Acetic acid denaturing pulsed field capillary electrophoresis for RNA separation

Based on our previous work of in‐capillary denaturing polymer electrophoresis, we present a study of RNA molecular separation up to 6.0 kilo nucleotide by pulsed field CE. This is the first systematic investigation of electrophoresis of a larger molecular mass RNA in linear hydroxyethylcellulose (HEC) under pulsed field conditions. The parameters that may influence the separation performance, e.g. gel polymer concentration, modulation depth and pulse frequency, are analyzed in terms of resolution and mobility. For denaturing and separating RNA in the capillary simultaneously, 2 M acetic acid was added into the HEC polymer to serve as separation buffer. Result shows that (i) in pulsed field conditions, RNA separation can be achieved in a wide range of concentration of HEC polymer, and RNA fragments between 0.3 and 0.6 kilo nucleotide are sensitive to the polymer concentration; (ii) under certain pulsed field conditions, RNA fragments move linearly as the modulation depth increases; (iii) 12.5 Hz is the resonance frequency for RNA reorientation time and applied frequency.     

Flow-based and sieving matrix-free DNA differentiation by a miniaturized field flow fractionation device

DNA separation is typically done by gel electrophoresis based on its charge property. In our previous work, we reported that dielectrophoresis could be used to manipulate polystyrene nanoparticles' motion by using a miniaturized electrical field flow fractionation device (micro-EFFF) with a segmented electrode operated under a pulsed voltage (PV). In this work, we report the manipulation and separation of DNA molecules using the micro-EFFF. DNA motion was in situ visualized inside the device. Results revealed that dielectrophoresis governed DNA motion, which was strongly correlated with the pulse frequency but not the duty cycle of a PV. A longer retention time of DNA molecules was measured under a PV. The retention time increased with the length of DNA molecules. As the micro-EFFF is flow-based and sieving-matrix-free, it has a potential to be applied to sample preparation in a micrototal analysis system or when fractionated molecules are needed for downstream analysis.     

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.     

Orientation of DNA and the agarose gel matrix in pulsed electric fields

Transient electric birefringence has been used as an analytical tool to study the orientation of DNA in agarose gels, and to study the orientation of the matrix alone. The sign of the birefringence of DNA oriented in an agarose gel is negative, as observed in free solution, indicating that the DNA molecules orient parallel to the direction of the electric field. If the median pore diameter of the gel is larger than the contour length of the DNA molecule, the DNA effectively does not see the matrix and the birefringence relaxation time is the same as observed in free solution. However, if the median pore diameter of the gel is smaller than the contour length of the DNA, the DNA molecule becomes stretched as well as oriented. For DNA molecules of moderate size (less than or equal to 4 kb), stretching in the gel causes the birefringence relaxation times to increase to the values expected for fully stretched molecules. Complete stretching is not observed for larger DNA molecules. The orientation and stretching of DNA molecules in the gel matrix indicates that end-on migration, or reptation, is a likely mechanism for DNA electrophoresis in agarose gels. When the electric field is rapidly reversed in polarity, very little change in the orientation of the DNA is observed if the DNA molecules were completely stretched and had reached their equilibrium orientation before the field was reversed in direction. Hence completely stretched, oriented DNA molecules are able to reverse their direction of migration in the electric field with little or no loss of orientation. However, if the DNA molecules were not completely stretched or if the equilibrium orientation had not been reached, substantial disorientation of the DNA molecules is observed at field reversal. The forced rate of disorientation in the reversing field is faster than the field-free rate of disorientation. Complicated patterns of reorientation can be observed after field reversal, depending on the degree of orientation in the original field direction. The effect of pulsed electric fields on the orientation of the agarose gel matrix itself was also investigated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reorientational Dynamics of Water Confined in Zeolites

We present a detailed molecular‐dynamics study of water reorientation and hydrogen‐bond dynamics in a strong confinement situation, within the narrow pores of an all‐silica Linde type A (LTA) zeolite. Two water loadings of the zeolite are compared with the bulk case. Water dynamics are retarded in this extreme hydrophobic confinement and the slowdown is more pronounced at higher water loading. We show that water reorientation proceeds mainly by large‐amplitude angular jumps, whose mechanism is similar to that determined in the bulk. The slowdown upon hydrophobic confinement arises predominantly from an excluded‐volume effect on the large fraction of water molecules lying at the interface with the zeolite matrix, with an additional minor contribution coming from a structuring effect induced by the confinement.     

