一种可逆键合电泳微芯片的制作及在蛋白质分离中的应用

阐述了一种可逆键合电泳微芯片的制作方法, 以及电泳微芯片在蛋白质分离、临床尿蛋白检测方面的应用. 用标准光刻腐蚀技术在石英基片上腐蚀泳道, 清洗腐蚀好的基片和盖片后, 在真空条件下实现键合. 此种方法键合制作的电泳微芯片可重复键合使用, 制得的电泳微芯片成功地用于标准蛋白质分离以及临床尿蛋白分析.     

Hot embossing of electrophoresis microchannels in PMMA substrates using electric heating wires

A simple method based on electric heating wires has been developed for the rapid fabrication of poly(methyl methacrylate) (PMMA) electrophoresis microchips in ordinary laboratories without the need for microfabrication facilities. A piece of stretched electric heating wire placed across the length of a PMMA plate along its midline was sandwiched between two microscope slides under pressure. Subsequently, alternating current was allowed to pass through the wire to generate heat to emboss a separation microchannel on the PMMA separation channel plate at room temperature. The injection channel was fabricated using the same procedure on a PMMA sheet that was perpendicular to the separation channel. The complete microchip was obtained by bonding the separation channel plate to the injection channel sheet, sealing the channels inside. The electric heating wires used in this work not only generated heat; they also served as templates for embossing the microchannels. The prepared microfluidic microchips have been successfully employed in the electrophoresis separation and detection of ions in connection with contactless conductivity detection.     

A new soft lithographic route for the facile fabrication of hydrophilic sandwich microchips

Manufacturing materials are an essential element for the fabrication of microfluidic chips. PDMS, the most widely used polymeric material, is associated with apparent disadvantages such as hydrophobic nature, while other materials also suffer from some limitations. In this paper, a new soft lithographic route was proposed for the facile manufacturing of hydrophilic sandwich microchips, using bisphenol A based epoxy acrylate (BABEA) as a new patterning material. The BABEA copolymers are hydrophilic, highly transparent in visible range while highly untransparent when the wavelength is less than 290 nm, and of high replication fidelity. By combining with appropriate monomers, including glycidyl methacrylate, methylmethacrylate, and acrylic acid, the copolymers contain active functional groups, which allows for easy postmodification for desirable functional units. A fabrication procedure was proposed for manufacturing hybrid quartz/BABEA copolymer/quartz microchips. In the procedure, no micromachining equipments, wet etching, or imprinting techniques were involved, making the fabrication approach applicable in ordinary chemistry laboratories. The performance of the prepared microchips was demonstrated in terms of CIEF with UV-whole channel imaging detection. The hydrophilic microchannel ensures stable focusing while the polymeric middle layer acts as a perfectly aligned optical slit for whole channel UV absorbance detection.     

Solvent bonding of poly(methyl methacrylate) microfluidic chip using phase-changing agar hydrogel as a sacrificial layer

In this report, a solvent bonding method based on phase-changing agar hydrogel has been developed for the fabrication of poly(methyl methacrylate) (PMMA) microfluidic chips. Prior to bonding, the channels and the reservoir ports on PMMA channel plates were filled with molten agar hydrogel that could gelate to form solid sacrificial layers at room temperature. Subsequently, PMMA cover sheets were covered on the channeled plates and 1,2-dichlororethane was applied to the interspaces between them. The agar hydrogel in the channels could prevent the bonding solvent and the softened surface of the PMMA cover sheets from filling in the channels. After solvent bonding, the agar hydrogel in the channels and the reservoir ports was melted and removed under pressure. The sealed channels in the complete microchips had been examined by an optical microscope and a scanning electron microscope. The results indicated that high-quality bonding was achieved at room temperature. The prepared microfluidic microchips have been successfully employed in the electrophoresis separation and detection of three cations in combination with contactless conductivity detection.     

