Continuous microfluidic DNA and protein trapping and concentration by balancing transverse electrokinetic forces

Sample pre-concentration can be a critical element to improve sensitivity of integrated microchip assays. In this work a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields to assess the ability to concentrate a sample prior to other enzymatic modifications or capillary electrophoretic separations. Employing a pressure-driven flow to perfuse the microchannel, negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main daughter channel. Experimental results demonstrated that, depending of the buffer selection, different DNA migration and accumulation dynamics were seen. Charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility, high electrophoretic mobility, high ionic strength buffer or migrated towards an equilibrium position within the channel in a high electroosmotic mobility, high electrophoretic mobility, low ionic strength buffer. The various migration behaviours are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the bounding walls. Under continuous flow conditions, DNA samples were concentrated several-fold by balancing these transverse electrokinetic forces. The electrokinetic trapping technique presented here is a simple technique which could be expanded to concentrate or separate other analytes as a preconditioning step for downstream processes.     

Channel wall coating on a poly-(methyl methacrylate) CE microchip by thermal immobilization of a cellulose derivative for size-based protein separation

We demonstrate channel wall coating using a cellulose derivative on a poly-(methyl methacrylate) (PMMA) CE microchip to eliminate EOF disturbing protein separation. The channel walls were modified by preconditioning with a solution containing the cellulose derivative and then thermally evaporating the solution to produce hydrophilic channel walls which prevent adsorption of analytes via a hydrophobic interaction. When the PMMA substrate was coated with the cellulose derivative hydroxypropylmethylcellulose (HPMC) 90SH, the water contact angle on the coated substrate was decreased (up to 15 degrees ) and EOF was significantly suppressed (up to 4.0 x 10(-6) cm2.V(-1)s(-1)). Three proteins (20.5, 68.0, and 114.6 kDa) were successfully separated on the 0.15% HPMC 90SH-coated channel walls with good reproducibility of migration time (RSD <1.75%) and high efficiency (theoretical plate number per meter: 2.62 x 10(5)).     

Electrochromatography on acrylate‐based monolith in cyclic olefin copolymer microchip: A cost‐effective and easy‐to‐use technology

This article shows that there is great interest in using an electrochromatographic microchip made of hexyl acrylate (HA) based porous monolith cast within the channel of a cyclic olefin copolymer (COC) device. The monolith is simultaneously in situ synthesized and anchored to the inner walls of the channel in less than 10 min. By appropriate choice of light intensity used during the synthesis, the separation efficiency obtained for nonpolar solutes such as polycyclic aromatic hydrocarbons (PAH) is increased up to 250 000 plates/m. The performance of this HA‐filled COC microchip was investigated for a wide range of analytes of varying nature. The reversed‐phase separation of four aflatoxins is obtained in less than 2 min. The baseline separation of a mixture of neurotransmitters including six amino acids and two catecholamines is possible thanks to the superimposition of the differences in electrophoretic mobility on the chromatographic process. The durability of the system at pH 13 allows the separation of five biogenic amines and the quantitative determination of two of them in numerous wine samples. The feasibility of on‐line preconcentration is also demonstrated. Hydrophilic surface modification of COC channel via UV‐photografting with poly(ethylene glycol) methacrylate (PEGMA) before in situ synthesis of HA, is necessary to reduce the adsorption of very hydrophobic solutes such as PAH during enrichment. The detection limit of fluoranthene is decreased down to less than 1 ppb with a preconcentration of 4.5 h on the HA‐filled PEGMA functionalized COC microchip.     

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.     

Rinse and evaporation coating of poly(methyl methacrylate) microchip for separation of sodium dodecyl sulfate-protein complex

We developed a novel channel wall coating on a poly(methyl methacrylate) (PMMA) microchip using methylcellulose (MC) as a coating reagent to suppress electroosmotic flow (EOF) following the strong analytes adsorption via hydrophobic interaction with channel walls of PMMA. Our coating was obtained by first rinsing channel walls with MC-containing aqueous solution followed by evaporation. The coating made the hydrophilic channel wall lowering EOF by two orders of magnitude (1.2 x 10(-5)cm(2)V(-1)s(-1)) as well as reducing the hydrophobic adsorption. On the coated channel walls, we successfully separated sodium dodecyl sulfate-protein complexes with high reproducibility and efficiency using dextran as a lower viscosity protein separation medium.     

