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.     

Experimental characterization of end-column electrochemical detection in conjunction with nonaqueous capillary electrophoresis

An end-column electrochemical detector arrangement for capillary electrophoresis (CE) based on a 75-microm-i.d. capillary and a 25-microm microdisk electrode is characterized. The investigations were carried out using a nonaqueous (acetonitrile-based) buffer and ferrocene model compounds which offer high reliability for voltammetric measurements. The positioning of the microdisk electrode relative to the capillary outlet is the most important parameter for optimization of detection performance as it determines the characteristics of mass transport toward the electrode and the effect of ohmic potential drop resulting from the electrophoretic current on the actual detection potential. On the basis of spatially resolved studies, it was concluded that for the detection system used the microdisk electrode should be placed in a central position relative to the capillary outlet at a distance within the range of 75-100 microm. The presence of a high-voltage electric field had no negative effect on baseline noise, which was demonstrated by comparison of capillary flow injection based on gravity flow and CE experiments. Even a faster stabilization of the baseline was observed by increasing the separation voltage.     

An integrated decoupler for capillary electrophoresis with electrochemical detection: application to analysis of brain microdialysate.

An approach to capillary electrophoresis with electrochemical detection (CE-EC) suitable for determination of dopamine in 1-min brain microdialysate samples is described. The CE-EC system includes an electrochemical detection cell that permits easy, precise, and permanent alignment of a carbon fiber microelectrode with a separation capillary (30-micron i.d., 75-cm length). Amperometric detection was performed at a constant applied potential of 600 mV with respect to a Ag/AgCl reference electrode. Decoupling of the electrophoretic current from the amperometric detector was accomplished with an integrated end-column decoupler prepared by etching the capillary outlet with HF. The decoupler produces baseline noise of 50 fA, or less, in the presence of 10-20-muA current in the separation capillary. The low baseline noise affords low mass (attomoles) and low concentration (nanomolar) detection limits for dopamine and 4-methylcatechol. A peak attributable to dopamine was identified in electropherograms of brain microdialysate samples obtained from anesthetized rats. Identification of the dopamine peak was confirmed by pharmacological methods. Dopamine was readily detected in 1-min brain microdialysate samples. The dopamine concentration in 1-min brain microdialysis samples was significantly altered by drug treatments and by brief electrical stimulation of dopaminergic axons.     

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.     

Electrochemical detection method for nonelectroactive and electroactive analytes in microchip electrophoresis

In this work, we establish an indirect amperometric detection method via mounting a single carbon fiber disk working electrode in the end part of a microchannel. This in-channel configuration for microchip capillary electrophoresis brings about that the potential of the working electrode in the case of electrochemical reduction reaction is coupled by the separation electric field, while the potential of the working electrode in the case of electrochemical oxidation reaction is not coupled by the separation electric field. Such a special performance provides a convenient and sensitive approach for indirectly detecting nonelectroactive analytes that relies on amperometric response of dissolved oxygen in solution and directly detecting electroactive analytes based on their own amperometric response on the carbon fiber electrode. This method has shown its essential importance in the analysis of inorganic cations, biomolecules, and electroosmotic flow rates. Based on preliminary results, a detection limit of 1.0 microM for K(+) and Na(+) have been achieved.     

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.     

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 microchip electrophoresis device with integrated electrochemical detection: a direct comparison of constant potential amperometry and sinusoidal voltammetry

A microchip electrophoresis system with integrated electrochemical detection is described in this work. The hybrid device utilizes poly(dimethylsiloxane) as the electrophoresis channel substrate and a planar gold electrode lithographically fabricated onto a glass slide for electrochemical detection. The system is characterized by the separation and detection of various neurotransmitters. The gold working electrode is placed just inside the separation channel without adverse effects on the detection sensitivity, due to the electrical decoupling of the detection and electrophoresis systems. The close proximity of the working electrode to the exit of the separation channel results in symmetric peak shapes and efficient separations (50,000-100,000 plates/m). A direct comparison between the frequency-based electrochemical technique, sinusoidal voltammetry, and the more commonly used constant potential (DC) amperometry is made. Sinusoidal voltammetry is found to be roughly an order of magnitude more sensitive than DC amperometry, with calculated mass detection limits (S/N = 3) of 12 amol and 15 amol for dopamine and isoproterenol, respectively.     

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.     

