Nanoband electrode for high-performance in-channel amperometric detection in dual-channel microchip capillary electrophoresis

A nanoband electrode detector integrated with a dual-channel polydimethylsiloxane microchip is proposed for in-channel amperometric detection in microchip capillary electrophoresis. Gold nanoband electrodes, which were fabricated on SU-8 substrates with a 100-nm-width gold layer, were introduced into the dual-channel microchip to be an electrochemical detector. Due to the nano-sized width of the detector, the noise of the amperometric detection was significantly reduced, and a high separation resolution was achieved for monitoring the analytes. The detection sensitivity of the system was improved by high signal-to-noise ratio, and a low detection limit on microchip was obtained for p-aminophenol (2.09 nM). Because of the high resolution in measuring half-peak width, the plate number that is used to evaluate the separation efficiency was 1.5-fold higher than that using 50-μm-width electrochemical detector. The effect of sample injection time and data acquisition time on separation efficiency was investigated, and an attractive separation efficiency was achieved with a plate number up to 17,500.     

Microchip capillary electrophoresis/electrochemistry

Microfabricated fluidic devices have generated considerable interest over the past ten years due to the fact that sample preparation, injection, separation, derivatization, and detection can be integrated into one miniaturized device. This review reports progress in the development of microfabricated analytical systems based on microchip capillary electrophoresis (CE) with electrochemical (EC) detection. Electrochemical detection has several advantages for use with microchip electrophoresis systems, for example, ease of miniaturization, sensitivity, and selectivity. In this review, the basic components necessary for microchip CEEC are described, including several examples of different detector configurations. Lastly, details of the application of this technique to the determination of catechols and phenols, amino acids, peptides, carbohydrates, nitroaromatics, polymerase chain reaction (PCR) products, organophosphates, and hydrazines are described.     

Poly(methylmethacrylate) and Topas capillary electrophoresis microchip performance with electrochemical detection

A capillary electrophoresis (CE) microchip made of a new and promising polymeric material: Topas (thermoplastic olefin polymer of amorphous structure), a cyclic olefin copolymer with high chemical resistance, has been tested for the first time with analytical purposes, employing an electrochemical detection. A simple end-channel platinum amperometric detector has been designed, checked, and optimized in a poly-(methylmethacrylate) (PMMA) CE microchip. The end-channel design is based on a platinum wire manually aligned at the exit of the separation channel. This is a simple and durable detection in which the working electrode is not pretreated. H(2)O(2) was employed as model analyte to study the performance of the PMMA microchip and the detector. Factors influencing migration and detection processes were examined and optimized. Separation of H(2)O(2) and L-ascorbic acid (AsA) was developed in order to evaluate the efficiency of microchips using different buffer systems. This detection has been checked for the first time with a microchip made of Topas, obtaining a good linear relationship for mixtures of H(2)O(2) and AsA in different buffers.     

Plastified poly(ethylene terephthalate) (PET)-toner microfluidic chip by direct-printing integrated with electrochemical detection for pharmaceutical analysis

Rapid separation and determination of acetaminophen and its hydrolysate with end-channel electrochemical (EC) detection integrated on a plastified poly(ethylene terephthalate) (PET)-toner microchip capillary electrophoresis (CE) system was investigated. In this separation and detection system, a Pt ultramicroelectrode integrated on a three-dimensional adjustor was used as working electrode. Factors influencing the separation and detection were investigated and optimized. Results show that acetaminophen and p-aminophenol can be well separated within 84 s with R.S.D. < 1% for migration time and R.S.D. < 3.6% for detection current for both analytes. Detection limits for both analytes are determined to be 5.0 μM (S/N = 3). This method has been successfully applied to the detection of trace p-aminophenol in paracetamol tablets. The results demonstrate that the PET-toner microchips can obtain better performance than PDMS microfluidic devices but at much lower cost.     

