Decreased protein peak asymmetry and width due to static capillary coating with hydrophilic derivatives of polydimethylacrylamide

We have recently described [1] a fast and simple method for the "adsorbed static" coating of capillaries in capillary zone electrophoresis (CZE) with epoxy-poly(dimethylacrylamide) (EPDMA). Protein CZE peaks in the EPDMA-coated capillaries exhibited a peak asymmetry similar to that obtained in capillaries with "covalent static" coating of polyacrylamide, suggesting a similar degree of adsorption of the protein onto the coating [2]. Instability of such coating at very low ionic strength and its stripping from the capillary in the presence of sodium dodecyl sulfate (SDS) also indicated a hydrophilic bonding of EPDMA to the silanol surface of the capillary, while its stripping in the CZE of "carboxylate-modified" polystyrene suggested a competition between carboxylate and EPDMA for the hydrophilic bonds to silanol. To test those propositions, a number of EPDMA-derived coating agents with increased hydrophilicity were synthesized. Of a number of the hydrophilic coating agents tested (Table 1) only two, 2% hydrolyzed EPDMA (HPDMA) hydrolyzed in sulfuric acid to effect the conversion of the epoxy groups to diols (Table 1, No. 38), and 20% EPDMA (Table 1, No. 44) exhibited for representative proteins a decreased peak asymmetry and width while the stability of the suppression of electroosmotic flow (EOF), and the stability of mobility in consecutive CZE runs was reduced relative to EPDMA. Coating agents which were more highly hydrophilic than those two (Table 1, No. 49) or less hydrophilic than 2% EPDMA (Table 1, Nos. 57, 53, 46) provided no stable static coating.     

Rapid capillary coating by epoxy-poly-(dimethylacrylamide): performance in capillary zone electrophoresis of protein and polystyrene carboxylate

A fast and simple method for the internal coating of capillaries in capillary zone electrophoresis (CZE) is that with epoxy-poly(dimethylacrylamide) (EPDMA). Duration of coating by that method is 30 min, compared with that of 24 h when using uncross-linked polyacrylamide (PA) under otherwise identical conditions. Under the conditions used for the CZE of proteins (pH 9.0, 2% polyethylene glycol), the capillary coating with EPDMA is stable for at least 50 consecutive runs as judged by the constancy of low electroosmotic flow, equalling the stability of coating achieved by PA. Protein mobilities and protein peak asymmetry (suggestive of reversible interaction with the capillary wall) are also found to be the same in EPDMA and PA coated capillaries. Differences between EPDMA and PA coating also exist: The former is unstable upon lowering the ionic strength of the buffer to 0.003, upon the addition of sodium dodecyl sulfate (SDS) to the buffer and in application to the hydrophobic analyte, polystyrene carboxylate.     

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip

We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.     

Underivatized cyclic olefin copolymer as substrate material and stationary phase for capillary and microchip electrochromatography

We report, for the first time, the use of underivatized cyclic olefin copolymer (COC, more specifically: Topas) as the substrate material and the stationary phase for capillary and microchip electrochromatography (CEC), and demonstrate chromatographic separations without the need of coating procedures. Electroosmotic mobility measurements in a 25 microm id Topas capillary showed a significant cathodic EOF that is pH-dependent. The magnitude of the electroosmotic mobility is comparable to that found in glass substrates and other polymeric materials. Open-tubular CEC was employed to baseline-separate three neutral compounds in an underivatized Topas capillary with plate heights ranging from 5.3 to 12.7 microm. The analytes were detected using UV absorbance at 254 nm, thus taking advantage of the optical transparency of Topas at short wavelengths. The fabrication of a Topas-based electrochromatography microchip by nanoimprint lithography is also presented. The microchip has an array of pillars in the separation column to increase the surface area. The smallest features that were successfully imprinted were around 2 microm wide and 5 microm high. No plasma treatment was used during the bonding, thus keeping the surface properties of the native material. An RP microchip electrochromatography separation of three fluorescently labeled amines is demonstrated on the underivatized microchip with plate heights ranging from 3.4 to 22 microm.     

