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We demonstrate a unique parameter for biomolecule separation that results from the nonlinear response of long, charged polymers to electrophoretic fields and apply it to extraction and concentration of nucleic acids from samples that perform poorly under conventional methods. Our method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is nonlinear. Consequently, we are able to concentrate DNA while rejecting high concentrations of contaminants. We demonstrate one application of this method by extracting DNA from challenging samples originating in the Athabasca oil sands. concentration | DNA | electrophoresis | purification | SCODA

Biological pores regulate the cellular traffic of a large variety of solutes, often with high selectivity and fast flow rates. These pores share several common structural features: the inner surface of the pore is frequently lined with hydrophobic residues, and the selectivity filter regions often contain charged functional groups. Hydrophobic, narrow-diameter carbon nanotubes can provide a simplified model of membrane channels by reproducing these critical features in a simpler and more robust platform. Previous studies demonstrated that carbon nanotube pores can support a water flux comparable to natural aquaporin channels. Here, we investigate ion transport through these pores using a sub-2-nm, aligned carbon nanotube membrane nanofluidic platform. To mimic the charged groups at the selectivity region, we introduce negatively charged groups at the opening of the carbon nanotubes by plasma treatment. Pressure-driven filtration experiments, coupled with capillary electrophoresis analysis of the permeate and feed, are used to quantify ion exclusion in these membranes as a function of solution ionic strength, pH, and ion valence. We show that carbon nanotube membranes exhibit significant ion exclusion that can be as high as 98% under certain conditions. Our results strongly support a Donnan-type rejection mechanism, dominated by electrostatic interactions between fixed membrane charges and mobile ions, whereas steric and hydrodynamic effects appear to be less important. biomimetic platform | ion channel | ion transport | nanofiltration

We use micrometer-sized fluidic channels to confine and measure electrophoresis of freely suspended individual microtubules. We measure orientation-dependent velocities of microtubules and the electro-osmotic flow mobility in our channels to infer the anisotropic electrophoretic mobility of microtubules under physiological conditions. We discuss the difference between electrophoresis and purely hydrodynamic motion and its implications for interpreting mobility measurements. We show that the mobility anisotropy is a factor of 0.83, clearly different from the well known anisotropy factor of 0.5 in Stokes drag coefficients for cylindrical objects. We also show that the velocity is independent of microtubule length, which would be different for hydrodynamic motion. We demonstrate that the electric force on the counterions has important consequences for the interpretation of electrophoresis experiments and that ignoring this can lead to an underestimation of the effective charge by orders of magnitude. From the electrophoresis measurements, we calculate an effective surface-charge density of -36.7 [+ or -] 0.4 mC/[m.sup.2] for microtubules. Electrophoretic measurements of subtilisin-digested microtubules, which have the negatively charged C termini on the outer surface removed, show a 24% decrease in mobility and, correspondingly, in surface charge, but no change in anisotropy.

This paper presents the design, fabrication and testing of a new structure of dielectrophoresis (DEP) chip having a three-dimensional (3-D) electric field gradient and an asymmetric distribution of the electric field in the vertical plane. This achievement was possible due to the special configuration of the electrodes: a bulk silicon electrode and a thin amorphous silicon electrode. The thick electrode defines, at the same time, the walls, while the two glass dies form the ceiling and floor of the microfluidic channel. The top glass die presents two etch-through inlet/outlet holes of the microfluidic channel. In the bottom glass die, isotropic via-holes are performed through the glass for the lead-outs. For this reason, the lead-outs does not generate fluidic leakage. Using a single material for the electrodes, the electrochemical effect for conventional multilayer metal electrodes is eliminated. The proposed DEP structure, with thin and thick electrodes, generates in the vertical plane an asymmetric distribution of the electric field and, therefore, an enhanced electric field gradient. As a result, for positive DEP, the particles are trapped near the thin electrode. while for negative DEP the particles are levitated. Compared with typical planar DEP devices, the proposed DEP structure, presents an increased DEP force in the vertical direction. As a result, the same trapping or levitation effect can be achieved at a lower voltage and, in this way, with a reduced heating of the solution. Using the bulk electrode for definition of the microfluidic channel, the need for a separate channel wall material is eliminated. The DEP device is wafer level packaged (being fully fabricated at wafer level using batch processes) therefore, can be consider as a low cost solution. The good isolating properties of the glass confer the opportunity of working at a high frequency range. Yeast cells have been used to successfully test the performance of the device: the trapping using positive DEP occurred on the bottom of the channel near the thin electrode, while the negative DEP generate a suspension of the cells 35-40 [micro]m from the bottom. Index Terms--Dielectrophoresis DEP), microfluidic device, silicon electrodes, three-dimensional (3-D) electric field gradient.

