Electrochemical response and separation in cyclic electric field-flow fractionation

Electric field-flow fractionation (EFFF) is a separation technique that couples a lateral electric field with axial Poiseuille flow to separate particles on the basis of size and/or mobility. In unidirectional EFFF, the field rapidly decreases in time due to charging of the double layer. The field strength could be increased by performing EFFF with cyclic electric fields. In cyclic electric field-flow fractionation (CEFFF), a periodic voltage, which can be either sinusoidal or square-wave, is applied in the lateral direction. In this paper, we measure the electrochemical response of CEFFF, i.e., the current-time response for a given time-dependent voltage and then utilize this electrochemical response in a transport model to predict separation. The CEFFF device studied here comprises two gold-coated glass plates separated by a spacer. The transient current profiles are measured for a step change and cyclic square-shaped voltage. The current profile is compared with the equivalent circuit model, and is fitted to a sum of two decaying exponentials. The dependence of the electrochemical response on voltage, frequency, channel thickness, and salt concentration is studied. Next, the electrochemical data are utilized in the convection-diffusion equation to develop a model for separation by CEFFF. The equations are solved by using a combination of analytical and numerical techniques to determine the mean velocity and the dispersion coefficient of molecules, and to determine the effect of various parameters on the separation efficiency of the EFFF device. Also, the model predictions are compared with experimental data available in the literature.     

Separation of charged colloids by a combination of pulsating lateral electric fields and poiseuille flow in a 2D channel

Separation of colloidal particles of different sizes is becoming increasingly important due to rapid developments in the area of proteomics, genetic engineering, drug discovery, etc. In particular, there is a need to accomplish these separations on a microscale in 'lab-on-a-chip' devices. In this paper, we propose a new method for accomplishing separation of charged colloids of different sizes in a microchannel. This method involves a combination of pulses of lateral electric fields and Poiseuille flow in the axial direction. We develop a model for this separation technique and obtain closed form solutions for the mean velocity and the dispersion coefficient for a pulse of molecules introduced into the channel. These expressions are then utilized to determine the channel length and the separation time. For reasonable value of design constants, the proposed technique can separate molecules of different sizes that have diffusivities of 10(-10) and 0.5 x 10(-10) m2/s in 15.7 s in a 3.7 mm long channel. The length and the time increase to 5.45 cm and 231 s if the ratio of the diffusivities is reduced from 2 to 1.2, i.e., the latter diffusivity is increased to 0.835 x 10(-10) m2/s, while keeping all the other parameters the same. If the diffusivities are about 10(-9) m(2)/s, the length and the time for separation are 1 cm and 17.5 s for D1/D2=2, and 16 cm and 269 s for D1/D2=1.2.     

Continuous separation of biomolecules by the laterally asymmetric diffusion array with out-of-plane sample injection

The laterally asymmetric diffusion array, a biomolecule sorting device, was used to continuously separate a mixture of T2 and T7 coliphage DNA molecules into its constituents. A two-dimensional array of obstacles (in the presence of an average flow v) can be used to rectify the Brownian motion of particles (in this case DNA molecules) so that they diffuse preferentially in one direction, and perpendicular to the direction of the applied field (in this case an electric field). This type of device had not yet been used for actual fractionation of biomolecules, due to difficulties in injection of the sample. Here we show that with a new injection strategy a well-defined, narrow and continuous stream of molecules can be injected into the separation channel, thus enabling this separation technique to be used in a working device. We expect this type of device could now be employed for separation of a variety of different biomolecules, ranging from long dsDNA to small proteins.     

Nanoparticle-filled capillary electrophoresis for the separation of long DNA molecules in the presence of hydrodynamic and electrokinetic forces

We report the analysis of long DNA molecules by nanoparticle-filled capillary electrophoresis (NFCE) under the influences of hydrodynamic and electrokinetic forces. The gold nanoparticle (GNP)/polymer composites (GNPPs) prepared from GNPs and poly(ethylene oxide) were filled in a capillary to act as separation matrices for DNA separation. The separations of lambda-DNA (0.12-23.1 kbp) and high-molecular-weight DNA markers (8.27-48.5 kbp) by NFCE, under an electric field of -140 V/cm and a hydrodynamic flow velocity of 554 microm/s, were accomplished within 5 min. To further investigate the separation mechanism, the migration of lambda-DNA was monitored in real time using a charge-coupled device (CCD) imaging system. The GNPPs provide greater retardation than do conventional polymer media when they are encountered during the electrophoretic process. The presence of interactions between the GNPPs and the DNA molecules is further supported by the fluorescence quenching of prelabeled lambda-DNA, which occurs through an energy transfer mechanism. Based on the results presented in this study, we suggest that the electric field, hydrodynamic flow, and GNPP concentration are the three main determinants of DNA separation in NFCE.     

