Disposable polyester-toner electrophoresis microchips for DNA analysis

Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215,000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.     

Amperometric detector designs for capillary electrophoresis microchips

Electrochemical (EC) detection is a sensitive and miniaturisable detection mode for capillary electrophoresis (CE) microchips. Detection cell design is very important in order to ensure electrical isolation from the high separation voltage. Amperometric detectors with different designs have been developed for coupling EC detection to CE-microchips. Different working electrode alignment: in-channel or end-channel has been tested in conjunction with several materials: gold, platinum or carbon. The end-channel detector was based on a platinum or gold wire manually aligned at the exit of the separation channel. Thick- (screen-printed carbon electrode) and thin-film (sputtered gold film) electrodes have also been employed with this configuration, but with a different design that allowed the rapid replacement of the electrode. The in-channel detector was based on a gold film within the separation channel. A gold-based dual electrode detector, which combined for the first time in- and end-channel detection, has been also tested. These amperometric detectors have been evaluated in combination to poly(methylmethacrylate) (PMMA) and Topas (thermoplastic olefin polymer of amorphous structure) CE-microchips. Topas is a new and promising cyclic olefin copolymer with high chemical resistance. Relevant parameters of the polymer microchip separation such as precision, efficiency or resolution and amperometric detection were studied with the different detector designs using p-aminophenol and L-ascorbic acid as model analytes in Tris-based buffer pH 9.0.     

Wall coating for capillary electrophoresis on microchips

This review article with 116 references describes recent developments in the preparation of wall coatings for capillary electrophoresis (CE) on a microchip. It deals with both dynamic and permanent coatings and concentrates on the most frequently used microchip materials including glass, poly(methyl methacrylate), poly(dimethyl siloxane), polycarbonate, and poly(ethylene terephthalate glycol). Characterization of the channel surface by measuring electroosmotic mobility and water contact angle of the surface is included as well. The utility of the microchips with coated channels is demonstrated by examples of CE separations on these chips.     

Affinity capillary electrophoresis on microchips

The present study shows that the application of the method of affinity capillary electrophoresis (ACE) to investigate interactions between ligands and their substrates can be realized on microchips. With ACE it is possible to characterize non-covalent molecular interactions (complexation and partition equilibria). Binding constants (K(B)) provide a measured value of the affinity of a ligand molecule to a substrate, which is basic information for the understanding of hormones, drugs and their targets, e.g. receptors in the human body. A microchip electrophoresis instrument equipped with a UV-detector and a home-built chip-station with electrochemical detection were used. ACE could be achieved with model solutions of neurotransmitters using sulfated beta-cyclodextrin (sCD) as substrate in a background buffer. This paper describes the advantages of microchip-ACE (MC-ACE) to traditional affinity capillary electrophoresis on a capillary. The results show that MC-ACE has great potential as a tool for fast scanning of interactions and to calculate binding constants of ligands with their substrates.     

Home-made capillary array electrophoresis for high-throughput amino acid analysis

A capillary array electrophoresis (CAE) with confocal rotary scanner for high-throughput carboxytetramethylrhodamine succinimidyl ester (TAMRA)-labeled amino acid (AA) analysis is presented. Performance of the CAE setup was evaluated with AA samples. Up to 128 capillaries could be detected in parallel. For the first time, the device was applied to separate the enantiomers of isoleucine, cysteine, and glutamic acid with cyclodextrin-modified electrolytes by capillary zone electrophoresis. Baseline separation of seven AAs is also demonstrated using micellar electrokinetic chromatography method.     

New silanization coating for DNA fragment analysis by capillary electrophoresis

We propose a new coating technique for fused silica capillaries using silanization with trimethylchlorosilane and diethylamine as a mediating agent in DNA separation using capillary electrophoresis. The proposed coating technique is simple and stable at a high pH. Capillaries coated by the new preparation method give excellent reproducibility for DNA fragment analysis with a good relative standard deviation of less than 0.7% for 150 runs and good stability at pH 8.2. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1405–1420, 2002     