Fabrication and evaluation of single- and dual-channel (Pi-design) microchip electrophoresis with electrochemical detection

A new dual-channel microchip capillary electrophoresis (MCE) has been developed on glass substrates for the first time with electrochemical detection. Dual-channel (called Pi-design) as well as single-channel microchips have been fabricated on soda-lime glass using photolithography, wet etching and thermal bonding. Moreover, a laser writing system has been applied for the fabrication of photomasks with the different microchip designs (single- and dual-channel configurations). The microfabricated channels have been characterized by optical, confocal and scanning electron microscopy. The resulting single- and dual-channel microchips have been evaluated using an end-channel amperometric detector based on one (single-channel) or two (dual-channel) 100-mum gold wires aligned at the outlet of the separation channel. Parameters affecting the separation of several phenolic compounds (dopamine, p-aminophenol and hydroquinone) have been studied in the glass microchips. Thus, the influence of separation voltage, detection potential and background electrolyte has been examined in the single-channel microchip. Different total length microchannel has been compared. Furthermore, the possibility of carrying out two simultaneous measurements has been demonstrated in the new dual-channel microchip electrophoresis. The injection format has been checked and resulted to be critical, in such a way that a special and new form is employed for obtaining simultaneous signals at both channels. Analytical characteristics, such as sensitivity and reproducibility have been evaluated and resulted very adequate.     

A method for UV-bonding in the fabrication of glass electrophoretic microchips.

This paper presents an approach for the development of methodologies amenable to simple and inexpensive microchip fabrication, potentially applicable to dissimilar materials bonding and chip integration. The method involves a UV-curable glue that can be used for glass microchip fabrication bonding at room temperature. This involves nothing more than fabrication of glue "guide channels" into the microchip architecture that upon exposure to the appropriate UV light source, bonds the etched plate and cover plate together. The microchip performance was verified by capillary zone electrophoresis (CZE) of small fluorescent molecules with no microchannel surface modification carried out, as well as with a DNA fragment separation following surface modification. The performance of these UV-bonded electrophoretic microchips indicates that this method may provide an alternative to high temperature bonding.     

Rapid separation of antimicrobial metabolites by microchip electrophoresis with UV linear imaging detection

This research examines microchip electrophoresis with linear imaging UV detection for the analysis of antimicrobial metabolites, monoacetylphloroglucinol (MAPG) and 2,4-diacetylphloroglucinol (2,4-DAPG) from Pseudomonas fluorescens F113. Initial results show the separation of MAPG, 2,4-DAPG and resorcinol in less than 20 s. This was achieved using a quartz microchip with a separation channel length of 25 mm. In order to quantitate the amount of MAPG and 2,4-DAPG in a microbial cultured supernatant sample, on-chip sample introduction in a methanol/buffer matrix was investigated. Sample introduction/injection parameters were optimized to improve sensitivity and thus decrease the limit of detection (LOD). The amount of antimicrobial metabolites present was quantitated with a separation time of 15 s. A previously developed capillary electrophoretic method was compared to the microchip method in relation to speed, efficiency, precision, linear range and limit of detection. This investigation shows the fastest separation so far of these antimicrobial metabolites with high efficiency.     

Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection

A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300?nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.     

Electrophoresis microchips with sharp inlet tips, for contactless conductivity detection, fabricated by in-situ surface polymerization

A novel method based on in-situ surface polymerization of methyl methacrylate (MMA) has been developed for rapid fabrication of poly(methyl methacrylate) (PMMA) electrophoresis microchips with sharp inlet tips. Prepolymerized MMA containing an ultraviolet (UV) initiator was directly sandwiched between a nickel template and a PMMA plate. The image of the relief on the nickel template was precisely replicated in the synthesized PMMA layer on the surface of the commercially available PMMA plate during UV-initiated polymerization at room temperature. The chips were subsequently assembled by thermal bonding of channel plates and cover sheets. The sample was directly introduced into the separation channel through a sharp inlet tip, which was placed in the sample vial, without use of an injection cross. The attractive performance of the novel PMMA microchips has been demonstrated by using contactless conductivity detection for determination of several inorganic ions. Such rapid and simple sample introduction leads to highly reproducible signals with relative standard deviations of less than 5% for peak responses. These new approaches significantly simplify the process of fabricating PMMA devices and show great promise for high-speed microchip analysis.      

DNA analysis on electrophoretic microchips: effect of operational variables

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.     

Fabrication and performance of fiber electrophoresis microchips

A method based on the in situ polymerization of methyl methacrylate (MMA) has been developed for the rapid fabrication of a novel separation platform, fiber electrophoresis microchip. To demonstrate the concept, prepolymerized MMA molding solution containing a UV initiator was sandwiched between a poly(methyl methacrylate) (PMMA) cover plate and a PMMA base plate bearing glycerol-permeated fiberglass bundles and was exposed to UV light. During the UV-initiated polymerization, the fiberglass bundles were embedded in the PMMA substrate to form fiberglass-packed microchannels. When the glycerol in the fiberglass bundles was flushed away with water, the obtained porous fiberglass-packed channels could be employed to perform electrophoresis separation. Scanning electron micrographs (SEMs) and microscopic images offered insights into the fiber electrophoresis microchip. The analytical performance of the novel microchip has been demonstrated by separating and detecting dopamine and catechol in connection with end-column amperometric detection. The fiber-based microchips can be fabricated by the new approach without the need for complicated and expensive lithography-based microfabrication techniques, indicating great promise for the low-cost production of microchips, and should find a wide range of applications.     

Integrated isotachophoretic preconcentration with zone electrophoresis separation on a quartz microchip for UV detection of flavonoids

A quartz microchip integrated isotachophoretic (ITP) preconcentration with zone electrophoresis (ZE) separation was fabricated using a novel multi-point pressure method featured in normal temperature and lower pressure during bonding process. ITP followed by subsequential ZE of two flavonoids, quercetin and isorhamnetin on the microchip was performed consecutively on the homemade microfluidic workstation with UV detection, resulting in a decreased detectable concentration of 32-fold, compared to the ZE mode only, and their detection limits decreased down to 0.2 microg/mL and 1.2 microg/mL, respectively.     

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.     

Ultraviolet thermal lensing detection of amino acids

Thermal lensing (TL) permits ultra-sensitive measurements of optical absorption of analytes in very small liquid volumes. We report the construction and use of a TL detector based on pulsed ultraviolet (UV) laser excitation (266 nm). We applied this detector to quantitate amino acids using capillary electrophoresis (CE) as a means of separation. Sixteen individual amino acids are readily detected, but the signal has a complex dependence on intensity caused by the combination of (1) one-photon absorption; (2) two-photon absorption (TPA); and (3) photodestruction of amino acid molecules in the focus of the laser beam. An aqueous solution containing tyrosine, tryptophan, and cysteine is electrophoretically separated and the individual amino acids are detected by UV TL. The estimated limit of detection is 7 microM for tyrosine, 2.5 microM for tryptophan and 33 microM for cysterine, which translates into 0.35 fmol for tyrosine, 0.125 fmol for tryptophan, and 1.65 fmol for cysteine in the 140pL detection volume. It is found that two-photon absorption of water and the formation of color centers in the fused silica walls of the flowcell can contribute a significant, drifting background signal, but this interference can be minimized by selecting an appropriate focus condition and excitation-detection geometry. We suggest that as UV laser sources become available, UV TL may become a method of choice for measuring the concentrations of many analytes in different separation formats in which the volume is highly limited.     

Capillary electrophoresis on microchip

Capillary electrophoresis and related techniques on microchips have made great strides in recent years. This review concentrates on progress in capillary zone electrophoresis, but also covers other capillary techniques such as isoelectric focusing, isotachophoresis, free flow electrophoresis, and micellar electrokinetic chromatography. The material and technologies used to prepare microchips, microchip designs, channel geometries, sample manipulation and derivatization, detection, and applications of capillary electrophoresis to microchips are discussed. The progress in separation of nucleic acids and proteins is particularly emphasized.     

Conductivity detection in capillary electrophoresis

Among electrochemical detection methods in capillary electrophoresis, conductometric methods are of specific interest for the determination of inorganic species. This is due to the fact that inorganic ions exhibit a high equivalent conductivity which corresponds to the analytical signal in conductivity detection. Indirect optical absorption methods, which are widely used in capillary electrophoretic ion analyses, become less sensitive with smaller capillary dimensions and thus have disadvantages in electrokinetic chip separation technologies.Conductivity detection for capillary electrophoresis is performed either through galvanic contact or in a contactless mode. Techniques using a galvanic contact of the sample ions with the measuring electrode are performed either on-capillary without decoupling of the separation high voltage, or off-capillary after grounding the separation voltage in order to avoid interferences. This technology is specifically important when a suppressor is used prior to detection. Most contactless methods use oscillometric techniques in order to obtain the analytical signal.This review discusses the theoretical and instrumental background of conductivity detection in capillary electrophoresis and reports on recent aspects and applications using conductometric detection methods for capillary zone electrophoresis.     

Numerical analysis of an electrokinetic double-focusing injection technique for microchip CE

The injection techniques in electrophoresis microchips play an important role in the sample-handling process, whose characteristics determine the separation performance achieved, and the shape of a sample plug delivered into the separation channel has a great impact on the high-quality separation performance as well. This paper describes a numerical investigation of different electrokinetic injection techniques to deliver a sample plug within electrophoresis microchips. A novel double-focusing injection system is designed and fabricated, which involves four accessory arm channels in which symmetrical focusing potentials are loaded to form a unique parallel electric field distribution in the intersection of injection channel and separation channel. The parallel electric field effectuates virtual walls to confine the spreading of a sample plug at the intersection and prevents sample leakage into separation channel during the dispensing step. The key features of this technique over other injection techniques are the abilities to generate regular and nondistorted shape of sample plugs and deliver the variable-volume sample plugs by electrokinetic focusing. The detection peak in the proposed injection system is uniform regardless of the position of the detection probe in the separation channel, and the peak resolution is greatly enhanced. Finally, the double-focusing injection technique shows the flexibility in detection position and ensures improved signal sensitivity with good peak resolution due to the delivered high-quality sample plug.     

Rapid enantioseparation of 1-aminoindan by microchip electrophoresis with linear-imaging UV detection.

Chiral separations of 1-aminoindan (AI) by cyclodextrin electrokinetic chromatography (CDEKC) were investigated on microfluidic quartz chips. By using a microchip electrophoresis (MCE) instrument equipped with a linear-imaging UV detector, the separation process of the enantiomeric compounds was observed. When sulfated beta-cyclodextrin was employed as a chiral selector, the baseline separation of AI could be achieved within 1 min with a high repeatability. The relative standard deviation of the migration time was less than 6%. The fastest separation was achieved in 14 s, utilizing a separation length of only 6.1 mm. These results show that the MCE analysis employing a linear imaging UV detector has a significant potential for fast chiral analysis.     

Micro flow modules with combined fluid flow channel and optical detection waveguide - hyper Rayleigh scattering as a case study

Micro flow modules with optical detection have been fabricated in a way which enables optical waveguiding inside and a defined interaction length along the fluid channel. Because of the usually lower refractive index of the solution compared with that of the substrate, so-called "leaky" optical wave-guiding must be employed. The combination of the fluid flow channel function with that of the optical waveguide has advantages for all miniaturized optical detection cells. It has been shown for hyper Rayleigh scattering (HRS) that improvement of the analytical principle is inherent in the miniaturization. The detection limit can be enhanced by at least a factor of 20. The applied HRS measurement procedure also enables simultaneous detection of two photon absorption (TPA) fluorescence. The severe boundary conditions of capillary electrophoresis were used as micro flow module design constraints to enable the transfer of the approach to other types of analysis.     

Electrochemical Detection for Capillary Electrophoresis Microchips: A Review

Electrochemistry detection offers considerable promise for capillary‐electrophoresis (CE) microchips, with features that include remarkable sensitivity, portability, independence of optical path length or sample turbidity, low cost and power requirements, and high compatibility with modern micromachining technologies. This article highlights key strategies in controlled‐potential electrochemical detectors for CE microchip systems, along with recent advances and directions. Subjects covered include the design of the electrochemical detection system, its requirements and operational principles, common electrode materials, isolation from the separation voltage, derivatization reactions, typical applications, and future prospects. It is expected that electrochemical detection will become a powerful tool for CE microchip systems and will lead to the creation of truly portable (and possibly disposable) devices.