A rejuvenation method for poly(N,N-dimethylacrylamide)-coated glass microfluidic chips

As microfluidic chips come to integrate the higher levels of functionality required for the implementation of advanced bioanalytical protocols, a crucial factor is that of cost. Although glass chips provide advantages in multilayer integrations, their cost is far higher than that of polymer chips. However, a simple and effective rejuvenation protocol for glass microchips may enable higher levels of integration and functionality on glass microchips. Here we present a method to rejuvenate glass microchips that had been used for capillary electrophoresis to the extent that their performance was degraded. This degradation was due to one of the two mechanisms: (i) a deterioration of the polymer coating on the inner surface of the microchannel or (ii) an aging of the glass substrate. Using the method presented here, we have rejuvenated more than 50 such "aged" microchips. The performance of these microchips was fully restored after the rejuvenation and lasted for hundreds of DNA separation runs. Our experiments indicate that the loss of resolution in microchip separations was not associated with glass aging, but was due to the degradation of the polymer coating on the inner surface of microchannels. This suggests that it is possible to extend the microchip lifetime "forever" using the rejuvenation protocol and that the exploration of higher levels of integration and functionality on glass microchips (or of hybrid structures involving materials capable of withstanding the reagents and elevated temperatures used) is feasible.     

Immobilization of trypsin in the layer-by-layer coating of graphene oxide and chitosan on in-channel glass fiber for microfluidic proteolysis

In this report, trypsin was immobilized in the layer-by-layer (LBL) coating of graphene oxide (GO) and chitosan on a piece of glass fiber to fabricate microchip bioreactor for efficient proteolysis. LBL deposition driven by electrostatic forces easily took place on the surface of the glass fiber, providing mild environmental conditions so that the denaturation and autolysis of the immobilized trypsin was minimized. Prior to use, a piece of the prepared trypsin-immobilized glass fiber was inserted into the channel of a microchip to form a core-changeable bioreactor. The novel GO-based bioreactor can be regenerated by changing its fiber core. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin, myoglobin, cytochrome c, and hemoglobin and the digestion time was significantly reduced to less than 10 s. The obtained digests were identified by MALDI-TOF MS. The digestion performance of the core-changeable GO-based microchip bioreactor was comparable to that of 12-h in-solution tryptic digestion. The novel microchip bioreactor is simple and efficient, offering great promise for high-throughput protein identification.     

Immobilization of trypsin on silica-coated fiberglass core in microchip for highly efficient proteolysis

In this report, trypsin was immobilized on silica-coated fiberglass core in microchip to form a core-changeable bioreactor for highly efficient proteolysis. To prepare the fiber core, a layer of organic-inorganic hybrid silica coating was prepared on the surface of a piece of glass fiber by a sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, trypsin was immobilized on the coating with the aid of glutaraldehyde. Prior to use, the enzyme-immobilized fiber was inserted into the channel of a microchip to form an in-channel fiber bioreactor. The novel bioreactor can be regenerated by changing its fiber core. The scanning electron microscopy images of the cross-section of a trypsin-immobilized fiber indicated that a layer of ∼1 μm thick film formed on the glass substrate. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c) and the digestion time was significantly reduced to less than 10 s. The digests were identified by MALDI-TOF MS with sequence coverages of 45% (BSA) and 77% (Cyt-c) that were comparable to those obtained by 12-h conventional in-solution tryptic digestion. The fiber-based microchip bioreactor provides a promising platform for the high-throughput protein identification.     

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.     

Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis

An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.     

Viability study of HL60 cells in contact with commonly used microchip materials

This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (R(ap)) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower R(ap) as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24 h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing.     

