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.     

Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.     

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.     

Improving the compatibility of contact conductivity detection with microchip electrophoresis using a bubble cell

A new approach for improving the compatibility between contact conductivity detection and microchip electrophoresis was developed. Contact conductivity has traditionally been limited by the interaction of the separation voltage with the detection electrodes because the applied field creates a voltage difference between the electrodes, leading to unwanted electrochemical reactions. To minimize the voltage drop between the conductivity electrodes and therefore improve compatibility, a novel bubble cell detection zone was designed. The bubble cell permitted higher separation field strengths (600 V/cm) and reduced background noise by minimizing unwanted electrochemical reactions. The impact of the bubble cell on separation efficiency was measured by imaging fluorescein during electrophoresis. A bubble cell four times as wide as the separation channel led to a decrease of only 3% in separation efficiency at the point of detection. Increasing the bubble cell width caused larger decreases in separation efficiency, and a 4-fold expansion provided the best compromise between loss of separation efficiency and maintaining higher field strengths. A commercial chromatography conductivity detector (Dionex CD20) was used to evaluate the performance of contact conductivity detection with the bubble cell. Mass detection limits (S/N = 3) were as low as 89 +/- 9 amol, providing concentration detection limits as low as 71 +/- 7 nM with gated injection. The linear range was measured to be greater than 2 orders of magnitude, from 1.3 to 600 microM for sulfamate. The bubble cell improves the compatibility and applicability of contact conductivity detection in microchip electrophoresis, and similar designs may have broader application in electrochemical detection as the expanded detection zone provides increased electrode surface area and reduced analyte velocity in addition to the reduction of separation field effects.     

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.     

Simple and sensitive electrode design for microchip electrophoresis/electrochemistry

A simple and sensitive electrode design for microchip capillary electrophoresis/electrochemistry (CE-EC) is presented. The system employs metal microwires as the working electrodes for electrochemical detection. Two general approaches for integration of electrodes in microchip CE-EC are commonly used, end-channel and microfabrication. The end-channel approach allows electrode cleaning and the use of chemically modified electrodes; however, the designs generally lack portability and the ability to incorporate multiple electrodes. Microfabrication allows the incorporation of multiple electrodes on-chip and is easily made portable; however, it requires the use of expensive metallization and clean room facilities, and integration of more than one electrode material is challenging. The reported approach aligns a solid metal microwire through the separation channel allowing integration of multiple electrodes and the use of different electrode materials without sacrificing the portability. A detection limit of 100 nM for dopamine was achieved without the use of a decoupler as a result of a higher collection efficiency with the new design.     

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.     

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.     

Parallel-opposed dual-electrode detector with recycling amperometric enhancement for capillary electrophoresis

The assembly and characterization of dual-electrode amperometric detection for capillary electrophoresis are described. The detector consists of a disk electrode and an integrated on-capillary electrode fabricated by depositing a gold film onto the end of the separation capillary. The two electrodes are brought together, aligned, and fixed in position using a pair of acrylic plates with a straight groove on one of the plates, the same design as that of a conventional end-column detector. A portion of the on-capillary electrode is parallel-opposed to the disk electrode in a thin-layer geometry. In this region, the redox cycling established between these two electrodes significantly enhances the amperometric signals of electrochemically reversible analytes. For measurements of dopamine in pH 6.9 phosphate electrolyte with a 12.5-μm-i.d. capillary, such a configuration is 10-fold more sensitive than conventional end-column detection. The linear range exceeds 4 orders of magnitude (1.2 mM-50 nM) and the detection limit is 12 nM (4.2 amol, S/N = 3). Various modes of potential settings for the dual-electrode detection are also discussed.     

Trace analysis of DNA: preconcentration, separation, and electrochemical detection in microchip electrophoresis using Au nanoparticles

We have developed a simple and sensitive on-chip preconcentration, separation, and electrochemical detection (ED) method for trace analysis of DNA. The microchip comprised of three parallel channels: the first two are for the field-amplified sample stacking and subsequent field-amplified sampled injection steps, while the third one is for the microchip gel electrophoresis (MGE) with ED (MGE-ED). To improve preconcentration and separation performances of the method, the stacking and separation buffers containing the hydroxypropyl cellulose (HPC) matrix were modified with gold nanoparticles (AuNPs). The formation of AuNPs and HPC/AuNP-modified buffers were characterized by UV-visible spectroscopy and TEM experiments. The conducting polymer-modified electrode was also modified with AuNPs to enhance detection performances of the electrode. The conducting polymer/AuNP layers act as electrocatalysts for the direct detection of DNA based on their oxidation in a solution phase. The total sensitivity was improved by approximately 25 000-fold when compared with a conventional MGE-ED analysis. The calibration plots were linear (r2 = 0.9993) within the range of 0.003-1.0 pg/microL for a 20-bp DNA sample. The sensitivity was 0.20 nA/(fg/microL), with a detection limit of 5.7 amol in a 50-microL sample, based on S/N = 3. The applicability of the method for the analysis of 13 fragments present in a 100-bp DNA ladder was successfully demonstrated.     

