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To improve point-of-care quantification using microchip capillary electrophoresis (MCE), the chip-to-chip variabilities inherent in disposable, single-use devices must be addressed. This work proposes to integrate an internal standard (ISTD) into the microchip by adding it to the background electrolyte (BGE) instead of the samplethus eliminating the need for additional sample manipulation, microchip redesigns, and/or system expansions required for traditional ISTD usage. Cs and Li ions were added as integrated ISTDs to the BGE, and their effects on the reproducibility of Na quantification were explored. Results were then compared to the conclusions of our previous publication which used Cs and Li as traditional ISTDs. The in-house fabricated microchips, electrophoretic protocols, and solution matrixes were kept constant, allowing the proposed method to be reliably compared to the traditional method. Using the integrated ISTDs, both Cs and Li improved the Na peak area reproducibility approximately 2-fold, to final RSD values of 2.2-4.7% (n = 900). In contrast (to previous work), Cs as a traditional ISTD resulted in final RSDs of 2.5-8.8%, while the traditional Li ISTD performed poorly with RSDs of 6.3-14.2%. These findings suggest integrated ISTDs are a viable method to improve the precision of disposable MCE devicesgiving matched or superior results to the traditional method in this study while neither increasing system cost nor complexity. [ABSTRACT FROM AUTHOR]

Considering the important functions of cellular Na+ and K+ together with their cooperative efforts on various biological processes, it is significant to simultaneously detect Na+ and K+ at a single-cell level. Here, we present a novel method to discriminate and quantify simultaneously Na+ and K+ in single cells using a new near-infrared fluorescent probe associated with microchip electrophoresis. The fluorescent probe selectively responds to both Na+ and K+. The microchip electrophoresis allows accurate single-cell manipulation and effective distinction of Na+ and K+. Based on the method, the concentration of Na+ and K+ in single normal and cancer cells was compared, and the variation of Na+ and K+ in single cancer cells during the early stage of apoptotic volume decrease was monitored, which would help us to better understand the critical roles of Na+ and K+ in malignant cells and apoptosis. This method has paved a new way for the research of the synergistic function of Na+ and K+ in the regulation of various biological processes at a single-cell level. [ABSTRACT FROM AUTHOR]

A dual-channel sequential injection microchip capillary electrophoresis system with pressure-driven injection is demonstrated for simultaneous separations of anions and cations from a single sample. The poly(methyl methacrylate) (PMMA) microchips feature integral in-plane contactless conductivity detection electrodes. A novel, hydrodynamic "split-injection" method utilizes background electrolyte (BGE) sheathing to gate the sample flows, while control over the injection volume is achieved by balancing hydrodynamic resistances using external hydrodynamic resistors. Injection is realized by a unique flow-through interface, allowing for automated, continuous sampling for sequential injection analysis by microchip electrophoresis. The developed system was very robust, with individual microchips used for up to 2000 analyses with lifetimes limited by irreversible blockages of the microchannels. The unique dual-channel irreversible blockages of the microchannels. The unique dual-channel geometry was demonstrated by the simultaneous separation of three cations and three anions in individual microchannels in under 40 s with limits of detection (LODs) ranging from 1.5 to 24 μM. From a series of 100 sequential injections the %RSDs were determined for every fifth run, resulting in %RSDs for migration times that ranged from 0.3 to 0.7 (n = 20) and 2.3 to 4.5 for peak area (n = 20). This system offers low LODs and a high degree of reproducibility and robustness while the hydrodynamic injection eliminates electrokinetic bias during injection, making it attractive for a wide range of rapid, sensitive, and quantitative online analytical applications. [ABSTRACT FROM AUTHOR]

We report analysis of N-glycans derived from disease-free individuals and patients with Barrett's esophagus, high-grade dysplasia, and esophageal adenocarcinoma by microchip electrophoresis with laser-induced fluorescence detection. Serum samples in 10 μL aliquots are enzymatically treated to cleave the N-glycans that are subsequently reacted with 8-aminopyrene-1,3,6-trisulfonic acid to add charge and a fluorescent label. Separations at 1250 V/cm and over 22 cm yielded efficiencies up to 700?000 plates for the N-glycans and analysis times under 100 s. Principal component analysis (PCA) and analysis of variance (ANOVA) tests of the peak areas and migration times are used to evaluate N-glycan profiles from native and desialylated samples and determine differences among the four sample groups. With microchip electrophoresis, we are able to distinguish the three patient groups from each other and from disease-free individuals. [ABSTRACT FROM AUTHOR]

It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to eight samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to eight samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/rnL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid, and high-throughput analysis. [ABSTRACT FROM AUTHOR]