Capillary electrophoresis coupled with electrochemiluminescence detection using porous etched joint

A new setup to couple capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection is described in which the electrical connection of CE is achieved through a porous section at a distance of 7 mm from the CE capillary outlet. Because the porous capillary wall allowed the CE current to pass through and there was no electric field gradient beyond that section, the influence of CE high-voltage field on the ECL procedure was eliminated. The porous section formed by etching the capillary with hydrofluoric acid after only one side of the circumference of 2-3 mm of polyimide coating of the capillary was removed, while keeping the polyimide coating on the other part to protect the capillary from HF etching makes the capillary joint much more robust since only a part of the circumference of it is etched. A standard three-electrode configuration was used in experiments with Pt wire as a counter electrode, Ag/AgCl as a reference electrode, and a 300-microm diameter Pt disk as a working electrode. Compared with CE-ECL conventional decoupler designs, the present setup with a porous joint has no added dead volume created. Moreover, the dead volume can be increasingly decreased by shortening the distance ( approximately 100 microm) between the working electrode and the end of the separation capillary. The versatility in choice of capillaries and separation buffers within this design is the main advantage over the use of small i.d. capillary and low conductivity buffer in some CE-ECL systems. The performance of this setup is illustrated by the analyses of tripropylamine and proline.     

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.     

Integrated Capillary Electrophoresis/Electrochemical Detection with Metal Film Electrodes Directly Deposited onto the Capillary Tip

The practical application of electrochemical detection in capillary electrophoresis has been hampered by irreproducibility and inconvenience related to capillary/electrode alignment. In order to eliminate these problems, a simple, flexible method by which the capillary and the working electrode were integrated into a single operational unit was devised and evaluated. The electrodes were formed by sputtering a thin conductive layer of Au or Pt onto the exit tip of the capillary. Depending on the size of the capillary used (i.e., both inner and outer diameters), Au on-capillary electrodes (OCEs) gave detection limits at the micromolar level and slightly below for the test analytes dopamine and catechol. More important, operation of the OCEs required no alignment procedures beyond immersion in the CE buffer reservoir/detector cell. OCEs used in this manner exhibited relative standard deviations of 2-4% for repeated injections even if removed from solution between runs. Finally, the Au and Pt OCEs could themselves be modified further by conventional electrochemical procedures. Here, Cu OCEs, formed by electrodeposition onto Au, were used to detect carbohydrate compounds; and an enzyme OCE, formed by adsorption of glucose oxidase onto Pt, was used to detect glucose.     

Micromachined electrophoresis chips with thick-film electrochemical detectors

A capillary electrophoresis (CE) microsystem, based on the combination of microphotolithographically fabricated separation chips and thick-film electrochemical detector strips, is described. The microsystem consists of a planar screen-printed carbon line electrode mounted perpendicular to the flow direction. Such coupling obviates the need for permanent attachment of the detector, to allow easy and fast replacement of the working electrode. Variables influencing the separation efficiency and amperometric response, including the channel-electrode spacing, separation voltage, or detection potential, are assessed and optimized. The versatility, simplicity, and low-cost advantages of the new design are coupled to an attractive performance, with submicromolar detection limits, and good precision. Applicability for assays of mixtures of nitroaromatic explosives or catecholamines is demonstrated. Such use of screen-printed detectors should also benefit conventional CE systems, particularly in applications requiring a frequent replacement of the working electrode.     

In-channel electrochemical detection for microchip capillary electrophoresis using an electrically isolated potentiostat

A new electrode configuration for microchip capillary electrophoresis (CE) with electrochemical (EC) detection is described. This approach makes it possible to place the working electrode directly in the separation channel. The "in-channel" EC detection was accomplished without the use of a decoupler through the utilization of a specially designed, electrically isolated potentiostat. The effect of the working electrode position on the separation performance (in terms of plate height and peak skew) of poly(dimethylsiloxane)-based microchip CEEC devices was evaluated by comparing the more commonly used end-channel configuration with this new in-channel approach. Using catechol as the test analyte, it was found that in-channel EC detection decreased the total plate height by a factor of 4.6 and lowered the peak skew by a factor of 1.3. A similar trend was observed for the small, inorganic ion nitrite. Furthermore, a fluorescent and electrochemically active amino acid derivative was used to directly compare the separation performance of in-channel EC detection to that of a widely used laser-induced fluorescence (LIF) detection scheme. In this case, it was found that the plate height and peak skew for both detection schemes were essentially equal, and the separation performance of in-channel EC detection is comparable to LIF detection.     