Brute Force Orientation of Matrix‐Isolated Molecules: Reversible Reorientation of Formaldehyde in an Argon Matrix toward Perfect Alignment

Brute force orientation by an electric field is a promising way of controlling the orientation of polar molecules in the gas phase, but its application to condensed‐phase molecules has been very limited. We studied the reorientation of formaldehyde molecules in a solid Ar matrix under the influence of a strong electric field using reflection absorption infrared spectroscopy. Asymptotically perfect alignment of the formaldehyde molecules along the field was achieved at field strengths exceeding 1×10⁸ V m⁻¹. The vibrational bands of the aligned molecules exhibited a unidirectional Stark shift proportional to the field strength. The reorientation of the molecules was reversible despite the cryogenic solid environment of the system.     

Measurement of electroosmotic and electrophoretic velocities using pulsed and sinusoidal electric fields

In this work, we explore two methods to simultaneously measure the electroosmotic mobility in microchannels and the electrophoretic mobility of micron‐sized tracer particles. The first method is based on imposing a pulsed electric field, which allows to isolate electrophoresis and electroosmosis at the startup and shutdown of the pulse, respectively. In the second method, a sinusoidal electric field is generated and the mobilities are found by minimizing the difference between the measured velocity of tracer particles and the velocity computed from an analytical expression. Both methods produced consistent results using polydimethylsiloxane microchannels and polystyrene micro‐particles, provided that the temporal resolution of the particle tracking velocimetry technique used to compute the velocity of the tracer particles is fast enough to resolve the diffusion time‐scale based on the characteristic channel length scale. Additionally, we present results with the pulse method for viscoelastic fluids, which show a more complex transient response with significant velocity overshoots and undershoots after the start and the end of the applied electric pulse, respectively.     

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.     

Multiple dielectric relaxations in liquid crystals, their analysis and interpretation

Dielectric measurements on laterally substituted molecules were carried out in the frequency range 10 Hz to 10 MHz. In these systems, the main part of the molecules exhibits only a very small dipole moment in the direction of the para-axis, whereas the lateral substituent is strongly polar. Depending on the position of the dipole (o,m,p), a more or less strong dipolar correlation in the parallel direction was detected. The absorption data at higher frequencies were fitted to two Cole-Cole mechanisms. The low frequency relaxation was interpreted as angular vibration of the dipole moment of the lateral group, and the high frequency one as the reorientation about the para-axis of the main part of the molecule and of the lateral group. The appearance of two high frequency mechanisms is unexpected and demonstrates that in complicated molecules the dynamics also become differentiated.     

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.     

Combined AC‐electrokinetic effects: Theoretical considerations on a three‐axial ellipsoidal model

AC fields induce charges at the structural interfaces of particles or biological cells. The interaction of these charges with the field generates frequency‐dependent forces that are the basis for AC‐electrokinetic effects such as dielectrophoresis (DEP), electrorotation (ROT), electro‐orientation, and electro‐deformation. The effects can be used for the manipulation or dielectric single‐particle spectroscopy. The observation of a particular effect depends on the spatial and temporal field distributions, as well as on the shape and the dielectric and viscoelastic properties of the object. Because the effects are not mutually independent, combined frequency spectra are obtained, for example, discontinuous DEP and ROT spectra with ranges separated by the reorientation of nonspherical objects in the linearly and circularly polarized DEP and ROT fields, respectively. As an example, the AC electrokinetic behavior of a three‐axial ellipsoidal single‐shell model with the geometry of chicken‐red blood cells is considered. The geometric and electric problems were separated using the influential‐radius approach. The obtained finite‐element model can be electrically interpreted by an RC model leading to an expression for the Clausius–Mossotti factor, which permits the derivation of force, torque, and orientation spectra, as well as of equations for the critical frequencies and force plateaus in DEP and of the characteristic frequencies and peak heights in ROT. Expressions for the orientation in linearly and circularly polarized fields, as well as for the reorientation frequencies were also derived. The considerations suggested that the simultaneous registration of various AC‐electrokinetic spectra is a step towards the dielectric fingerprinting of single objects.     