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.     

Bead-based electrochemical immunoassay for bacteriophage MS2

Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus. beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL.     

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.     

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.     

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.     

Temporal resolution in electrochemical imaging on single PC12 cells using amperometry and voltammetry at microelectrode arrays

Carbon-fiber-microelectrode arrays (MEAs) have been utilized to electrochemically image neurochemical secretion from individual pheochromocytoma (PC12) cells. Dopamine release events were electrochemically monitored from seven different locations on single PC12 cells using alternately constant-potential amperometry and fast-scan cyclic voltammetry (FSCV). Cyclic voltammetry, when compared to amperometry, can provide excellent chemical resolution; however, spatial and temporal resolution are both compromised. The spatial and temporal resolution of these two methods have been quantitatively compared and the differences explained using models of molecular diffusion at the nanogap between the electrode and the cell. A numerical simulation of the molecular flux reveals that the diffusion of dopamine molecules and electrochemical reactions both play important roles in the temporal resolution of electrochemical imaging. The simulation also reveals that the diffusion and electrode potential cause the differences in signal crosstalk between electrodes when comparing amperometry and FSCV.     

Performance of pyrolyzed photoresist carbon films in a microchip capillary electrophoresis device with sinusoidal voltammetric detection

Pyrolyzed photoresist films (PPF) are introduced as planar carbon electrodes in a PDMS-quartz hybrid microchip device. The utility of PPF in electroanalytical applications is demonstrated by the separation and detection of various neurotransmitters. PPF is found to form a stable, low-capacitance, durable layer on quartz, which can then be used in conjunction with a microchip capillary electrophoretic device. Sinusoidal voltammetric detection at PPF electrodes is shown to be very sensitive, with a detection limit (S/N = 3) of 100 nM for dopamine, corresponding to a mass detection limit (S/N = 3) of 2 amol. The selectivity of analysis in the frequency domain is demonstrated by isolating each individual signal in a pair of analytes that are chromatographically unresolved. Effectively decoupling the electrophoresis and electrochemical systems allows the electrodes to be placed just inside the separation channel, which results in efficient separations (80 000-100 000 plates/m).     

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.     

Nanofabrication of bio-self assembled monolayer and its electrochemical property for toxicant detection

Cyclic voltammetry (CV) has been used to investigate the electrochemical behavior of a glutathione (GSH) self assembled monolayer on modified gold electrodes (Bio-SAM). The GSH monolayer exhibits an influence on electrode surface activity. Electrochemically immobilized dsDNA onto a Cyt c/GSH-SAM/Au electrode, which is useful for the fabrication of a nanobiosensing device. The immobilized Cyt c followed by dsDNA immobilized films maintained its surface activity and finally dsDNA/Cyt c/GSH-SAM/Au electrode, targeted for the detection of toxicants. The films were characterized by CV, DPV, and AFM. The differential pulse voltammetry (DPV) technique was applied to detect three kinds of common toxins, 2-aminoanthracene (2-AA), 3-bromobenzanthrone (3-BBA) and bisphenol A (BPhA). The electrochemical signals showed good inverse relationship with the increase of concentrations of toxicants. Our proposed system based on electrochemical method with nanoscale film technology can be applied at highly sensitive biosensor for detecting various toxic chemicals.     

A nano-structured Ni(II)–chelidamic acid modified gold nanoparticle self-assembled electrode for electrocatalytic oxidation and determination of methanol

A nano-structured Ni(II)–chelidamic acid (2,6-dicarboxy-4-hydroxypyridine) film was electrodeposited on a gold nanoparticle–cysteine–gold electrode. The morphology of Ni(II)–chelidamic acid gold nanoparticle self‐assembled electrode was investigated by scanning electron microscopy (SEM). Electrocatalytic oxidation of methanol on the surface of modified electrode was studied by cyclic voltammetry and chronoamperometry methods. The hydrodynamic amperometry at a rotating modified electrode at constant potential versus reference electrode was used for detection of methanol. Under optimized conditions the calibration plots are linear in the concentration range 0–50 mM with a detection limit of 15 μM. The formed matrix in our work possessed a 3D porous network structure with a large effective surface area, high catalytic activity and behaved like microelectrode ensembles. The modified electrode indicated reproducible behavior and a high level stability during the experiments, making it particularly suitable for analytical purposes.