Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.     

End-labeled free-solution electrophoresis of DNA

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.     

Free-flow zone electrophoresis and isoelectric focusing using a microfabricated glass device with ion permeable membranes

This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow isoelectric focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow isoelectric focusing could be established. Furthermore, the application of hydrodynamic focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow isoelectric focusing employing a transversal voltage of up to 150 V across the separation chamber.     

Thermophoresis of single stranded DNA

The manipulation and analysis of biomolecules in native bulk solution is highly desired; however, few methods are available. In thermophoresis, the thermal analog to electrophoresis, molecules are moved along a microscopic temperature gradient. Its theoretical foundation is still under debate, but practical applications for analytics in biology show considerable potential. Here we measured the thermophoresis of highly diluted single stranded DNA using an all‐optical capillary approach. Temperature gradients were created locally by an infrared laser. The thermal depletion of oligonucleotides of between 5 and 50 bases in length were investigated by fluorescence at various salt concentrations. To a good approximation, the previously tested capacitor model describes thermophoresis: the Soret coefficient linearly depends on the Debye length and is proportional to the DNA length to the power of 0.35, dictated by the conformation‐based size scaling of the diffusion coefficient. The results form the basis for quantitative DNA analytics using thermophoresis.     

Influence of electric field intensity, ionic strength, and migration distance on the mobility and diffusion in DNA surface electrophoresis

In order to increase the separation rate of surface electrophoresis while preserving the resolution for large DNA chains, e.g., genomic DNA, the mobility and diffusion of Lambda DNA chains adsorbed on flat silicon substrate under an applied electric field, as a function of migration distance, ionic strength, and field intensity, were studied using laser fluorescence microscope. The mobility was found to follow a power law with the field intensity beyond a certain threshold. The detected DNA peak width was shown to be constant with migration distance, slightly smaller with stronger field intensity, but significantly decreased with higher ionic strength. The molecular dynamics simulation demonstrated that the peak width was strongly related with the conformation of DNA chains adsorbed onto surface. The results also implied that there was no diffusion of DNA during migration on surface. Therefore, the Nernst-Einstein relation is not valid in the surface electrophoresis and the separation rate could be improved without losing resolution by decreasing separation distance, increasing buffer concentration, and field intensity. The results indicate the fast separation of genomic DNA chains by surface electrophoresis is possible.     

Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique—isoelectric focusing of proteins and a single-stranded DNA fragment

Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm⁻¹ and 422 V cm⁻¹, depending on the application.     

Universal interpolating function for the dispersion coefficient of DNA fragments in sieving matrices

The separation of DNA fragments by (slab or capillary) gel electrophoresis has been studied extensively. To characterize the separation achieved by such systems, one needs to understand the impact (and their dependency upon the experimental quantities) of two physical parameters: the electrophoretic mobility mu and the diffusion coefficient D. Three different regimes have been shown to exist for both mu and D: the Ogston regime, the reptation regime and the reptation with orientation regime (note that separation is only possible for the first two regimes). In the small electric field limit, both mu and D are apparently well described by theories for all three regimes. Unfortunately this results in disjointed scaling laws and no theory-based general equations can apply to all regimes. Recently, an empirical interpolating formula has been proposed that adequately fits the low electric field mobility mu of dsDNA fragments across all three regimes and is compatible with accepted theories. In this article we review and clarify the current state of knowledge regarding the size dependence of the mobility and the diffusion coefficient and propose an interpolating formula for molecular size dependence of the low field diffusion coefficient D. With formulas for both the mobility and the diffusion coefficient as a function of the experimental conditions one could, in principle, optimize any gel/polymer matrix-based electrophoresis system for a wide range of DNA molecular sizes.     

Infrared Diffusion-Ordered Spectroscopy Reveals Molecular Size and Structure**

Inspired by ideas from NMR, we have developed Infrared Diffusion-Ordered Spectroscopy (IR-DOSY), which simultaneously characterizes molecular structure and size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes–Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient and tracking its equilibration in an IR-frequency resolved manner. Analogous to NMR-DOSY, a two-dimensional IR-DOSY spectrum has IR frequency along one axis and diffusion coefficient (or equivalently, size) along the other, so the chemical structure and the size of a compound are characterized simultaneously. In an IR-DOSY spectrum of a mixture, molecules with different sizes are nicely separated into distinct sets of IR peaks. Extending this idea to higher dimensions, we also perform 3D-IR-DOSY, in which we combine the conformation sensitivity of femtosecond multi-dimensional IR spectroscopy with size sensitivity.     