Probing the electric field alignment of a thermotropic liquid crystalline polymer by synchrotron radiation

The orientation of a cyclic side-chain thermotropic liquid crystalline material in an AC field was monitored in real-time using synchrotron radiation. Monitoring the realignment processes in the millisecond-to-minute time-scale was made possible by the high X-ray flux. Orientation parameters and response times were calculated as a function of temperature and frequency. Response times decreased exponentially with temperature due to a decrease in the viscosity. Very little dependence of the response time on frequency was observed, except at low temperatures, where a switch from homeotropic to planar alignment of the molecules was detected. This reorientation of the director was studied in real-time and the resulting complex diffraction patterns were due to equal but opposite director rotations from an alignment parallel to the applied electric field to an alignment perpendicular to the applied electric field. The orientation parameters were highest in the central portion of the mesophase temperature range. At temperatures near clearing, the net degree of orientation diminished. Cooling through the mesophase with an applied electric field resulted in much larger orientation parameters than could be obtained by aligning at a fixed temperature in the mesophase.     

MECHANISM OF ORIENTATION AND LINEAR DICHROISM OF PHOTOSYNTHETIC PARTICLES IN ELECTRIC FIELDS: CHLOROPLASTS AND CHLOROPHYLL-PROTEIN COMPLEXES

Abstract— The mechanisms of orientation in pulsed and alternating electric fields of thylakoids (derived from the sonication of spinach chloroplasts) and of light-harvesting chlorophyll a/b-protein complexes (CPII) were investigated by utilizing linear dichroism techniques. Comparisons of the linear dichroism spectra of thylakoids and CPII particles suggest that the latter are oriented with their directions of largest electronic polarizabilities (and thus probably their largest dimensions) within the thylakoid membrane planes. At low electric field strengths (< 12 V cm^?1), and at low frequencies of alternating electric fields (< 0.25 Hz), thylakoid membranes tend to align with their normals parallel to the direction of the applied electric field; the mechanism of orientation involves a permanent dipole moment of the thylakoids which is oriented perpendicular to the planes of the membranes. However, at high field strengths and high frequencies of the applied alternating electric fields, the thylakoids tend to orient with their planes parallel to the applied field, thus exhibiting an inversion of the sign of the linear dichroism as the electric field strength is increased. At the higher frequencies and at higher field strengths, the orientation mechanisms of the thylakoids involve induced dipole moments related to anisotropies in the electronic polarizabilities. The polarizability is higher within the plane than along a normal to the plane, thus accounting for the inversion of the dichroism as the electric field strength is increased. The CPII particles align with their largest dimension parallel to the applied field at all field strength, indicating that the induced dipole moment dominates the orientation mechanisms in pulsed electric fields. The magnitude of the absolute linear dichroism of CPII suspensions increases with increasing dilution, indicating that aggregates of lower symmetry are formed at higher concentrations of the CPII complexes.     

Dielectrophoretic field‐flow fractionation of electroporated cells

We describe the development and testing of a setup that allows for DEP field‐flow fractionation (DEP‐FFF) of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells based on their different polarizabilities. We first optimized the channel and electrode dimensions, flow rate, and electric field parameters for efficient DEP‐FFF separation of moderately heat‐treated CHO cells (50°C for 15 min) from untreated ones, with the former used as a uniform and stable model of electroporated cells. We then used CHO cells exposed to electric field pulses with amplitudes from 1200 to 2800 V/cm, yielding six groups containing various fractions of nonporated, reversibly porated, and irreversibly porated cells, testing their fractionation in the chamber. DEP‐FFF at 65 kHz resulted in distinctive flow rates for nonporated and each of the porated cell groups. At lower frequencies, the efficiency of fractionation deteriorated, while at higher frequencies the separation of individual elution profiles was further improved, but at the cost of cell flow rate slowdown in all the cell groups, implying undesired transition from negative into positive DEP, where the cells are pulled toward the electrodes. Our results demonstrate that fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells is feasible at a properly selected frequency.     

A single molecule detection method for understanding mechanisms of electric field-mediated interstitial transport of genes

The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. In order to characterize and improve the interstitial transport, we developed a reproducible single molecule detection method to observe the electromobility of DNA in a range of pulsed, high field strength electric fields typically used during electric field-mediated gene delivery. Using agarose gel as an interstitium phantom, we investigated the dependence of DNA electromobility on field magnitude, pulse duration, pulse interval, and pore size in the interstitial space. We observed that the characteristic electromobility behavior, exhibited under most pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, and pulse interval of the applied pulse sequences, as well as the pore size of the fibrous matrix through which the DNA migrated. Our data also suggest the existence of a minimum pulse amplitude required to initiate electrophoretic transport. These results are useful for understanding the mechanisms of DNA electromobility and improving interstitial transport of genes during electric field-mediated gene delivery.     

