Analysis of amino acids and proteins using a poly(methyl methacrylate) microfluidic system

Plastic microchips are very promising analytical devices for the high-speed analysis of biological compounds. However, due to its hydrophobicity, their surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in a significant uncontrolled adsorption on the channel walls. This paper describes the migration of fluorescence-labeled amino acids and proteins using the poly(methyl methacrylate) microchip. A cationic starch derivative significantly decreases the adsorption of analytes on the channel walls. The migration time of the analytes was related to their molecular weight and net charge or pI of the analytes. FITC-BSA migrated within 2 min, and the theoretical plate number of the peak reached 480,000 plates/m. Furthermore, proteins with a wide range of pI values and molecular weights migrated within 1 min using the microchip.     

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.     

Protein adsorption in static microsystems: effect of the surface to volume ratio

A numerical model for the adsorption kinetics of proteins on the walls of a microchannel has been developed using the finite element method (FEM) to address the coupling with diffusion phenomena in the restricted microchannel volume. Time evolutions of the concentration of one species are given, both in solution and on the microchannel walls. The model illustrates the adsorption limitation sometimes observed when the microdimensions of these systems induce a global depletion of the bulk solution. A new non-dimensional parameter is introduced to predict the final value of the coverage of any microsystem under static adsorption. A working curve and a criteria (h/K[Gamma](max) > 10) are provided in order to choose, for given adsorption characteristics, the value of the volume-to-surface ratio (i.e. the channel height h) avoiding depletion effects on the coverage (relative coverage greater than 90% of the theoretical one). Simulations were compared with confocal microscopy measurements of IgG antibody adsorption on the walls of a PET microchannel. The fit of the model to the experimental data show that the adsorption is under apparent kinetic control.     

Thermoplastic microchannel fabrication using carbon dioxide laser ablation

We report the procedures of machining microchannels on Vivak co-polyester thermoplastic substrates using a simple industrial CO(2) laser marker. To avoid overheating the substrates, we develop low-power marking techniques in nearly anaerobic environment. These procedures are able to machine microchannels at various aspect ratios. Either straight or serpent channel can be easily marked. Like the wire-embossed channel walls, the ablated channel surfaces become charged after alkaline hydrolysis treatment. Stable electroosmotic flow in the charged conduit is observed to be of the same order of magnitude as that in fused silica capillary. Typical dynamic coating protocols to alter the conduit surface properties are transferable to the ablated channels. The effects of buffer acidity on electroosmotic mobility in both bare and coated channels are similar to those in fused silica capillaries. Using video microscopy we also demonstrate that this device is useful in distinguishing the electrophoretic mobility of bare and latex particles from that of functionalized ones.     

Two simple and rugged designs for creating microfluidic sheath flow

A simple design capable of 2-dimensional hydrodynamic focusing is proposed and successfully demonstrated. In the past, most microfluidic sheath flow systems have often only confined the sample solution on the sides, leaving the top and bottom of the sample stream in contact with the floor and ceiling of the channel. While relatively simple to build, these designs increase the risk of adsorption of sample components to the top and bottom of the channel. A few designs have been successful in completely sheathing the sample stream, but these typically require multiple sheath inputs and several alignment steps. In the designs presented here, full sheathing is accomplished using as few as one sheath input, which eliminates the need to carefully balance the flow of two or more sheath inlets. The design is easily manufactured using current microfabrication techniques. Furthermore, the sample and sheath fluid can be subsequently separated for recapture of the sample fluid or re-use of the sheath fluid. Designs were demonstrated in poly(dimethylsiloxane) (PDMS) using soft lithography and poly(methyl methacrylate) (PMMA) using micromilling and laser ablation.     

Separation of platelets from whole blood using standing surface acoustic waves in a microchannel

Platelet separation from blood is essential for biochemical analyses and clinical diagnosis. In this article, we propose a method to separate platelets from undiluted whole blood using standing surface acoustic waves (SSAWs) in a microfluidic device. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with interdigitated transducer (IDT) electrodes patterned on a piezoelectric substrate. To avoid shear-induced activation of platelets, the blood sample flow was hydrodynamically focused by introducing sheath flow from two side-inlets and pressure nodes were designed to locate at side walls. By means of flow cytometric analysis, the RBC clearance ratio from whole blood was found to be over 99% and the purity of platelets was close to 98%. Conclusively, the present technique using SSAWs can directly separate platelets from undiluted whole blood with higher purity than other methods.     