Conditions for purification of proteins by free-flow zone electrophoresis

The separation of proteins by free-flow zone electrophoresis is generally impaired by a number of secondary effects which cause spreading of the protein streams with resultant loss in resolution. A strategy is outlined, based on experimental observations and numerical modeling, which allows the operating conditions and separation chamber dimensions to be chosen so as to obtain complete separation of two proteins of known mobility. This approach takes into account some dispersive phenomena such as molecular diffusion, electroosmosis and residence time gradients. In some cases, the right operating conditions cannot be achieved on earth and electrophoresis separations in microgravity may then be justified.     

Diffusion constant of a nonspecifically bound protein undergoing curvilinear motion along DNA

The mechanism of a protein's diffusion along a DNA segment is a subject of much current interest because of the involvement of this diffusion in numerous biological processes, including the recognition of DNA sequences and chemical modifications of DNA. In this work we present a theoretical derivation of the diffusion coefficient of a nonspecifically bound protein, assuming that the protein follows a helical track along the DNA. It is shown that, for protein-sized molecules, the principal contribution to the total translational friction comes from the curvilinear motion along the helix, and this contribution is given by 6pietaRR(oc)(2) + 8pietaR(3), where R is the protein radius, ROC is the distance of separation between the center of mass of the protein and the helical axis of DNA, and eta is the viscosity of the medium. The translational diffusion of the protein along the helical track of DNA is thus predicted to have a nearly R(-3) size dependence, not the R(-1) dependence characterizing simple translational diffusion. It is shown that this expression gives rather good estimates of the translational diffusion coefficient measured in single molecule experiments.     

A review of the zone broadening contributions in free-flow electrophoresis

The broadening of analyte streams, as they migrate through a free-flow electrophoresis (FFE) channel, often limits the resolving power of FFE separations. Under laminar flow conditions, such zonal spreading occurs due to analyte diffusion perpendicular to the direction of streamflow and variations in the lateral distance electrokinetically migrated by the analyte molecules. Although some of the factors that give rise to these contributions are inherent to the FFE method, others originate from non-idealities in the system, such as Joule heating, pressure-driven crossflows, and a difference between the electrical conductivities of the sample stream and background electrolyte. The injection process can further increase the stream width in FFE separations but normally influencing all analyte zones to an equal extent. Recently, several experimental and theoretical works have been reported that thoroughly investigate the various contributions to stream variance in an FFE device for better understanding, and potentially minimizing their magnitudes. In this review article, we carefully examine the findings from these studies and discuss areas in which more work is needed to advance our comprehension of the zone broadening contributions in FFE assays.     

Simultaneous measurements of the electrophoretic mobility,diffusion coefficient and orientation of dsDNA during electrophoresis in polymer solutions

We determined simultaneously the electrophoretic mobility, diffusion coefficient D and molecular orientation during electrophoresis of dsDNAs in polymer solutions ranging from the dilute to the semidilute regime. We established, for the first time, master scaling laws for the diffusion coefficient showing a universal behavior. A model found in the literature designed for the dilute regime allows, surprisingly, to describe the mobility data over the whole range of concentrations studied and at the same time the biased reptation with fluctuations (BRF) failed for the semidilute regime, even when constraint release of the network was taken into account. These quantitative determinations of D are of practical interest to evaluate band broadening during capillary electrophoresis and provide data for stimulating investigation of the physics of DNA electrophoretic motion.     

Towards an integrated device that utilizes adherent cells in a micro-free-flow electrophoresis chip to achieve separation and biosensing

We immobilized adherent human embryonic kidney (HEK) cells—which are able to trace adenosine triphosphate (ATP) —inside a microfluidic free-flow electrophoresis (μFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 μM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.     

