Profiling pH gradients across nanocapillary array membranes connecting microfluidic channels

Nanocapillary array membranes (NCAMs), comprised of thin (d approximately 5-10 microm) nuclear track-etched polycarbonate sheets containing approximately 10(8) cm(-2) nearly parallel nanometer-diameter capillaries, may act to gate fluid transport between microfluidic channels to effect, for example, sample collection. There is interest in H+-transport across these NCAMs because there is significant practical interest in being able to process analyte-containing samples under different pH conditions in adjacent layers of an integrated microfluidic circuit and because protons, with their inherently high mobility, present a challenge in separating microfluidic environments with different properties. To evaluate the capability of NCAMs to support pH gradients, the proton transport properties of NCAMs were studied using laser scanning confocal fluorescence microscopy (LSCFM). Spatiotemporal maps of [H+] in microfluidic channels adjacent to the NCAMs yield information regarding diffusive and electrokinetic transport of protons. The NCAMs studied here are characterized by a positive zeta potential, zeta > 0, so at small nanocapillary diameters, the overlap of electrical double layers associated with opposite walls of the nanocapillary establish an energy barrier for either diffusion or electrokinetic transport of cations through the nanometer-diameter capillaries due to the positive charge on the nanocapillary surface. Proton transfer through an NCAM into microchannels is reduced for pore diameters, d < or = 50 nm and ionic strengths I < or = 50 mM, while for large pore diameters or solution ionic strengths, the incomplete overlap of electric double layer allows more facile ionic transfer across the membranes. These results establish the operating conditions for the development of multilevel integrated nanofluidic/microfluidic architectures which can support multidimensional chemical analysis of mass-limited samples requiring sequential operations to be implemented at different pH values.     

Direct immobilization of Fab' in nanocapillaries for manipulating mass-limited samples

Interfacing nanoscale elements into a microfluidic device enables a new range of fluidic manipulations. Nanocapillary array membranes (NCAMs), consisting of thin (5 microm < d < 20 microm) membranes containing arrays of nanometer diameter (10 nm < a < 500 nm) pores, are a convenient method of interfacing vertically separated microchannels in microfluidic devices that allow the external control of analyte transport between microfluidic channels. To add functionality to these nanopores beyond simple fluid transport, here we incorporate an antibody-based molecular recognition element onto the pore surface that allows selective capture, purification, and release of specific analytes from a mixture. The pores are fabricated by electroless plating of gold into the nanopores of an NCAM (Au-NCAM). An antibody is then immobilized on the Au-NCAM via gold-thiol chemistry as a thiolated fragment of antigen-binding (Fab') prepared by direct digestion of the antibody followed by reduction of the disulfide linkage on the hinge region. The successful immobilization and biological activity of the resultant Fab' through this protocol is verified on planar gold by fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. Selective capture and release of human insulin is verified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The relative mass spectral peak intensities for insulin versus nonantigenic peptides increase more than 20-fold after passing through the Fab'-Au-NCAM relative to the control Au-NCAM. The affinity-tagged Au-NCAM can be incorporated into microfluidic devices to allow the concentration, capture, and characterization of analytes in complex mixtures with high specificity.     

Electrokinetically driven fluidic transport in integrated three-dimensional microfluidic devices incorporating gold-coated nanocapillary array membranes

Electrokinetically driven fluid transport was evaluated within three-dimensional hybrid nanofluidic-microfluidic devices incorporating Au-coated nanocapillary array membranes (NCAMs). Gold NCAMs, prepared by electroless gold deposition on polymeric track-etched membranes, were susceptible to gas bubble formation if the interfacial potential difference exceeded approximately 2 V along the length of the gold region. Gold membranes were etched to yield 250 mum wide coated regions that overlap the intersection of two orthogonal microfluidic channels in order to minimize gas evolution. The kinetics of electrolysis of water at the opposing ends of the gold region was modeled and found to be in satisfactory agreement with experimental measurements of the onset of gas bubble formation. Conditions to achieve electrokinetic injection across Au-coated NCAMs were identified, with significant reproducible injections being possible for NCAMs modified with this relatively thin gold stripe. Continuous gold films led to suppressed injections and to a variety of ion enrichment/depletion effects in the microfluidic source channel. The suppression of injections was understood through finite element modeling which revealed the presence of a significant electrophoretic velocity component in opposition to electroosmotic flow at the edge of the Au-dielectric regions.     