A new, commercial hybrid ceramic polymer, Ormocomp, was introduced to the fabrication of microfluidic separation chips using two independent techniques, UV lithography and UV embossing. Both fabrication methods provided Onnocomp chips with stable cathodic electro- osmotic flow which enabled examination of the Ormocomp biocompatibility by means of microchip capillaiy electrophoresis (MCE) and (intrinsic) fluorescence detection. The hydrophobic/hydrophilic properties of Onnocomp were examined by screening its interactions with bovine serum albumin and selected amino acids of varying hydrophobicity. The results show that the ceramic, organic-inorganic polymer structure natively resists bio-fouling on microchannel walls even so that the Ormocomp microchips can be used in intact protein analysis without prior surface modification. With theoretical separation plates approaching 104 m-1 for intact proteins and 106 m' for amino acids and peptides, our results suggest that Onnocomp microchips hold record-breaking performance as microfluidic separation platforms. In addition, Ormocomp was shown to be suitable for optical fluorescence detection even at near-UV range (ex 355 run) with detection limits atananomolar level (∼200 nM) for selected inherently fluorescent pharmaceuticals. [ABSTRACT FROM AUTHOR]

The performance of microfluidic glass devices with a monolithically integrated nanospray tip have been evaluated. The nanospray tip is generated directly on the edge of a microfluidic glass chip by a pulling step followed by HF-etching for directed formation of a sharp tip with defined emitter area. For a fair judgment of the MS- detection sensitivity, we compared the detection performance with commercial nanospray needles. For that purpose the effect of the emitter opening on the sensitivity was studied in detail for different microfluidic chips as well as for commercial nanospray needles. A comparison of the chip-nanospray device with commercial nanospray needles revealed that a comparable spray performance is obtained at similar emitter diameters. A stable electrospray could be generated at such tapered tips without any need for hydrodynamic or electroosmotic pumping. The nanospray chips were successfully applied for coupling microchip electrophoresis and mass spectrometry. For improved performance, the separation channel of the microdevice was flushed with hydroxypropylmethylcellulose (HPMC) in order to reduce analyte-wall interactions and electroosmotic flow. [ABSTRACT FROM AUTHOR]

Microchip electrophoresis is an emerging analytical technology with several useful attributes including rapid separation lime, small sample requirements, and automation. In numerous potential applications, such as chemical monitoring or high-throughput screening, it may be desirable to use a system for many analyses without operator intervention; however, long-term operation of microchip electrophoresis systems has received little attention. We have developed a microchip electrophoresis system that can automatically inject samples at 6 s intervals for 24 h resulting in collection of 14400 assays in one session. Continuous operation time of a prototype of the device was limited to 2 h due to degradation of reagents and electrophoresis buffers on the chip; however, modification so that all reagents were continuously perfused into reservoirs on the device ensured fresh reagents were always used for analysis and enabled extended operating sessions. The electrophoresis chip incorporated a cell perfusion chamber and reagent addition channels to allow chemical monitoring of fluid around cells cultured on the chip by serial electrophoretic immunoassays. The immunoassay had detection limits of 0.4 nM for insulin and generated ~4% relative standard deviation over an entire 24 h period with no evidence of signal drift. The combined system was used to monitor insulin secretion from single islets of Langerhans for 6-39 h. The monitoring experiments revealed that islets have secretion dynamics that include spontaneous oscillations after extended nonoscillating periods and possible ultradian rhythms. [ABSTRACT FROM AUTHOR]

A scalable and rather inexpensive solution to producing microanalytical systems with "on-chip" three-dimensional (3D) microelectrodes is presented in this study, along with applicability to practical electrochemical (EC) detection scenarios such as preconcentration and interferant removal. This technique to create high-aspect-ratio (as much as 4:1) gold microstructures in constrained areas involved the modification of stud bump geometry with microfabricated silicon molds via an optimized combination of temperature, pressure, and time. The microelectrodes that resulted consisted of an array of square pillars ~18 μm tall and 20 μm wide on each side, placed at the end of a microfabricated electrophoresis channel. This technique increased the active surface area of the microelectrodes by as much as a factor of 50, while mass transfer and, consequently, preconcentration collection efficiencies were increased to ~400%, compared to ~30% efficiency for planar nonmodified microelectrodes (samples that were used included the neurotransmitters dopamine and catechol). The 3D microelectrodes were used both in a stand-alone configuration, for direct EC detection of model catecholamine analytes, and, more interestingly, in dual electrode configurations for EC sample processing prior to detection downstream at a second planar electrode. In particular, the 3D electrodes were shown to be capable of performing coulometry or complete (100%) redox conversion of analyte species over a wide range of concentrations, from 4.3 μM to 4.4 mM, in either plug-flow or continuous-flow formats. [ABSTRACT FROM AUTHOR]

We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37 °C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 mm (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies. [ABSTRACT FROM AUTHOR]

