Continuous-flow separation of nanoparticles by electrostatic sieving at a micro-nanofluidic interface

Continuous-flow separation of nanoparticles (NPs) (15 and 39 nm) is demonstrated based on electrostatic sieving at a micro-nanofluidic interface. The interface is realized in a poly(dimethylsiloxane) device with a nanoslit of 525 nm laterally spanning the microfluidic channel (aspect ratio of 540:1). Within this nanoslit, the Debye layers overlap and generate an electrostatic sieve. This was exploited to selectively deflect and sort NPs with a sorting purity of up to 97%. Because of the continuous-flow operation, the sample is continuously fed into the device, immediately separated, and the parameters can be adapted in real time. For bioanalytical purposes, we also demonstrate the deflection of proteins (longest axis 6.8 nm). The continuous operation mode and the general applicability of this separation concept make this method a valuable addition to the current Lab-on-a-Chip devices for continuous sorting of NPs and macromolecules.     

An electrokinetic/hydrodynamic flow microfluidic CE-ESI-MS interface utilizing a hydrodynamic flow restrictor for delivery of samples under low EOF conditions

A hydrodynamic flow restrictor (HDR) that is used to combine electrokinetic and hydrodynamic flow streams has been fabricated in a microfluidic channel by laser micromachining. Combining electrokinetic and hydrodynamic flow streams is challenging in microfluidic devices, because the hydrodynamic flow often overpowers the electrokinetic flow, making it more difficult to use low electroosmotic flow in the electrokinetic portion of the system. The HDR has been incorporated into a capillary electrophoresis-mass spectrometry interface that provides continuous introduction of a make-up solution and negates the hydrodynamic backpressure in the capillary electrophoresis channel to the extent that low EOF can be utilized. Moreover, the hydrodynamic backpressure is sufficiently minimized to allow coatings that minimize EOF to be used in the electrokinetically driven channel. Such coatings are of great importance for the analysis of proteins and other biomolecules that adsorb to charged surfaces.     

Design of an interface to allow microfluidic electrophoresis chips to drink from the fire hose of the external environment

An interface design is presented that facilitates automated sample introduction into an electrokinetic microchip, without perturbing the liquids within the microfluidic device. The design utilizes an interface flow channel with a volume flow resistance that is 0.54-4.1 x 10(6) times lower than the volume flow resistance of the electrokinetic fluid manifold used for mixing, reaction, separation, and analysis. A channel, 300 microm deep, 1 mm wide and 15-20 mm long, was etched in glass substrates to create the sample introduction channel (SIC) for a manifold of electrokinetic flow channels in the range of 10-13 microm depth and 36-275 microm width. Volume flow rates of up to 1 mL/min were pumped through the SIC without perturbing the solutions within the electrokinetic channel manifold. Calculations support this observation, suggesting a leakage flow to electroosmotic flow ratio of 0.1:1% in the electrokinetic channels, arising from 66-700 microL/min pressure-driven flow rates in the SIC. Peak heights for capillary electrophoresis separations in the electrokinetic flow manifold showed no dependence on whether the SIC pump was on or off. On-chip mixing, reaction and separation of anti-ovalbumin and ovalbumin could be performed with good quantitative results, independent of the SIC pump operation. Reproducibility of injection performance, estimated from peak height variations, ranged from 1.5-4%, depending upon the device design and the sample composition.     

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.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity

Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.     

Recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices

AC electrokinetics is a generic term that refers to an induced motion of particles and fluids under nonuniform AC electric fields. The AC electric fields are formed by application of AC voltages to microelectrodes, which can be easily integrated into microfluidic devices by standard microfabrication techniques. Moreover, the magnitude of the motion is large enough to control the mass transfer on the devices. These advantages are attractive for biomolecular analysis on the microfluidic devices, in which the characteristics of small space and microfluidics have been mainly employed. In this review, I describe recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices. The applications include fluid pumping and mixing by AC electrokinetic flow, and manipulation of biomolecules such as DNA and proteins by various AC electrokinetic techniques. Future prospects for highly functional biomolecular analysis on microfluidic devices with the aid of AC electrokinetics are also discussed.     

