Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip

Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method.     

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.     

Elimination of suction effect in interfacing microchip electrophoresis with inductively coupled plasma mass spectrometry using porous monolithic plugs

A suction-free interfacing method was developed for microchip electrophoresis hyphenated with inductively coupled plasma mass spectrometry (MCE-ICP-MS). The hyphenated system was composed of a microchip, a demountable capillary microflow nebulizer (d-CMN) combined with a heated single pass spray chamber, a negative pressure sampling device, a high voltage power supply, a syringe pump and an ICP-MS. To eliminate the nebulizer suction generated by the pneumatic nebulizer and to ensure that the makeup solution flowed into the nebulizer, two porous polymer plugs were fabricated in the microchip. As a result, reasonably true electropherograms were obtained when compared to the CE separation performed in the traditional MCE-ICP-MS mode without porous polymer plugs. Electrophoretic separation of I(-) and IO(3)(-) was achieved within 25 s in a microchip with an effective separation length of only 15 mm at an electric field of 857 V cm(-1) using 10 mmol L(-1) borate (pH 9.2) as the running buffer. A resolution of 1.3 was obtained and the absolute detection limits for I(-) and IO(3)(-) were 0.12 and 0.13 fg, respectively. The precisions (RSD, n = 10) of the migration time and peak height for I(-) and IO(3)(-) were in the range of 1.1-1.6% and 2.5-2.8%, respectively. Two table salt samples were analyzed by an external calibration method. The iodate contents were in accordance with their labeled values. The recoveries of I(-) and IO(3)(-) in the table salt samples were in the range of 92-105%.     

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.     

Integration of a nanoporous platinum thin film into a microfluidic system for non-enzymatic electrochemical glucose sensing.

In this paper, we describe an amperometric-type enzymeless glucose sensing system based on a nanoporous platinum (Pt) electrode embedded in a microfluidic chip. This microchip system is comprised of a microfluidic transport channel network and a miniaturized electrochemical cell for nonenzymatic glucose sensing. Sample and buffer solutions were transferred to the cell by programmed electroosmotic flow (EOF). A nanoporous Pt electrode with the roughness factor of 200.6 was utilized to determine glucose concentrations in phosphate buffered saline (PBS) by the direct oxidation of glucose, without any separation process. The sensitivity of the developed system is 1.65 microA cm-2 mM-1 in the glucose concentration range from 1-10 mM in PBS.     

Ionspray microchip

An ionspray microchip is introduced. The chip is based on the earlier presented nebulizer microchip that consists of glass and silicon plates bonded together. A liquid inlet channel, nebulizer gas inlet, and nozzle are etched on the silicon plate and a platinum heater is integrated on the glass plate. The nebulizer microchip has been previously used in atmospheric pressure chemical ionization, atmospheric pressure photoionization, sonic spray ionization, and thermospray ionization modes. In this work we show that the microchip can be operated also in ionspray mode by introducing high voltage to the silicon plate of the microchip. The effects of operation parameters (voltage, nebulizer gas pressure, sample solution flow rate, solvent composition, and analyte concentration) on the performance of the ion spray microchip were studied. Under optimized conditions the microchip provides efficient ionization of small and large compounds and good quantitative performance. The feasibility of the ion spray microchip in liquid chromatography/mass spectrometry (LC/MS) was demonstrated by the analysis of tryptic peptides of bovine serum albumin. Copyright © 2010 John Wiley & Sons, Ltd.     

Shah convolution differentiation Fourier transform for rear analysis in microchip capillary electrophoresis

This paper first reports the application of Shah convolution differentiation Fourier transform for rear analysis. Rear analysis eliminates the need to create a well-defined and reproducible sample plug, thus making the operation simpler. The number of solution reservoirs, for microchip capillary electrophoresis (CE), could be reduced from the usual four to three. Sample bias in CE could be avoided too. The separation channel was first filled with the fluorescent sample solution, and subsequently flushed out with the buffer. The rear of each analyte zone gives rise to its flight of sigmoid-shaped steps in the time-domain. The time-domain detector signal was first differentiated and then Fourier transform was performed. The Fourier transform results were represented in the form of a magnitude plot. It is proposed that this would be as equally applicable to other separation techniques (e.g., chromatography) and detection methods (e.g., absorption).     

Design of separation length and electric field strength for high-speed DNA electrophoresis

Gel-based DNA separation on microchip will play an important role in future genomic analysis due to its potential for high-efficiency and high-speed. Optimal design of microchip and separation condition is essential to take full advantage of high-speed separation on microchip. Separation length L and electric field strength E, which are crucial for design of microchip system, are focused on in this paper. Simultaneous optimization of L and E was carried out to achieve the most rapid separation. It was shown that the condition of L and E and the shortest separation time is closely related to the shape of resolution Rs surface in a three-dimensional space with axes E, L, and Rs. This surface was investigated, taking sample injection, detector, diffusion, and Joule heating into account. Thermal gradient broadening due to Joule heating helps to produce camber or ridge shape of Rs surface, which is essential for the shortest separation length and separation time. Sample plug length and detection volume should be more carefully controlled in microchip. The property of diffusion coefficient was shown to play a key role in determining Rs surface.     