This paper describes a novel on-line sample preconcentration and separation technique named transient trapping (tr-trapping), which improves the efficiencies of separation and concentration by using a partially injected short micellar plug in microchip electrophoresis. Although a longer separation length often provides a better resolution of complexed or closely migrating analytes, our proposed theoretical model indicated that a trap-and-release mechanism enables a short micellar zone, which was partially injected into the separation channel, to work as an effective concentration and separation field. Application of the tr-trapping technique to microchip micellar electrokinetic chromatography (MCMEKC) was performed on a newly fabricated 5-way-cross microchip by using sodium dodecyl sulfate and rhodamine dyes as test micelle and analytes, respectively. When the injection times of micelle (tinj,M) and sample solution (tinj,S) were 1.0 and 2.0 s, respectively, both the preconcenfration and separation of the dyes were completely finished within only 3.0 s. At tinj,S of 8.0 s, a 393-fold improvement of the detectability was achieved in comparison with conventional MCMEKC. The resolution obtained with tr-trapping-MCMEKC was also better than that with conventional MCMEKC in spite of the 160-fold shorter length of the injected micellar zone at tinj,M of 1.0 s. These results clearly demonstrated that the tr-trapping technique in MCMEKC provides a rapid, high-resolution and detectability analysis even in the short separation channel on the microchips. [ABSTRACT FROM AUTHOR]

We report a dynamic cross-linking effect of Mg2+ that enhances the sieving properties of low-viscosity poly(vinylpyrrolidone) (PVP) solutions. A low-viscosity PVP solution was applied to nondenaturing microchip electrophoresis of protein samples using microchips made of poly(methyl methacrylate). The separation resolution of nondenatured protein markers in 1.8% PVP solution was improved by adding 1-20 mM MgC2. We studied the effect of the ratio of cross-linking agent on mobility of protein samples and showed that protein retardation (In μ/μ0) is correlated with the ratio of cross-linking agent to PVP ([cMg2+/cPVP]) as ln μ/μ0 = A′[cMg2+/cPVP]b′. A′ was related to the protein radius (R), and b′ was found to be 0.72 for proteins with R = 2.4 nm and 0.82 for proteins with R = 1.85 nm. A structural study of PVP in semidilute solutions using dynamic light scattering showed that incremental increases of Mg2+ ion concentration from 5 to 20 mM in 1.8% PVP solution increased the hydrodynamic radius of PVP polymers by 20%. [ABSTRACT FROM AUTHOR]

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 °C and 100 V/cm with a 20 μM affinity capture probe (TM = 57.7 °C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (~150 000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors. [ABSTRACT FROM AUTHOR]

This paper demonstrates for the first time the use of adaptive functional nickel nanowires for switching on-demand operation of microfluidic devices. Controlled reversible magnetic positioning and orientation of these nanowires at the microchannel outlet offers modulation of the detection and separation processes, respectively. The former facilitates switching between active and passive detection states to allow the microchip to be periodically activated to perform a measurement and reset it to the passive (‘off’) state between measurements. Fine magnetic tuning of the separation process (postchannel broadening of the analyte zone) is achieved by reversibly modulating the nanowire orientation (i.e., detector alignment) at the channel outlet. The concept can be extended to other microchip functions and stimuli-responsive materials and holds great promise for regulating the operation of microfluidic devices in reaction to specific needs or unforeseen scenarios. [ABSTRACT FROM AUTHOR]

A fully packed capillary electrochromatographic (CEC) microchip showing improved solution and chip handling was developed. Microchannels for the CEC microchip were patterned on a cyclic olefin copolymer substrate by injection molding and packed fully with 0.8-μm monodisperse colloidal silica beads utilizing a sell-assembly packing technique. The silica packed chip substrate was covered and thermally press-bonded. After fabrication, the chip was filled with buffer solution by sell-priming capillary action. The self-assembly packing at each channel served as a built-in nanofilter allowing quick loading of samples and running buffer solution without filtration. Because of a large surface area-to-volume ratio of the silica packing, reproducible control of electroosmotic flow was possible without leveling of the solutions in the reservoirs resulting 1.3% rsd in migration rate. The capillary electrophoretic separation characteristics of the chip were studied using fluorescein isothiocyanate (FITC)-derivatized amino acids as probe molecules. A mixture of FITC and four FITC-derivatized amino acids was successfully separated with 2-mm separation channel length. [ABSTRACT FROM AUTHOR]

