtained agreed well with conventional liquid chromatography, an EPA recommended procedure.     

Thin- and thick-film real-time gamma radiation detectors

The cruel reality of today's world forces mankind to be alert and prepared for all kinds of terrorist threats, including exposure to radiation. There is a need for cost-effective alternatives to existing commercially available real-time gamma radiation dosimeters. In this paper, numerous oxides such as NiO, CeO/sub 2/, TeO/sub 2/, In/sub 2/O/sub 3/, SiO, MnO, etc., and their mixtures in different proportions were used as the basis for such a substitute. Thin- and thick-film devices were made in the form of resistors and capacitors, structures with interdigitated electrodes, and pn-junctions. These, in different mixtures, were found to change their sensitivity when exposed to /spl gamma/-rays. In general, thin-film devices were found to be more sensitive to lower doses of radiation than the counterpart thick films. However, it was experimentally demonstrated that it is possible to fabricate a device that would satisfy the requirement of a particular application, such as the sensitivity to /spl gamma/-radiation exposure and working dose regions. Based on the above data, these structures might be regarded as a cost-effective alternative for room-temperature real-time /spl gamma/-radiation dosimetry.     

Microchip capillary electrophoresis with electrochemical detection

A novel microchip capillary electrophoresis system with electrochemical detection, using the replaceable microelectrode, is first reported. This kind of electrode can be fabricated in general laboratories and can be replaced quickly with electrodes of different materials according to the requirements of experiments. The end-column electrochemical detection on microchip CE was achieved by fixing the working electrode (such as carbon fiber, Pt, or Au, etc.) through a guide tube on the end of the separation channel. The experiment results indicate that the alignment of the electrode with the channel outlet can be carried out accurately and reproducibly, and therefore, the detection device has low noise and good reproducibility. The detection limit of dopamine is 2.4 x 10(-7) M, which is the lowest result reported so far. The separation and detection of dopamine, 5-hydroxytryptamine and epinephrine using carbon fiber and Pt microdisk electrodes within 50 s was successfully performed.     

Characteristic electrochemical responses of polymer microchannel-microelectrode chips

Polymer microchannel chips (dimension of the channel, 20 microm (depth) x 100 microm (width)) integrated with dual microband electrodes were fabricated by an imprinting method, and their characteristic electrochemical responses were elucidated in detail. A silicon micromachined template fabricated by photolithography and dry etching techniques was used for imprinting. An aqueous solution of a ferrocene derivative was brought into the microchannel by pressure-driven flow, and the electrode responses were studied on the basis of voltammetry and chronoamperometry. A linear sweep voltammetry of 1-hydroxyethylferrocene (FeCp-OH) in water demonstrated that the electrode responses in the microchannel chip were best characterized by one-dimensional diffusion along the channel length, reflecting the structural dimension of the channel. In generation-collection mode experiments, furthermore, a collection efficiency as high as approximately 90% was attained in the microchannel owing to both restricted space and characteristics of solution flow in the channel. It was confirmed that diffusion and solution flow made molecular transport very efficient in the microchannel. The experimental results were also compared with those predicted by computer simulations.     

Signal-to-noise ratio in microelectrode-array-based electrochemical detectors

The signal-to-noise ratio at an electrode array depends on the electrode area, the perimeter-to-area ratio of the electroactive portion of the surface, the mass transfer coefficient of the analyte-electrode combination, the measurement bandwidth, and the sources and magnitudes of the noises. Simple models for chronoamperometry with an array in quiescent solution and for hydrodynamic current at an array in one wall of a rectangular conduit through which analyte-containing solution is following are given. Noises from seven sources, including environmental noises, are considered in a noise model. The signal and noise models are combined to yield a model for signal-to-noise ratio at array-based electrochemical detectors. There exists an optimum array density for a given area that depends on the noise power, noise resistance, the current density at a sparse array, and the current density at a solid electrode of the same area. Approximations that lead to simple expressions for the optimum electroactive area fraction and noise resistance lead to results that are in good agreement with more complex and less approximate calculations. Electrodes of millimeter dimensions consisting of about 1% active surface with electroactive "pieces" of micrometer dimensions are anticipated to yield detection limits of about 1 fmol injected into a typical packed-column liquid chromatograph. This corresponds to about 10(-10) M analyte in the detector and about an order of magnitude improvement over solid electrodes.     