Integration of microchip electrophoresis with electrochemical detection using an epoxy-based molding method to embed multiple electrode materials

This paper describes the use of epoxy-encapsulated electrodes to integrate microchip-based electrophoresis with electrochemical detection. Devices with various electrode combinations can easily be developed. This includes a palladium decoupler with a downstream working electrode material of either gold, mercury/gold, platinum, glassy carbon, or a carbon fiber bundle. Additional device components such as the platinum wires for the electrophoresis separation and the counter electrode for detection can also be integrated into the epoxy base. The effect of the decoupler configuration was studied in terms of the separation performance, detector noise, and the ability to analyze samples of a high ionic strength. The ability of both glassy carbon and carbon fiber bundle electrodes to analyze a complex mixture was demonstrated. It was also shown that a PDMS-based valving microchip can be used along with the epoxy-embedded electrodes to integrate microdialysis sampling with microchip electrophoresis and electrochemical detection, with the microdialysis tubing also being embedded in the epoxy substrate. This approach enables one to vary the detection electrode material as desired in a manner where the electrodes can be polished and modified as is done with electrochemical flow cells used in liquid chromatography.     

In-channel modification of electrochemical detector for the detection of bio-targets on microchip

The coulometric efficiency (C_eff) of an amperometric detector integrated on PDMS/glass capillary electrophoresis microfluidic device (microchip) has been enhanced by in-channel electrochemical modification. In-channel electrochemical deposition of gold particles was performed in order to vertically increase the surface area of the Au sensing microelectrode. The roughness of the electrodes was characterized using scanning electron microscopy and profilometric analysis. The degree of electrode modification was also characterized by roughness factor determination. Separation processes including detection potential was optimized and the analytical performance of the microchip was tested using a mixture of dopamine (DA) and catechol (CA). The modified electrochemical detector provided well-resolved separation of DA and CA in less than 60 s with enhanced sensitivity; no peak broadening was observed. The limit of detection using in-channel modification of working electrode for DA and CA are 60 and 110 nM, respectively. Thus, in-channel electrochemical deposition of metallic particles should be used to enhance the C_eff of integrated amperometric detection of analytes with good redox properties in order to obtain lower LODs.     

Recent developments in electrochemical detection for microchip capillary electrophoresis

Significant progress in the development of miniaturized microfluidic systems has occurred since their inception over a decade ago. This is primarily due to the numerous advantages of microchip analysis, including the ability to analyze minute samples, speed of analysis, reduced cost and waste, and portability. This review focuses on recent developments in integrating electrochemical (EC) detection with microchip capillary electrophoresis (CE). These detection modes include amperometry, conductimetry, and potentiometry. EC detection is ideal for use with microchip CE systems because it can be easily miniaturized with no diminution in analytical performance. Advances in microchip format, electrode material and design, decoupling of the detector from the separation field, and integration of sample preparation, separation, and detection on-chip are discussed. Microchip CEEC applications for enzyme/immunoassays, clinical and environmental assays, as well as the detection of neurotransmitters are also described.     

Design and fabrication of integrated solid-phase extraction-zone electrophoresis microchip

Integrated solid-phase extraction-zone electrophoresis (SPE-ZE) device has been designed and fabricated on microchip. The structures were fabricated by using multiple layers of SU-8 polymer with a novel technique that enables easy alignment and high yield of the chips. SU-8 adhesive bonding has two major advantages: it enables bonding of high aspect ratio pillars and it results in fully SU-8 microchannels with uniform electrokinetic flow properties. The SPE-ZE device has a fluidic reservoir with 15:1 high aspect ratio pillars for bead filters that act as a SPE part in the chip structure. The separation unit is a 25 mm long electrophoresis channel starting from the outlet of SPE reservoir. Argon laser-induced fluorescence (LIF) detector was used to monitor simultaneously the SPE reservoir and the detection site at the end of the electrophoresis channel. Flow characteristics and electric field distributions were simulated with Femlab software. Fluorescein was used as the analyte for detecting the operational performance of the chip. Adsorption, bead rinsing, elution and detection were tested to verify functioning of the chip design.     