Development of an analytical method using microchip capillary electrophoresis for the measurement of fluorescein-labeled salivary components in response to exercise stress

We have developed an analytical method using microchip capillary electrophoresis (microchip CE) for the high-speed separation of fluorescein-labeled salivary components in response to exercise stress. Optimal separation was obtained using a borate buffer at pH 9.5 containing 10 mM beta-cyclodextrin and 1.0% (w/v) methylcellulose. To minimize individual differences in human saliva, such as viscosity, conductivity, and contaminants, the concentration of methylcellulose in the analytical conditions played a key factor. The optimized separation conditions produced identical electropherograms successfully despite of the use of different microchips made from quartz glass or poly-methylmethacrylate (PMMA). In addition, a practical application of bicycle ergometer stress was performed. Some components in human saliva showed a marked decrease after exercise stress.     

A design of nanosized PEGylated-latex mixed polymer solution for microchip electrophoresis

We report here advanced microchip electrophoresis using a nanoparticle doped polymer solution that enables greater separation of DNA. The proposed system is simple and effective without any new apparatus or complicated procedures. Various amounts and sizes (80 nm, 110 nm, and 193 nm) of polymer nanoparticle solutions (PEGylated-latex) were mixed with a conventional polymer solution for microchip electrophoresis. When a 0.49 wt% hydroxyl propyl methyl cellulose (HPMC) buffer solution was mixed with a 2.25 wt% 80 nm-PEGylated-latex a higher separation efficiency and a higher mobility of a wider molecular range of dsDNA (10 bp to 2 kbp) was achieved under low viscosity conditions (<5.5 cP) than in conventional 0.7% HPMC. The separation performance was dependent on the particle size and concentration. Furthermore, the effectiveness of the larger PEGylated-latex (193 nm) was not as high as the smaller one (80 to 110 nm). The observed separation improvement by polymer solution with latex-nanoparticles seems to derive from the balance between wider polymer mesh size and the structural obstacles of particles in the buffer.     

Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis

In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.     

Conformational separation of monosaccharides of glycoproteins labeled with 2-aminoacrydone using microchip electrophoresis

The conformational separation of monosaccharides labeled with fluorescent 2-aminoacrydone (AMAC) was performed by electrophoresis on a plastic microchip with light-emitting diode confocal fluorescence detection. The AMAC-labeled five neutral monosaccharide mixture (D-glucose (Glc), D-mannose, D-galactose, L-fucose, and D-xylose) or two amino monosaccharide mixture (N-acetyl-D-glucosamine and N-acetyl-D-galactosamine) were well separated at pH 8.5 and 0.5% w/v methylcellulose of 200 mM borate buffer conditions using microchip electrophoresis. The separation was successfully performed considering the difference in stability of the complex between the hydroxyl residue of the monosaccharide and borate ions, and we found that 200 mM and pH 8.5 of borate buffer conditions were critical. High-speed separation for the neutral monosaccharides (50 s) and for amino monosaccharides (70 s) was attained at a 400 V/cm of electric field condition, showing all peak resolutions were greater than 0.9% and RSD of mobility were less than 1.9%. The detection limits of 0.86 microM for Glc and <1 microM for all other monosaccharides were enhanced with the addition of 0.5% w/v methylcellulose to the buffer. These attainments are fully compatible with conventional CE. The analysis of the subtle differences in the conformational stability and the value of the hydroxyl residue of the borate complex allowed the development of an efficient prospective tool for attaining high-resolution separation of monosaccharide mixtures having complicated and analogous conformations.     

Influence of the pH on separating DNA by high-speed microchip electrophoresis.

Various factors are critical in resolving DNA molecules at high speed, including the separation medium, concentration, composition, and pH of the buffer, as well as the electric field strength. To this study, considered the composition of a buffer and the difference in the pH, while paying attention to whether the separation ability changes in the microchip electrophoresis of DNA. DNA separation was particularly affected by both the buffer composition and the pH. Under the optimal microchip electrophoresis conditions that were determined in this study, an improved resolution of a wider range of DNA fragment sizes was achieved. Moreover, the total separation time decreased from 240 s to 100 s. Thus, by simplifying and improving the DNA electrophoresis in the microchip, this technique is now widely applicable to several different scientific fields.     

Chiral separation of NBD-amino acids by ligand-exchange micro-channel electrophoresis.