Electrokinetic molecular separation in nanoscale fluidic channels

This report presents a study of electrokinetic transport in a series of integrated macro- to nano-fluidic chips that allow for controlled injection of molecular mixtures into high-density arrays of nanochannels. The high-aspect-ratio nanochannels were fabricated on a Si wafer using interferometric lithography and standard semiconductor industry processes, and are capped with a transparent Pyrex cover slip to allow for experimental observations. Confocal laser scanning microscopy was used to examine the electrokinetic transport of a negatively charged dye (Alexa 488) and a neutral dye (rhodamine B) within nanochannels that varied in width from 35 to 200 nm with electric field strengths equal to or below 2000 V m-1. In the negatively charged channels, nanoconfinement and interactions between the respective solutes and channel walls give rise to higher electroosmotic velocities for the negatively charged dye than for the neutral dye, towards the negative electrode, resulting in an anomalous separation that occurs over a relatively short distance (<1 mm). Increasing the channel widths leads to a switch in the electroosmotic transport behavior observed in microscale channels, where neutral molecules move faster because the negatively charged molecules are slowed by the electrophoretic drag. Thus a clear distinction between "nano-" and "microfluidic" regimes is established. We present an analytical model that accounts for the electrokinetic transport and adsorption (of the neutral dye) at the channel walls, and is in good agreement with the experimental data. The observed effects have potential for use in new nano-separation technologies.     

Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry

A novel microfabricated device for isoelectric focusing (IEF) incorporating an optimized electrospray ionization (ESI) tip was constructed on polycarbonate plates using laser micromachining. The IEF microchip incorporated a separation channel (50 micro x 30 micro x 16 cm), three fluid connectors, and two buffer reservoirs. Electrical potentials used for IEF focusing and electrospray were applied through platinum electrodes placed in the buffer reservoirs, which were isolated from the separation channel by porous membranes. Direct ESI-mass spectrometry (MS) using electrosprays produced directly from a sharp emitter "tip" on the microchip was evaluated. The results indicated that this design can produce a stable electrospray and that performance was further improved and made more flexible with the assistance of a sheath gas and sheath liquid. Error analysis of the spectral data showed that the standard deviation in signal intensity for an analyte peak was less than approximately 5% over 3 h. The production of stable electrosprays directly from microchip IEF device represents a step towards easily fabricated microanalytical devices. Microchannel IEF separations of protein mixtures were demonstrated for uncoated polycarbonate microchips. Direct microchannel IEF-ESI-MS was demonstrated using the microfabricated chip with an ion-trap mass spectrometer for characterization of protein mixtures.     

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.     

Multiple flow profiles for two-phase flow in single microfluidic channels through site-selective channel coating

An approach to control two-phase flow systems in a poly(dimethylsiloxane) (PDMS) microfluidic device using spatially selective surface modification is demonstrated. Side-by-side flows of ethanol?:?water solutions containing different polymers are used to selectively modify both sides of a channel by laminar flow patterning. Introduction of air pockets during modification allows for control over the length of the channel section that is modified. This approach makes it possible to achieve slug flow and side-by-side flow of water : 1-octanol simultaneously within the same PDMS channel, without the need of additional structural elements. A key finding is that conditioning of the PDMS channels with 1-octanol before polymer deposition is crucial to achieving stable side-by-side flows.     

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.     

Quantitative Online Detection of Low‐Concentrated Drugs via a SERS Microfluidic System

Herein, quantitative online monitoring of concentration fluctuations of different interesting drugs, namely, the phenothiazine promethazine as well as the anti‐cancer agent mitoxantrone via surface enhanced Raman scattering assay based on a microfluidic device is demonstrated. With the applied liquid/liquid two‐phase‐segmented flow system we succeed in preventing the adhesion of nanoparticle aggregates to the channel walls which is necessary for a quantitative analysis. Even after repeated cycles no carry‐over due to sedimentation of colloid particles is observed. To the best of our knowledge these are the first measurements applying a combination of a microfluidic device with SERS detection for quantitative online monitoring of fluctuations in drug concentrations over hours without use of aggressive chemicals for rinsing the chip surfaces prior to each measurement.     