A one-dimensional transient model of electrical field flow fractionation

A well-developed classical theory is available for constant-voltage electrical field flow fractionation (EFFF). Recent experimental research, however, has demonstrated that pulsed fields may enhance retention in some cases. A generalized mathematical approach is presented for the prediction of retention ratios for any field type, pulsed or constant. The methodology is applied and demonstrated for a square wave protocol. Complex concentration profiles arise wherein particles are focused either towards the walls or into the channel center. The computational results indicate that pulsation can either increase retention time or decrease retention time by manipulating the effective electric field and suggest that separation resolution may also be improved.     

Separation of different physical forms of plasmid DNA using a combination of low electric field strength and flow in porous media: effect of different field gradients and porosity of the media

The retention of different physical forms of DNA by an electric field in a chromatography system was studied. We were able to effectively separate the supercoiled and the open circular forms of plasmid DNA using this type of electrochromatography system. Chromatography columns were packed with porous beads, and an axial electric field was applied so that convective buffer flow opposed the direction of electrophoresis of the DNA. A model system composed of approximately equal amounts of the super-coiled and open circular forms of the plasmid pBR 322 (4322 base pairs) was used to test the separation. Chromatography beads (agarose-based) with different porosities were used to determine the effect of the stationary phase on the separation. The porous media did not have a major effect on the separation, but the best separations were obtained using porous chromatography media made with the highest agarose concentration (10% agarose). Selective elution of plasmid DNA with different forms was obtained by either increasing the flow rates or decreasing the electric field strength (by steps or a gradient). In all the separations, the more compact supercoiled form of the plasmid was retained less strongly than either the open circular form (nicked) or the linear form. High molecular weight host genomic DNA was more strongly retained than the plasmid DNA. Increasing the ionic strength of the buffer improved resolution and capacity. The capacity of the separation was determined by injecting increasing amounts of plasmid DNA. Satisfactory separation was obtained at sample loading of up to 360 microg of total DNA on a column with dimensions of 2.5 by 11 cm (bed volume of 54 mL). The retention of DNA depends upon a counter-current flow of electrophoresis and convective flow and could be regarded as a type of field flow fractionation. The retention of the DNA by the electric field and flow is discussed in relation to the diffusion coefficients of the DNA.     

Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis

A microchip structure for field amplification stacking (FAS) was developed, which allowed the formation of comparatively long, volumetrically defined sample plugs with a minimal electrophoretic bias. Up to 20-fold signal gains were achieved by injection and separation of 400 microm long plugs in a 7.5 cm long channel. We studied fluidic effects arising when solutions with mismatched ionic strengths are electrokinetically handled on microchips. In particular, the generation of pressure-driven Poiseuille flow effects in the capillary system due to different electroosmotic flow velocities in adjacent solution zones could clearly be observed by video imaging. The formation of a sample plug, stacking of the analyte and subsequent release into the separation column showed that careful control of electric fields in the side channels of the injection element is essential. To further improve the signal gain, a new chip layout was developed for full-column stacking with subsequent sample matrix removal by polarity switching. The design features a coupled-column structure with separate stacking and capillary electrophoresis (CE) channels, showing signal enhancements of up to 65-fold for a 69 mm long stacking channel.     

Trapping DNA with a high throughput microfluidic device

Long strands of DNA can be trapped and concentrated near the inlet of a microfluidic channel by applying a pressure gradient and an opposing electric field. The mechanism for trapping involves a migration of DNA perpendicular to both the fluid flow and the electric field. Migration leads to a highly nonuniform distribution of DNA within a cross section of the channel, with the bulk of the DNA concentrated in a thin (10 μm) layer next to the walls of the channel. This highly concentrated layer generates an electrophoretic flux toward the inlet to the device, despite the much larger fluid flow in the opposite direction. In this paper, the extent to which DNA can be trapped and concentrated by this means has been characterized by fluorescence measurements. At short times (<2 hours) nearly all the incoming DNA remains trapped within the device until the electric field is turned off. The DNA largely accumulates near the inlet, but after 30–60 minutes additional DNA starts to accumulate deeper into the channel. Eventually DNA leaks from the device itself, but ≈80% of the incoming DNA can be retained for up to 5 hours. Optimizing the electric field strength can increase the amount of DNA that can be trapped, but the efficiency is not affected by the channel cross‐section.     