Screen‐Printed Contactless Conductivity Detector for Microchip Capillary Electrophoresis

A new method for mass fabrication of silver ink conductivity detector electrodes for poly(methylmethacrylate) (PMMA) microchip electrophoretic systems has been developed based on screen‐printing technology. Printing of silver conductivity electrodes was performed through a patterned stencil on thin PMMA sheets. Following the electrode fabrication, the PMMA sheets are cut into cover sheets, and are aligned and sealed to the channel plate thus establishing a complete microchip separation device. The effects of the electrode width and spacing on the response and resolution have been investigated and the optimized electrode performance was compared to commonly used aluminum electrodes in the determination of ammonium, methyl ammonium, and sodium. The utility of the screen‐printed contactless conductivity detector (SPCCD) electrodes is further demonstrated for the separation and detection of organic acids with excellent reproducibility (RSD values of 3.7% and 4.1% for oxalate and tartrate, respectively). The thick‐film fabrication of the electrode material demonstrates the ability to mass‐fabricate detection devices with total process of device fabrication requiring less than 4 h (including the fabrication of channel plate, cover sheet with the electrodes, and subsequent bonding). The fabrication method described here is convenient and does not compromise the detector performance, hence offers great promise for producing single use field deployable analytical microsystems.     

A novel Au–Ag–Pt three-electrode microchip sensing platform for chromium(VI) determination

A simple, rapid and portable electrochemical microchip sensing platform has been successfully constructed for chromium(VI) determination. Gold–silver–platinum (Au–Ag–Pt) three-material electrodes (gold as working electrode, silver as reference electrode and platinum as counter electrode) were integrated on one poly(methyl methacrylate) (PMMA) substrate by polymer compatible photolithography process. The three-electrode microchip sensing platform was used for Cr(VI) determination for the first time, and exhibited high sensitivity and good reproducibility. A wide linear range from 2 to 200 μM with a good linear correlation (R² = 0.998) was obtained, and the detection limit was 0.9 μM. In addition, the practical analytical application of the sensing micro-platform was assessed by determination of Cr(VI) in real water samples with satisfactory results. Armed with the remarkable advantages, such as ease of use, low analyte consumption, inexpensive cost and fast response time, the microchip sensing platform may hold great potential for the high-throughput and in-field environmental monitoring Cr(VI) pollutant.     

Improved separation efficiency of neurotransmitters on a native printed capillary electrophoresis microchip simply by manipulating electroosmotic flow

Separation and determination of dopamine and epinephrine with end-channel electrochemical (EC) detection integrated on a native printed microchip capillary electrophoresis (CE) system was investigated. Factors influencing the separation and detection were investigated and optimized. Results show manipulating EOF, which can be easily achieved by adjusting buffer pH, is a simple and effective way to achieve the baseline separation of dopamine and epinephrine in native polymeric microchips. Without surface modification of microchannel, printed microchips with advantages of low cost and easy preparation can achieve high performance like other microfluidic devices.     

Determination of cefotaxime and desacetylcefotaxime in cerebrospinal fluid by solid-phase extraction and high-performance liquid chromatography

A microfluidic analytical system for the separation and detection of organic peroxides, based on a microchip capillary electrophoresis device with an integrated amperometric detector, was developed. The new microsystem relies on the reductive detection of both organic acid peroxides and hydroperoxides at -700 mV (vs. Ag wire/AgCl). Factors influencing the separation and detection processes were examined and optimized. The integrated microsystem offers rapid measurements (within 130 s) of these organic-peroxide compounds, down to micromolar levels. A highly stable response for repetitive injections (RSD 0.35-3.12%; n = 12) reflects the negligible electrode passivation. Such a "lab-on-a-chip" device should be attractive for on-site analysis of organic peroxides, as desired for environmental screening and industrial monitoring.     

Simultaneous microchip enzymatic measurements of blood lactate and glucose

A miniaturized capillary electrophoretic (CE) microchip device for the simultaneous measurements of lactate and glucose is described. The new microchip bioassay protocol integrates an electrophoretic separation of lactate and glucose, post-column enzymatic reactions of these metabolites with their respective oxidase enzymes, and an amperometric (anodic) detection of enzymatically-liberated hydrogen peroxide at a gold-coated thick-film carbon detector. Factors influencing the response have been examined and optimized, and the analytical performance has been characterized. Applicability of the microchip assay to clinical samples, such as serum and blood, is demonstrated. The microchip protocol obviates cross enzymatic reactions and interferences from major oxidizable constituents common to dual glucose-lactate enzyme electrodes. Such ability to rapidly separate and quantitate lactate and glucose on a small microchip platform should find important clinical and biotechnological applications.     