Ionic conductance of nanopores in microscale analysis systems: where microfluidics meets nanofluidics

In this tutorial review we illustrate the origin and dependence on various system parameters of the ionic conductance that exists in discrete nanochannels as well as in nanoporous separation and preconcentration units contained as hybrid configurations, membranes, packed beds, or monoliths in microscale liquid phase analysis systems. A particular complexity arises as external electrical fields are superimposed on internal chemical and electrical potential gradients for tailoring molecular transport. It is demonstrated that the variety of geometries in which the microfluidic/nanofluidic interfaces are realized share common, fundamental features of coupled mass and charge transport, but that phenomena behind the key steps in a particular application can be significantly tuned, depending on the morphology of a material. Thus, the understanding of morphology-related transport in internal and external electrical potential gradients is critical to the performance of a device. This addresses a variety of geometries (slits, channels, filters, membranes, random or regular networks of pores, etc.) and applications, e. g., the gating, sensing, preconcentration, and separation in multifunctional miniaturized devices. Inherently coupled mass and charge transport through ion-permselective (charge-selective) microfluidic/nanofluidic interfaces is analyzed with a stepwise-added complexity and discussed with respect to the morphology of the charge-selective spatial domains. Within this scenario, the electrostatics and electrokinetics in microfluidic and nanofluidic channels, as well as the electrohydrodynamics evolving at microfluidic/nanofluidic interfaces, where microfluidics meets nanofluidics, define the platform of central phenomena.     

Design and fabrication of a multilayered polymer microfluidic chip with nanofluidic interconnects via adhesive contact printing

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.     

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.     

Sample transport and electrokinetic injection in a microchip device for chemical cytometry

Sample transport and electrokinetic injection bias are well characterized in capillary electrophoresis and simple microchips, but a thorough understanding of sample transport on devices combining electroosmosis, electrophoresis, and pressure-driven flow is lacking. In this work, we evaluate the effects of electric fields from 0 to 300 V/cm, electrophoretic mobilities from 10(-4) to 10(-6) cm(2)/Vs, and pressure-driven fluid velocities from 50 to 250 μm/s on sample injection in a microfluidic chemical cytometry device. By studying a continuous sample stream, we find that increasing electric field strength and electrophoretic mobility result in improved injection and that COMSOL simulations accurately predict sample transport. The effects of pressure-driven fluid velocity on injection are complex, and relative concentration values lie on a surface defined by pressure-driven flow rates. For high-mobility analytes, this surface is flat, and sample injection is robust despite fluctuations in flow rate. For lower mobility analytes, the surface becomes steeper, and injection depends strongly on pressure-driven flow. These results indicate generally that device design must account for analyte characteristics and specifically that this device is suited to high-mobility analytes. We demonstrate that for a suitable pair of peptides fluctuations in injection volume are correlated; electrokinetic injection bias is minimized; and electrophoretic separation is achieved.     

The effect of photomask resolution on separation efficiency on microfabricated devices

Separation quality on glass microfluidic devices fabricated from photomasks of different optical resolutions was compared by measuring the dispersion (apparent diffusion) coefficients of a set of standard compounds separated on these devices. Currently, the channel manifolds of most microfluidic devices are patterned using chrome photomasks. A much cheaper, more robust alternative to chrome photomasks are laser photoplotted masks. The primary disadvantage to using laser photoplots is that the optical resolution of these masks is not as high as that of chrome masks, and this feature increases the side-wall roughness of etched channel manifolds patterned using such masks. The increased wall roughness may affect the fluid flow within the channels and, therefore, the separation quality. To determine the effect of increased sidewall channel roughness, microchip channel manifolds were patterned in soda lime glass using a chrome photomask and laser photoplots printed at resolutions of 620, 1240, 3100 and 6200 dots per centimetre (dpc). Separations were performed on these devices using dilute solutions of fluorescently labeled amino acids. The peak variances of the amino acids were calculated at increasing distances down the separation channel and plotted as a function of migration time. From this plot, dispersion coefficients of the analytes were measured. This allowed for a reliable, relatively easy, direct separation analysis among microchips fabricated from the various photomasks. After multiple separations using microchips fabricated from each resolution mask, we found that the change in sidewall surface roughness did not significantly affect the dispersion coefficients measured, and thus the separation quality. The lower mask resolution limit, rather, was governed by the fidelity to which the mask could capture the original CAD design.     

A simple mechanism for reliable particle sorting in a microdevice with combined electroosmotic and pressure-driven flow

Selective transport and sorting of particles in microfluidic devices by electroosmosis is complicated due to superposition of uncontrolled hydrodynamic pressure contributions on the electroosmotic force. In this paper, we present a microfluidic concept for the reliable and simple separation and sorting of particles in a microchip by electroosmosis combined with pressure-driven flow. The presented device allows fluid quantities to be switched and particles to be sorted within a channel manifold using only a single power supply with fixed voltage and an electric switch. Consequently, chip operation and fluid switching procedure are greatly simplified compared to a situation, in which several independent power sources are used for flow balancing, as is the common procedure. With the triple-T channel design presented, backpressure flow disturbing the electrokinetic fluid and particle separation process is eliminated by introducing controlled opposed hydrodynamic flow of buffer from side channels. This pressure-driven flow is generated on-chip by setting up differences in the reservoir pressures in a defined manner. A detailed flow analysis based on the equivalence of fluid flow and electric current is performed and the conditions for reliable chip function are worked out.     

