On‐chip protein isoelectric focusing using a photoimmobilized pH gradient†

An immobilized pH gradient was directly constructed on the inner wall of a microfluidic chip channel by photoimmobilizing focused carrier ampholytes onto the wall. A mixture of carbonic anhydrase, myoglobin, and trypsin inhibitor was successfully isoelectric‐focused and separated with good linearity between the pI values of proteins and the location of the focused bands. Furthermore, coating methods for the resistance of protein nonselective adsorption and simultaneously for pH gradient photocoupling were screened. The PEG‐silane coating method was found to be better than the cross‐linked polyacrylamide coating and aminosilane modification methods. Finally, based on the open tubular column mode of carrier ampholytes’ immobilization and effective antiadsorption coating, the immobilized pH gradient was reused and the chip was recycled for the first time. By virtue of its remarkable features including simplicity, convenience, high efficiency of protein enrichment and separation, and potential for coupling site‐selective IEF with other analytical or separation techniques, this novel method promises to be useful in several applications related with zwitterionic biomolecules.     

Microfluidic system for dielectrophoretic separation based on a trapezoidal electrode array

This paper presents a novel microfluidic device for dielectrophoretic separation based on a trapezoidal electrode array (TEA). In this method, particles with different dielectric properties are separated by the device composed of the TEA for the dielectrophoretic deflection of particles under negative dielectrophoresis (DEP) and poly(dimethylsiloxane)(PDMS) microfluidic channel with a sinuous and expanded region. Polystyrene microparticles are exposed to an electric field generated from the TEA in the microfluidic channel and are dielectrophoretically focused to make all of them line up to one sidewall. When these particles arrive at the region of another TEA for dielectrophoretic separation, they are separated having different positions along the perpendicular direction to the fluid flow due to their different dielectrophoretic velocities. To evaluate the separation process and performance, both the effect of the flow rate on dielectrophoretic focusing and the influence of the number of trapezoidal electrodes on dielectrophoretic separation are investigated. Now that this method utilizes the TEA as a source of negative DEP, non-specific particle adhering to the electrode surface can be prevented; conventional separation approaches depending on the positive DEP force suffer from this problem. In addition, since various particle types are continuously separated, this method can be easily applicable to the separation and analysis of various dielectric particles with high particle recovery and selectivity.     

Mucin (MUC5AC) expression by lung epithelial cells cultured in a microfluidic gradient device

We have developed a microfluidic gradient device for controlling mucin gene expression of NCI-H292 epithelial cells derived from lung tissues. We hypothesized that gradient profiles would control mucin gene expression of lung epithelial cells. However, it was not possible to generate various stable gradient profiles using conventional culture methods. To address this limitation, we used a microfluidic gradient device to create various gradient profiles (i.e. non-linear, linear, and flat) in a temporal and spatial manner. NCI-H292 lung epithelial cells were exposed to concentration gradients of epidermal growth factor in a microfluidic gradient device with continuous medium perfusion. We demonstrated an effect of gradient profiles on mucin expression of lung epithelial cells cultured in the microfluidic gradient device. It was revealed that NCI-H292 lung epithelial cells exposed to the flat gradient profile of the epidermal growth factor exhibited high expression of mucin as compared with cells exposed to non-linear and linear gradient profiles. Therefore, this microfluidic gradient device could be a potentially useful tool for regulating the mucin expression of lung epithelial cells exposed to chemokine gradient profiles.     

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.     

Micro-/nanofluidic device for tunable generation of a concentration gradient: application to Caenorhabditis elegans chemotaxis

In this study, we propose a novel micro-/nanofluidic device that can generate a chemical concentration gradient using a parallel nanochannel as gradient generator. This device is easy to fabricate, showing high reproducibility. Its main feature is the multiple-nanochannel-based gradient generator, which permits the diffusion of small molecules and tunably generates concentration gradients. The nanopattern for the nanochannels can be rapidly and easily fabricated by wrinkling a diamond-like carbon thin film which is deposited on a polydimethylsiloxane substrate; the generation of the concentration gradient can be adjusted by controlling the dimensions of the nanochannels. The developed gradient generator is embedded into a microfluidic device to study chemotaxis in the nematode Caenorhabditis elegans, which has a highly developed chemosensory system and can detect a wide variety of chemical molecules. This device shows good performance for rapid analysis of C. elegans chemotaxis under sodium chloride stimuli.

Field-effect flow control in a polydimethylsiloxane-based microfluidic system.