Integrated electrophoresis chips/amperometric detection with sputtered gold working electrodes

An on-chip electrochemical detector for micromachined capillary electrophoresis (CE) systems, based on sputtering a gold working electrode directly onto the capillary outlet, is described. The new on-chip detector preparation requires no microfabrication or alignment procedures nor a decoupling mechanism. The attractive performance of the integrated electrophoresis chips/amperometric detection was demonstrated for the anodic detection of neurotransmitters. The response for dopamine was linear from 20 to 200 μM, with a LOD of 1.0 μM and a sensitivity of 52 pA/μM. Such intimate coupling of capillary electrophoresis chips and electrochemical detection facilitates the realization of complete integrated microanalytical devices.     

Capillary electrophoresis microchip with a carbon nanotube-modified electrochemical detector

Significant improvements in the performance of a capillary electrophoresis (CE) microchip with an electrochemical detector are observed using a carbon nanotube (CNT)-modified working electrode. The CNT-modified electrode allows CE amperometric detection at significantly lower operating potentials and yields substantially enhanced signal-to-noise characteristics. The electrocatalytic detection is coupled to resistance to surface fouling and hence enhanced stability. Such advantages are illustrated in connection with several classes of hydrazine, phenol, purine, and amino acid compounds. Substantial minimization of surface fouling effects has been demonstrated in connection with the monitoring of phenol and tyrosine. Factors affecting the performance of the new CNT detector were assessed and optimized. CNTs from different sources are evaluated, and the effect of an anodic pretreatment is explored. The broad and significant catalytic activity exhibited by CNT-based CE detectors indicates great promise for a wide range of bioanalytical and environmental applications.     

Rudimentary capillary-electrode alignment for capillary electrophoresis with electrochemical detection

A capillary-electrode holder was constructed for electrochemical detection in capillary electrophoresis (CE). The device allows for positioning of the working electrode at the end of the capillary column without the aid of micropositioners or microscopes. The design facilitates the exchange of electrodes and capillaries without the need of refabricating the entire capillary-electrode setup. The system can be assembled in a very short period of time. Alignment with the self-guided system proved to be reproducible for the electrodes used (carbon, nickel, copper). The advantages of reduced downtime and low cost, make the device very attractive for the routine analysis of electroactive species by CE with electrochemical detection.     

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.     

A chip-based electrophoresis system with electrochemical detection and hydrodynamic injection

A simple capillary electrophoresis system in the planar format that uses a new, hydrodynamic injection principle is described. The system was realized with poly(dimethylsiloxane)-glass chips and microdisk electrodes for amperometric detection. Using a double-tee injector, no precise voltage control of the electrolyte reservoirs was needed, thus making the microchip CE system more user-friendly. The analytical characteristics of chip-based CE-EC were evaluated using ascorbic acid as the model analyte. The reproducibility of migration time and signal height was expressed by relative standard deviations of 1.2% and 5.1%, respectively (n = 5). The limit of detection for ascorbic acid was approximately 5 microM at a signal-to-noise ratio of 3. Practical application concerning the determination of physiologically relevant compounds such as noradrenaline and L-dopa are discussed.     

Integrated on-capillary electrochemical detector for capillary electrophoresis

An on-capillary electrochemical detector for capillary electrophoresis is described. It consists of a gold wire mounted permanently at the end of the capillary perpendicular to the direction of flow. This mode of detection eliminates the need for the micromanipulators or specially machined cell holders for alignment that are used for in-capillary detection modes. It also makes it possible to perform relatively fast CEEC separations using very short capillaries. The use of this detector for both off-column detection of catecholamines and end-column detection of carbohydrates by CE-PAD is described.     

On-column electrochemical detection for microchip capillary electrophoresis

The development of a cellulose acetate decoupler for on-column electrochemical detection in microchip capillary electrophoresis is presented. The capillary based laser-etched decoupler is translated to the planar format to isolate the detector circuit from the separation circuit. The decoupler is constructed by aligning a series of 20 30-microm holes through the coverplate of the microchip with the separation channel and casting a thin film of cellulose acetate within the holes. The decoupler shows excellent isolation of the detection circuit for separation currents up to 60 microA, with noise levels at or below 1 pA at a carbon fiber electrode. Detection limits of 25 nM were achieved for dopamine. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.     