A robust cross-linked polyacrylamide coating for microchip electrophoresis of dsDNA fragments

Surface derivatization plays an important role in microchip electrophoresis. It not only enhances the resolution, but also improves the reproducibility. So far, the most popularly used derivatization method for glass microchannels is to covalently attach a layer of linear polyacrylamide (LPA) to the channel surfaces. However, LPA coating has two problems: incomplete coverage and limited lifetime. To address these issues, we have recently developed a cross-linked polyacrylamide (CPA) derivatization protocol and demonstrated it for high-resolution protein separations by CIEF, CGE, and CZE. In this report, we used this protocol to coat microchip channels and exhibited the reliability and robustness of CPA coating for microchip electrophoresis of DNA molecules. dsDNA fragments were used as our test samples. High resolutions were obtained for fragments ranging from 100 bp to 10 kpb. After more than 800 runs, the CPA-coated microchannels still performed well and comparable resolutions were maintained throughout these runs.     

Microchip PCR

Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties     

Property of ionic liquid in electrophoresis and its application in chiral separation on microchips

The aqueous solution of a kind of room-temperature ionic liquids (RTILs), 1-ethyl-3-methylimidazolium-tetrafluoroborate (1E-3MI-TFB), demonstrated its exclusive electroosmotic property in microchip electrophoresis. It was applied as the working electrolyte for chiral separation in glass microchip electrophoresis. Compared with boric acid buffer, 1E-3MI-TFB aqueous solution exhibited a broader separation window for enantiomers of dipeptides. Then the influences of chiral selector, pH and concentration on efficiency of chiral separation were discussed in detail. The unique mechanism of the generation of EOF was explored in a glass microchannel using 1E-3MI-TFB aqueous solution as working electrolyte. A possible status of 1E-3MI cation in water was suggested at the first time, which facilitated the explanation of EOF and its characteristics in glass microchannel. Additionally, microchip electrophoresis using 1E-3MI-TFB aqueous solution was successfully applied to the chiral separation of complex enantiomers of dipeptides. RTILs aqueous solution, as the electrolyte for the separation of complicated optical isomers, could lead to a revolution in the analytical methods of chiral or conformational analysis for biomolecules.     

In-situ grafting hydrophilic polymer on chitosan modified poly(dimethylsiloxane) microchip for separation of biomolecules

In this paper, a simple and green modification method is developed for biomolecules analysis on poly(dimethylsiloxane) (PDMS) microchip with successful depression of nonspecific biomolecules adsorption. O-[(N-succinimdyl)succiny]-o'-methyl-poly(ethylene glycol) was explored to form hydrophilic surface via in-situ grafting onto pre-coated chitosan (Chit) from aqueous solution in the PDMS microchannel. The polysaccharide chains backbone of Chit was strongly attracted onto the surface of PDMS via hydrophobic interaction combined with hydrogen bonding in an alkaline medium. The methyl-poly(ethylene glycol) (mPEG) could produce hydrophilic domains on the mPEG/aqueous interface, which generated brush-like coating in this way and revealed perfect resistance to nonspecific adsorption of biomolecules. This strategy could greatly improve separation efficiency and reproducibility of biomolecules. Amino acids and proteins could be efficiently separated and successfully detected on the coated microchip coupled with end-channel amperometric detection at a copper electrode. In addition, it offered an effective means for preparing biocompatible and hydrophilic surface on microfluidic devices, which may have potential use in the biological analysis.     

Highly efficient dynamic modification of plastic microfluidic devices using proteins in microchip capillary electrophoresis

New dynamic coating agents were investigated for the manipulation of electroosmotic flow (EOF) in poly(methylmethacrylate) (PMMA) microchips. Blocking proteins designed for enzyme-linked immunosorbent assay (ELISA) applications (e.g. Block Ace and UltraBlock), and egg-white lysozyme were proposed in this study. The EOF could be enhanced, suppressed or its direction could be reversed, depending on the buffer pH and the charge on the proteins. The coating procedure is simple, requiring only filling of the microchannels with a coating solution, followed by a rinse with a running buffer solution prior to analysis. One major advantage of this method is that it is not necessary to add the coating agent to the running buffer solution. Block Ace and UltraBlock coatings were stable for at least five runs in a given microchannel without the need to condition the coating between runs other than replenishing the buffer solution after each run, i.e. the RSD values of EOF (n=5) were less than 4.3%, and there was no significant change in the EOF after 5 runs. The reproducibility of the coating procedures was found from the channel-to-channel RSD values of the EOF, and were less than 5.0% when using HEPES-Na buffer (pH 7.4) as the running buffer. Several examples of electrophoretic separations of amino acids and biogenic amines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) are demonstrated in this paper. The dynamic coating method has the potential for a broad range of applications in microchip capillary electrophoresis (microchip CE) separations.     

Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.     

Microfluidic routing of aqueous and organic flows at high pressures: fabrication and characterization of integrated polymer microvalve elements

This paper presents the first systematic engineering study of the impact of chemical formulation and surface functionalization on the performace of free-standing microfluidic polymer elements used for high-pressure fluid control in glass microsystems. System design, chemical wet-etch processes, and laser-induced polymerization techniques are described, and parametric studies illustrate the effects of polymer formulation, glass surface modification, and geometric constraints on system performance parameters. In particular, this study shows that highly crosslinked and fluorinated polymers can overcome deficiencies in previously-reported microvalve architectures, particularly limited solvent compatibility. Substrate surface modification is shown effective in reducing the friction of the polymer-glass interface and thereby facilitating valve actuation. A microchip one-way valve constructed using this architecture shows a 2 x 10(8) ratio of forward and backward flow rates at 7 MPa. This valve architecture is integrated on chip with minimal dead volumes (70 pl), and should be applicable to systems (including chromatography and chemical synthesis devices) requiring high pressures and solvents of varying polarity.     

A rigid poly(dimethylsiloxane) sandwich electrophoresis microchip based on thin-casting method

We present a novel concept of glass/poly(dimethylsiloxane) (PDMS)/glass sandwich microchip and developed a thin-casting method for fabrication. Unlike the previously reported casting method for fabricating PDMS microchip, several drops of PDMS prepolymer were first added on the silanizing SU-8 master, then another glass plate was placed over the prepolymer as a cover plate, and formed a glass plate/PDMS prepolymer/SU-8 master sandwich mode. In order to form a thin PDMS membrane, a weight was placed on the glass plate. After the whole sandwich mode was cured at 80 degrees C for 30 min, the SU-8 master was easily peeled and the master microstructures were completely transferred to the PDMS membrane which was tightly stuck to the glass plate. The microchip was subsequently assembled by reversible sealing with the glass cover plate. We found that this PDMS sandwich microchip using the thin-casting method could withstand internal pressures of >150 kPa, more than 5 times higher than that of the PDMS hybrid microchip with reversible sealing. In addition, it shows an excellent heat-dissipating property and provides a user-friendly rigid interface just like a glass microchip, which facilitates manipulation of the microchip and fix tubing. As an application, PDMS sandwich microchips were tested in the capillary electrophoresis separation of fluorescein isothiocyanate-labeled amino acids.     

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.     

Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection

The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF). Using a commercial "double-T" glass microchip, the sample was electrokinetically loaded in the "double-T" intersection and then subjected to electrophoretic separation along the main separation channel. In order to decrease interactions of mitochondria with channel walls during the analysis, poly(vinyl alcohol) was used as a dynamic coating. This procedure eliminates the need for complicated covalent surface modifications within the channels that were previously used in capillary electrophoresis methods. For analysis, mitochondria, isolated from bovine liver tissue, were selectively labelled using 10-nonyl acridine orange (NAO). The results consist of electropherograms where each mitochondrial event is a narrow spike (240 +/- 44 ms). While the spike intensity is representative of its NAO content, its migration time is used to calculate and describe its electrophoretic mobility, which is a property still largely unexplored for intracellular organelles. The five-fold decrease in separation time (4 min for microchip versus 20 min for capillary electrophoresis) makes microchip electrophoretic separations of organelles a faster, sensitive, low-sample volume alternative for the characterization of individual organelle properties and for investigations of subcellular heterogeneity.