We demonstrate electrophoresis in I-shaped microchannels with a new design and operation principle. Unlike the conventional T- or cross-shaped microchannels, the simple I-shaped design makes it straightforward to integrate parallel microchannels with electrodes onto a microchip. The operation of the I-shaped microchannels has been enabled by the autonomous solution filling technique, which exploits the high gas solubility in poly- (dimethylsiloxane) (PDMS). We fabricated an I-shaped microchannel array (IMA) chip by integrating 12 independent microchannels and 2 electrodes onto a 3 cm × 2 cm area in a PDMS-glass hybrid microchip. For autonomous regulation of stable sample plugs in all the microchannels, we discovered that O2 plasma treatment of the PDMS-made reservoirs is effective. On the IMA chip, size-dependent separation of double-stranded (ds) DNA and sequence-specific separation of single-stranded DNA were achieved. Specifically, 10 fragments in a 100-1000-bp dsDNA ladder were separated using hydroxy-ethylcellulose as a sieving matrix within a separation length of 2 mm, and polymerase chain reaction products of the wild-type K-ras gene and its point mutant were separated using a probe DNA-poly(dimethylacrylamide) conjugate on the basis of affinity capillary electrophoresis. The IMA chip presented here opens up a new possibility of large-scale integration of microchannels for high-throughput electrophoretic analyses. [ABSTRACT FROM AUTHOR]

A poly(ethylene glycol)-functionalized acrylic copolymer was developed for fabrication of microfluidic devices that are resistant to protein and peptide adsorption. Planar microcapillary electrophoresis (µCE) devices were fabricated from this copolymer with the typical cross pattern to facilitate sample introduction. In contrast to most methods used to fabricate polymeric microchips, the photopolyinerization-based method used with the copolymer reported in this work was of the soft lithography type, and both patterning and bonding could be completed within 10 min. In a finished microdevice, the cover plate and patterned substrate were bonded together through strong covalent bonds. Additionally, because of the resistance of the copolymer to adsorption, fabricated microfluidic devices could be used without surface modification to separate proteins and peptides. Separations of fluorescein isothiocyanate-labeled protein and peptide samples were accomplished using these new polymeric µCE microchips. Separation efficiencies as high as 4.7 ×104 plates were obtained in less than 40 s with a 3.5-cm separation channel, yielding peptide and protein peaks that were symmetrical. [ABSTRACT FROM AUTHOR]

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 µM deep and 60 µM 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 µM wide with a 5 µM 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 elecirophoresis (p-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (μ-CEC). The separations were per- formed with an electric field (E) of 90 V/cm and were completed in less than 4 mm 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 µM for alanine. The separation efficiency was found to be 6.4 × 104, 2.0 × 10³, 4.8 × 10³, and 3.4 × 102 plates for the p-CEC of the oligonucleotides and μ-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 μ-CEC of the oligonucleotides and μ-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. [ABSTRACT FROM AUTHOR]

In this work, a semihydrodynamic (SHD) injection method was introduced and coupled with high salt stacking and electrokinetic chromatography for the analysis of estrogen and estrogen binding using a simple cross microchannel. The SHD method allows all samples to be hydrodynamically injected and focused into the separation channel at a relatively high flow rate and without splitting and diffusion, leading to reproducible bias-free injections of larger sample volumes (up to 50 nL) within 3 s. Moreover, the injection method is initiated without voltage switching, leading to a reduced mixing effect. Such advantages are well suited for performing stacking and sweeping on a microchip. We investigated the stacking effect under continuous and discontinuous co-ion conditions as well as under sweeping conditions. Micellar sweeping effect alone was relatively weak (7-8 times), partly due to a lower sodium cholate concentration (30 mM) used for the running buffer. By combining the sweeping effect with high salt stacking, however, up to a 200-300-fold enhancement factor could be achieved, and the high-salt and low-surfactant contents for the running buffer were favorable for binding study under nonequilibrium conditions. To the best of our knowledge, this is the first demonstration of the hydrodynamic injection used for high salt sample stacking on a microchip, also for further combining micellar eleclrochromatography and affinity separation for the analysis of hydrophobic ligand binding using microchip electrophoresis. [ABSTRACT FROM AUTHOR]

This paper demonstrates the ability to use capillary electrophoresis (CE) separation coupled with laser-induced fluorescence for analyzing the contents of single femtoliter-volume aqueous droplets. A single droplet was formed using a T-channel (3 μm wide by 3 μm tall) connected to microinjectors, and then the droplet was fluidically moved to an immiscible boundary that isolates the CE channel (50 μm wide by 50 μm tall) from the droplet generation region. Fusion of the aqueous droplet with the immiscible boundary effectively injects the droplet content into the separation channel. In addition to injecting the contents of droplets, we found aqueous samples can be introduced directly into the separation channel by reversibly penetrating. and resealing the immiscible partition. Because droplet generation in channels requires hydrophobic surfaces, we have also investigated the advantages to using all hydrophobic channels versus channel systems with patterned hydrophobic and hydrophilic regions. To fabricate devices with patterned surface chemistry, we have developed a simple strategy based on differential wetting to deposit selectively a hydrophilic polymer (poly(styrenesulfonate)) onto desired regions of the microfluidic chip. Finally, we applied our device to the separation of a simple mixture of fluorescein-labeled amino acids contained within a ∼-10-fL droplet. [ABSTRACT FROM AUTHOR]