Microchip capillary electrophoresis coupled with a boron-doped diamond electrode-based electrochemical detector

The attractive behavior and advantages of a diamond electrode detector for a micromachined capillary electrophoresis (CE) system are discussed. A chemically vapor-deposited boron-doped diamond (BDD) film band (0.3 x 6.0 mm) electrode is used for end-column amperomettic detection. The favorable performance of the diamond electrode microchip detector is indicated from comparison to a commonly used thick-film carbon detector. The diamond electrode offers enhanced sensitivity, lower noise levels, and sharper peaks for several groups of important anaytes (nitroaromatic explosives, organophosphate nerve agents, phenols). The favorable signal-to-background characteristics of the BDD-based CE detector are coupled with a greatly improved resistance to surface fouling and greater isolation from high separation voltages. The enhanced stability is indicated from a RSD of 0.8% for 60 repetitive measurements of 5 ppm 2,4,6-trinitrotoluene (vs RSD of 10.8% at the thick-film carbon electrode). A highly linear response is obtained for the explosives 1,3-dinitrobenzene and 2,4-dinitrotoluene over the 200-1,400 ppb range, with detection limits of 70 and 110 ppb, respectively. Factors influencing the performance of the BDD detector are assessed and optimized. The attractive properties of BDD make it very promising material for electrochemical detection in CE microchip systems and other micromachined flow analyzers.     

Boron-doped diamond microelectrodes for use in capillary electrophoresis with electrochemical detection

The fabrication and characterization of boron-doped diamond microelectrodes for use in electrochemical detection coupled with capillary electrophoresis (CE-EC) is discussed. The microelectrodes were prepared by coating thin films of polycrystalline diamond on electrochemically sharpened platinum wires (76-, 25-, and 10-microm diameter), using microwave-assisted chemical vapor deposition (CVD). The diamond-coated wires were attached to copper wires (current collectors), and several methods were explored to insulate the cylindrical portion of the electrode: nail polish, epoxy, polyimide, and polypropylene coatings. The microelectrodes were characterized by scanning electron microscopy, Raman spectroscopy, and cyclic voltammetry. They exhibited low and stable background currents and sigmoidally shaped voltammetric curves for Ru(NH3)6(3+/2+) and Fe(CN)6(3-/4-) at low scan rates. The microelectrodes formed with the large diameter Pt and sealed in polypropylene pipet tips were employed for end-column detection in CE. Evaluation of the CE-EC system and the electrode performance were accomplished using a 10 mM phosphate buffer, pH 6.0, run buffer, and a 30-cm-long fused-silica capillary (75-microm i.d.) with dopamine, catechol, and ascorbic acid serving as test analytes. The background current (approximately 100 pA) and noise (approximately 3 pA) were measured at different detection potentials and found to be very stable with time. Reproducible separation (elution time) and detection (peak current or area) of dopamine, catechol, and ascorbic acid were observed with response precisions of 4.1% or less. Calibration curves constructed from the peak area were linear over 4 orders of magnitude, up to a concentration between 0.1 and 1 mM. Mass limits of detection for dopamine and catechol were 1.7 and 2.6 fmol, respectively (S/N = 3). The separation efficiency was approximately 33,000, 56,000, and 98,000 plates/m for dopamine, catechol, and ascorbic acid, respectively. In addition, the separation and detection of 1- and 2-naphthol in 160 mM borate buffer, pH 9.2, was investigated. Separation of these two analytes was achieved with efficiencies of 118,000 and 126,000 plates/m, respectively.     

Capillary electrophoresis with electrochemical detection for chiral separation of optical isomers

The enantiomers of two amine derivatives were directly separated by capillary electrophoresis (CE), employing β-cyclodxtrin (β-CD) as a chiral additive in strongly alkaline solutions. The analytes were detected by electrochemistry, using a copper disk electrode at +675 mV vs Ag/AgCl reference electrode. Both the free enantiomers and the enantiomer-cyclodxtrin inclusion complexes could be detected using this approach, although the complexed forms gave lower oxidation currents than the free forms. Factors affecting the chiral CE separation of the analytes, such as working potential, concentration of running buffer and β-CD, and applied voltage, were extensively investigated. Under the optimum conditions, baseline separation of the enantiomers could be accomplished in less than 18 min. In addition, a successful application of the method to the enantiomeric purity determination confirmed its validity and practicability.