Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips

MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.     

High efficiency micellar electrokinetic chromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips

This paper describes a simple method for the effective and rapid separation of hydrophobic molecules on polydimethylsiloxane (PDMS) microfluidic devices using Micellar Electrokinetic Chromatography (MEKC). For these separations the addition of sodium dodecyl sulfate (SDS) served two critical roles - it provided a dynamic coating on the channel wall surfaces and formed a pseudo-stationary chromatographic phase. The SDS coating generated an EOF of 7.1 x 10(-4) cm(2) V(-1) s(-1) (1.6% relative standard deviation (RSD), n = 5), and eliminated the absorption of Rhodamine B into the bulk PDMS. High efficiency separations of Rhodamine B, TAMRA (6-carboxytetramethylrhodamine, succinimidyl ester) labeled amino acids (AA), BODIPY FL CASE (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester) labeled AA's, and AlexaFluor 488 labeled Escherichia coli bacterial homogenates on PDMS chips were performed using this method. Separations of Rhodamine B and TAMRA labeled AA's using 25 mM SDS, 20% acetonitrile, and 10 mM sodium tetraborate generated efficiencies > 100,000 plates (N) or 3.3 x 10(6) N m(-1) in <25 s with run-to-run migration time reproducibilities <1% RSD over 3 h. Microchips with 30 cm long serpentine separation channels were used to separate 17 BODIPY FL CASE labeled AA's yielding efficiencies of up to 837,000 plates or 3.0 x 10(6) N m(-1). Homogenates of E. coli yielded approximately 30 resolved peaks with separation efficiencies of up to 600,000 plates or 2.4 x 10(6) N m(-1) and run-to-run migration time reproducibilities of <1% RSD over 3 h.     

High electric field strength two-dimensional peptide separations using a microfluidic device

New instrumentation has been developed to improve the resolution, efficiency, and speed of microfluidic 2D separations using MEKC coupled to high field strength CE. Previously published 2D separation instrumentation [Ramsey, J. D. et al., Anal. Chem. 2003, 75, 3758-3764] from our group was limited to a maximum potential difference of 8.4 kV, resulting in an electric field strength of only approximately 200 V/cm in the first dimension. The circuit described in this report has been designed to couple a higher voltage supply with a rapidly switching, lower voltage supply to utilize the best features of each. Voltages applied in excess of 20 kV lead to high electric field strength separations in both dimensions, increasing the separation resolution, efficiency, and peak capacity while reducing the required analysis time. Detection rates as high as six peptides per second (based on total analysis time) were observed for a model protein tryptic digest separation. Additionally, higher applied voltages used in conjunction with microfluidic chips with longer length channels maintained higher electric field strengths and produced peak capacities of over 4000 for some separations. Total separation time in these longer channel devices was comparable to that obtained in short channels at low field strength; however, resolving power improved approximately threefold.     

Cover Picture: Electrophoresis 2'2011

Issue no. 2 is a regular issue assembled of 16 solid and original research articles distributed over 3 distinct parts. Part I is on novel trends in fundamentals and methodologies including theoretical models for selectivity of charged solutes in MEKC, system peaks in indirect detection, measuring epimerization constants by MEEKC, bundled CE using micro‐structured fibers, 2‐D separations by coupling CIEF and CEC, high speed DNA CE, MCE of N‐glycans and mucin expression in a microfluidic gradient device. Part II is concerned with detection, sensitivity enhancement, on‐column preconcentration and microdialysis sampling involving the design of continuous full filling CEC‐ESI‐MS using nanoparticles, CE‐fluorescence using tapered optical fiber, CZE separation of pesticide residues in water samples with acid‐assisted on‐column preconcentration and CE‐LIF to detect neurotransmitter amino acids and carbamathione in brain microdialysis samples. Novel methods for the separation and profiling of various proteins and large nucleic fragments are described in 4 consecutive papers grouped in part III. Featured articles include: Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography (( 10.1002/elps.201000405 )) Bundled capillary electrophoresis using microstructured fibres ( 10.1002/elps.201000442 )) Two‐dimensional separation system by on‐line hyphenation of capillary isoelectric focusing with pressurized capillary electrochromatography for peptide and protein mapping ( 10.1002/elps.201000419 )) Microchip electrophoresis of N‐glycans on serpentine separation channels with asymmetrically tapered turns ( 10.1002/elps.201000461 ))     