Unsteady transport phenomena in free-flow electrophoresis--prerequisite of ultrafast sample cleaning in microfluidic devices

The evolution partial differential equations describing the transport processes induced by hydrodynamic flow in free-flow electrophoresis (FFE) are solved by the generalized dispersion theory. Our theoretical analysis demonstrates that the central injection of solutes into a relatively fast hydrodynamic flow enables to transport them to the channel outlet well before they are spread through the width of the channel and their migration is negatively affected by a contact with walls. In this case, the axial zone spreading decreases by increasing the linear velocity of hydrodynamic flow. The resulting dependencies of convective and dispersion coefficients on the velocity of flow and parameters of the separation channel show the optimum separation conditions with respect to resolution and analysis time. Due to the unsteady character of transport processes, effective FFE separations can potentially be performed in a microfluidic device in seconds. This is a reasonable time to separate low-molecular mass impurities in the electric field. Thus, a fast and efficient sample cleaning before subsequent analysis by electrospray ionization-mass spectrometry (ESI-MS) or another separation method can be performed.     

Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels

The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.     

Monolithic integration of well-ordered nanoporous structures in the microfluidic channels for bioseparation

Gel electrophoresis and capillary gel electrophoresis are widely used for the separation of biomolecules. With increasing demand in the miniaturized devices such as lab-on-a-chip, it is necessary to integrate such a separation component into a chip format. Here, we describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures inside the microchannels for the separation of DNA molecules. In our approach, the colloidal crystals were first grown inside the microchannel using evaporation assisted self-assembly process. Then the void spaces among the colloidal crystals were filled with epoxy-based negative tone photoresist (SU-8). UV radiation was used to cure the photoresist at the desired area inside the microchannel. After subsequent development and nanoparticle removal, the well-ordered nanoporous structures inside the microchannel were obtained. Our results indicated that it was possible to construct periodic porous nanostructures inside the microchannels with cavity size around 300 nm and interconnecting pores around 30 nm. The mobility of large DNA molecules with different sizes was measured as a function of the applied electric field in the nanoporous materials. It was also demonstrated that 1 kilo-base pair (kbp) DNA ladders could be separated in such an integrated system within 10 min under moderate electric field.     

Non-orthogonal micro-free flow electrophoresis: From theory to design concept

Micro-free flow electrophoresis (μFFE) is a technique that facilitates continuous separation of molecules in a shallow channel with a hydrodynamic flow and an electric field at an angle to the flow. We recently developed a general theory of μFFE that suggested that an electric field non-orthogonal to the flow could improve resolution. Here, we used computer modeling to study resolution as a function of the electric field strength and the angle between the electric field and the hydrodynamic flow. In addition we used our general theory of μFFE to investigate other important influences on resolution, which include the velocity of the hydrodynamic flow, the height of the separation channel, and the magnitude and direction of the electroosmotic flow. Finally, we propose four designs that could be used to generate non-orthogonal electric fields and discuss their relative merits.     

Brownian dynamics simulations of electrophoretic DNA separations in a sparse ordered post array

We use Brownian dynamics simulations to analyze the electrophoretic separation of λ-DNA (48.5 kbp) and T4-DNA (169 kbp) in a hexagonal array of 1 μm diameter posts with a 3 μm center-to-center distance. The simulation method takes advantage of an efficient interpolation algorithm for the non-uniform electric field to reach an ensemble size (100 molecules) and simulation length scale (1 mm) that produces meaningful results for the average electrophoretic mobility and effective diffusion (dispersion) coefficient of these macromolecules as they move through the array. While the simulated electrophoretic mobility for λ-DNA is close to the experimental data, the simulation underestimates the magnitude of the corresponding dispersion coefficient. The simulations predict baseline resolution in a 15 mm device after 7 min using an electric field around 30 V/cm, with the resolution increasing exponentially as the electric field further decreases. The mobility and dispersivity data point out two essential phenomena that have been overlooked in previous models of DNA electrophoresis in post arrays: the relaxation time between collisions and simultaneous collisions with multiple posts.     

Effect of electric field switching on the electrophoretic mobility of single-stranded DNA molecules in polyacrylamide gels

We have examined the effects of pulsed electric fields on the separation of single-stranded DNA molecules in polyacrylamide sequencing gels. Using different electric field pulsing regimens, the mobilities of single-stranded DNA molecules can be retarded or increased as compared to conventional electrophoresis. These results indicated that pulsed field techniques can be applied to gel electrophoresis of small single-stranded DNA molecules.     

