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.     

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.     

Indirect fluorescence detection of amino acids on electrophoretic microchips

Microfabricated devices enable rapid separations of a variety of clinically significant analytes, including DNA, proteins, and amino acids. However, absorbance detection has been difficult to achieve on these devices, prohibiting analysis of nonfluorophore-bearing or nonfluorescently tagged analytes. An alternative detection technique exploiting indirect fluorescence has been adapted to the electrophoretic microchip to provide fast analysis of amino acids, bypassing the need for absorbance detection or fluorescence derivitization procedures. Nineteen of the standard amino acids could be detected with an average detection limit of 32.9 microM (approximately 1.6 amol). Despite the fact that the detection sensitivity was lower than that achievable by labeling the amino acids with fluorescein isothiocyanate (approximately 1 nM), circumventing sample preparation and the difficulties inherent with tagging complex samples make this technique attractive for a variety of assays where sensitivity is not critical. To demonstrate the applicability to real sample matrixes, the analysis of urine with elevated amino acid levels is used as a model system where the elevated levels are indicative of a variety of pathologies including amino acid metabolism disorders and kidney malfunction. The minimal sample handling and rapid separations achievable by employing indirect detection on microchips provides the potential for high-throughput applications for certain amino acid analyses.     

毛细管电泳非接触电导检测器的研制

非接触电导检测法已经成为毛细管电泳芯片中一种通用的物质分离检测方法．本文提出了一种微型化的电容耦合非接触电导分析系统,采用新颖的三明治电极结构减小了电极之间的寄生电容．这种电极结构能够有效地减小电极的长度,克服了非接触电导检测中电极易于折断的缺点．当采用电极宽度1mm、电极间距1mm、MES／His缓冲溶液浓度为20mmol／L和激励电压20Vpp、90kHz的条件时,检测器具有最好的分离效果．在最佳的分离效果,实现了1mmol／L的K^＋和Mg^2＋混合无机阳离子的分离检测．     

Attenuated total internal reflectance infrared microspectroscopy as a detection technique for capillary electrophoresis

A novel detector for capillary electrophoresis (CE) using single-bounce attenuated total internal reflectance (ATR) Fourier transform infrared (FT-IR) microspectroscopy is presented. The terminus of the CE capillary is placed approximately 1 microm from the internal reflectance crystal at the focus of an ATR infrared microscope. Using pressure driven flow injection, concentration and volume detection limits have been determined for 25- and 10-microm-i.d. silica capillaries. Upon injection of 820 pL of succinylcholine chloride in a 10-microm capillary, a concentration detection limit of approximately 0.5 parts per thousand (ppt), or 410 pg, is found. The injection volume detection limit using a 108 ppt solution is 2.0 pL (216 pg). Sample separations using a programmed series of pressure, voltage, and again pressure on 25-, 50-, and 75-microm-i.d. capillaries are shown. CE separations of citrate and nitrate, as well as succinylcholine chloride with sodium salicylate using acetone as a neutral marker, are demonstrated. Several advantages of this CE-FT-IR technique include: (1) minimization of postcolumn broadening as a result of a small detector volume; (2) the ability to signal average spectra of the same aliquot, thereby improving the signal-to-noise in a stopped-flow environment; and (3) simplicity of design.     

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.     

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).     

Capillary electrophoresis and contactless conductivity detection of ions in narrow inner diameter capillaries

Capillary electrophoresis and conductometry represent a combination of a high-resolution separation method with a sensitive detection principle for the analysis of ionic species. In this paper, results are reported that are obtained with a contactless conductivity detector. This device works without a galvanic contact of the electrolyte and the electrodes. The conductivity sensor is based on two metal tubes that act as cylindrical capacitors. These electrodes are both placed around a fused-silica capillary with a detection gap of 1 mm left in between. When a high audio or low ultrasonic oscillation frequency between 40 and 100 kHz is applied to one of the electrodes, a signal is produced as soon as an analyte zone with a different conductivity compared to the background electrolyte passes the detection gap. An amplifier and rectifier is connected to the other electrode where the signal is further processed. Limits of detection for lithium and fluoride are 4 and 13 ppb, respectively, with a linear range over 4 orders of magnitude from 90 ppb up to more than 1000 ppm for both anions and cations. Furthermore, it is demonstrated that for species with lower equivalent conductivities, such as organic ions, indirect conductivity detection is a sensitive alternative to indirect optical detection methods. Limits of detection of 50 ppb and below are obtained for organic acids.     

On-chip contactless four-electrode conductivity detection for capillary electrophoresis devices

In this contribution, a capillary electrophoresis microdevice with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-microm-wide, and 20-microm-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection. Contactless conductivity detection was performed in either a two- or a four-electrode configuration. Experimental results confirmed the improved characteristics of the four-electrode configuration over the classical two-electrode detection setup. The four-electrode configuration allows for sensitive detection for varying carrier-electrolyte background conductivity without the need for adjustment of the measurement frequency. Reproducible electrophoretic separations of three inorganic cations (K+, Na+, Li+) and six organic acids are presented. Detection as low as 5 microM for potassium was demonstrated.     