A high-performance polycarbonate electrophoresis microchip with integrated three-electrode system for end-channel amperometric detection

A fully integrated polycarbonate (PC) microchip for CE with end-channel electrochemical detection operated in an amperometric mode (CE-ED) has been developed. The on-chip integrated three-electrode system consisted of a gold working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, which was fabricated by photo-directed electroless plating combined with electroplating. The working electrode was positioned against the separation channel exit to reduce post-channel band broadening. The electrophoresis high-voltage (HV) interference with the amperometric detection was assessed with respect to detection noise and potential shifts at various working-to-reference electrode spacing. It was observed that the electrophoresis HV interference caused by positioning the working electrode against the channel exit could be diminished by using an on-chip integrated reference electrode that was positioned in close proximity (100 microm) to the working electrode. The CE-ED microchip was demonstrated for the separation of model analytes, including dopamine (DA) and catechol (CA). Detection limits of 132 and 164 nM were achieved for DA and CA, respectively, and a theoretical plate number of 2.5x10(4)/m was obtained for DA. Relative standard deviations in peak heights observed for five runs of a standard solution containing the two analytes (0.1 mM for each) were 1.2 and 3.1% for DA and CA, respectively. The chip could be continuously used for more than 8 h without significant deterioration in analytical performance.     

Skin‐worn Soft Microfluidic Potentiometric Detection System

A flexible skin‐mounted microfluidic potentiometric device for simultaneous electrochemical monitoring of sodium and potassium in sweat is presented. The wearable device allows efficient natural sweat pumping to the potentiometric detection chamber, containing solid‐contact ion‐selective Na⁺ and K⁺ electrodes, during exercise activity. The fabricated microchip electrolyte‐sensing device displays good analytical performance and addresses sweat mixing and carry‐over issues of early epidermal potentiometric sensors. Such soft skin‐worn microchip platform integrates potentiometric measurement, microfluidic technologies with flexible electronics for real‐time wireless data transmission to mobile devices. The new fully integrated microfluidic electrolyte‐detection device paves the way for practical fitness and health monitoring applications.     

High performance liquid chromatography coupled to an optical fiber detector coated with laccase for screening catecholamines in plasma and urine

An analytical method based on separation by high performance liquid chromatography (HPLC) and detection by optical fiber (OF) coated with an enzyme (laccase), has been developed for separation and quantification of catecholamines, namely epinephrine, dopamine and norepinephrine. The application of OF as a detector in this analytical system relies on the variation of the reflected optical power detected when the catecholamines eluted from the HPLC column act as the substrate of the laccase immobilized on a tip of a single-mode OF. The developed method shows a high linearity in a range between 5 and 125 pg/mL and detection limits of 3.5, 2.9 and 3.3 pg/mL for epinephrine, dopamine and norepinephrine, respectively. The analytical performance of the proposed method was compared with a classical analytical method, namely high performance liquid chromatography-electrochemical detector (HPLC-ED) regarding catecholamines detection, showing great analytical advantages such as low cost of equipment. Additionally, the proposed method was applied to catecholamines determination in actual samples of plasma and human urine.     

Design and development of microfabricated capillary electrophoresis devices with electrochemical detection