Controlling adsorption and release of drug and small molecules by organic functionalization of mesoporous materials

A series of mesoporous nanosphere materials that are functionalized with various terminal and bridging organic groups were synthesized. They have improved adsorption capacity and different release properties for drug and small molecules. The materials contained terminal vinyl, 3-mercaptopropyl, 3-aminopropyl, and secondary amine functional groups and bridging ethane, ethene, and benzene groups within their mesopore channel walls. The samples containing mercaptopropyl and vinyl groups showed greater adsorption capacity and better controlled release behavior for rhodamine 6G molecules. On the other hand, mesoporous matrices containing amine functional groups showed higher adsorption capacity and better release properties for ibuprofen molecules. Further studies revealed that the bridging organic groups in the mesopore channel walls also improved the adsorption capacity and release properties of the materials compared to the corresponding samples containing no bridging organic groups. Such improved adsorption and controlled release properties of molecules by simple changes of functional groups on mesoporous materials are important for the development of nanomaterial drug delivery vehicles and for controlled release of drugs over long time periods at specific targeted sites in the body. By judicious choice of organic groups and by systematic design and synthetic approaches, nanoporous materials having different adsorption capacity and release properties for many other drug molecules can also be achieved.     

Timing controllable electrofusion device for aqueous droplet-based microreactors

This paper describes an electrofusion device for controlling the precise moment of fusion between droplets by applying an electric field. This device allows (i) accurate determination of the start of chemical/biological reactions, (ii) minimum contact of reactants with channel walls--eliminating surface absorption problems, (iii) easy fabrication and (iv) continuous observation of initiated reaction. We demonstrated the fusion of beta-galactosidase and fluorescein di-beta-D-galactopyranoside (FDG) droplets, and observed the enzymatic reaction using fluorescence microscopy. In addition, sequential fusion of pico-litre droplets was also accomplished.     

Foam flow through porous media

There are two parts to the interaction of foam with porous media. How the foam interacts with the surface and the flow within the substrate, which is the focus of this review. Flow-through porous media has been investigated experimentally with the main focus in literature being on enhanced oil recovery and remediation. Recently, investigation of the flow of foam through a deformable substrate for dishwashing application has led to the development of mathematical models. It has been proposed that foam flow through pore channels is similar to the behaviour observed within microchannels. Meaning that to investigate the effects these properties have on foam flow it is best to observe them within a model channel then build up to a 3D structure of interlinking channels to resemble porous media. In this review, it is highlighted that a large amount of work is needed in understanding the interaction of foam and/or liquid within porous networks. Methods that can be applied to better represent foam and liquid flow in porous media are discussed within this review, including both using microchannels to simulate individual pores and using these systems to build up to a 3D structure of interlinking pores. In addition, more advanced imaging techniques to observe the flow through porous materials are discussed, including computed tomography scanning nuclear magnetic resentence and confocal microscopy. There is still more work required to fully understand the flow within porous media, including observing the affect of dead-end pores, closed loops and rough channel walls have on the flow.     

Pretreatment of Corn Stover with Twin-Screw Extrusion Followed by Enzymatic Saccharification

Pretreatment of biomass before subjecting it to enzyme saccharification is crucial with regards to facilitating access of enzyme to biomass. Extrusion, as a continuous and cost-effective pretreatment method, combines heating with high shear and mixing opening cell walls at the microscopic scale, thus largely increasing the specific surface area (SSA) of biomass for enzyme adsorption. The objective of this study was to examine the effect of extrusion as a pretreatment method and the underlying factors ruling the improvement of sugar yields. The optimum glucose, xylose, and combined sugar recoveries were 48.79%, 24.98%, and 40.07%, respectively, at 27.5% moisture content and 80 rpm screw speed. These yields were 2.2, 6.6, and 2.6 times higher than those for untreated corn stover. X-ray diffraction analysis showed that the crystallinity index was not a good indicator of sugar yield. However, scanning electron microscopy showed that the cellulose network was exposed due to the destruction of the lignin sheath. The Langmuir adsorption model was shown to be an effective tool for the estimation of the SSA of corn stover. The SSA of pretreated samples was significantly amplified over the control, revealing that extrusion can open the cell wall at the microscopic scale, which was especially favorable on sugar yields.     