Fast parallel detection of feline panleukopenia virus DNA by multi‐channel microchip electrophoresis with programmed step electric field strength

A multi‐channel microchip electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge‐coupled device camera were used to simultaneously detect the separations in three parallel channels using laser‐induced fluorescence detection. The parallel separations of a 100‐bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M_r = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single‐run and one‐step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel electrophoresis. The proposed multi‐channel microchip electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease‐related DNA fragments in parallel with high speed, throughput, and accuracy.     

Separation of carbohydrates on electrophoresis microchips with controlled electrolysis

This study reports the separation of fructose, galactose, glucose, lactose and sucrose on glass microchip electrophoresis (ME) devices using a microfluidic platform adapted with external reservoirs for controlling the electrolysis phenomenon. The connections between external reservoirs and microfluidic platform were performed by saline bridges created using silicone tubing filled with BGE. The separation conditions were optimized and the best results were achieved using a BGE containing 75 mmol/L NaOH and 15 mmol/L trisodium phosphate. Electrophoretic separations were monitored using a capacitively coupled contactless conductivity detection system. The controlled electrolysis has successfully allowed the application of a higher voltage on the separation channel promoting the baseline separation of five carbohydrates within 180 s with great run‐to‐run repeatability (RSD < 1%). The achieved efficiencies ranged from 45 000 ± 6000 to 70 000 ± 3000 plates/m demonstrating a performance better than ME devices without controlled electrolysis. The proposed system offered good linearity from 1 to 10 mmol/L and LODs between 150 and 740 μmol/L. The use of external tubes for controlling the electrolysis phenomenon on ME devices has solved common problems associated to run‐to‐run repeatability and analytical reliability required for routine and quantitative analysis.     

Continuous dielectrophoretic separation of particles in a spiral microchannel

Particle separation is a fundamental operation in the areas of biology and physical chemistry. A variety of force fields have been used to separate particles in microfluidic devices, among which electric field may be the most popular one due to its general applicability and adaptability. So far, however, electrophoresis‐based separations have been limited primarily to batchwise processes. Dielectrophoresis (DEP)‐based separations require in‐channel micro‐electrodes or micro‐insulators to produce electric field gradients. This article introduces a novel particle separation technique in DC electrokinetic flow through a planar double‐spiral microchannel. The continuous separation arises from the cross‐stream dielectrophoretic motion of particles induced by the non‐uniform electric field inherent to curved channels. Specifically, particles are focused by DEP to one sidewall of the first spiral, and then dielectrophoretically deflected toward the other sidewall of the second spiral at a particle‐dependent rate, leading to focused particle streams along different flow paths. This DEP‐based particle separation technique is demonstrated in an asymmetric double‐spiral microchannel by continuously separating a mixture of 5/10 μm particles and 3/5 μm particles.     

Microfluidic preparative free‐flow isoelectric focusing in a triangular channel: System development and characterization

A preparative scale free‐flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular‐shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion‐permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50×70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free‐flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule‐heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370±20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.     

Negative dielectrophoresis-based particle separation by size in a serpentine microchannel

Dielectrophoresis has been widely used to focus, trap, concentrate, and sort particles in microfluidic devices. This work demonstrates a continuous separation of particles by size in a serpentine microchannel using negative dielectrophoresis. Depending on the magnitude of the turn-induced dielectrophoretic force, particles travelling electrokinetically through a serpentine channel either migrate toward the centerline or bounce between the two sidewalls. These distinctive focusing and bouncing phenomena are utilized to implement a dielectrophoretic separation of 1 and 3 μm polystyrene particles under a DC-biased AC electric field of 880 V/cm on average. The particle separation process in the entire microchannel is simulated by a numerical model.     