Our efforts have been focused on developing a self-contained and transportable microfabricated electrophoresis (CE) system with integrated electrochemical detection (ED). The current prototype includes all necessary electrodes “on-chip” and utilizes miniaturized CE and ED supporting electronics custom designed for this purpose. State-of-the-art design/modeling tools and novel microfabrication procedures were used to create recessed platinum electrodes with complex geometries and the CE/ED device from two patterned ultra-flat glass substrates. The electrodes in the bottom substrate were formed by a self-aligned etch and deposition technique followed by a photolithographic lift-off process. The microchannels (20 μm deep×65 μm wide (average)) were chemically etched into the top substrate followed by thermal bonding to complete the microchip device. CE/ED experiments were performed using 0.02 M phosphate buffer (pH 6), an analyte/buffer solution (2.2 mM dopamine, 2.3 mM catechol) and varying separation voltages (0-500 V) with a custom electronics unit interfaced to a laptop computer for data acquisition. Detection limits (S/N=3) were found to be at the micromolar level and a linear detection response was observed up to millimolar concentrations for dopamine and catechol. The microchip CE/ED system injected 50 pl volumes of sample, which corresponded to mass detection limits on the order of 200 amol. For the first time, an integrated “on-chip” multi-electrode array CE/ED device was successfully designed, fabricated and tested. The majority of the electrodes (six out of eight) in the array were capable of detecting dopamine with the amplitude of the signal (i.e., peak heights) decreasing as the electrode distance from the channel exit increased.     

Fabrication and evaluation of a carbon-based dual-electrode detector for poly(dimethylsiloxane) electrophoresis chips

The first carbon-based dual-electrode detector for microchip capillary electrophoresis (CE) is described. The poly(dimethylsiloxane) (PDMS)-based microchip CE devices were constructed by reversibly sealing a PDMS layer containing separation and injection channels to another PDMS layer containing carbon fiber working electrodes. End-channel amperometric detection was employed and the performance of the chip was evaluated using catechol. The response was found to be linear between 1 and 600 microM with an experimentally determined limit of detection (LOD) of 500 nM and a sensitivity of 30 pA/microM. Collection efficiencies for catechol ranged from 36.0 to 43.7% at field strengths of 260-615 V/cm. The selectivity that can be gained with these devices is demonstrated by the first CE-based dual-electrode detection of a Cu(II) peptide complex. These devices illustrate the potential for a rugged and easily constructed microchip CE system with an integrated carbon-based detector of similar scale.     

Feasibility of SU-8-based capillary electrophoresis-electrospray ionization mass spectrometry microfluidic chips for the analysis of human cell lysates

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface and an ESI emitter were developed to improve the speed and throughput of proteomics analyses. Validation of the microchip method was performed based on peptide mass fingerprinting and single peptide sequencing of selected protein standards. Rapid, yet reliable identification of four biologically important proteins (cytochrome C, β-lactoglobulin, ovalbumin and BSA) confirmed the applicability of the SU-8 microchips to ambitious proteomic applications and allowed their use in the analysis of human muscle cell lysates. The characteristic tryptic peptides were easily separated with plate numbers approaching 10(6), and with peak widths at half height as low as 0.6 s. The on-chip sheath flow interface was also exploited to the introduction of an internal mass calibrant along with the sheath liquid which enabled accurate mass measurements by high-resolution Q-TOF MS. Additionally, peptide structural characterization and protein identification based on MS/MS fragmentation data of a single tryptic peptide was obtained using an ion trap instrument. Protein sequence coverages exceeding 50% were routinely obtained without any pretreatment of the proteolytic samples and a typical total analysis time from sampling to detection was well below ten minutes. In conclusion, monolithically integrated, dead-volume-free, SU-8 microchips proved to be a promising platform for fast and reliable analysis of complex proteomic samples. Good analytical performance of the microchips was shown by performing both peptide mass fingerprinting of complex cell lysates and protein identification based on single peptide sequencing.     

Carbon paste-based electrochemical detectors for microchip capillary electrophoresis/electrochemistry

The first reported use of a carbon paste electrochemical detector for microchip capillary electrophoresis (CE) is described. Poly(dimethylsiloxane) (PDMS)-based microchip CE devices were constructed by reversibly sealing a PDMS layer containing separation and injection channels to a separate PDMS layer that contained carbon paste working electrodes. End-channel amperometric detection with a single electrode was used to detect amino acids derivatized with naphthalene dicarboxaldehyde. Two electrodes were placed in series for dual electrode detection. This approach was demonstrated for the detection of copper(II) peptide complexes. A major advantage of carbon paste is that catalysts can be easily incorporated into the electrode. Carbon paste that was chemically modified with cobalt phthalocyanine was used for the detection of thiols following a CE separation. These devices illustrate the potential for an easily constructed microchip CE system with a carbon-based detector that exhibits adjustable selectivity.