Gradient elution isotachophoresis for enrichment and separation of biomolecules

A novel format for performing capillary isotachophoresis (ITP) is described -- gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 microm. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10,000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100,000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.     

Condensation nucleation light scattering detection for capillary electrophoresis

We describe two means for interfacing condensation nucleation light scattering detection to capillary electrophoresis (CE). With the first method, a fused-silica capillary was used for the separation and the CE was grounded through a Nafion membrane that also connected the system to a microconcentric pneumatic nebulizer. Limits of detection (LODs) for underivatized amino acids were at the low microgram per milliliter level, and separation efficiencies were ～9 times lower than the optimum predicted for these species based on the injection plug width and axial dispersion by diffusion. LODs were limited by background nonvolatiles resulting from dissolution of fused silica at the high pHs used for the separations. An alternate system employed PEEK capillaries which acted as the separation capillary and also as the inner nebulizer capillary. In this case, the exit end of the capillary was coated with conductive paint which extended to the tip of the nebulizer, was in contact with the CE buffer, and was grounded to complete the CE circuit. Response was nonlinear and the separation efficiency of this system was somewhat lower than that for the Nafion membrane system. Response as peak heights for all of the amino acids and peptides studied was nearly identical on a mass basis. With this system, much lower background signals were obtained, and as a result, LODs for underivatized amino acids and peptides were below the 1 μg/mL level, corresponding to less than 10 pg or less than 100 fmol injected. Both systems were fairly simple, effective means to generate aerosols with the low flows of CE and should be applicable to interfacing of other aerosol-based detectors with CE.     

Integrated capillary electrophoresis on flexible silicone microdevices: analysis of DNA restriction fragments and detection of single DNA molecules on microchips

Microchips for integrated capillary electrophoresis systems were produced by molding a poly(dimethylsiloxane) (PDMS) silicone elastomer against a microfabricated master. The good adhesion of the PDMS devices on clean planar surfaces allows for a simple and inexpensive generation of networks of sealed microchannels, thus removing the constraints of elaborate bonding procedures. The performance of the devices is demonstrated with both fast separations of φX-174/HaeIII DNA restriction fragments labeled with the intercalating dye YOYO-1 and fluorescently labeled peptides. Detection limits in the order of a few zeptomoles (10(-)(21) mol) have been achieved for each injected DNA fragment, corresponding to a mass detection limit of ～2 fg for the 603 base pair fragment. Single λ-DNA molecules intercalated with YOYO-1 at a base pair-to-dye ratio of 10:1 could be detected with an uncomplicated laser-induced fluorescence detection setup. High single-molecule detection efficiency (>50%) was achieved under electrophoretically controlled mass transport conditions in PDMS microchannels.     

Real-Time Imaging through Optical Fiber Array-Assisted Laser-Induced Fluorescence of Capillary Electrophoretic Enantiomer Separations

An advanced detection system based on laser-induced fluorescence imaging for capillary electrophoresis (CE) is presented. An optical fiber array was constructed for collection and transportation of the emitted fluorescent light to the charge-coupled device (CCD) camera. The fiber array makes the setup compact compared with a setup where the capillary is imaged through a camera objective. The imaging detector captures the sample zones in motion during the migration through the capillary. This allows unique studies on dynamic events otherwise unrevealed. During the study, unexplained nonlinear migration behavior was revealed. Enantiomer separations of dansylated amino acids using cyclodextrins, imaged between 1.5 and 12 cm of a 28-cm-long 50-μm i.d. capillary, were used for evaluation of the system. Comparing the optical fiber array with a camera lens system, the signal-to-noise-ratio (S/N) was 10 times higher. This is due to a combination of both higher signal and lower noise levels. To improve the S/N ratio further, a computer program for signal processing was designed. Using dichlorofluorescein, a concentration limit of detection (CLOD) of 350 pM was achieved and improved 10 times to 35 pM with computer postprocessing using 79 images. This is equal to 400 zeptomole for a 3-mm-long sample zone in a 50-μm i.d. capillary.     

An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications

This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.     

Microfabricated capillary electrophoresis amino acid chirality analyzer for extraterrestrial exploration.