Recent progress in DNA analysis by capillary electrophoresis

A number of recent developments in DNA analysis by capillary electrophoresis are here reviewed. They include capillary arrays for fast, parallel DNA sequencing as well as microfabricated capillary arrays. Microfluidic chips for DNA sizing and quantitation are also covered, as well as microdevices containing arrays of regular obstacles acting as size-separators during DNA migration. Screening of DNA point mutations by two much improved techniques is also reported: in one case, such mutations are detected (but only on relative short, ca. 60-70 base-long fragments) by free electrophoresis in rather acidic (pH ca. 3) buffers; in the case of single-strand chain polymorphism, an improved technique is described based on near-neutral pH buffers with mixtures of Tris/MES cations/zwitterions. When studying the behavior of inorganic and organic cations in the Debye-Hückel layer of DNA, it was found that the latter (especially a large number of Good's buffers and other zwitterions, such as His) would bind to the DNA filament not only via charge interaction, but also via additional bonds, notably hydrogen bonds, thus altering the electrophoretic (and possibly the biological) behavior of DNA molecules. However, whether or not borate ions would bind to DNA remains still an unsettled question. Finally, capillary electrophoresis was found to be instrumental in measuring fine physicochemical parameters pertaining to DNA polyelectrolytes, such as their free mobility and their translational diffusion coefficients.     

A new absorbed coating for DNA fragment analysis by capillary electrophoresis

A fully automated coating procedure was devised based on adsorption of a new polymer, poly(dimethylacrylamide-co-allyl glycidyl ether) onto the capillary surface. The whole procedure takes less than 30 min and does not require the use of organic solvents, viscous solutions, or elevated temperature. The coating is stable even under harsh conditions such as alkaline pH, elevated temperature, and denaturant conditions that destroy most other presently available adsorbed coatings. This new adsorbed coating is highly stable and easy to produce in quantity, making it quite unique, and further making it possible to operate with any DNA sieving matrix. Finally, the above-mentioned properties facilitate coating regeneration by a simple wash with a strongly alkaline solution, thus extending the lifetime of the capillary, a highly desirable property for any coating used in biopolymer separations.     

Microfabricated capillary array electrophoresis DNA analysis systems

The equation of motion for a peak in a gas chromatography experiment is shown to be exact whenever the column outlet pressure is approximately zero. Based on steady-state assumptions, this equation is solved for an isothermal analysis with a single-ramp inlet-pressure or flow-rate program. Theoretical calculations of retention times are confirmed experimentally for two n-alkanes using a capillary column connected to a mass-selective detector. A slight but consistent discrepancy between theoretical and measured results is interpreted as a failure of the steady-state assumption when the magnitude of the rate of change of inlet pressure is large.     

Electrochemical detector based on sol-gel-derived carbon composite material for capillary electrophoresis microchips

An on-chip disk electrode based on sol-gel-derived carbon composite material could be easily and reproducibly fabricated. Unlike other carbon-based electrodes reported previously, this detector is rigid, convenient to fabricate, and amenable to chemical modifications. Based on the stable and reproducible characters of this detector, a copper particle-modified detector was developed for the detection of carbohydrates which extends the application of the carbon-based electrode. In our experiments, the performance of the new integrated detector for rapid on-chip measurement of epinephrine and glucose was illustrated. Experimental procedures including the fabrication of this detector, the configuration of separation channel outlet and electrode verge, and the performance characteristics of this new electrochemical detector were investigated.     

DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system

We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDA) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDA system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis.     

Polymeric matrices for DNA sequencing by capillary electrophoresis

We review the wide range of polymeric materials that have been employed for DNA sequencing separations by capillary electrophoresis. Intensive research in the area has converged in showing that highly entangled solutions of hydrophilic, high molar mass polymers are required to achieve high DNA separation efficiency and long read length, system attributes that are particularly important for genomic sequencing. The extent of DNA-polymer interactions, as well as the robustness of the entangled polymer network, greatly influence the performance of a given polymer matrix for DNA separation. Further fundamental research in the field of polymer physics and chemistry is needed to elucidate the specific mechanisms by which DNA is separated in dynamic, uncross-linked polymer networks.     