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.     

Detection of nitrated benzodiazepines by indirect laser-induced fluorescence detection on a microfluidic device

Recently there has been concern regarding the use of flunitrazepam and other low-dose benzodiazepines in drug-facilitated sexual assault. These compounds are placed in drinks of unsuspecting victims and produce a sedative effect with anterorgrade amnesia. Chip-based microfluidic systems can provide a quick and disposable procedure for the detection of flunitrazepam and other nitrated benzodiazepines used in these crimes. This paper describes the application of indirect quenching of cyanine dye (Cy5) for detection of nitrated benzodiazepines. The separation is performed on a microfluidic device with a separation channel 8 cm long and 50 microm wide and utilizes indirect fluorescence detection with 635 nm laser excitation. The optimization of the separation using micellar electrokinetic chromatography with organic modifiers is described. A borate buffer containing 2.6 microM Cy5 dye, 15 mM sodium dodecyl sulphate (SDS) and 20% methanol is used. Complete separation of four target drugs occurs in under 2 min with limits of detection in the low microg/ml range. Overall the method provides a rapid and simple analysis for the presence of nitrated benzodiazepines in beverages and other similar preparations.     

Integrated continuous microfluidic liquid-liquid extraction

We describe continuous flow liquid-liquid phase separation in microfluidic devices based on capillary forces and selective wetting surfaces. Effective liquid-liquid phase separation is achieved by using a thin porous fluoropolymer membrane that selectively wets non-aqueous solvents, has average pore sizes in the 0.1-1 microm range, and has a high pore density for high separation throughput. Pressure drops throughout the microfluidic network are modelled and operating regimes for the membrane phase separator are determined based on hydrodynamic pressure drops and capillary forces. A microfluidic extraction device integrating mixing and phase separation is realized by using silicon micromachining. Modeling of the phase separator establishes the operating limits. The device is capable of completely separating several organic-aqueous and fluorous-aqueous liquid-liquid systems, even with high fractions of partially miscible compounds. In each case, extraction is equivalent to one equilibrium extraction stage.     

Continuous-Flow Nanoparticle Trapping Driven by Hybrid Electrokinetics in Microfluidics

We introduce herein an efficient microfluidic approach for continuous transport and localized collection of nanoparticles via hybrid electrokinetics, which delicately combines linear and nonlinear electrokinetics driven by a composite DC-biased AC voltage signal. The proposed technique utilizes a simple geometrical structure, in which one or a series of metal strips serving as floating electrode (FE) are attached to the substrate surface and arranged in parallel between a pair of coplanar driving electrodes (DE) in a straight microchannel. On application of a DC-biased AC electric field across the channel, nanoparticles can be transported continuously by DC bulk electroosmotic flow, and then trapped selectively onto the metal strips due to AC-field induced-charge electrokinetic (ICEK) phenomenon, which behaves as counter-rotating micro-vortices around the ideally polarizable surfaces of FE. Finite-element simulation is carried out by coupling the dual-frequency electric field, flow field and sample mass transfer in sequence, for guiding a practical design of the microfluidic nanoparticle concentrator. With the optimal device geometry, the actual performance of the technique is investigated with respect to DC bias, AC voltage amplitude, and field frequency by using both latex nanospheres (∼500 nm) and BSA molecules (∼10 nm). Our experimental observation indicates nanoparticles are always enriched into a narrow bright band on the surface of each FE, and a horizontal concentration gradient even emerges in the presence of multiple metal strips, which therefore permits localized analyte enrichment. The proposed trapping method is supposed to guide an elaborate design of flexible electrokinetic frameworks embedding FE for continuous-flow analyte manipulation in modern microfluidic systems.     