Pharmaceutical applications of isoelectric focusing on microchip with imaged UV detection

For the first time, the application of a commercial Shimadzu microchip electrophoresis system MCE-2010 equipped with an imaging UV detector for isoelectric focusing (IEF) of therapeutic proteins is reported. By proper adjustment of the pH gradient, samples with pI values ranging from 2.85 to 10.3 can be focused to the imaged part of the separation channel. Three therapeutic proteins (hirudin, erythropoietin, and bevacizumab) have been successfully focused on the microchip, and the results have been compared to conventional capillary IEF in terms of peak profile, pI values, and reproducibility.     

Flow manipulation for sweeping with a cationic surfactant in microchip capillary electrophoresis

Flow manipulation in sweeping microchip capillary electrophoresis (CE) is complicated by the free liquid communication between channels at the intersection, especially when the electroosmotic flows are mismatched in the main channel. Sweeping in traditional CE with cationic micelles is an effective way to concentrate anionic analytes. However, it is a challenge to transfer this method onto microchip CE because the dynamic coating process on capillary walls by cationic surfactants is interrupted when the sample solution free of surfactants is introduced into the microchip channels. This situation presents a difficulty in the sample loading, injection and dispensing processes. By adding surfactant at a concentration around the critical micelle concentration and by properly designing the voltage configuration, the flows in a microchip were effectively manipulated and this sweeping method was successfully moved to microchip CE using tetradecyltrimethylammonium bromide (TTAB). The sweeping effect of cationic surfactant in the sample solution was discussed theoretically and studied experimentally in traditional CE. The flows in a microchip were monitored with fluorescence imaging, and the injection and sweeping processes were studied by locating the detection point along the separation channel. A detection enhancement of up to 500-fold was achieved for 5-carboxyfluorescein.     

Sensitivity enhancing injection from a sample reservoir and channel interface in microchip electrophoresis

The stacking of a cationic analyte (i.e., rhodamine B) at the interface between a sample reservoir and channel in a microchip electrophoresis device is described for the first time. Stacking at negative polarity was by micelle to solvent stacking where the dye was prepared in a micellar solution (5 mM sodium dodecyl sulfate in 25 mM phosphoric acid, pH 2.5) and the channel was filled with high methanol content background solution (70% methanol in 50 mM phosphoric acid, pH 2.5). The injection of the stacked dye into the channel was by simple reversal of the voltage polarity with the sample solution and background solution at the anodic and cathodic reservoirs of the straight channel, respectively. The enrichment of rhodamine B at the interface and injection of the stacked dye into the channel was clearly visualized using an inverted fluorescence microscope. A notable sensitivity enhancement factor of up to 150 was achieved after 2 min at 1 kV of micelle to solvent stacking. The proposed technique will be useful as a concentration step for analyte mixtures in simple and classical cross‐channel microchip electrophoresis devices or for the controlled delivery of enriched reagents or analytes as narrow plugs in advanced microchip electrophoresis devices.     

蛋白质的微芯片电泳分离及其与脱氧核糖核酸迁移规律的比较

在自制的聚二甲基硅氧烷(PDMS)微芯片上,使用十二烷基磺酸钠(SDS)无胶筛分电泳分离体系(10 g/L的羟乙基纤维素(HEC), 1 g/L的SDS, 40 mmol/L磷酸盐缓冲溶液,pH 7.0),采用在线自校正激光诱导荧光检测方法,在6.4 min内高效分离了异硫氰酸荧光素(FITC)衍生的6种蛋白质标准样品,连续6次电泳所得迁移时间的相对标准偏差(RSD)均小于10%。用自主建立的脱氧核糖核酸(DNA)定量分离模型对蛋白质迁移数据进行拟合,发现SDS-蛋白质复合物迁移规律与DNA相似,但迁移淌度与相对分子质量及电场强度之间的线性关系明显变差,可见原DNA分离模型要扩展到蛋白质范围必须对一些参数进行校正。     

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser‐induced fluorescence detection

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK_a determination of small molecule analytes is demonstrated with the multilane microchip.     

Spatio-temporally resolved detection on a microfluidic chip for monitoring the dynamic processes of molecular events

Detection is an essential aspect in analytical approaches. In liquid phase separations, many attempts have been focused on the capability to detect a partial or an entire column. However, detection in both spatial and temporal resolutions has not gained much attention yet. Here we present the concept of spatio-temporally resolved detection (STRD) and a proof-of-the-concept microchip electrophoresis (MCE)-STRD system. The MCE-STRD system was mainly composed of a microchip and an STRD unit, which were designed completely based on the requirements for spatial and temporal resolutions. In the STRD unit, a linear light beam expanded from a UV LED light source was employed to illuminate the whole separation channel of the microchip while a linear CCD sensor that has an identical effective length as the separation channel and more pixels per unit length was used to detect the absorbance signals through the separation channel. As each pixel of the CCD sensor can detect a corresponding channel space in real time, the CCD provides both spatial and temporal resolutions. A significant advantage of STRD over conventional detection schemes is its capability for monitoring the dynamic processes of molecular events occurring in the separation channel. This was demonstrated through the monitoring of the dynamic processes of protein-DNA and protein-drug interactions in chip isoelectric focusing (chip IEF). The MCE-STRD system provided not only whole pictures of the entire dynamic processes at-a-glance but also quantitative kinetic information (dissociation rate constants) of the dynamic processes. With further development, we anticipate that STRD could be a promising tool for the characterization of biomolecular interactions and the observation of migration behaviours of analytes.     