The application of the field-effect for direct control of electroosmosis in a polydimethylsiloxane (PDMS)-based microfluidic system, constructed on a silicon wafer with a 2.0 microm electrically insulating layer of silicon dioxide, is demonstrated. This microfluidic system consists of a 2.0 cm open microchannel fabricated on a PDMS slab, which can reversibly adhere to the silicon wafer to form a hybrid microfluidic device. Aside from mechanically serving as a robust bottom substrate to seal the channel and support the microfluidic system, the silicon wafer is exploited to achieve field-effect flow control by grounding the semiconductive silicon medium. When an electric field is applied through the channel, a radial electric potential gradient is created across the silicon dioxide layer that allows for direct control of the zeta potential and the resulting electroosmotic flow (EOF). By configuring this microfluidic system with two power supplies at both ends of the microchannel, the applied electric potentials can be varied for manipulating the polarity and the magnitude of the radial electric potential gradient across the silicon dioxide layer. At the same time, the longitudinal potential gradient through the microchannel, which is used to induce EOF, is held constant. The results of EOF control in this hybrid microfluidic system are presented for phosphate buffer at pH 3 and pH 5. It is also demonstrated that EOF control can be performed at higher solution pH of 6 and 7.4 by modifying the silicon wafer surface with cetyltrimethylammonium bromide (CTAB) prior to assembly of the hybrid microfluidic system. Results of EOF control from this study are compared with those reported in the literature involving the use of other microfluidic devices under comparable solution conditions.     

Epithelial‐to‐mesenchymal transition of human lung alveolar epithelial cells in a microfluidic gradient device

Epithelial‐to‐mesenchymal transition (EMT), a process in which epithelial cells undergo phenotypic transitions to fibrotic cells, is induced by stimulants including transforming growth factor‐beta1 (TGF‐β1). In the present study, we developed a microfluidic gradient device to reproduce EMT in A549 human lung alveolar epithelial cells in response to TGF‐β1 gradients. The device was directly mounted on the cells that had grown in cell culture plates and produced a stable concentration gradient of TGF‐β1 with negligible shear stress, thereby providing a favorable environment for the anchorage‐dependent cells. A549 cells elongated with the characteristic spindle‐shaped morphological changes with upregulation of alpha‐smooth muscle actin, a mesenchyme marker, in a gradient‐dependent manner, suggestive of EMT progression. We observed that at higher TGF‐β1 concentrations ranging from 5 to 10 ng/mL, the cultures in the microfluidic device allowed to quantitatively pick up subtle differences in the EMT cellular response as compared with plate cultures. These results suggest that the microfluidic gradient device would accurately determine the optimal concentrations of TGF‐β1, given that epithelial cells of different tissue origins greatly vary their responses to TGF‐β1. Therefore, this microfluidic device could be a powerful tool to monitor EMT induced by a variety of environmental stresses including cigarette smoke with high sensitivity.     

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 ))     

Specific Transport of Target Molecules by Motor Proteins in Microfluidic Channels

Direct transport powered by motor proteins can alleviate the challenges presented by miniaturization of microfluidic systems. There have been several recent attempts to build motor‐protein‐driven transport systems based on simple capturing or transport mechanisms. However, to achieve a multifunctional device for practical applications, a more complex sorting/transport system should be realized. Herein, the proof of concept of a sorting device employing selective capture of distinct target molecules and transport of the sorted molecules to different predefined directions is presented. By combining the bottom‐up functionality of biological systems with the top‐down handling capabilities of micro‐electromechanical systems technology, highly selective molecular recognition and motor‐protein‐based transport is integrated in a microfluidic channel network.     

Sampling and analysis of nanoparticles with cold fibre SPME device

A new approach is described to capture nano‐size aerosols on internally‐cooled micro tubing of the solid‐phase microextraction (SPME) device followed by convenient introduction of the collected analytes into analytical instrument. Particles were generated using an aerosol formation by homogeneous nucleation of an organic vapor, and subsequent growth to nano‐size particles by coagulation of decanedioic acid, bis[2‐ethylhexyl] ester (DEHS). The approach was validated by using carbon dioxide‐cooled micro tubing to collect the nanosize DEHS particles followed by analyses on GC‐flame ionization detector (FID). Particle size ranged from 150 to 590 nm. Temperature difference between the SPME device and DEHS particles mixture created a temperature gradient and resulted in thermophoretic effect that was determining the extraction rate. SPME device was cooled to as low as –75°C, while the DEHS remained close to room temperature. Several aspects of nanoparticle sampling were tested to demonstrate the principle of the sampling approach. These included the effects of thermal gradient, sample flow rate, sampling time, CO₂ delivery mode (constant coolant delivery vs. constant temperature), and particle size. Results were normalized to measure particulate concentrations using direct sampling with PTFE filters. Nanoparticle extractions of DEHS mass were proportional to sampling time. Normalized mass of DEHS extracted increased with increase in temperature gradient and with increase of the cross flow velocity. Preliminary results indicate that the variation of heat transfer boundary layer caused by the variation in the cross flow velocity produce self‐compensating effect at constant coolant delivery, indicating that this approach could be used for field determinations including the time‐weighted average sampling of nanoparticles. Thus, it may be possible to develop simple device based on this concept for field applications.     