Mass transport in nanofluidic devices

Nanofluidics is a recent appearing research field, introduced in 1995 as an analogue of the field of microfluidics, and has been becoming popular in the past few years. The proximity of the channel dimension, the Debye length, and the size of biomolecules such as DNA and proteins gives the unique features of nanofluidic devices. Of various unique properties of the nanofluidics, mass transport in nanochannel plays determining roles in fundamental reaches and practical applications of nanofluidic device. Thus, much work including numerical and experimental researches has been performed to investigate the mass transport behaviors in nanofluidic devices. This review summarizes the fabrication technologies for nanofluidic devices, the mass transport behaviors in nanochannel, and their applications in bioanalysis. The main focus will be laid on the effects of nanochannel size and surface charge on mass transport including electrokinetic transport of charged analytes, diffusion of electric neutral molecules, ionic current rectification, concentration polarization, nonlinear electrokinetic flow at the micro-nanofluidic interfaces.     

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10-100 fold in comparison with conventional IEF.     

Temperature-controlled flow switching in nanocapillary array membranes mediated by poly(N-isopropylacrylamide) polymer brushes grafted by atom transfer radical polymerization

We report actively controlled transport that is thermally switchable and size-selective in a nanocapillary array membrane (NCAM) prepared by grafting poly(N-isopropylacrylamide) (PNIPAAm) brushes onto the exterior surface of a Au-coated polycarbonate track-etched membrane. A smooth Au layer on the membrane surface, which is key to obtaining a uniform polymer film, was prepared by thermal evaporation of approximately 50 nm Au on both exterior surfaces. After evaporation, the inner diameter of the pore is reduced slightly, but the NCAM retains a narrow pore size distribution. PNIPPAm brushes with 10-30 nm (dry film) thickness were grafted onto the Au surface through surface-initiated atom transfer radical polymerization (ATRP) using a disulfide initiator, (BrC(CH3)2COO(CH2)11S)2. Molecular transport through the PNIPAAm polymer brush-modified NCAMs was investigated by real-time fluorescence measurements using fluorescein isothiocyanate (FITC)-labeled dextrans ranging from 4.4 to 282 kDa in membranes with variable initial pore diameters (80, 100, and 200 nm) and different PNIPAAm thicknesses. Manipulating the temperature of the NCAM through the PNIPAAm lower critical solution temperature (LCST) causes large, size-dependent changes in the transport rates. Over specific ranges of probe size, transport is completely blocked below the LCST but strongly allowed above the LCST. The combination of the highly uniform PNIPAAm brush and the monodisperse pore size distribution is critical in producing highly reproducible switching behavior. Furthermore, the reversible nature of the switching raises the possibility of using them as actively controlled filtration devices.     

Effects of liquid conductivity differences on multi-component sample injection, pumping and stacking in microfluidic chips

As an increasing number of processes are being integrated into Lab-on-a-chip devices, there is an increasing need for flexible and accurate sample manipulation techniques for effective transport and separation. Conductivity differences between running buffer and analyte samples can arise as a product of on-chip processing, or by design. The two situations studied here are sample pumping (where bulk transport is increased and separation of charged analytes is delayed using a relatively high conductivity sample), and sample stacking (where bulk transport is decreased and separation of charged analytes is expedited using a relatively low conductivity sample). A recently developed dynamic loading method for on-chip sample injection in a straight-cross channel configuration is applied here to both pumping and stacking cases. A key characteristic of the dynamic loading method is the ability to inject samples of high concentration density and uniformity of any length. By employing the conductivity differences alone, the effectiveness of either sample transport or sample separation are shown to improve over the uniform conductivity case. Then it is demonstrated that increasing the sample length, through dynamic loading, greatly increases the effectiveness of sample pumping, evidenced in an eight-fold increase in peak height as well as a decrease in total sample length at a downstream detector. Dynamic loading in the sample stacking case was shown to also increase peak intensity height (three-fold) in rapid separations. These results demonstrate that the dynamic loading technique, used in conjunction with strategic conductivity differences, significantly extends the capabilities of microfluidic chips.     