Hydrodynamic size and charge of polyelectrolyte complexes

Polyelectrolyte complexes have a wide range of applications for surface modification and flocculation and sorption of organic molecules from solutions. As an example, complexes between poly(diallyl dimethyl ammonium chloride) and poly(styrene sulfonate) have been investigated by diffusion and electrophoresis NMR. The formation of primary or soluble complexes is monitored. The hydrodynamic size is characterized by the hydrodynamic radius, calculated from the diffusion coefficient determined by pulsed field gradient NMR. In the combination with electrophoresis NMR, the effective charge of the molecules and complexes is determined. The hydrodynamic size of the primary complex is smaller than that of the pure polyelectrolyte of the larger molecular weight, in the present case poly(styrene sulfonate), in solution, since charges are compensated by the oppositely charged polyelectrolyte and hence the repelling forces diminish. The effective charge of the complexes is drastically reduced.     

Preparative continuous separation of biological particles by means of free-flow magnetophoresis in a free-flow electrophoresis chamber.

For sorting, cells or cellular components can specifically be labeled by antibody-coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow-chamber of a free-flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free-flow electrophoresis system, the particle fractions are collected in different vials. On-line control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 X 10(8) particles per hour. In experiments with blood cells, a high enrichment of either B-or-T-lymphocytes was obtained. In contrast to free-flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.     

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.     

Correlation of transverse and rotational diffusion coefficient: a probe of chemical composition in hydrocarbon oils

Measurements of relaxation time and diffusion coefficient by nuclear magnetic resonance are well-established techniques to study molecular motions in fluids. Diffusion measurements sense the translational diffusion coefficients of the molecules, whereas relaxation times measured at low magnetic fields probe predominantly the rotational diffusion of the molecules. Many complex fluids are composed of a mixture of molecules with a wide distribution of sizes and chemical properties. This results in correspondingly wide distributions of measured diffusion coefficients and relaxation times. To first order, these distributions are determined by the distribution of molecular sizes. Here we show that additional information can be obtained on the chemical composition by measuring two-dimensional diffusion-relaxation distribution functions, a quantity that depends also on the shape and chemical interactions of molecules. We illustrate this with experimental results of diffusion-relaxation distribution functions on a series of hydrocarbon mixtures. For oils without significant amounts of asphaltenes, the diffusion-relaxation distribution functions follow a power-law behavior with an exponent that depends on the relative abundance of saturates and aromatics. Oils with asphaltene deviate from this trend, as asphaltene molecules act as relaxation contrast agent for other molecules without affecting their diffusion coefficient significantly. In waxy oils below the wax appearance temperature a gel forms. This is reflected in the measured diffusion-relaxation distribution functions, where the restrictions due to the gel network reduce the diffusion coefficients without affecting the relaxation rates significantly.     

A theoretical method to analyze diffusion of probe molecules in nanostructured fluids by fluorescence correlation spectroscopy

In this paper, we study the fluorescence fluctuation correlation function in structured fluids where the diffusion coefficients of probe molecules have different values depending on the distance from initial position, and we derive two simple expressions. Both of them reproduce the exact numerical results rather accurately. One of the expressions contains a time-dependent diffusion coefficient and has a clear physical meaning. We show a procedure to analyze experimental data using the time-dependent diffusion coefficient which results from crossover from free diffusion inside a mesh to hindered diffusion through mesh structures.     

Size and shape of fast-tumbling bicelles as determined by translational diffusion

In this study, the size and shape of an isotropic bicelle have been determined by measuring the translational diffusion as a function of the volume fraction of the lipids. A linear relation between the diffusion coefficients is obtained for both DMPC and DHPC in the bicelles. The slope of this linear function, which is strongly shape-dependent, is found to be different for the two molecules. This difference is direct evidence that the two molecules are not fully mixed in the bicelle. The shape- combined with the size-dependence of the diffusion coefficient allows us to calculate both the size and shape of the bicelle.     

Transport coefficients for hard-chain fluid

Diffusion coefficient and shear viscosity are calculated for fluids containing molecules modelled as chains of tangent hard spheres. A formula for the Stokes–Einstein relation is proposed for hard chain fluids to calculate the shear viscosity from the diffusion coefficient. The numerical results show a good agreement between theoretical values and molecular dynamics results