Impact of reservoir potentials on the analyte behavior in microchip electrophoresis: computer simulation and experimental validation for DNA fragments

Fundamental understanding of the impact of reservoir potentials on the analyte behavior on the microfluidic chips is an important issue in microchip electrophoresis (MCE) for suitable injection and separation of analytes, since the applied potentials may significantly affect the shape of sample plug, sample leakage from the injection channel to the separation channel, injected sample amount, and separation efficiency. This study addressed this issue for the case of a conventional cross-geometry microchip with four reservoirs using computer simulations, the results of which were verified by the analysis of DNA fragments. For the microchip with a definite structure and migration distance, the injected sample amount was shown to be the vital parameter for improving the limit of detection and resolution. During injection, the shape of the sample plug could be adjusted by varying the reservoir potentials. It was demonstrated that a "magnified injection" (applying high voltage on the three reservoirs to the sample reservoir) is useful to enhance the detection sensitivity depending on the analyte composition, although such injection was previously avoided because of introducing too large amounts of the analyte in comparison with two established modes, floating and pinched injection. Optimal magnified injection was proved to improve the sensitivity for about 4 times over that of pinched injection for the analysis of DNA step ladders using microchip gel electrophoresis (MCGE). Sample leakage of DNA fragments could be suppressed by applying a high positive voltage on injection channel during separation, but the voltage degraded the injected amount and resolution.     

Concurrent determination and separation of inorganic cations and anions in microchip electrophoresis with precisely controlled high‐voltage

An improved method for the concurrent determination and separation of cations and anions by microchip electrophoresis with capacitively coupled contactless conductivity detection (ME‐C⁴D) is described. Two kinds of microchip structures were designed. The first microchip has a long bent separation channel. And for the defects of the first microchip, the second microchip with a Y‐type separation channel has been proposed. The background electrolyte (BGE) composed of 20 mm His/MES and 0.01 mm CTAB was optimized for inhibiting the electroosmotic flow (EOF). Due to the low electroosmotic flow, the cations and anions migrate in opposite directions and can be separated from each other. With the precisely controlled high‐voltage, cations and anions can be migrated in microchannels according to our requirements and sequentially detected by a C⁴D detector built in‐house. Samples containing K⁺, Na⁺, Li⁺, Cl⁻, F⁻ and PO₄³⁻ were analyzed simultaneously in a single run (within 140 s) by both methods. The reproducibility obtained by both methods remained below 5% for migration time and within 3.5–9.1% for peak areas. The proposed concurrent determination methods are inexpensive, simple, fast, ease of operation, high degree of integration.     

Combination of large‐volume sample stacking with an electroosmotic flow pump with field‐amplified sample injection on cross‐channel chips

A combination of two online sample concentration techniques, large‐volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field‐amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed‐polarity voltages on a cross‐channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP‐FASI. The applied pressure generated a counter‐flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520‐fold sensitivity increase was obtained in the LVSEP‐FASI analysis of a standard dye, which was 33‐times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100‐fold sensitivity increase was achieved with the 75‐μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d ‐leucine.     

On-line chemiluminescence detection for isoelectric focusing of heme proteins on microchips

In this paper we present a sensitive chemiluminescence (CL) detection of heme proteins coupled with microchip IEF. The detection principle was based on the catalytic effects of the heme proteins on the CL reaction of luminol-H2O2 enhanced by para-iodophenol. The glass microchip and poly(dimethylsiloxane) (PDMS)/glass microchip for IEF were fabricated using micromachining technology in the laboratory. The modes of CL detection were investigated and two microchips (glass, PDMS/glass) were compared. Certain proteins, such as cytochrome c, myoglobin, and horseradish peroxidase, were focused by use of Pharmalyte pH 3-10 as ampholytes. Hydroxypropylmethylcellulose was added to the sample solution in order to easily reduce protein interactions with the channel wall as well as the EOF. The focused proteins were transported by salt mobilization to the CL detection window. Cytochrome c, myoglobin, and horseradish peroxidase were well separated within 10 min on a glass chip and the detection limits (S/N=3) were 1.2x10(-7), 1.6x10(-7), and 1.0x10(-10) M, respectively.     