Effect of quasi‐interpenetrating network of polyacrylamide and poly(N,N‐dimethylacrylamide) on separation of dsDNA fragments and basic protein using CE

Quasi‐interpenetrating network (quasi‐IPN) of linear polyacrylamide (LPA) with low molecular mass and poly(N,N‐dimethylacrylamide) (PDMA), which is shown to uniquely combine the superior sieving ability of LPA with the coating ability of PDMA, has been synthesized for application in dsDNA and basic protein separation by CE. The performance of quasi‐IPN on dsDNA separation was determined by polymer concentration, electric field strength, LPA molecular masses and different acrylamide (AM) to N,N‐dimethylacrylamide (DMA) ratio. The results showed that all fragments in Φ×174/HaeIII digest were achieved with a 30 cm effective capillary length at –6 kV at an appropriate polymer solution concentration in bare silica capillaries. Furthermore, EOF measurement results showed that quasi‐IPN exhibited good capillary coating ability, via adsorption from aqueous solution, efficiently suppressing EOF. The effect of the buffer pH values on the separation of basic proteins was investigated in detail. The separation efficiencies and analysis reproducibility demonstrated the good potentiality of quasi‐IPN matrix for suppressing the adsorption of basic proteins onto the silica capillary wall. In addition, when quasi‐IPN was used both as sieving matrix and dynamic coating in bare silica capillaries, higher peak separation efficiencies, and better migration time reproducibility were obtained.     

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild‐type DNA. Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K‐ras oncogene. LDR‐FSCE enabled electrophoretic resolution of these 19 LDR‐FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR‐FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K‐ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR, and detection all on one platform.     

Travelling‐wave ion mobility and negative ion fragmentation of high‐mannose N‐glycans

The isomeric structure of high‐mannose N‐glycans can significantly impact biological recognition events. Here, the utility of travelling‐wave ion mobility mass spectrometry for isomer separation of high‐mannose N‐glycans is investigated. Negative ion fragmentation using collision‐induced dissociation gave more informative spectra than positive ion spectra with mass‐different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling‐wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N‐glycans released from the well‐characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross‐sectional data, details of the negative ion collision‐induced dissociation spectra of all resolved isomers are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

Simulation and Evaluation of the Silicon-Micromachined Columns for Gas Chromatography

In this paper, the impact of airflow velocity and pressure in the two popular configuration gas chromatography (GC) column channels (the serpentine channel and the spiral channel) was simulated and evaluated using ANSYS dynamic analysis. The simulated result of airflow velocity and pressure distribution in the Gas chromatography (GC) channel shows that the impact in the spiral channel corner is smaller than that in the serpentine channel corner. So, the spiral channel columns were fabricated based on the MEMS technology and the stationary phase OV‐1 was coated using a dynamic procedure. The separation performance of the 3 m non‐polar GC column shows perfect separation efficiency for the non‐polar components, the microfabricated GC column yields 7100 theoretical plate, and the analysis time is less than 200 s.     

Disposable polyester-toner electrophoresis microchips for DNA analysis

Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215,000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.     

System-oriented dispersion models of general-shaped electrophoresis microchannels

This paper presents a system-oriented model for analyzing the dispersion of electrophoretic transport of charged analyte molecules in a general-shaped microchannel, which is represented as a system of serially connected elemental channels of simple geometry. Parameterized analytical models that hold for analyte bands of virtually arbitrary initial shape are derived to describe analyte dispersion, including both the skew and broadening of the band, in elemental channels. These models are then integrated to describe dispersion in the general-shaped channel using appropriate parameters to represent interfaces of adjacent elements. This lumped-parameter system model offers orders-of-magnitude improvement in computational efficiency over full numerical simulations, and is verified by results from experiments and numerical simulations. The model is used to perform a systematic parametric study of serpentine channels consisting of a pair of complementary turn microchannels, and the results indicate that dispersion in a particular turn can contribute to either an increase or decrease of the overall band broadening. The efficiency and accuracy of the system model is further demonstrated by its application to general-shaped channels that occur in practice, including a serpentine channel with multiple complementary turns and a multi-turn spiral-shaped channel. The results indicate that our model is an accurate and efficient simulation tool useful for designing optimal electrophoretic separation microchips.     