Three-dimensional (3D) hydrodynamic focusing for continuous sampling and analysis of adherent cells

A simple three-dimensional (3D) hydrodynamic focusing microfluidic device integrated with continuous sampling, rapid dynamic lysis, capillary electrophoretic (CE) separation and detection of intracellular content is presented. One of the major difficulties in microfluidic cell analysis for adherent cells is that the cells are prone to attaching to the channel surface. To solve this problem, a cross microfluidic chip with three sheath-flow channels located on both sides of and below the sampling channel was developed. With the three sheath flows around the sample solution-containing cells, the formed soft fluid wall prevents the cells from adhering to the channel surface. Labeled cells were 3D hydrodynamically focused by the sheath-flow streams and smoothly introduced into the cross-section one by one. The introduction of sheath-flow streams not only ensured single-cell sampling but avoided blockage of the sampling channel by adherent cells as well. The maximum rate for introduction of individual cells into the separation channel was about 151 cells min(-1). With electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 400 ms at the entry of the channel by sodium dodecylsulfate (SDS) added in the sheath-flow solution. The microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single HepG2 cells. The average analysis throughput of ROS and GSH in single cells was 16-18 cells min(-1).     

Molecular transport in narrow channels

The micro-hydrodynamic method is applid to the calculation of the molecular transport in narrow channels in case of capillary condensation, at the flow anisotropy resulted from the potential of the wall surface and/or of boundary vapor and fluid phases. The mechanisms of molecular transport in the one-phase and two-phase fluid flows as a dependence of fluid density and adsorption potential of channel walls are discussed.     

Single-step fabrication and characterization of photonic crystal biosensors with polymer microfluidic channels

A method for simultaneously integrating label-free photonic crystal biosensor technology into microfluidic channels by a single-step replica molding process is presented. By fabricating both the sub-micron features of the photonic crystal sensor structure and the >10 microm features of a flow channel network in one step at room temperature on a plastic substrate, the sensors are automatically self-aligned with the flow channels, and patterns of arbitrary shape may be produced. By measuring changes in the resonant peak reflected wavelength from the photonic crystal structure induced by changes in dielectric permittivity within an evanescent field region near its surface, detection of bulk refractive index changes in the fluid channel or adsorption of biological material to the sensor surface is demonstrated. An imaging detection instrument is used to characterize the spatial distribution of the photonic crystal resonant wavelength, gathering thousands of independent sensor readings within a single fluid channel.     

Polymer adsorption onto a micro-sphere from optical tweezers electrophoresis

We explore the design and operation of an optical-tweezers electrophoresis apparatus to resolve polymer adsorption dynamics onto a single micro-sphere in a micro-fluidic environment. Our model system represents a broader class of micro-fluidic electrophoresis experiments for biosensing and fundamental colloid and surface science diagnostics. We track the adsorption of 100 kDa poly(ethylene oxide) homopolymer onto a colloidal silica sphere that is optically trapped in a crossed parallel-plate micro-channel. The adsorption dynamics are probed on the ～1 μm particle length scale with ～1 s temporal resolution. Because the particle electrophoretic mobility and channel electro-osmotic flow are exquisitely sensitive to the polymer layer hydrodynamic thickness, particle dynamics can be complicated by polymer adsorption onto the micro-channel walls. Nevertheless, using experiments and a theoretical model of electro-osmotic flow in channels with non-uniform wall ζ-potentials, we show that such influences can be mitigated by adopting a symmetrical flow configuration. The equilibrium hydrodynamic layer thickness of 100 kDa poly(ethylene oxide) on colloidal silica is ～10 nm at polymer concentrations ?10 ppm (weight percent), with the dynamics reflecting polymer solution concentration, flow rate, and polydispersity.