We report here on the use of a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing using dye terminator chemistry. The CFTC chip consisted of a 20-loop spiral microfluidic channel hot-embossed into polycarbonate (PC) that had three well-defined temperature zones poised at 95, 55, and 60 °C for denaturation, renaturation, and DNA extension, respectively. The sequencing cocktail was hydrodynamically pumped through the microreactor channel at different linear velocities ranging from 1 to 12 mm/s. At a linear velocity of 4 mm/s resulting in a 36-s extension time, a read length of >600 bp could be obtained in a total reaction time of 14.6 min. Further increases in the flow rate resulted in a reduction in the total reaction time but also produced a decrease in the sequencing read length. The CFTC chip could be reused for subsequent sequencing runs (>30) with negligible amounts of carryover contamination or degradation in the sequencing read length. The CFTC microchip was subsequently coupled to a solid-phase reversible immobilization (SPRI) microchip made from PC for purification of the DNA sequencing ladders (i.e., removal of excess dye-labeled dideoxynucleotides, DNA template, and salts) prior to gel electrophoresis. Coupling of the CFTC chip to the SPRI microchip showed read lengths similar to that obtained from bench-top instruments but did not require manual manipulation of the cycle sequencing reactions following amplification. [ABSTRACT FROM AUTHOR]

We describe a novel class of DNA separation media for microchip electrophoresis, ‘physically cross-linked’ block copolymer networks, which provide rapid (<4.5 min) and remarkably enhanced resolution of DNA in a size range critical for genotyping. Linear poly(acrylamide-co-dihexylacrylamide) (LPA-co-DHA) comprising as little as 0.13 mol % dihexylacrylamide yields substantially improved electrophoretic DNA separations compared to matched molar mass linear polyacrylamide. Single-molecule videomicroscopic images of DNA electrophoresis reveal novel chain dynamics in LPA-co-DHA matrixes, resembling inchworm movement, to which we attribute the increased DNA resolution. Substantial improvements in DNA peak separation are obtained, in particular, in LPA-co-DHA solutions at polymer/copolymer concentrations near the interchain entanglement threshold. Higher polymer concentrations yield enhanced separations only for small DNA molecules (< 120 base pairs). Hydrophobically cross-linked networks offer advantages over conventional linear polymers based on enhanced separation performance (or speed) and over chemically cross-linked gels because hydrophobic cross-links can be reversibly broken, allowing facile microchannel loading. [ABSTRACT FROM AUTHOR]

Previous reports describing sample stacking on microchip capillary electrophoresis (µCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for µCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of µCE in the emerging miniaturized analytical instrumentation. [ABSTRACT FROM AUTHOR]

We have developed a novel approach for interfacing ionically conductive membranes with microfluidic systems using phase-changing sacrificial layers. Imprinted microchannels in a polymer substrate are filled with a heated liquid that solidifies at room temperature, a monomer solution is placed over the protected channels and polymerized to form a rigid semipermeable copolymer, and then the protective layer is melted and removed, leaving an open microchannel interfaced with a polymer membrane. We have applied this method in miniaturizing electric field gradient focusing (EFGF) and carrying out on-chip protein preconcentration. A semipermeable copolymer in the EFGF microchips fills a region of changing cross-sectional area, which allows a gradient in electric field to be established when an electrical potential is applied. Our technique provides microchip EFGF devices that offer 3-fold improved resolution in protein focusing compared with capillary-based systems. In addition, these EFGF microchips can separate peptide samples with resolution similar to what is obtained in capillary electrophoresis microdevices, and the micro-EFGF systems enrich analytes by a factor of > 150. Finally, we have fabricated membrane-integrated microfluidic devices that can concentrate protein samples (R-phycoerythrin) over 10 000-fold to facilitate microchip capillary electrophoresis. Interfacing microchannels with ion-permeable membranes has great potential to enhance microchip analysis of biomolecules. [ABSTRACT FROM AUTHOR]

We present a simple and robust isotachophoresis (ITP) method that can be integrated with microchip-based capillary electrophoresis (CE) devices to achieve million- fold sample stacking. We performed an experimental parametric study to show the effects of initial sample ion concentration, leading ion concentration, and trailing ion concentration on ITP stacking. We also discuss the usefulness and limitations of a simple one-dimensional nondispersive model and a scaling analysis for dispersion rate. We found that a single-column ITP configuration together with electroosmotic flow suppression and high leading ion concentration provide high-performance ITP and can be integrated readily with CE separation. We demonstrated detection of trace of 100 fM Alexa Fluor 488 (signal-to-noise ratio of 11) with a concentration increase of a factor of 2 × 106. Application of our ITP/ CE protocol to the stacking and separation of negatively charged fluorescent tracers (Alexa Fluor 488 and bodipy) resulted in a concentration increase of 6.4 × 10´ and a signal increase of 4.5 × 105. The ITP/CE protocol can be performed with a standard microchannel cross design or simple flow control. The method can be implemented with available off-the-shelf chip systems using off-the-shelf voltage control systems and buffer chemistries. [ABSTRACT FROM AUTHOR]

This work describes the fabrication and evaluation of a poly(dimethyl)siloxane (PDMS)-based device that enables the discrete injection of a sample plug from a continuous-flow stream into a microchannel for subsequent analysis by electrophoresis. Devices were fabricated by aligning valving and flow channel layers followed by plasma sealing the combined layers onto a glass plate that contained fittings for the introduction of liquid sample and nitrogen gas. The design incorporates a reduced-volume pneumatic valve that actuates (on the order of hundreds of milliseconds) to allow analyte from a continuously flowing sampling channel to be injected into a separation channel for electrophoresis. The injector design was optimized to include a pushback channel to flush away stagnant sample associated with the injector dead volume. The effect of the valve actuation time, the pushback voltage, and the sampling stream flow rate on the performance of the device was characterized. Using the optimized design and an injection frequency of 0.64 Hz showed that the injection process is reproducible (RSD of 1.77%, n = 15). Concentration change experiments using fluorescein as the analyte showed that the device could achieve a lag time as small as 14 s. Finally, to demonstrate the potential uses of this device, the microchip was coupled to a microdialysis probe to monitor a concentration change and sample a fluorescein dye mixture. [ABSTRACT FROM AUTHOR]

A study comparing the electrophoretic separation performance attainable from microchips molded by masters fabricated using conventional CNC machining techniques with commercial microchips, wire imprinted microchips, and microchips from LIGA molding devices is presented. An electrophoresis-based detection system using fluorescence microscopy was used to determine the analytical utility of these microchips. The separation performance of CNC microchips was comparable to commercially available microchips as well as those fabricated from LIGA masters. The important feature of the CNC machined masters is that they have rapid design-to-device times using routinely available machining tools. This low-cost prototyping approach provides a new entry point for researchers interested in thermoplastic microchips and can accelerate the development of polymer-based lab-on-a-chip devices. [ABSTRACT FROM AUTHOR]

A method for the fast analysis of lipoproteins by microchip electrophoresis with light-emitting diode confocal fluorescence detection has been developed. Lipoproteins labeled with BODIPY FL C5-ceramide are found to strongly adsorb on the bare surface of a poly(methyl methacrylate) (PMMA) microchip. Sodium dodecyl sulfate and cetyltrimethylammonium bromide were therefore utilized to alter lipoproteins and channel surface to make them bear the same type of charge. After modification, the peak shape of lipoproteins was greatly improved, demonstrating lipoprotein adsorption on a PMMA chip dramatically reduced due to electrostatic repulsion. In addition, polymers were added into the running buffer to suppress electroosmotic flow and to serve as a sieving matrix. As a result, lipoprotein separation was manipulated by both electrophoretic mobilities and particle sizes. Various separation parameters including surfactant concentration, buffer pH, and polymer concentration as well as on-line concentration were investigated systematically. Under optimal conditions, two baseline separations of standard lipoproteins including high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein were achieved with different selectivity. This method affords high separation speed (within 100 s) and high reproducibility. The intraassay and interassay RSDs of lipoprotein migration times were in the range of 0.90- 1.9%, indicating this method is highly reliable. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A recent report describes a reversible valve that can be used in series to achieve diaphragm pumping on chip (Grover, W. H.; Skelley, A. M.; Liu, C. N.; Lagally, E. T.; and Mathies, R. A. Sen Actuators, B 2003,89,315- 323). Here, the functionality of an integrated diaphragm pump on a hybrid PDMS-glass microchip to perform pressure injections for electrophoretic separations is demonstrated. A chip design that can perform both pressure and electrokinetic (ER) injection is described, and a mixture of fluorescein and ROX dyes in borate buffer is utilized as a model sample system. Multiple electrophoretic separations of sample injected with pres- sure and voltage are compared. Over multiple EK injections, an electrophoretic bias is observed and the injected analytes are not representative of the sample, with the peak area ratio changing 20% after 20 runs. Over multiple pressure injections, however, the sample composition is maintained, with a 3.6% CV over 20 runs. The data presented show the ability to alternate between injection types and pressure-inject a representative sample volume after a bias has already been observed with multiple EK injections. Multiple pressure injections have been performed on sample volumes as low as 500 nL while maintaining sample composition, supporting its use in integrated systems for small-volume sampling. [ABSTRACT FROM AUTHOR]