The chiral separation of amino acid derivatives by ligand-exchange electrophoresis in a microchannel chip was performed for the first time. A Cu(II) complex with L-prolinamide was used as a chiral selector. The migration behaviors of eleven NBD-DL-amino acids were investigated by ligand-exchange capillary electrophoresis (LE-CE). The enantiomer of five NBD-amino acids (Ser, Thr, Val, Phe and His) could be separated by LE-CE using a 20 mM ammonium acetate buffer (pH 9.0) containing 10 mM copper acetate, 20 mM L-prolinamide and 1 mM SDS. NBD-His was eluted in the order D-form and L-form, while the elution order of another enantiomers was L-form and D-form. Under this condition, the enantioseparation of these five NBD-amino acids by ligand-exchange microchip electrophoresis (LE-ME) was investigated using a glass microchip. The enantioseparation of NBD-Ser, -Thr and -His could be successfully accomplished by LE-ME. LE-ME was superior to LE-CE in terms of the short migration time and a good enantiomeric separation.     

Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresis

Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.     

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.     

Study of the influence of surfactants on the transfer of gases into liquids by inverse gas chromatography

Analyses of amino acids and peptides were performed using a quartz microchip and an interface for microchip electrophoresis-electrospray ionization mass spectrometry (MCE-ESI-MS). In MCE-ESI-MS, negative pressure caused by ESI increased band broadening and deteriorated separation. We tried to suppress the negative pressure and improve separation using a microchip with a long separation channel. Separations of peptide standards were compared using two microchips with long separation channel (58.9 mm) and short one (22.9 mm). Theoretical plate numbers and resolution were improved significantly using the former. The theoretical plate numbers of [Val4]angiotensin was 8600 using the former and 1700 using the latter. When background electrolytes of low pH were used in an uncoated quartz microchip, electrokinetic injection was difficult because of weak electroosmotic flow. The use of successive multiple ionic polymer layers coating of the microchip channel stabilized electrokinetic injection and permitted analysis of amino acids and peptides even under low pH conditions. Separation of amino acids was successfully performed using formic acid solution (pH 2.5) as background electrolyte.     

Fast and simultaneous detection of heavy metals using a simple and reliable microchip-electrochemistry route: An alternative approach to food analysis

This paper reports, for the first, the fast and simultaneous detection of prominent heavy metals, including: lead, cadmium and copper using microchip CE with electrochemical detection. The direct amperometric detection mode for microchip CE was successfully applied to these heavy metal ions. The influences of separation voltage, detection potential, as well as the concentration and pH value of the running buffer on the response of the detector were carefully assayed and optimized. The results clearly show that reliable analysis for lead, cadmium, and copper by the degree of electrophoretic separation occurs in less than 3min using a MES buffer (pH 7.0, 25mM) and l-histidine, with 1.2kV separation voltage and -0.8V detection potential. The detection limits for Pb(2+), Cd(2+), and Cu(2+) were 1.74, 0.73 and 0.13microM (S/N=3). The %R.S.D. of each peak current was <6% and migration times <2% for prolonged operation. To demonstrate the potential and future role of microchip CE, analytical possibilities and a new route in the raw sample analysis were presented. The results obtained allow the proposed microchip CE-ED acts as an alternative approach for metal analysis in foods.     

Rapid analysis of oligosaccharides derived from glycoproteins by microchip electrophoresis

A novel method for fast profiling of complex oligosaccharides released from glycoproteins based on microchip electrophoresis (mu-CE) is presented here. The characterization of separation conditions, i.e., the composition, concentration and pH of running buffer as well as the applied voltage, has been performed using maltose (G2), cellobiose ( G2'), maltriose (G3) and panose (G3') as oligosaccharide isomer models. In mu-CE, much better separation of oligosaccharide isomers and oligosaccharide ladder was obtained in phosphate buffer than in borate buffer over a wide pH range. Under optimal conditions, high-performance separation of the N-linked complex oligosaccharides released from ribonuclease B, fetuin, alpha1-acid glycoprotein (AGP) and IgG was achieved using polymethylmethacrylate (PMMA) microchips with an effective separation channel of 30 mm. These results represent the first reported analysis of the N-linked oligosaccharides derived from glycoproteins by mu-CE, indicating that the present mu-CE-based method is a promising alternative for characterization of the N-linked oligosaccharides in glycoproteins.     

Open channel-based microchip electrophoresis interfaced with mass spectrometry via electrostatic spray ionization

The coupling between open channel-based microchip electrophoresis and mass spectrometry via electrostatic spray ionization is proposed for in situ detection of fractionated analytes. Electrophoretic separation is performed in an open channel fabricated in a plastic substrate. The solvent of background electrolyte is evaporated from the open channel because of Joule heating during electrophoresis, leaving the dried electrophoretic bands to be directly analyzed by mass spectrometry via scanning electrostatic spray ionization. Proof-of-concept results are obtained with fluorescent dyes and antibiotics as the test samples, demonstrating an efficient on-chip detection platform based on the electrophoresis and electrostatic spray ionization mass spectrometry.