This paper shows that proteins display an unexpectedly wide range of behaviors in buffers containing moderate (0.1-10 mM) concentrations of SDS (complete unfolding, formation of stable intermediate states, specific association with SDS, and various kinetic phenomena); capillary electrophoresis provides a convenient method of examining these behaviors. Examination of the dynamics of the reponse of proteins to SDS offers a way to differentiate and characterize proteins. Based on a survey of 18 different proteins, we demostrate that proteins differ in the concentrations of SDS at which they denature, in the rates of unfolding in SDS, and in the profiles of the denaturation pathways. We also demonstrate that these differences can be exploited in the analysis of mixtures. capillary electrophoresis | surfactant | intermediates | kinetics

Icosahedral bacteriophages pack their double-stranded DNA genomes to near-crystalline density and achieve one of the highest levels of DNA condensation found in nature. Despite numerous studies, some essential properties of the packaging geometry of the DNA inside the phage capsid are still unknown. We present a different approach to the problems of randomness and chirality of the packed DNA. We recently showed that most DNA molecules extracted from bacteriophage P4 are highly knotted because of the cyclization of the linear DNA molecule confined in the phage capsid. Here, we show that these knots provide information about the global arrangement of the DNA inside the capsid. First, we analyze the distribution of the viral DNA knots by high-resolution gel electrophoresis. Next, we perform Monte Carlo computer simulations of random knotting for freely jointed polygons confined to spherical volumes. Comparison of the knot distributions obtained by both techniques produces a topological proof of nonrandom packaging of the viral DNA. Moreover, our simulations show that the scarcity of the achiral knot 41 and the predominance of the torus knot [5.sub.1] over the twist knot [5.sub.2] observed in the viral distribution of DNA knots cannot be obtained by confinement alone but must include writhe bias in the conformation sampling. These results indicate that the packaging geometry of the DNA inside the viral capsid is writhe-directed. bacteriophage | DNA condensation | DNA electrophoresis | Monte Carlo simulation | DNA writhe

We demonstrate a complete nanotube electrophoresis system (nanotube radii in the range of 50 to 150 nm) based on lipid membranes, comprising DNA injection, single-molecule transport, and single-molecule detection. Using gel-capped electrodes, electrophoretic single-file transport of fluorescently labeled dsDNA molecules is observed inside nanotubes. The strong confinement to a channel of molecular dimensions ensures a detection efficiency close to unity and identification of DNA size from its linear relation to the integrated peak intensity. In addition to constituting a nanotechnological device for identification and quantification of single macromolecules or biopolymers, this system provides a method to study their conformational dynamics, reaction kinetics, and transport in cell-like environments. electrophoresis | lipid | conformation

In this paper, we have designed and fabricated a microfluidic channel to focus biological cells using dielectrophoresis for cytometry applications. The device consists of an elliptic-like channel fabricated by isotropic etching of soda lime glass wafers and a subsequent wafer-bonding process. Microelectrodes are patterned on the circumference of the channel to generate ac fringing fields that result in negative dielectrophoretic forces directing cells from all directions to the center of the channel. An analysis using a thin shell model and experiments with microbeads and human leukemia HL60 cells indicate that biological cells can be focused using an ac voltage of an amplitude up to 15 [V.sub.p-p] and a frequency below 100 kHz, respectively. This design eliminates the sheath flow and the fluid control system that makes conventional cytometers bulky, complicated, and difficult to operate, and offers the advantages of a portable module that could potentially be integrated with on-chip impedance or optical sensors into a micro total analysis system. [1219] Index Terms--Cytometry, dielectrophoresis, microcytometer, microfluids, 3-D focusing.

Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins. capillary electrophoresis | fluorescence | MS | protease

Byline: S. L. Grokhovsky (1,2), S. A. Strel'tsov (1), A. L. Zhuze (1) Keywords: camptothecin; topotecan; circular supercoiled DNA; single- and double-strand breaks in DNA; agarose gel electrophoresis Abstract: A study was made of the temperature, concentration, and time dependences for the emergence of breaks in the sugar-phosphate backbone of a circular supercoiled DNA (scDNA) in the presence of a campto-thecin derivative topotecan (TPT) and in the absence of DNA topoisomerase I (topo I). The experiments were carried out in low ionic strength solutions (10 mM sodium cacodylate) at neutral pH (6.8). The incubation time necessary for the appearance of double-strand breaks in scDNA in the presence of TPT correlated with the time of formation of strong TPT--DNA complex. This is the first demonstration that molecules of the camptothecin family can cause double-strand breaks in scDNA in the absence of the enzyme. A model is suggested for the complex composed of two crossed DNA duplexes bound through a bridge of two dimers of the TPT lactone form. According to this model, two carbonyl groups of D rings of different TPT dimers form hydrogen bonds with 2-amino groups of guanines located in the neighboring base pairs of different strands of one DNA duplex. At the same time, two other carbonyl groups of D rings of TPT dimers form hydrogen bonds with 2-amino groups of guanines 5 bp apart in one and the same strand of the second DNA duplex. Author Affiliation: (1) Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia (2) University of Oslo Medical Research Center, Moscow, 119991, Russia Article History: Registration Date: 12/10/2004

Capillary electrophoresis performed on microchips has made great progress toward practical applications in recent years. This article describes the use of microchip-based capillary electrophiresis, with and without the integrated function [...]

Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures.

Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated [Lambda]-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed.

The environment and the circumstances leading to the Sephadex group of products for gel filtration (SEC, size exclusion chromatography) are described. The importance of Porath's and the author's experience from zone electrophoresis is stressed. To be able to get high resolution the technique for packing columns was of decisive importance for electrophoresis. It paved the way for the observation of the separation properties of dextran gels. The synthesis of dextran-based gels at AB Pharmacia and the development of varieties of them for varying applications are described together with the project leading to their marketing in 1959. The work of the author on gel filtration is summarized. The story is based on publication and memory. Aids to the latter have been laboratory notebooks and other documents still in the possession of the author., INTRODUCTION In 1950, after having finished a fil.kand. (B.Sc) exam at the University of Uppsala, I approached Professor Arne Tiselius at the Institute of Biochemistry there to become a graduate [...]

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

Excellent electrophoretic separations of a variety of biological molecules can be accomplished by using uncharged, triblock copolymers as the 'gel' media. These copolymers form uncrosslinked, lyotropic liquid crystalline phases of large micelles between which molecules must travel. Unlike crosslinked hydrogels in common use, these alternative media have highly ordered internal structures. Pluronic F127, representative of the copolymer class, contains poly(ethylene oxide) (EO) and poly(propylene oxide) (PO) units with an approximate molecular formula [(EO).sub.106][(PO).sub.70][(EO).sub.106]. Concentrated (18-30%) solutions of Pluronic F127 are freely flowing liquids at low temperature (0-5 [degrees] C) but form gel-like, cubic liquid crystals of large, spherical micelles when warmed. The utility of these media is illustrated by separations of linear, double-stranded DNA up to 3,000 bp long by conventional electrophoresis, and of single-stranded DNAs from 4 to 60 nt long by capillary electrophoresis. Extraordinary separations of supercoiled DNAs were also obtained by capillary electrophoresis. The versatility, availability, and ease of use of Pluronic polymers offer major advantages over conventional media for preparative and high performance analytical separations of nucleic acids and other biomolecules. Mechanisms of molecular transport and separation operating in polymer liquid crystals must differ in fundamental ways from those in crosslinked gels. Lyotropic polymer liquid crystals are unique systems for elucidating mechanisms of macromolecule migration in ordered, dense media, and provide opportunities in separations science.

Capillary electrophoresis (CE) is a usefool tool in separating and detecting free amino carboxylic acids such as the synthetic multidentate chelating agents, EDTA (ethylenediaminetetraacetic acid) and NTA (nitrilotriacetic acid), and their Co(II) and Co(III) complexes in a single run without prior pretreatment. Equilibrium calculations of the CE results obtained indicate that Co(III) are thermodynamically and kinetically more stable and easier to detect and separate than Co(II) complexes.

In vitro kinase assay studies of cell extracts of c-Mil, the avian homologue of mammalian serine/threonine kinase c-Raf-1, indicate phosphorylation of c-Jun by immunoaffinity-purified c-Mil/Raf. Tryptic phosphopeptide analysis of the c-Jun/c-Mil complex situated in the cytoplasm reveals a reduction in electrophoretic mobility of c-Jun due to phosphorylation. This implies the possibility of phosphorylating c-Jun in a mitogen-activated protein kinase-independent manner.

DNA solutions subjected to an electric field exhibit an instability that leads to DNA segregation in aggregates tilted with regard to the field. With the use of epifluorescence videomicroscopy, the evolution of DNA patterns in capillaries as a function of DNA concentration, DNA size, field strength, and field frequency was studied. The field threshold for segregation was decreased when the frequency was lowered or when the DNA molecular weight or concentration was increased. Aggregation is attributed to an electrohydrodynamic instability triggered by the dipole-dipole interaction. This phenomenon explains the failure of earlier attempts to separate large DNA in capillaries., Molecular genetics relies heavily on the electrophoretic separation of DNA molecules by size. Until recently, this was achieved mainly in slab gels, but the joint use of capillary electrophoresis (CE) [...]

A study analyzed the effect of a nonuniform electric field on the heat transfer features of boiling as a result of a small nichrome wire immersed in a dielectric fluid. It also examined the feasibility of employing the non-uniform field, which creates a dielectric force on the generated vapor bubbles, as a bubble removing mechanism in a microgravity environment. The dielectrophoresis force initially showed great promise as a tool for bubble detachment and downward acceleration. However, after the bubble reached its position of equilibrium, it remained stationary, resulting in the combination of the newly generated bubbles with the first bubble, and creating a vapor film between the electrodes.

Undergraduate students often graduate without having hands-on experience with the electrophoresis technique due to the time required to complete the procedure or the lack of equipment. An experiment was designed for students to learn biochemical concepts in addition to the technique and theory of SDS-PAGE.

An analysis of Capillary electrophoresis (CE) to separate, identifies, and quantitates cations in water samples are presented. Findings show that the variation of the relative standard deviation is mainly comes from sample ion concentrations that are at the low end of the calibration curve such as in the determination of sodium in river water and calcium in 1% seawater.