Reorientation behaviour of bent core molecules in response to an external electric field as detected by time-resolved FTIR spectroscopy

Time-resolved polarized Fourier transform infrared spectroscopy (FTIR) is employed to analyse the segmental orientation and mobility of achiral bent core molecules in response to an external electric field. By shearing the substance between ITO coated CaF2 windows two types of domain, racemic and homochiral, are formed in the high temperature B2 phase. Each of these domains is characterized by two spontaneous symmetry-breaking instabilities which yield a symmetric and an antisymmetric electro-optical response, respectively. Taking advantage of the specificity of IR spectroscopy, this switching behaviour is analysed on a molecular level for the moieties of the bent core liquid crystal materials. In this way, the electrically induced reorientation of the different segments on a cone and the suppression of the antiferroelectric structure at higher frequencies can be followed in detail. Furthermore the biased rotation of the two carbonyl groups around the molecular long axis is determined. It is shown that all molecular units move synchronously on the time scale of the experiment (10mus).     

Reorientation behaviour of bent core molecules in response to an external electric field as detected by time-resolved FTIR spectroscopy

Time-resolved polarized Fourier transform infrared spectroscopy (FTIR) is employed to analyse the segmental orientation and mobility of achiral bent core molecules in response to an external electric field. By shearing the substance between ITO coated CaF2 windows two types of domain, racemic and homochiral, are formed in the high temperature B2 phase. Each of these domains is characterized by two spontaneous symmetry-breaking instabilities which yield a symmetric and an antisymmetric electro-optical response, respectively. Taking advantage of the specificity of IR spectroscopy, this switching behaviour is analysed on a molecular level for the moieties of the bent core liquid crystal materials. In this way, the electrically induced reorientation of the different segments on a cone and the suppression of the antiferroelectric structure at higher frequencies can be followed in detail. Furthermore the biased rotation of the two carbonyl groups around the molecular long axis is determined. It is shown that all molecular units move synchronously on the time scale of the experiment (10mus).     

Pulsed field gel electrophoresis: studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system

We have studied the migration of DNA in pulsed field agarose gels under a variety of electrophoresis conditions. We have made use of an instrument which can generate electric fields of any orientation, magnitude, or duration to compare different separation techniques for DNA molecules of from 1 to several thousand kilobase pairs. We discuss the capabilities of the system and present results of gel runs in which electrophoresis conditions were changed individually or in combination. The mobility of DNA in pulsed field gels is shown to reflect a number of interdependent physical parameters.     

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.     

Nitrogen nuclear spin flips in nitroxide spin probes of different sizes in glassy o-terphenyl: possible relation with α- and β-relaxations

The pulsed electron-electron double resonance (ELDOR) technique was employed to study nitroxide spin probes of three different sizes dissolved in glassy o-terphenyl. A microwave pulse applied to the central hyperfine structure (hfs) component of the nitroxide electron paramagnetic resonance spectrum was followed by two echo-detecting pulses of different microwave frequency to probe the magnetization transfer (MT) to the low-field hfs component. The MT between hfs components is readily related to flips in the nitrogen nuclear spin, which in turn are induced by molecular motion. The MT on the time scale of tens of microseconds was observed over a wide temperature range, including temperatures near and well below the glass transition. For a bulky nitroxide, it was found that MT rates approach dielectric α (primary) relaxation frequencies reported for o-terphenyl in the literature. For small nitroxides, MT rates were found to match the frequencies of dielectric β (secondary) Johari-Goldstein relaxation. The most probable motional mechanism inducing the nitrogen nuclear spin flips is large-angle angular jumps, between some orientations of unequal occupation probabilities. The pulsed ELDOR of nitroxide spin probes may provide additional insight into the nature of Johari-Goldstein relaxation in glassy media and may serve as a tool for studying this relaxation in substances consisting of non-rigid molecules (such as branched polymers) and in heterogeneous and non-polar systems (such as a core of biological membranes).