Fabrication and performance of a three-dimensionally adjustable device for the amperometric detection of microchip capillary electrophoresis

A microchip CE-amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an AD cell containing a one-dimensionally adjustable disk detection electrode in a Plexiglas holder. It facilitates the precise 3-D alignment between the channel outlet and the detection electrode without a complicated 3-D manipulator. The performance of this unique system was demonstrated by separating five aromatic amines (1,4-phenyldiamine, aniline, 2-methylaniline, 4-chloroaniline, and 1-naphthylamine) of environmental concern. Factors influencing their separation and detection processes were examined and optimized. The five analytes have been well separated within 140 s in a 74 cm long separation channel at a separation voltage of +2500 V using a 10 mM phosphate buffer (pH 3.5). Highly linear response is obtained for the five analytes over the range 20-200 microM with the detection limits ranging from 0.46 to 1.44 microM, respectively. The present system demonstrated long-term stability and reproducibility with RSDs of less than 5% for the peak current (n = 9). The new approach for the microchannel-electrode alignment should find a wide range of applications in CE, flowing injection analysis, and other microfluidic analysis systems.     

On‐line sample preconcentration by polarity switching in floating electrode‐integrated microchannel

In this study, we found that the polarity switching was effective to enrich and separate fluorescent analytes which have weakly‐dissociated groups in a floating platinum electrode (width, 50 µm; thickness, 2.5 µm)‐integrated straight‐channel in microchip electrophoresis (MCE). In the straight channel filled with an Alexa Flour 488 (AF488) solution, a sharp peak was observed after the polarity inversion with a 530‐fold enhancement of the sensitivity relative to the conventional MCE analysis. By using a fluorescent pH indicator, we verified that a sharp high‐pH zone was generated nearby the floating electrode and moved toward the anode with maintaining the high pH, which induced the sample enrichment like a dynamic pH junction mechanism. In the floating electrode‐embedded channel, the mixture of AF488‐labeled proteins was also well concentrated and separated within 100 s.     

Poly(methylmethacrylate) microchip electrophoresis of proteins using linear-poly(acrylamide) solutions as separation matrix.

Poly(methylmethacrylate) (PMMA) microchip electrophoresis of sodium dodecyl sulfate-protein complexes (SDS-PC) using linear-poly(acrylamide) (L-PA) as a separation matrix was investigated. Prior to electrophoresis, channel walls of PMMA were modified with methylcellulose (MC) to prevent adsorption between channel walls and SDS-PC. Size-based protein separation (SBPS) was successfully performed using the MC-coated microchips with Ferguson plot-fittings. The entangled L-PA solution provided high resolution of peaks of SDS-PC when the concentration of L-PA was increased. Some investigations into the separation mechanism, such as the plot of the logarithm of mobility of each SDS-PC versus the logarithm of the molecular weight of the complex exhibiting linear behavior, indicated that the separation mechanism was dependent on mass discrimination, in accordance with Ogston model.     

Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence

A method, using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for analyzing chitin oligosaccharides is described. Chitin oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) via reductive amination at 37 degrees C for 16 h (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid-phosphate buffer or an alkaline borate buffer. The effects of buffer types, buffer pH values, and buffer concentrations on the separation were examined. The analytes were successfully separated by using a pH 4.6 citric acid-phosphate within 19 min. The APTS-derivatized chitin monosaccharide (D-glucosamine) migrated first. The analytes were also completely separated by using a pH 9.0 borate buffer within 24 min. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized APTS labeling procedure and the CE-LIF method was demonstrated.     

Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection

Microchip electrophoresis for the short-time analysis of amino acids in Japanese green tea was developed. The amino acids in Japanese green tea were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The derivatives were filtered and directly analyzed by electrophoresis on a plastic microchip with a 31-mm long separation channel with fluorescence detection. Amino acid analysis of Japanese green tea was improved by removing polyphenols using a polyvinylpolypyrrolidone pretreatment. Elution profiles of NBD-amino acids were examined under different running buffer conditions, and the sodium dodecyl sulphate in the running buffer exhibited a dramatically high-separation efficiency of amino acids by inhibiting their adsorption on the channel walls. Under the optimized conditions (5 mM phosphate buffer (pH 5.5) containing 0.05 mM sodium dodecylsulfate as running buffer), the main amino acids contained in Japanese green tea were well separated within 2 min, and theanine (1475 mg/100 g tea leaf), Arg (408 mg/100 g tea leaf) and Gln (217 mg/100 g tea leaf) were detected in Japanese green tea.