Multiple-channel microchips for high-throughput DNA analysis by capillary electrophoresis

Capillary electrophoresis on microfabricated multiple-channel chips has great potential for high-throughput analysis. This review focuses on multiple-channel chips used for high-throughput DNA analysis. It covers progress in the design and fabrication of multiple-channel chips and detection schemes used on these chips. Applications are concentrated on DNA fragment sizing, genotyping, and sequencing.     

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.     

Monomer surface modifications for rapid peptide analysis by capillary electrophoresis and capillary electrochromatography coupled to electrospray ionization-mass spectrometry

In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R(2) approximately 0.99) pH dependence with isoelectric points (pI) at 8.6-8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5 x 10(5) plates/m were performed.     

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.     

Fabrication of poly(methyl methacrylate) capillary electrophoresis microchips by in situ surface polymerization

A novel method based on in situ surface polymerization of methyl methacrylate (MMA) has been developed for the rapid fabrication of poly(methyl methacrylate) (PMMA) capillary electrophoresis (CE) microchips. MMA containing both thermal and ultraviolet (UV) initiators was allowed to prepolymerize in a water bath to form a fast curing molding solution that was subsequently sandwiched between a nickel template and a PMMA plate. The images of the raised microchannels on the nickel template were precisely replicated into the synthesized PMMA substrates during the UV-initiated polymerization of the molding solution within 30 min under ambient temperature. The attractive performances of the novel PMMA microchips have been demonstrated in connection with amperometric detection for the separation and detection of several model analytes. The new approach significantly simplifies the process for fabricating PMMA devices and could be applied to other materials that undergo light-initiated polymerization.     

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.     

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.     

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis

Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I-V) characteristic and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes-fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters.     

Design and simulation of sample pinching utilizing microelectrodes in capillary electrophoresis microchips

The paper proposed novel designs to pinch the transverse diffusion of the sample in the injection mode using microelectrodes to generate the potential difference at the channel intersection in the capillary electrophoresis (CE) microchip. A pair of microelectrodes was used to conduct the injection channel and the separation channel, which directly provided the potential to pinch the sample without using a power supply. These new designs of the CE microchip simplify the electric circuitry and improve performance. Simulations were performed using the CFD-ACE[trade mark sign] software. The mechanisms of diffusion and electrophoresis were employed in the numerical simulation. The injection and separation processes of the sample were simulated and the parameters of the present design were investigated numerically.     

Macroporous polymeric materials for bioanalytical microchips

Macroporous copolymers derived from 2-cyanoethyl methacrylate were prepared using low-molecular-weight and polymeric porogens. The influence of the polymerization conditions on the pore characteristics and surface morphology of the macroporous materials was examined. The possibility of efficiently using the synthesized polymeric matrices for protein assay was evaluated with a model biocomplementary couple as example.     

Direct electrochemical detection of glucose in human plasma on capillary electrophoresis microchips

We developed an electrochemical detector on a hybrid chip for the determination of glucose in human plasma. The microchip system described in this paper consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate. The copper microelectrode is fabricated by selective electroless deposition. The fabrication of the decoupler is performed by platinum electrochemical deposition on the metal film formed by electroless deposition. Factors influencing the performance, including detection potential, separation field strength, and buffer concentration, were studied. The electrodes exhibited good stability and durability in the analytical procedures. Under optimized detection conditions, glucose responded linearly from 10 microM to 1 mM. Finally, glucose in human plasma from three healthy individuals and two diabetics was successfully determined, giving a good prospect for a new clinical diagnostic instrument.     

A covalent capillary coating of diazoresin and polyglycerol dendrimer for protein analysis using capillary electrophoresis

Overcoming proteins adsorption on the inner surface of capillary has attracted increasing attention recently. By using the unique photochemistry reaction of diazoresin (DR), a new covalent capillary coating was prepared on the fused‐silica capillary through layer‐by‐layer self‐assembly of DR with polyglycerol (PG) dendrimer. The separation performance of covalently DR/PG‐dendrimer coated capillary noticeably exceeded the bare capillary and the noncovalently linked DR/PG‐dendrimer capillary. A baseline separation of lysozyme, myoglobin, bovine serum albumin, and ribonuclease A was achieved using CE within 20 min. Besides, the covalently linked DR/PG‐dendrimer coating has the remarkable stability and reproducibility. Especially, compared with the traditional method which use highly toxic and moisture‐sensitive silane coupling agent, this method seems to be a simple and environmental friendly way to prepare the covalently coated capillaries for CE.     

Fluorescent derivatization for low concentration protein analysis by capillary electrophoresis

Fluorescence derivatization can allow for the low concentration analysis of proteins by capillary electrophoresis. Major problems arising from inefficient chemistry and multiple derivatives must be overcome, however, for the method to be successful. A number of methods are discussed in this review.     

Transient isotachophoresis in carrier ampholyte-based capillary electrophoresis for protein analysis

Transient ITP (t-ITP) has been used in carrier ampholyte-based CE (CABCE) to enhance the sensitivity of protein analysis. The characteristics of carrier ampholytes (CAs) narrow pH cuts-based buffers, when used as BGEs in CE are compatible with t-ITP requirements. Indeed, being the sole buffering species of such solutions, CAs impose a pH close to their pI. Thus, in these solutions, the CAs possess low electrophoretic mobility. As a consequence, by adding an ionic component with high electrophoretic mobility either in the studied sample or in the BGE, a t-ITP step can be generated. This has first been demonstrated for protein test mixtures. Then, the combination of t-ITP with CABCE has been applied to study a real sample, the bovine milk.     

Non-covalent capillary coatings for protein separations in capillary electrophoresis

Adsorption of proteins, particularly basic proteins onto fused silica capillaries severely degrades capillary electrophoretic performance. This review provides a synopsis of the fundamentals underlying protein adsorption and its impact on CE performance. The efficacy of small molecule background electrolyte additives, surfactants, physically adsorbed polymers (dynamic and static), and successive multiple ionic-polymer layer coatings are evaluated using a number of performance metrics. Peak efficiency and migration time reproducibility are used as measures of reversible protein adsorption, while protein recovery, electroosmotic flow reproducibility and step changes in the baseline are used as indicators of irreversible protein adsorption.     

Liquid extraction surface analysis in-line coupled with capillary electrophoresis for direct analysis of a solid surface sample

A surface-sampling technique of liquid extraction surface analysis (LESA) was in-line coupled with capillary electrophoresis (CE) to expand the specimen types for CE to solid surfaces. The new direct surface analysis method of LESA–CE was applied to the determination of organophosphorus pesticides, including glufosinate-ammonium, aminomethylphosphonic acid, and glyphosate on the external surface of a fruit such as apple. Without any sample pretreatment, the analytes sprayed on the surface of a half apple were directly extracted into a liquid microjunction formed by dispensing the extractant from the inlet tip of a separation capillary. After extraction, the analytes were derivatized in-capillary with a fluorophore 4-fluoro-7-nitro-2,1,3-benzoxadiazole and analyzed with CE-laser induced fluorescence (LIF). The limits of detection for glufosinate-ammonium, aminomethylphosphonic acid, and glyphosate were 2.5, 1, and 10 ppb, respectively, which are at least 20 times lower than the tolerance limits established by the U.S. Environmental Protection Agency. Thus, we demonstrated that LESA–CE is a quite sensitive and convenient method to determine analytes on a solid surface avoiding the dilution from sample pretreatment procedures including homogenization of a bulk sample.