Star-shaped polymers for DNA sequencing by capillary electrophoresis

The separation and sequencing of DNA are the main objectives of the Human Genome Project, and this project has also been very useful for gene analysis and disease diagnosis. Capillary electrophoresis (CE) is one of the most common techniques for the separation and analysis of DNA. DNA separations are usually achieved using capillary gel electrophoresis (CGE) mode, in which polymer gel is packed into the capillary. Compared with a traditional CGE matrix, a hydrophilic polymer matrix, which can be adsorb by the capillary wall has numerous advantages, including stability, reproducibility and ease of automation. Various water-soluble additives, such as linear poly(acrylamide) (PAA) and poly(N,N-dimethylacrylamide) (PDMA), have been employed as media. In this study, different star-shaped PDMA polymers were designed and synthesized to achieve lower polymer solution viscosity. DNA separations with these polymers avoid the disadvantages of high viscosity and long separation time while maintaining high resolution (10 bp between 271 bp and 281 bp). The influences of the polymer concentration and structure on DNA separation were also determined in this study; higher polymer concentration yielded better separation performance, and star-like polymers were superior to linear polymers. This work indicates that modification of the polymer structure is a potential strategy for optimizing DNA separation.     

Numerical simulation of electrokinetic injection techniques in capillary electrophoresis microchips

The effective design and control of a capillary electrophoresis (CE) microchip requires a thorough understanding of the electrokinetic transport phenomena associated with its microfluidic injection system. The present study utilizes a numerical simulation approach to investigate these electrokinetic transport processes and to study the control parameters of the injection process. Injection systems with a variety of different configurations are designed and tested, including the cross-form, T-form, double-T-form, variable-volume focused flow cross-form, and variable-volume triple-T-form configuration. Each injection system cycles through a predetermined series of steps in which the magnitudes and distributions of the applied electric field are precisely manipulated in order to effectuate a virtual valve. This study investigates the sample leakage effect associated with each of the injection configurations and applies the double-L, pullback, and focusing injection techniques to minimize the sample leakage effect. The injection methods presented in this paper have the exciting potential for use in high-quality, high-throughput chemical analysis applications and throughout the micro-total-analysis systems field.     

Pesticide analysis by capillary electrophoresis

In this work, a critical and updated revision of the current situation of the analysis of pesticides by Capillary Electrophoresis (CE) is presented. The review has been written in two main sections. The first one presents a thorough revision of the various offline and on-line sample preconcentration procedures that have been used in conjunction with CE to analyze these compounds. The second part reviews the various detection strategies (i.e., UV, LIF, MS, and electrochemical) and CE modes that have been applied to the analysis of pesticides. Future trends that can be expected from this hot research area are also discussed.     

Cycling gradient capillary electrophoresis: a low-cost tool for high-throughput analysis of genetic variations

In the present work, we introduce a new type of DNA variation detection. This method represents a transfer of melting gel technique onto multicapillary electrophoresis DNA sequencing instrument with further improvements to achieve maximum sample throughput while maintaining a high performance. The main improvement comes from application of cycling (revolving) temporal temperature gradient in place of a single-sweep gradient, commonly used in similar gel-based techniques. This improvement enables utilization of multiple-injection technique, in which multiple samples are injected into the same capillary (or sets of capillaries) separated by predefined time intervals of partial electrophoresis. The periodic oscillation of the temperature results in identical separation conditions of all samples injected in such series. Using this novel approach, we demonstrate a dramatic increase in separation throughput by turning a standard commercial 96-capillary array instrument into a semicontinuous flow mutation detection system capable to screen over 15 000 samples in 24 h of operation on a single 96-capillary commercial instrument. This represents a 10-fold increase in sample throughput over the current comparable technology.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

Simplex maximization of the correlation coefficient for DNA sizing analysis by capillary electrophoresis

The separation of DNA molecules in polymeric solution by capillary electrophoresis involves the optimization of several variables, such as polymer solution concentration, electric field separation, temperature, etc. The optimization of each variable individually usually is a time-consuming process and the results may reach a false optimum point. Chemometric methods are suitable to be applied in such cases in which a number of variables can be optimized simultaneously. The simplex is a chemometric method that can perform such a task easily and efficiently. In this study, a simplex method was carried out to maximize the correlation coefficient (r(2)) of a logarithmic plot of mobility (mu) vs. base pair (bp), which was obtained from the separation of DNA fragments of size between 75 and 4072 bp. The simplex showed three vertexes with r(2) > 0.98 and the vertex 21 showing the highest resolution. For the fragments between 201 and 2036 bp, the r(2) increased to 0.992 with and relative standard deviation (RSD) lower than 0.2% (inter- and intra-day variation). The precision of the method in determining the size of a PCR DNA fragment was carried out using a 1 kbp DNA ladder. With the addition of an internal standard to the sample, the precision could be further improved.