Chiral separations of fluorescein isothiocyanate-labeled amino acids have been performed on a microfabricated capillary electrophoresis chip to explore the feasibility of using such devices to analyze for extinct or extant life signs in extraterrestrial environments. The test system consists of a folded electrophoresis channel (19.0 cm long x 150 microns wide x 20 microns deep) that was photolithographically fabricated in a 10-cm-diameter glass wafer sandwich, coupled to a laser-excited confocal fluorescence detection apparatus providing subattomole sensitivity. Using a sodium dodecyl sulfate/gamma-cyclodextrin pH 10.0 carbonate electrophoresis buffer and a separation voltage of 550 V/cm at 10 degrees C, baseline resolution was observed for Val, Ala, Glu, and Asp enantiomers and Gly in only 4 min. Enantiomeric ratios were determined for amino acids extracted from the Murchison meteorite, and these values closely matched values determined by HPLC. These results demonstrate the feasibility of using microfabricated lab-on-a-chip systems to analyze extraterrestrial samples for amino acids.     

CE/electrospray ionization-MS analysis of underivatized d/l-amino acids and several small neurotransmitters at attomole levels through the use of 18-crown-6-tetracarboxylic acid as a complexation reagent/background electrolyte

A new capillary electrophoresis/mass spectrometry technique is introduced for attomole detection of primary amines (including several neurotransmitters), amino acids, and their d/l enantiomers in one run through the use of a complexation reagent while using only approximately 1 nL of sample. The technique uses underivatized amino acids in conjunction with an underivatized capillary, which significantly reduces cost and analysis time. It was found that when (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TCA, MW 440) was used as the background electrolyte/complexation reagent during the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of underivatized amino acids, stable complexes were formed between the amino acids and the 18-C-6-TCA molecules. These complexes, which exhibited high ionization efficiencies, were detectable at attomole levels for most amino acids. The detection limits of the AA/18-C-6-TCA complexes were on the average more than 2 orders of magnitude lower than that of the free amino acids in solution. In addition to lower detection limits under CE/ESI-MS, a solution of 18-C-6-TCA in the concentration range of 5-30 mM provided high separation efficiency for mixtures of l-amino acids as well as mixtures of d/l-amino acids. By using a solution of 18-C-6-TCA as the background electrolyte in conjunction with an underivatized, 130-cm-long, 20-microm-i.d., 150-microm-o.d. fused-silica capillary and by monitoring the m/z range of the amino acid/18-C-6-TCA complexes (m/z 515-700), most of the standard amino acids and many of their enantiomers were separated and detected with high separation efficiency and high sensitivity (nanomolar concentration detection limits) in one run. The solutions of 18-C-6-TCA also worked well as the CE/ESI-MS BGE for low-level detection of several neurotransmitters and some of their d/l enantiomers as well as for the analysis of amino acids at endogenous levels in lysed red blood cells.     

Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.     

Rugged gap reactor device for postcolumn fluorescence detection in capillary electrophoresis

In this paper, the construction and performance of a rugged device for postcolumn derivatization in capillary electrophoresis (CE) are described. The device was based on a gap design, and a gap with a very small distance (<3 μm, estimated under microscope) could be easily constructed without micromanipulation. Addition of derivatizing reagents into the reaction capillary was attributable to gravity flow. The concentration of derivatizing reagents can be controlled through manipulating the electroosmotic flow in the reaction capillary and the height of the liquid levels from the derivatizing reagents to the buffer reservoirs. The device has been applied in fluorescence detection of amino acids using a mixture of o-phthaldialdehyde/2-mercaptoethanol as derivatizing reagent. Theoretical plate numbers for 11 amino acids separated in a pH 9.5 borate buffer were obtained in the order of 40?000-250?000. The detection limit for glycine (S/N = 2) was found to be 6.7 × 10(-)(7) mol/L using a commercial HPLC fluorescence detector modified for CE. Free amino acids in a wine sample were also determined. Because the device is quite stable, we believe that it can be used routinely in analytical laboratories.     

Detection of native amino acids and peptides utilizing sinusoidal voltammetry

Native amino acids and peptides were detected at a copper microelectrode using sinusoidal voltammetry (SV). Traditionally, these molecules can only be measured after derivatization with either a fluorescent or electroactive tag. In this work, an electrocatalytic oxidation reaction at copper is used to detect underivatized peptides and amino acids. The oxidation reaction is somewhat independent of peptide structure (i.e., it is not limited to the detection of aromatic amino acids) and is therefore able to produce nanomolar detection limits for all amino acids and peptides tested. A scanning technique, sinusoidal voltammetry, is used to provide the sensitivity of constant-potential techniques but also provide selectivity gained through utilization of the frequency domain. The frequency spectrum due to the oxidation of each molecule has a unique "fingerprint" response resulting from the kinetics of oxidation at the electrode surface. Through examination of the frequency spectra, even structurally similar molecules can be easily distinguished from one another. Flow injection analysis is used to demonstrate the sensitive and selective detection of a variety of amino acids and peptides. This technique can also be easily coupled to a separation step, i.e., high-performance liquid chromatography or capillary electrophoresis without electrode fouling from the adsorption of the analytes.