Reduction in sample injection bias using pressure gradients generated on chip

Sample injection in microchip-based capillary zone electrophoresis (CZE) frequently rely on the use of electric fields which can introduce differences in the injected volume for the various analytes depending on their electrophoretic mobilities and molecular diffusivities. While such injection biases may be minimized by employing hydrodynamic flows during the injection process, this approach typically requires excellent dynamic control over the pressure gradients applied within a microfluidic network. The current article describes a microchip device that offers this needed control by generating pressure gradients on-chip via electrokinetic means to minimize the dead volume in the system. In order to realize the desired pressure-generation capability, an electric field was applied across two channel segments of different depths to produce a mismatch in the electroosmotic flow rate at their junction. The resulting pressure-driven flow was then utilized to introduce sample zones into a CZE channel with minimal injection bias. The reported injection strategy allowed the introduction of narrow sample plugs with spatial standard deviations down to about 45 μm. This injection technique was later integrated to a capillary zone electrophoresis process for analyzing amino acid samples yielding separation resolutions of about 4–6 for the analyte peaks in a 3 cm long analysis channel.     

Microfluidic device for capillary electrochromatography-mass spectrometry

A novel microfabricated device that integrates a monolithic polymeric separation channel, an injector, and an interface for electrospray ionization-mass spectrometry detection (ESI-MS) was devised. Microfluidic propulsion was accomplished using electrically driven fluid flows. The methacrylate-based monolithic separation medium was prepared by photopolymerization and had a positively derivatized surface to ensure electroosmotic flow (EOF) generation for separation of analytes in a capillary electrochromatography (CEC) format. The injector operation was optimized to perform under conditions of nonuniform EOF within the microfluidic channels. The ESI interface allowed hours of stable operation at the flow rates generated by the monolithic column. The dimensions of one processing line were sufficiently small to enable the integration of 4-8 channel multiplexed structures on a single substrate. Standard protein digests were utilized to evaluate the performance of this microfluidic chip. Low- or sub-fmol amounts were injected and detected with this arrangement.     

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.     

Recent advances in single-molecule detection on micro- and nano-fluidic devices

Single-molecule detection (SMD) allows static and dynamic heterogeneities from seemingly equal molecules to be revealed in the studies of molecular structures and intra- and inter-molecular interactions. Micro- and nanometer-sized structures, including channels, chambers, droplets, etc., in microfluidic and nanofluidic devices allow diffusion-controlled reactions to be accelerated and provide high signal-to-noise ratio for optical signals. These two active research frontiers have been combined to provide unprecedented capabilities for chemical and biological studies. This review summarizes the advances of SMD performed on microfluidic and nanofluidic devices published in the past five years. The latest developments on optical SMD methods, microfluidic SMD platforms, and on-chip SMD applications are discussed herein and future development directions are also envisioned.     

A microfluidic device for performing pressure-driven separations

Microchannels in microfluidic devices are frequently chemically modified to introduce specific functional elements or operational modalities. In this work, we describe a miniaturized hydraulic pump created by coating selective channels in a glass microfluidic manifold with a polyelectrolyte multilayer (PEM) that alters the surface charge of the substrate. Pressure-driven flow is generated due to a mismatch in the electroosmotic flow (EOF) rates induced upon the application of an electric field to a tee channel junction that has one arm coated with a positively charged PEM and the other arm left uncoated in its native state. In this design, the channels that generate the hydraulic pressure are interconnected via the third arm of the tee to a field-free analysis channel for performing pressure-driven separations. We have also shown that modifications in the cross-sectional area of the channels in the pumping unit can enhance the hydrodynamic flow through the separation section of the manifold. The integrated device has been demonstrated by separating Coumarin dyes in the field-free analysis channel using open-channel liquid chromatography under pressure-driven flow conditions.     

Optical trapping in micro- and nanoconfinement systems: Role of thermo-fluid dynamics and applications

In this mini-review, recent advances on the role of a focused laser in micro- and nanofluidic systems is widely introduced with special interest in thermo-fluid dynamical aspects and their importance in optical manipulation. As a brief introduction to microfluidic systems, we describe the advantages and challenges of the use of micro- and nanoscale confinement in optical trapping, as well as standard fabrication techniques for micro- and nanofluidic systems. From thermo-fluid dynamical viewpoints, various phenomena that accompany a laser irradiation to fluidic devices, are explained in detail. These phenomena can affect the optical trapping of target materials significantly, and are classified into two categories: one that induces the fluid flow around the target and another that directly acts on it as an external force. These classes are reviewed by shedding light on some recent cutting-edge researches for optical manipulation. Some applications using thermo-fluid dynamics in microfluidic systems for the measurement of optical forces and for the separation, measurement, and detection of target materials are also introduced.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.