Improvements on the electrokinetic injection technique for microfluidic chips

This paper presents a T-form electrokinetic injection system for the discrete time-based loading and dispensing of samples of variable-volume in a microfluidic chip. A novel push-pull effect is produced during the loading and dispensing processes by the application of an appropriate control voltage distribution. The experimental and numerical results show that this push-pull loading technique produces compact sample plugs and hence improves the detection resolution of the microfluidic device. The injection system is integrated with a microflow switch, and a suitable voltage control scheme is proposed to guide the sample to the desired outlet port such that the microfluidic device can function as a microdispenser. The time-based variable-volume T-form injection method presented in this study is performed using a compact geometry and a simple control scheme and can be readily integrated with other microfluidic devices to form a microfluidic system capable of continuous monitoring and analysis of bioreactions in the life science and biochemistry fields.     

Surface enhanced Raman spectroscopy for microfluidic pillar arrayed separation chips

Numerous studies have addressed the challenges of implementing miniaturized microfluidic platforms for chemical and biological separation applications. However, the integration of real time detection schemes capable of providing valuable sample information under continuous, ultra low volume flow regimes has not fully been addressed. In this report we present a chip based chromatography system comprising of a pillar array separation column followed by a reagent channel for passive mixing of a silver colloidal solution into the eluent stream to enable surface enhanced Raman spectroscopy (SERS) detection. Our design is the first integrated chip based microfluidic device to combine pressure driven separation capability with real time SERS detection. With this approach we demonstrate the ability to collect distinctive SERS spectra with or without complete resolution of chromatographic bands. Computational fluidic dynamic (CFD) simulations are used to model the diffusive mixing behaviour and velocity profiles of the two confluent streams in the microfluidic channels. We evaluate the SERS spectral band intensity and chromatographic efficiency of model analytes with respect to kinetic factors as well as signal acquisition rates. Additionally, we discuss the use of a pluronic modified silver colloidal solution as a means of eliminating contamination generally caused by nanoparticle adhesion to channel surfaces.     

Optimal configuration of capillary electrophoresis microchip with expansion chamber in separation channel

This study develops a novel capillary electrophoresis (CE) microfluidic device featuring a conventional cross-form injection system and an expansion chamber located at the inlet of the separation channel. The combined injection system/expansion chamber arrangement is designed to deliver a high-quality sample band into the separation channel such that the detection performance of the device is enhanced. Numerical simulations are performed to investigate the electrokinetic transport processes in the microfluidic device and to establish the optimal configuration of the expansion chamber. The results indicate that an expansion chamber with an expansion ratio of 2.5 and an expansion length of 500 microm delivers a sample plug with the correct shape and orientation. With this particular configuration, the peak intensities of the sample are sharp and clearly distinguishable in the detection region of the separation channel. Therefore, this configuration is well suited for capillary electrophoresis applications which require a highly sensitive resolution of the sample plug. The novel CE microfluidic device developed in this study has an exciting potential for use in high-performance, high-throughput chemical analysis applications and in many other applications throughout the field of micro-total-analysis-systems.     

Analysis of electrokinetic transport of a spherical particle in a microchannel

Electrokinetically driven microfluidic devices that are used for biological cell/particle manipulation (e.g., cell sorting, separation) involve electrokinetic transport of these particles in microchannels whose dimension is comparable with particles' size. This paper presents an analytical study on electrokinetic transport of a charged spherical particle in a charged parallel-plate microchannel. Under the thin electric double-layer assumption, solutions in closed-form solutions for the particle velocity and disturbed electrical and fluid velocity fields are obtained for plane-symmetric (along the channel centerline) and asymmetric (off the channel centerline) motions of a sphere in a parallel-plate microchannel. The effects of relative particle size and eccentricity (i.e., off the centerline distance) on a particle's translational and rotational velocities are analyzed.     