Fabrication of a poly(dimethylsiloxane)-based electrochemiluminescence detection cell for capillary electrophoresis

An easy but effective technique is described here for quick fabrication of low-cost electrochemiluminescence detection cells for capillary electrophoresis. The technique is based on molding of poly(dimethylsiloxane) (PDMS) with a capillary column inserted into a pipet tip. Two access holes are left in the PDMS slab; they provide neat accommodations for the separation capillary and the working electrode made with the same type of tip. Since the access holes are well-aligned, the electrode and the capillary are automatically aligned; thus, end-column detection is easily obtained. Fabrication of the detection cell is straightforward; no micromechanical operation is included. Also the principle for the procedure makes it possible to efficiently batch production detection cells with good reproducibility. Because of the end-column scheme, the cell can be adopted for electrophoresis with electrochemical detection as well.     

Simplifying CE-MS operation. 2. Interfacing low-flow separation techniques to mass spectrometry using a porous tip

A robust, reproducible, and single-step interface design between low flow rate separation techniques, such as sheathless capillary electrophoresis (CE) and nanoliquid chromatography (nLC), and mass spectrometry (MS) using electrospray ionization (ESI), is introduced. In this design, the electrical connection to the capillary outlet was achieved through a porous tip at the capillary outlet. The porous section was created by removing 1-1.5 in. of the polyimide coating of the capillary and etching this section by 49% solution of HF until it is porous. The electrical connection to the capillary outlet is achieved simply by inserting the capillary outlet containing the porous tip into the existing ESI needle (metal sheath) and filling the needle with the background electrolyte. Redox reactions of water at the ESI needle and transport of these small ions through the porous tip into the capillary provides the electrical connection for the ESI and for the CE outlet electrode. The etching process reduces the wall thickness of the etched section, including the tip of the capillary, to 5-10 microm, which for a 20-30 microm i.d. capillary results in stable electrospray at approximately 1.5 kV. The design is suitable for interfacing a wide range of capillary sizes with a wide range of flow rates to MS via ESI, but it is especially useful for interfacing narrow (<30 microm i.d.) capillaries and low flow rates (<100 nL/min). The advantages of the porous tip design include the following: (1) its fabrication is reproducible, can be automated, and does not require any mechanical tools. (2) The etching process reduces the tip outer diameter and makes the capillary porous in one step. (3) The interface can be used for both nLC-MS and CE-MS. (4) If blocked or damaged, a small section of the tip can be etched off without any loss of performance. (5) The interface design leaves the capillary inner wall intact and, therefore, does not add any dead volume to the CE-MS or nLC-MS interface. (6) Bubble formation due to redox reactions of water at the high-voltage electrode is outside of the separation capillary and does not affect separation or MS performances. The performance of this interface is demonstrated by the analyses of amino acids, peptide, and protein mixtures.     

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.     

Elimination of high-voltage field effects in end column electrochemical detection in capillary electrophoresis by use of on-chip microband electrodes

The influence of the separation voltage on end column electrochemical detection (EC) in capillary electrophoresis (CE) has been investigated using an electrochemical detector chip based on an array of microband electrodes. It is shown, both theoretically and experimentally, that the effect of the CE electric field on the detection can be practically eliminated, without using a decoupler, by positioning the reference electrode sufficiently close to the working electrode. In the present study, this was demonstrated by using an experimental setup in which neighboring microband electrodes on a chip, positioned 30 microns from the end of the CE capillary, were used as working and reference electrodes, respectively. The short distance (i.e., 10 microns) between the working and reference electrode ensured that both of the electrodes were very similarly affected by the presence of the CE electric field. With this experimental setup, no significant influence of the CE voltage on the peak potentials for gold oxide reduction could be seen for CE voltages up to +30 kV. The detector noise level was also found to be reduced.     

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.     

Capillary electrophoresis to mass spectrometry interface using a porous junction

A new capillary electrophoresis interface to electrospray ionization mass spectrometry (CE/ESI-MS) is introduced in which the electrical connection to the CE capillary outlet/ESI electrode is achieved by transfer of small ions related to the background electrolyte (BGE) through a porous section near the CE capillary outlet. In this design, only a small section of the capillary wall is made porous. The porous section is created by first thinning a small section of the capillary wall by drilling a well into it and then etching the remaining thin wall porous. This design has two advantages over previous designs (in which the whole circumference of the capillary was made porous): first, the capillary interface is more robust because only a small section of it is made porous, and therefore, no liquid junction is needed to secure the porous section. The electrical connection is achieved simply by inserting the capillary outlet containing the porous junction into the existing ESI needle and filling the needle with the BGE. Second, the time required to make the fused silica porous is reduced from approximately 1 h to a few minutes. In addition, there is no dead volume associated with the porous design, and because the actual metal/liquid contact occurs outside of the CE capillary, bubble formation due to redox reactions of water at the electrode does not affect CE/ESI-MS performance. The performance of this interface is demonstrated by the analyses of peptide and protein mixtures.