Surface‐Tension‐Confined Microfluidics and Their Applications

This article is a brief overview of the emerging microfluidic systems called surface‐tension‐confined microfluidic (STCM) devices. STCM devices utilize surface energy that can control the movement of fluid droplets. Unlike conventional poly(dimethylsiloxane)‐based microfluidics which confine the movement of fluids by three‐dimensional (3D) microchannels, STCM systems provide two‐dimensional (2D) platforms for microfluidics. A variety of STCM devices have been prepared by various micro‐/nanofabrication strategies. Advantages of STCM devices over conventional microfluidics are significant reduction of energy consumption during device operation, facile introduction of fluids onto 2D microchannels without the use of a micropump, increased flow rate in a special type of STCM device, among others. Thus, STCM devices can be excellent alternatives for certain areas in microfluidics. In this Minireview, fabrication methods, operating modes, and applications of STCM devices are introduced.     

Detection of unlabeled particles in the low micrometer size range using light scattering and hydrodynamic 3D focusing in a microfluidic system

In this paper, we describe a microfluidic device composed of integrated microoptical elements and a two-layer microchannel structure for highly sensitive light scattering detection of micro/submicrometer-sized particles. In the two-layer microfluidic system, a sample flow stream is first constrained in the out-of-plane direction into a narrow sheet, and then focused in-plane into a small core region, obtaining on-chip three-dimensional (3D) hydrodynamic focusing. All the microoptical elements, including waveguides, microlens, and fiber-to-waveguide couplers, and the in-plane focusing channels are fabricated in one SU-8 layer by standard photolithography. The channels for out-of-plane focusing are made in a polydimethylsiloxane (PDMS) layer by a single cast using a SU-8 master. Numerical and experimental results indicate that the device can realize 3D hydrodynamic focusing reliably over a wide range of Reynolds numbers (0.5 < Re < 20). Polystyrene particles of three sizes (2, 1, and 0.5 μm) were measured in the microfluidic device with integrated optics, demonstrating the feasibility of this approach to detect particles in the low micrometer size range by light scattering detection.     

An electrochemically driven poly(dimethylsiloxane) microfluidic actuator: oxygen sensing and programmable flows and pH gradients

We describe the fabrication and performance of an integrated microelectrochemical reactor-a design possessing utility for multiple applications that include electrochemical sensing, the generation and manipulation of in-channel microfluidic pH gradients, and fluid actuation and flow. The device architecture is based on a three-electrode electrochemical cell design that incorporates a Pt interdigitated array (IDA) working (WE), a Pt counter (CE), and Ag pseudo-reference (RE) electrodes within a microfluidic network in which the WE is fully immersed in a liquid electrolyte confined in the channels. The microchannels are made from a conventional poly(dimethylsiloxane)(PDMS) elastomer, which serves also as a thin gas-permeable membrane through which gaseous reactants in the external ambient environment are supplied to the working electrode by diffusion. Due to the high permeability of oxygen through PDMS, the microfluidic cell supports significantly (>order of magnitude) higher current densities in the oxygen reduction reaction (ORR) than those measured in conventional (quiescent) electrochemical cells for the same electrode areas. We demonstrate in this work that, when operated at constant potential under mass transport control, the device can be utilized as a membrane-covered oxygen sensor, the response of which can be tuned by varying the thickness of the PDMS membrane. Depending on the experimental conditions under which the electrochemical ORR is performed, the data establish that the device can be operated as both a programmable pH gradient generator and a microfluidic pump.     