A new method for solvent bonding polymeric substrates to form microfluidic systems has been demonstrated. Prior to device sealing, channels in an embossed poly- (methyl methacrylate) (PMMA) piece are filled with a heated liquid (paraffin wax) that forms a solid sacrificial layer at room temperature. The sacrificial material prevents the bonding solvent (acetonitrile) and softened PMMA from fifing the channels. Once the sealing step is complete, the sacrificial layer is melted and removed, leaving enclosed microfluidic channels. We found that PMMA substrates welded together using this method could withstand internal pressures of >2250 psi, more than 1 order of magnitude higher than their thermally bonded counterparts. To demonstrate the usefulness of this method, microchip capillary electrophoresis (CE) devices in PMMA were created and tested. Amino acid and peptide mixtures were separated in <15 s, with >40 000 theoretical plates in a 2.5-cm separation distance. Electric fields as high as 1.5 kV/cm were applied in these microchips, and >300 CE runs were performed on a single device with no degradation of separation performance. The simplicity of the methods presented here and the improved robustness of the resulting devices should facilitate the broader implementation of polymer microchips in microfluidic analyses. [ABSTRACT FROM AUTHOR]

Microfluidic analysis systems are becoming an important technology in the field of analytical chemistry. An expanding area is concerned with the control of fluids and species in microchannels by means of an electric field. This paper discusses a new class of Si-compatible porous oxide interconnects for gateable transport of ions. The integration of such thin oxide films in microfluidics devices has been hampered in the past by the compatibility of oxides with silicon technology. A general fabrication method is given for the manufacture of silicon microsieve support structures by micromachining, on which a thin oxide layer is deposited by the spin-coating method. The deposition method was used for constructing γ-alumina, MCM-48 silica, and amorphous titania films on the support structures, from both water-based and solvent-based oxide sols. The final structures can be applied as microporous and mesoporous interconnecting walls between two microchannels. It is demonstrated that the oxide interconnects can be operated as ion-selective electrophoretic gates. The interconnects suppress Fick diffusion of both charged and uncharged species, so that they can be utilized as ionic gates with complete external control over the transport rates of anionic and cationic species, thus realizing the possibility for implementation of these Si-compatible oxide interconnects hi microchip analyses for use as dosing valves or sensors. [ABSTRACT FROM AUTHOR]

Fluorescently labeled proteins were electrophoretically concentrated on microfabricated devices prior to separation and laser-induced fluorescence detection on the same device. The proteins were concentrated using a porous silica membrane between adjacent microchannels that allowed the passage of buffer ions but excluded larger migrating molecules. Concentrated analytes were then injected into the separation column for analysis. Two basic microchip designs were tested that allowed sample concentration either directly in the sample injector loop or within the microchannel leading from the sample reservoir to the injector. Signal enhancements of ∼600-fold were achieved by on-chip preconcentration followed by SDS-CGE separation. Preconceniration for CE analysis in both coated and uncoated open channels was also demonstrated. Fluorescently labeled ovalbumin could be detected at initial concentrations as low as 100 fM by using a combination of field-amplified injection and preconcentration at a membrane prior to CE in coated channels. [ABSTRACT FROM AUTHOR]

Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methaciylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (μCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were I order of magnitude better with derivatized compared to untreated PMMA FACE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA μCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 × 104 plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses. [ABSTRACT FROM AUTHOR]

Integrating multiple analytical processes into microfluidic devices is an important research area required for a variety of microchip-based analyses. A microfluidic system is described that achieves preparative separations by intelligent fraction collection of attomole quantities of sample. The device consists of a main microfluidic channel used to perform electrophoresis, which is interconnected at 90° to two vertically displaced channels via a nanocapillary array membrane. The membrane interconnect contains nanometer-diameter pores that provide fluidic communication between the channels. Sample injection and analyte collection are controlled by application of an electrical bias between the microfluidic channels across the nanocapillary array. After the separation, the automated transfer of the FTTC-labeled Arg, Gln, and Gly bands occurs; a fluorescence detector located at the separation! collection channel interconnect is used to generate a triggering signal that initiates suitable voltages to allow near-quantitative transfer of analyte from the separation channel to the second fluidic layer. The ability to achieve such sample manipulations from mass-limited samples enables a variety of postseparation processing events. [ABSTRACT FROM AUTHOR]

Laser-patterning of nanoporous membranes at the junction of a cross channel in a microchip is used to integrate protein concentration with an electrokinetic injection scheme. Upon application of voltage, linear electrophoretic concentration of charged proteins is achieved at the membrane surface because buffer ions can easily pass through the membrane while proteins larger than the molecular weight cutoff of the membrane are retained. Sample preconcentration steps are ever-present components of analytical systems, since concentration of trace samples facilitates their detection.