Microchip free-flow electrophoresis on glass substrate using laser-printing toner as structural material

In this work, a microfluidic free-flow electrophoresis device, obtained by thermal toner transferring on glass substrate, is presented. A microdevice can be manufactured in only 1 h. The layout of the microdevice was designed in order to improve the fluidic and electrical characteristics. The separation channel is 8 microm deep and presents an internal volume of 1.42 microL. The deleterious electrolysis effects were overcome by using a system that isolates the electrolysis products from the separation channel. The Joule heating dissipation in the separation channel was found to be very efficient up to a current density of 8.83 mA/mm(2) that corresponds to a power dissipation per unit volume of running electrolyte of 172 mW/microL. Promising results were obtained in the evaluation of the microdevices for the separation of ionic dyes. The microfluidic device can be used for a continuous sample pretreatment step for micro total analysis system.     

A split microchannel design and analytical model to compensate for electroosmotic instabilities in micro-separations

Organic polymers offer many advantages as materials for the construction of microfluidic devices but suffer frequently from the limitation that the electrodynamic flow they support can exhibit considerable instability. This article describes a split-channel microfluidic device that can be used to compensate for changes in electroosmotic flow. The design of the separation system divides an analyte plug after injection between two separation channels of differing length. The two channels are later recombined for single point detection, eliminating the need for a scanning optical detection system. The utility of this simple design lies in the fact that the migration time of any analyte can be referenced to its twin in the parallel separation channel. This eliminates the need for a separate electroosmotic marker and allows mobilities measured in multiple devices to be compared quantitatively. Using a model adopted from the literature, the data from the split channel system can be used to precisely account for the drift that characterizes electrophoretic separations made in a polymer chip. The relative standard deviations of the analyte mobilities measured for replicate runs on multiple devices were reduced from values as high as 20% to ca. 1% RSD. This internal standardization procedure also appears to address other sources of drift in the electroosmotic flow (EOF) supported by the polymer microchannel, eliminating the need for careful monitoring of either the temperature or reservoir pH between separation runs.     

Fabrication of paper-based microfluidic sensors by printing

A novel method for the fabrication of paper-based microfluidic diagnostic devices is reported; it consists of selectively hydrophobizing paper using cellulose reactive hydrophobization agents. The hydrophilic–hydrophobic contrast of patterns so created has excellent ability to control capillary penetration of aqueous liquids in paper channels. Incorporating this idea with digital ink jet printing techniques, a new fabrication method of paper-based microfluidic devices is established. Ink jet printing can deliver biomolecules and indicator reagents with precision into the microfluidic patterns to form bio-chemical sensing zones within the device. This method thus allows the complete sensor, i.e. channel patterns and the detecting chemistries, to be fabricated only by two printing steps. This fabrication method can be scaled up and adapted to use high speed, high volume and low cost commercial printing technology. Sensors can be fabricated for specific tests, or they can be made as general devices to perform on-demand quantitative analytical tasks by incorporating the required detection chemistries for the required tasks.     

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.     

Monitoring spatial distribution of ethanol in microfluidic channels by using a thin layer of cholesteric liquid crystal

Monitoring spatial distribution of chemicals in microfluidic devices by using traditional sensors is a challenging task. In this paper, we report utilization of a thin layer of cholesteric liquid crystal for monitoring ethanol inside microfluidic channels. This thin layer can be either a polymer dispersed cholesteric liquid crystal (PDCLC) layer or a free cholesteric liquid crystal (CLC) layer separated from the microfluidic device by using a thin film of PDMS. They both show visible colorimetric responses to 4% of ethanol solution inside the microfluidic channels. Moreover, the spatial distribution of ethanol inside the microfluidic channel can be reflected as a color map on the CLC sensing layers. By using this device, we successfully detected ethanol produced from fermentation taking place inside the microfluidic channel. These microfluidic channels with embedded PDCLC or embedded CLC offer a new sensing solution for monitoring volatile organic compounds in microfluidic devices.