A continuous-flow,microfluidic fraction collection device

A microfluidic device is presented that performs electrophoretic separation coupled with fraction collection. Effluent from the 3.5 cm separation channel was focused via two sheath flow channels into one of seven collection channels. By holding the collection channels at ground potential and varying the voltage ratio at the two sheath flow channels, the separation effluent was directed to either specific collection channels, or could be swept past all channels in a defined time period. As the sum of the voltages applied to the two sheath flow channels was constant, the electric field remained at 275 V/cm during the separation regardless of the collection channel used. The constant potential in the separation channel allowed uninterrupted separation for late-migrating peaks while early-migrating peaks were being collected. To minimize the potential for carryover between fractions, the device geometry was optimized using a three-level factorial model. The optimum conditions were a 22.5° angle between the sheath flow channels and the separation channel, and a 350 μm length of channel between the separation outlet and the fraction channels. Using these optimized dimensions, the device performance was evaluated by separation and fraction collection of a fluorescently labeled amino acid mixture. The ability to fraction collect on a microfluidic platform will be especially useful during automated or continuous operation of these devices or to collect precious samples.     

Development and optimization of an integrated PDMS based‐microdialysis microchip electrophoresis device with on‐chip derivatization for continuous monitoring of primary amines

An all‐PDMS on‐line microdialysis‐microchip electrophoresis with on‐chip derivatization and electrophoretic separation for near real‐time monitoring of primary amine‐containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene‐2,3,‐dicarboxyaldehyde/cyanide (NDA/CN^?), and the separation was performed using a 15‐cm serpentine channel. The analytes were detected using LIF detection.     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

Poly(vinyl alcohol)-coated microfluidic devices for high-performance microchip electrophoresis

The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA-coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a distorted sample zone is injected using uncoated channels the application of PVA-coated channels results in an improved shape of the sample zone with less band broadening. Applying PVA-coated microchips for the separation of amines labeled with Alexa Fluor 350 even sub-second separations, utilizing a separation length of only 650 microm, could be obtained, while this was not possible using uncoated devices. By using PVA-coated devices rather than an uncoated chip a threefold increase in separation efficiencies could be observed. As the electroosmotic flow (EOF) was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Besides improved separation performance another important feature of the PVA-coated channels was the suppressed adsorption of fluorescent compounds in repetitive runs which results in an improved robustness and detection sensitivity. Applying PVA-coated channels, rinsing or etching steps could be omitted while this was necessary for a reliable operation of uncoated devices.     

Enhanced Microchip Electrophoresis of Neurotransmitters on Glucose Oxidase Modified Poly(dimethylsiloxane) Microfluidic Devices

In this paper, glucose oxidase (GOx) was employed to construct a functional film on the poly(dimethylsiloxane) (PDMS) microfluidic channel surface and apply to perform electrophoresis coupled with in‐channel electrochemical detection. The film was formed by sequentially immobilizing poly(diallyldimethylammonium chloride) (PDDA) and GOx to the microfluidic channel surface via layer‐by‐layer (LBL) assembly. A group of neurotransmitters (5‐hydroxytryptamine, 5‐HT; dopamine, DA; epinephrine, EP; dobuamine, DBA) as a group of separation model was used to evaluate the effect of the functional PDMS microfluidic devices. Electroosmotic flow (EOF) in the modified PDMS microchannel was well suppressed compared with that in the native one. Experimental conditions were optimized in detail. As expected, these analytes were efficiently separated within 110 s in a 3.7 cm long separation channel and successfully detected at a single carbon fiber electrode. Good performances were attributed to the decreased EOF and the interactions of analytes with the immobilized GOx on the PDMS surface. The theoretical plate numbers were 2.19×10⁵, 1.89×10⁵, 1.76×10⁵, and 1.51×10⁵ N/m at the separation voltage of 1000 V with the detection limits of 1.6, 2.0, 2.5 and 6.8 μM (S/N=3) for DA, 5‐HT, EP and DBA, respectively. In addition, the modified PDMS channels had long‐term stability and excellent reproducibility.