Trapping of DNA by dielectrophoresis

Under suitable conditions, a DNA molecule in solution will develop a strong electric dipole moment. This induced dipole allows the molecule to be manipulated with field gradients, in a phenomenon known as dielectrophoresis (DEP). Pure dielectrophoretic motion of DNA requires alternate current (AC) electric fields to suppress the electrophoretic effect of the molecules net charge. In this paper, we present two methods for measuring the efficiency of DEP for trapping DNA molecules as well as a set of quantitative measurements of the effects of strand length, buffer composition, and frequency of the applied electric field. A simple configuration of electrodes in combination with a microfluidic flow chamber is shown to increase the concentration of DNA in solution by at least 60-fold. These results should prove useful in designing practical microfluidic devices employing this phenomenon either for separation or concentration of DNA.     

Simultaneous measurements of the flow velocities in a microchannel by wide/evanescent field illuminations with particle/single molecules

A laser-induced fluorescence imaging method was developed to simultaneously measure flow velocities in the middle and near wall of a channel with particles or single molecules, by selectively switching from the wide field excitation mode to the evanescent wave excitation mode. Fluorescent microbeads with a diameter of 175 nm were used to calibrate the system, and the collisions of microbeads with channel walls were directly observed. The 175 nm microbeads velocities in the main flow and at 275 nm from the bottom of the channel were measured. The measured velocities of particles or single molecules in two positions in a microchannel were consistent with the calculated value based on Poiseuille flow theory when the diameter of a microbead was considered. The errors caused by Brownian diffusion in our measurement were negligible compared to the flow velocity. Single lambda DNA molecules were then used as a flowing tracer to measure the velocities. The velocity can be obtained at a distance of 309.0 +/- 82.6 nm away from bottom surface of the channel. The technique may be potentially useful for studying molecular transportation both in the center and at the bottom of the channel, and interactions between molecules and microchannel surfaces. It is especially important that the technique can be permitted to measure both velocities in the same experiment to eliminate possible experimental inconsistencies.     

Gaussian fluctuations in tethered DNA chains

In a recent work [Gao et al., Appl. Phys. Lett. 134, 113902 (2007)], we reported a novel DNA separation method by tethering DNA chains to a solid surface and then stretching the DNA chains with an electric field. The anchor is such designed that the critical force to detach a DNA chain is independent of its length. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA strands from the surface and thus DNA separation. Originally proposed for separation of long double-stranded DNA chains (>10 000 bps), this method has been proven useful also for short single-stranded DNA fragments (<100 bases) for which the fluctuation force induced by the solvent becomes significant. Here we show that the fluctuation force can be approximately represented by a gaussian model for tethered DNA chains. Analytical expressions have been derived to account for the dependence of the fluctuation force on the surface confinement, the polymer chain length, and the DNA tethering potential. The theoretical predictions are found to coincide with experiment.     

Electrophoretic ratcheting of spherical particles in well/channel microfluidic devices: Making particles move against the net field

We examine the electrophoresis of spherical particles in microfluidic devices made of alternating wells and narrow channels, including a system previously used to separate DNA molecules. Our computer simulations predict that such systems can be used to separate spherical particles of different sizes that share the same free-solution mobility. Interestingly, the electrophoretic velocity shows an inversion as the field intensity is increased: while small particles have higher velocities at low field, the situation is reversed at high fields with the larger particles then moving faster. The resulting nonlinearity suggests that asymmetric pulsed electric fields could be used to build separation ratchets: particles then have a net size-dependent velocity in the presence of a zero-mean external field. Exploiting the inversion mentioned above, we show how to design pulsed field sequences that make particles move against the mean field (an example of negative mobility). Finally, we demonstrate that it is possible to use pulsed fields to make particles of different sizes move in opposite directions, even though their charge have the same sign.     

Methods to electrophoretically stretch DNA: microcontractions, gels, and hybrid gel-microcontraction devices

The ability to controllably and continuously stretch large DNA molecules in a microfluidic format is important for gene mapping technologies such as Direct Linear Analysis (DLA). We have recently shown that electric field gradients can be readily generated in a microfluidic device and the resulting field is purely elongational. We present a single molecule fluorescence microscopy analysis of T4 DNA (169 kbp) stretching in the electric field gradients in a hyperbolic contraction microchannel. In addition, we are able to selectively pattern a crosslinked gel anywhere inside the microchannel. With an applied electric field, DNA molecules are forced to reptate through the gel and they moderately stretch as they exit the gel. By placing a gel immediately in front of the hyperbolic contraction, we bypass "molecular individualism" and achieve highly uniform and complete stretching of T4 DNA.