We have successfully achieved the integration of isothermal amplification and the subsequent analysis of specific gene fragments on poly(methyl methacrylate) microchips. In our experiments, loop-mediated isothermal amplification, which can offer higher specificity and efficiency than PCR, has been performed at a constant temperature (65 °C). After amplification, products could be either examined by the integrated microchip-based electrophoresis or directly observed by naked eye with SYBR Green I added into the reaction solution. By such an integrated micro system, the amplification and the subsequent analysis of prostate-specific antigen gene with template concentration at 23 fg/μL could be finished within 15 mm, which demonstrates its advantages of high specificity, good reproducibility, and fast speed in gene detection. [ABSTRACT FROM AUTHOR]

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 μm was determined to be the optimum decoupler size, with effective voltage isolation lasting for ∼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 μm 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/μM, r² = 0.9996) and epinephrine (2.1 μM, 2.6 pA/μM, r² = 0.9996) were obtained using the palladium decoupler in combination with a Pd working electrode. [ABSTRACT FROM AUTHOR]

The detection of human immunoglobulin M (IgM) was performed using capacitively coupled contactless conductivity detection (CCD) in electrophoresis carried out in conventional capillaries as well as on glass and poly(methyl methacrylate) (PMMA) microdevices. Also achieved was the analyses of IgG (an anti-human IgM) and the complex formed in the reaction between the two immunoreagents. It is demonstrated that CCD is a powerful tool suitable not only for the detection of antibodies but also for monitoring an inununological interaction. Conductivity measurements allow the direct determination of immunoreagents, and it is advantageous, since no labels are required. The immunoglobulin IgM has been taken as model analyte. The reproducibility of the analytical signal (RSD = 1%), sensitivity and limits of detection obtained for IgM (0.15 ng/mL in conventional capillaries and 34 ng/mL in microchips) are comparable to those previously obtained with amperometric detection. The immunological reaction was performed either in conventional microtiter plates as used in EUSA or in situ on the glass chip. [ABSTRACT FROM AUTHOR]

This paper presents an experimental and numerical investigation into the use of low-leakage injection techniques to deliver sample plugs within electrophoresis microchips. The study addresses the principal material transport mechanisms such as electrokinetic migration, fluid flow, and diffusion and gives detail analyses to the double-L injection technique, which employs electrokinetic manipulations to avoid sample leakage within the microchip. Electrical potential contour, velocity vector, and streamline distribution in the micro CE chip are successfully developed. Experimental and numerical testing results show the double-L injection technique is capable of reducing sample leakage within cross-form microfluidic chips. The current study confirms the double-L injection technique has an exciting potential for use in high-quality, high-throughput chemical analysis applications and in many other applications throughout the field of micro total analysis systems. [ABSTRACT FROM AUTHOR]

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 micromoldlng 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. [ABSTRACT FROM AUTHOR]

The separation and detection of underivatized carbohydrates, amino adds, and sulfur-containing antibiotics in an elecizophoretic microchip with pulsed amperometric detection (PAD) is described. This report also describes the development of a new chip configuration for microchip electrophoresis with PAD. The configuration consists of a layer of poly(dimethylsiloxane) that contains the microfluidic channels, reservoirs, and a gold microwire, sealed to a second layer of poly(dlmethylsiloxane). Example separations of carbohydrates, amino adds, and suffur-containing antibiotics are shown. The effect of the separation and injection potentials, buffer pH and composition, injection time, and PAD parameters were studied in an effort to optimize separations and detection. Detection limits ranging from 6 fmol (5 μM) for penicillin and ampicillin to 455 fmol (350 μM) for histidine were obtained. [ABSTRACT FROM AUTHOR]

Presents a novel pressurization technique for microchip electrophoresis that enables 15-s separation of protein mixtures extracted from biological samples. Impact of high pressures applied to separation channel before electrophoresis on the effective channel length and speed separation; Reduction in the migration time due to the pressurization technique; Comparison of the pressurization method with other pressure-driven methods.

Reports on developments in molecular diagnostics on electrophoretic microchips. Single-nucleotide polymorphisms; Coatings and sieving matrices; DNA sequencing.

Examines the high-voltage capacitively coupled contactless conductivity detection for microchip capillary electrophoresis. Conflict between separation voltage and electrochemical detection; Implementation of standard zone electrophoresis on microfabricated devices; Prevention of clogging on the narrow channel.

Describes the electrode configuration for microchip capillary electrophoresis (CE) with electrochemical (EC) detection. Utilization of isolated potentiostat; Advantages of integrating EC detection with microchip CE; Construction of decoupler.

Reports the fabrication of microchip devices for high-efficiency electrophoretic separation of dichlorofluoroscein (DFC). Relationship between the number of theoretical plates and applied voltage; Effects of channel geometry on the diffusion coefficient of DFC; Information on the micellar electrokinetic chromatography separation of several amino acids.

Demonstrates that a microchip background can be rejected with analyte velocity modulation in electrophoresis. Background rejection; Detection limit improvement; Modulation frequency dependence; Noise spectrum analysis; Laser excitation.

Applies cross-correlation electrophoresis on a microchip device. Comparison with traditional electrokinetic separations; Exhibition of a steady-state pseudorandon pattern in fluorescence intensity; Prevention of temporal broadening of correlogram features with modifications; Advantages of rapid migration times on microchip devices.

Presents a study which used microchip electrophoresis to resolve a binary mixture. Design of the microchip; Details on the strength of the electric field; Methodology used to conduct the study; Results of the study.