Electrochemical Analysis of Glycoprotein Samples Prepared on a Pneumatically‐controlled Microfluidic Device

In this work, we designed and manufactured a microfluidic device for isolation and purification of glycoprotein samples. The conceived sample preparation device was fabricated in polycarbonate by micro‐milling. The flow control and the fluid dosage into the micro‐channels was solved by equipping the device with integrated pneumatic valves. The biochemical functionality was provided by beaded support modified by molecules with affinity to glycoproteins which was stacked inside the micro‐channel reminiscent of packed affinity columns used in glycoprotein lectin assays. Unlabeled glycoproteins, namely fetuin, asialofetuin, and prostate‐specific antigen, were voltammetrically analyzed using catalytic peak H at silver amalgam electrode.     

SERS-based immunoassay using a gold array-embedded gradient microfluidic chip

Here we report the development of a programmable and fully automatic gold array-embedded gradient microfluidic chip that integrates a gradient microfluidic device with gold-patterned microarray wells. This device provides a convenient and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay platform for cancer biomarkers. We used hollow gold nanospheres (HGNs) as SERS agents because of their highly sensitive and reproducible characteristics. The utility of this platform was demonstrated by the quantitative immunoassay of alpha-fetoprotein (AFP) model protein marker. Our proposed SERS-based immunoassay platform has many advantages over other previously reported SERS immunoassay methods. The tedious manual dilution process of repetitive pipetting and inaccurate dilution is eliminated with this process because various concentrations of biomarker are automatically generated by microfluidic gradient generators with N cascade-mixing stages. The total assay time from serial dilution to SERS detection takes less than 60 min because all of the experimental conditions for the formation and detection of immunocomplexes can be automatically controlled inside the exquisitely designed microfluidic channel. Thus, this novel SERS-based microfluidic assay technique is expected to be a powerful clinical tool for fast and sensitive cancer marker detection.     

外加场分离技术与微流控技术联用在微纳尺度物质分离中的研究进展

微纳尺度物质的分离和分选在精准医学、材料科学和单细胞分析等研究中至关重要。精准、高效和快速的分离微纳尺度物质能够为癌症的早期诊断、生物样品检测和细胞筛选提供重要帮助,其中基于外加场分离技术的分离微纳尺度物质因可以对微纳尺度物质高效在线分离和分选,被广泛应用于微纳米颗粒、外泌体以及生物细胞的分离工作中,而目前多数外加场分离技术存在装备繁琐和样品消耗大等问题。微流控技术是一种通过制作微通道和微流控芯片操纵微小流体对微纳尺度样品组分进行分离的技术,因具有快速检测、高通量、在线分离、集成性高、成本低等优势现被应用于微纳尺度物质分离分析中,是一种微纳尺度物质分离的有效方法,通过在微流控芯片上设计不同的通道及外部配件提高主动场对微纳尺度物质分离效率。外加场分离技术与微流控技术联用可以实现微纳尺度物质的无损、高效、在线分离。该综述主要概述了近年来在微流控芯片上依托流动场、电场、磁场及声场等外加场分离技术来提高对微纳尺度物质分离效率的研究现状,并将各个外力场对单细胞、微颗粒等微纳尺度物质的分离进行分类介绍,总结各自的优缺点及发展应用,最后展望了外加场分离技术与微流控技术联用在应用于癌细胞的早期筛查、精确分离微尺度物质领域的未来发展前景,并提出联用技术的优势和未来应用等。     

Mixed‐mode chromatography integrated with high‐performance liquid chromatography for protein analysis and separation: Using bovine serum albumin and lysozyme as the model target

A type of mixed‐mode chromatography was integrated with high‐performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed‐mode column, the silica beads were activated with γ‐(2,3‐epoxypropoxy)‐propytrimethoxysilane and coupled with 4‐mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.     

Arrayed isoelectric focusing using photopatterned multi‐domain hydrogels

Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from hours in traditional benchtop IEF to minutes, the enclosed devices hinder post‐separation access to the sample for downstream analysis. The two‐layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post‐separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self‐indexed photomasks for alignment‐free fabrication of multi‐domain hydrogels. We leverage this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.     

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization,Direct RBC Contact,and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC‐derived ATP. These studies incorporate on‐chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC‐EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum‐black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8±0.2 nM NO per endothelial cell (average±SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil‐induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92±0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non‐adherent cell lines) and incorporate other detection schemes.     

Quality by design: A systematic and rapid liquid chromatography and mass spectrometry method for eprosartan mesylate and its related impurities using a superficially porous particle column

The present work describes the systematic development of a robust, precise, and rapid reversed‐phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality‐by‐design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters—column temperature, gradient time, and flow rate using eight experiments—to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused‐core C₁₈ (100 mm × 2.1 mm, 2.6 μm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry.