Dual-electrode electrochemical detection for poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips

The development of a poly(dimethylsiloxane)-based (PDMS-based) microchip electrophoresis system employing dual-electrode electrochemical detection is described. This is the first report of dual-electrode electrochemical detection in a microchip format and of electrochemical detection on chips fabricated from PDMS. The device described in this paper consists of a top layer of PDMS containing the separation and injection channels and a bottom glass layer onto which gold detection electrodes have been deposited. The two layers form a tight reversible seal, eliminating the need for high-temperature bonding, which can be detrimental to electrode stability. The channels can also be temporarily removed for cleaning, significantly extending the lifetime of the chip. The performance of the chip was evaluated using catechol as a test compound. The response was linear from 10 to 500 microM with an LOD (S/N = 3) of 4 microM and a sensitivity of 45.9 pA/microM. Collection efficiencies for catechol ranged from 28.7 to 25.9% at field strengths between 200 and 400 V/cm. Dual-electrode detection in the series configuration was shown to be useful for the selective monitoring of species undergoing chemically reversible redox reactions and for peak identification in the electropherogram of an unresolved mixture.     

Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bpy)3(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 microM (S/N = 3) and a linear range from 5 to 600 microM.     

Amperometric detection of carbohydrates with a portable silicone/quartz capillary microchip by designed fracture sampling

A silicone/quartz capillary microchip (SQCM) coupled with an ultranarrow sampling fracture was for the first time constructed without any micromachining. The SQCM could be used for direct determination of carbohydrates at a detection potential of +0.8 V (vs Ag/AgCl) with a copper microdisk electrode. The ultranarrow sampling fracture could be conveniently formed on a quartz capillary, which was fixed by a frame of poly(dimethylsiloxane) (PDMS). The designed fracture sampling suppressed the leakage of sample, thus simplifying the power supply. Furthermore, it thinned the sample plug for enhancing the resolution. The quartz capillary reduced the adsorption of analytes on the separation channel wall compared with a general PDMS microchip, thus enhanced the separation efficiency up to 239 000 plates/m for carbohydrates. This proposed system could satisfactorily separate eight carbohydrates within 180 s with good reproducibility and sensitively detect them in the linear ranges from 1 microM to 0.5 mM for trehalose and sucrose, 2.5 microM to 0.5 mM for lactose, galactose, glucose, and mannose, and 2.5 microM to 1.5 mM for fructose and xylose with the detection limit down to 90 amol. The designed microchip was successfully applied to detect carbohydrates in a practical acacia honey sample.     

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.     

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.     

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.     

Autonomous polymer loading and sample injection for microchip electrophoresis

We have developed an extremely simple method for microchip electrophoresis. Loading of a sieving polymer solution and injection of a sample solution are autonomously executed by a microchip fabricated in poly(dimethylsiloxane) (PDMS). In advance, the energy for the fluid pumping is stored in bulk PDMS by evacuating air dissolved in PDMS, and the information for the sample plug regulation is coded into the microchannel design. Besides the simplicity, our method brings about an advantageous effect: sample compaction due to the discontinuous electrophoretic mobility at the sample/polymer interface. The sample compaction effect was moderate in ordinary size-dependent separation for double-stranded DNA and was extreme in affinity electrophoresis for single-stranded DNA (ssDNA). In the latter separation mode, ssDNA components were sequence-specifically separated by difference in affinity to a probe oligonucleotide immobilized to the sieving polymer matrix. We separated up to 60-mer ssDNA mixtures based on single-base substitutions. The separation processes included typically 100-fold sample compaction and were completed within 15-30 s. This technology provides easy, simple, and sensitive method for detection of gene point mutations and typing of single-nucleotide polymorphisms.     

Electrogenerated chemiluminescence detection of amino acids based on precolumn derivatization coupled with capillary electrophoresis separation

A novel method for highly sensitive detection of primary and secondary amino acids with selective derivatization using acetaldehyde as a new derivatization reagent was proposed by capillary electrophoresis (CE) coupled with electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(II). The precolumn derivatization of these amino acids with acetaldehyde was performed in aqueous solution at room temperature for 1 h. Upon optimized derivatization, the ECL intensities and detection sensitivities of the amino acids were significantly enhanced by 20-70 times. Using four amino acids, arginine, proline, valine, and leucine, as model compounds, their derivatives could be completely separated by CE and sensitively detected by ECL within 22 min. The linear ranges were 0.5-100 microM for arginine and proline and 5-1000 microM for valine and leucine with the detection limits of 1 x 10(-7) (0.5 fmol, arginine), 8 x 10(-8) (0.4 fmol, proline), 1 x 10(-6) (5 fmol, valine), and 1.6 x 10(-6) M (8 fmol, leucine) at a signal-to-noise ratio of 3. The derivatization reactions and ECL process of amino acids were also proposed based on in situ Fourier transform infrared and ultraviolet spectrometric analyses.     

Microchip capillary electrophoresis with electrochemical detection of thiol-containing degradation products of V-type nerve agents

A microchip protocol for the capillary electrophoresis separation and electrochemical detection of thiol-containing degradation products of V-type nerve agents is described. The microchip assay relies on the derivatization reaction of 2-(dimethylamino)ethanethiol (DMAET), 2-(diethylamino)ethanethiol (DEAET), and 2-mercaptoethanol (ME) with o-phthaldialdehyde in the presence of the amino acid valine along with amperometric monitoring of the isoindole derivatives. Both off-chip and on-chip derivatization reactions have led to highly sensitive and rapid detection of the thiol degradation products. Various parameters influencing the derivatization, separation, and detection processes were examined and optimized. These include the amino acid co-reagent, reagent-mixing ratio, reaction time, injection time, separation voltage, and detection potential. The chip microsystem offers a rapid (<4 min) simultaneous detection of micromolar concentrations of DMAET, DEAET, and ME. Linear calibration plots were observed for the V-type nerve agent thiol degradation products, along with good stability and reproducibility (RSD < 8.0%). Detection limits of 5 and 8 microM were obtained for the off-chip reaction of DMAET and DEAET, respectively, following a 2-s injection. The suitability for assays of environmental matrixes was demonstrated for the determination of DMAET and DEAET in untreated tap and river water samples. The favorable analytical performance makes the new microfluidic device attractive for addressing the needs of various security scenarios.     

Microchip devices for high-efficiency separations

We have fabricated a 25-cm-long spiral-shaped separation channel on a glass microchip with a footprint of only 5 cm x 5 cm. Electrophoretic separation efficiencies for dichlorofluoroscein (DCF) on this chip exceeded 1,000,000 theoretical plates and were achieved in under 46 s at a detection point 22.2 cm from the injection cross. The number of theoretical plates increased linearly with the applied voltage, and at a separation field strength of 1,170 V/cm, the rate of plate generation was approximately 21,000 plates/s. The large radii of curvature of the turns minimized the analyte dispersion introduced by the channel geometry as evidenced by the fact that the effective diffusion coefficient of DCF was within a few percent of that measured on a microchip with a straight separation channel over a wide range of electric field strengths. A micellar electrokinetic chromatography separation of 19 tetramethylrhodamine-labeled amino acids was accomplished in 165 s with an average plate number of 280,000. The minimum resolution between adjacent peaks for this separation was 1.2.     

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.     

Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.     

Investigation of the mixing efficiency of a chaotic micromixer using thermal lens spectrometry

This work investigates the efficiency of a chaotic micromixer using thermal lens spectrometry. The outlet of the mixing device was connected to a thermal lens detection head integrating the probe beam optical fibers and the sample capillary. The chaotic micromixer consisted of a Y-shaped poly(dimethylsiloxane) (PDMS) microchip in which ribbed herringbone microstructures were etched on the floor of the main channel. Due to the solvent composition dependence of the thermal lens response, the photothermal method was shown to be highly sensitive to nonhomogeneous mixing compared to fluorescence detection. The apparatus was applied to the determination of Fe2+ with 1,10-phenanthroline using flow injection analysis; a limit of detection of 11 microg L(-1) of iron was obtained.     

An integrated fritless column for on-chip capillary electrochromatography with conventional stationary phases

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.     

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.     

Electrochromatography in microchips: reversed-phase separation of peptides and amino acids using photopatterned rigid polymer monoliths

A microfabricated glass chip containing fluidic channels filled with polymer monolith has been developed for reversed-phase electrochromatography. Acrylate-based porous polymer monoliths were cast in the channels by photopolymerization to serve as a robust and uniform stationary phase. UV light-initiated polymerization allows for patterning of polymer stationary phase in the microchip, analogous to photolithography, using a mask and a UV lamp for optimal design of injection, separation, and detection manifolds. The monoliths are cast in situ in less than 10 min, are very reproducible with respect to separation characteristics, and allow easy manipulation of separation parameters such as charge, hydrophobicity, and pore size. Moreover, the solvent used to cast the polymer enables electroosmotic flow, allowing the separation channel to be conditioned without need for high-pressure pumps. The microchip was used for separation of bioactive peptides and amino acids labeled with a fluorogenic dye (naphthalene-2,3-dicarboxaldehyde) followed by laser-induced fluorescence detection using a Kr+ ion laser. The microchip-based separations were fast (six peptides in 45 s), efficient (up to 600,000 plates/m), and outperformed the capillary-based separations in both speed and efficiency. We have also developed a method for complete removal of polymer from the channels by thermal incineration to regenerate the glass chips.     

Multichannel microchip electrophoresis device fabricated in polycarbonate with an integrated contact conductivity sensor array

A 16-channel microfluidic chip with an integrated contact conductivity sensor array is presented. The microfluidic network consisted of 16 separation channels that were hot-embossed into polycarbonate (PC) using a high-precision micromilled metal master. All channels were 40 microm deep and 60 microm wide with an effective separation length of 40 mm. A gold (Au) sensor array was lithographically patterned onto a PC cover plate and assembled to the fluidic chip via thermal bonding in such a way that a pair of Au microelectrodes (60 microm wide with a 5 microm spacing) was incorporated into each of the 16 channels and served as independent contact conductivity detectors. The spacing between the corresponding fluidic reservoirs for each separation channel was set to 9 mm, which allowed for loading samples and buffers to all 40 reservoirs situated on the microchip in only five pipetting steps using an 8-channel pipettor. A printed circuit board (PCB) with platinum (Pt) wires was used to distribute the electrophoresis high-voltage to all reservoirs situated on the fluidic chip. Another PCB was used for collecting the conductivity signals from the patterned Au microelectrodes. The device performance was evaluated using microchip capillary zone electrophoresis (mu-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (mu-CEC). The separations were performed with an electric field (E) of 90 V/cm and were completed in less than 4 min in all cases. The conductivity detection was carried out using a bipolar pulse voltage waveform with a pulse amplitude of +/-0.6 V and a frequency of 6.0 kHz. The conductivity sensor array concentration limit of detection (SNR = 3) was determined to be 7.1 microM for alanine. The separation efficiency was found to be 6.4 x 10(4), 2.0 x 10(3), 4.8 x 10(3), and 3.4 x 10(2) plates for the mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins, respectively, with an average channel-to-channel migration time reproducibility of 2.8%. The average resolution obtained for mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins was 4.6, 1.0, 0.9, and 1.0, respectively. To the best of our knowledge, this report is the first to describe a multichannel microchip electrophoresis device with integrated contact conductivity sensor array.     

Contactless conductivity detector for microchip capillary electrophoresis

A microfabricated electrophoresis chip with an integrated contactless conductivity detection system is described. The new contactless conductivity microchip detector is based on placing two planar sensing aluminum film electrodes on the outer side of a poly(methyl methacrylate) (PMMA) microchip (without contacting the solution) and measuring the impedance of the solution in the separation channel. The contactless route obviates problems (e.g., fouling, unwanted reactions) associated with the electrode-solution contact, offers isolation of the detection system from high separation fields, does not compromise the separation efficiency, and greatly simplifies the detector fabrication. Relevant experimental variables, such as the frequency and amplitude of the applied ac voltage or the separation voltage, were examined and optimized. The detector performance was illustrated by the separation of potassium, sodium, barium, and lithium cations and the chloride, sulfate, fluoride, acetate, and phosphate anions. The response was linear (over the 20 microM-7 mM range) and reproducible (RSD = 3.4-4.9%; n = 10), with detection limits of 2.8 and 6.4 microM (for potassium and chloride, respectively). The advantages associated with the contactless conductivity detection, along with the low cost of the integrated PMMA chip/detection system, should enhance the power and scope of microfluidic analytical devices.     

Fabrication of integrated microelectrodes for electrochemical detection on electrophoresis microchip by electroless deposition and micromolding in capillary technique

A new method for the fabrication of an integrated microelectrode for electrochemical detection (ECD) on an electrophoresis microchip is described. The pattern of the microelectrode was directly made on the surface of a microscope slide through an electroless deposition procedure. The surface of the slide was first selectively coated with a thin layer of sodium silicate through a micromolding in capillary technique provided by a poly(dimethylsiloxane) (PDMS) microchannel; this left a rough patterned area for the anchoring of catalytic particles. A metal layer was deposited on the pattern guided by these catalytic particles and was used as the working electrode. Factors influencing the fabrication procedure were discussed. The whole chip was built by reversibly sealing the slide to another PDMS layer with electrophoresis microchannels at room temperature. This approach eliminates the need of clean room facilities and expensive apparatus such as for vacuum deposition or sputtering and makes it possible to produce patterned electrodes suitable for ECD on microchip under ordinary chemistry laboratory conditions. Also once the micropattern is ready, it allows the researchers to rebuild the electrode in a short period of time when an electrode failure occurs. Copper and gold microelectrodes were fabricated by this technique. Glucose, dopamine, and catechol as model analytes were tested.     

Narrow sample channel injectors for capillary electrophoresis on microchips

In microchip CE, sample injection is generally achieved through cross, double-tee, or tee injector structures. In these reported approaches, channel width and depth are uniform at the injection intersection. Here, we present cross and tee injectors having narrow sample channels. Using a cross injector with reduced sample channel width, resolution, column efficiency, and sensitivity are remarkably improved. Furthermore, no leakage control is required in both injection and separation phases, making the microchip CE system more user-friendly. Good resolution can also be obtained using tee injectors with narrow sample channels, which would otherwise be impossible using conventional tee injectors. Using the narrow sample channel tee injector instead of conventional cross and double-tee injectors, the number of reservoirs in multiplexed systems can be reduced to N + 2 (N, the number of paralleled CE systems), the real theoretical limit. The virtues of the novel injectors were demonstrated with poly(dimethylsiloxane)-glass chips incorporating eight parallel CE channels.