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This Review describes how in the period 1940-1959 cell electrophoresis (in the earlier literature often referred to as 'microelectrophoresis') was used to explore the surface chemistry of cells. Using the erythrocyte as a suitable model for the study of biological membranes, the early investigators were agreed on the presence of negatively charged groups at the surface of this cell. The contemporary dogma was that these were phosphate groups associated with phospholipids. Work in the 1960s, particularly on changes in the electrokinetic properties of erythrocytes following treatment with proteolytic enzymes, lead to the realization that the negatively charged groups at the red cell surface are predominantly due to sialic acids carried on glycoproteins. It quickly became apparent from cell electrophoresis that sialic acids have a ubiquitous presence on the surface of animal cells. This finding required that any complete model of the plasma membrane must include glycosylated molecules at the cell periphery, thus laying the foundations for the field termed 'Glycobiology of the Cell Surface'.

In this paper we describe progress in using the prodigious data-collecting ability of multilane microelectrophoresis instruments to bear on problems in scaled nucleic acid assays. We emphasize compound stacking and solid-support loading as means to concentrate < 100 pg samples for direct injection. Reaction Mapping is applied to readout quantitative polymerase chain reaction gene-expression and as a way to practically overcome difficulty in interpreting amplification curves of multiplexed quantitative polymerase chain reaction at 20―50 gene/well complexity. We demonstrate multiplexed readout of gene expression over an abundancy range of 9 Log 2 units starting with reverse-transcribed samples as small as five molecules in each sample.

Mobilities of different chromatographic particles obtained from two electrokinetic methods were determined and compared. The particles were all based on porous silica, between 3 and 15 microm diameter, and were either native, or derivatized. As intermediate of chemical modification 3-mercaptopropyl-modified silica particles (TP-silica) are obtained. These particles were finally transformed into weakly basic anion exchangers with O-9-(tert-butylcarbamoyl)quinine (tBuCQN) as chiral selector. The electrophoretic mobility of the particles was determined from their migration velocity in an electric field using microelectrophoresis. Electrokinetic chromatography with a capillary column packed with the same particles was used to measure the electroosmotic flow generated. All measurements were carried out in background electrolytes of equal ionic strength (10(-2) mol/L), at pH varying between 3.5 and 9.5. From these data a rough estimation of the zeta-potential was made, taking Helmholtz-Smoluchowski conditions into consideration. With both methods the zeta-potential of the native silica particles is negative throughout, and its value increases with pH. The weakly basic tBuCQN particles have positive zeta-potentials at pH lower than about 7.5, but exhibit a negative zeta-potential above this pH, indicating the dominating effect of residual silanol groups at the silica surface. The zeta-potential for these anion-exchange particles ranged between +30 and -40 mV. The zeta-potentials derived with electrophoresis and electroosmosis agree, showing the adequacy of the approach, although many limitations must be taken into account in the treatment of the electrokinetic phenomena in such porous systems. These restrictions in interpreting mobility and zeta-potential were discussed.

The adsorption and depletion of the anionic polymer poly(styrene sulfonate) (PSS) on fresh human red blood cells (RBC) were investigated by measuring RBC electrophoretic mobility as a function of polymer molecular mass (48-2610 kDa), ionic strength (15 and 150 mM NaCl) and polymer concentration (

Chemical and structural complexity of bacterial cell surfaces complicate accurate quantification of cell surfaces properties. The presence of fibrils, fimbriae or other surface appendages on bacterial cell surfaces largely influence those properties and would therefore play a major function in interfacial phenomena as aggregation and adhesion. The electrophoretic softness and fixed charge density in the polyelectrolyte layer of nine Streptococcus mitis strains, usually carrying long sparsely distributed fibrils, were determined by the soft particle analysis using measured electrophoretic mobilities as a function of the ionic strength. In general, S. mitis cell surfaces are electrophoretically soft (1.0-2.5 nm) with a fixed negative charge density of -1.2 to -4.3 x 10(6) Cm-3. Further, a comparison with surfaces of other bacterial strains that are reported to be soft indicates that the Ohshima soft layer model does not provide information on the surface morphology causing the softness. The most likely reason is that the electroosmotic flow occurs only in the very outer region of thick extracellular surface layers. Nevertheless, determining the surface softness is essential for proper characterization of the cell surface electrostatics.

Emerging need for large-scale, high-resolution analysis of biopolymers, such as DNA sequencing polymerase chain reaction, (PCR) product sizing, single nucleotide polymorphism (SNP) hunting and analysis of protein molecules necessitated the development of automated and high-throughput gel electrophoresis based methods enabling rapid, high-performance separations in a wide molecular weight range. Scaling down electric field mediated separation processes supports higher throughput due to the applicability of higher voltages, thus speeding up analysis time. Indeed, efforts in miniaturization resulted in faster, easier, less costly and more convenient analyses, fulfilling the needs of the emerging biotechnology industry for microscale and massively parallel assays. The two primary approaches in miniaturizing electrophoresis dimensions are the capillary and microslab formats. This latter one evolved towards ultrathin-layer gel electrophoresis which is, except from the thickness of the separation platform, slightly in the upper side of the scale, resulting in considerably easier handling. Ultrathin-layer gel electrophoresis combines the advantages of conventional slab-gel electrophoresis (multilane format) and capillary gel electrophoresis (rapid, high-efficiency separations). It is readily automated, automatic versions of it have been extensively used for large-scale DNA sequencing in the Human Genome Project and more recently became popular in high throughput DNA fragment analysis. Ultrathin-layer techniques are the first step towards the wider use of electrophoresis microchips in perfecting a user-friendly interface between the user and the microdevice.

We present a rapidly fabricated micro-capillary electrophoresis chip that utilizes surface-tension-driven flow for sample injection and extraction of DNA. Surface-tension-driven flow (i.e. passive pumping) [G. M. Walker et al., Lab. Chip. 2002, 2, 131-134] injects a fixed volume of sample that can be predicted mathematically. Passive pumping eliminates the need for tubing, valves, syringe pumps, and other equipment typically needed for interfacing with microelectrophoresis chips. This method requires a standard micropipette to load samples before separation, and remove the resulting bands after analysis. The device was made using liquid phase photopolymerization to rapidly fabricate the chip without the need of special equipment typically associated with the construction of microelectrophoresis chips (e.g. cleanroom) [A. K. Agarwal et al., J. Micromech. Microeng. 2006, 16, 332-340; S. K. Mohanty et al., Electrophoresis 2006, 27, 3772-3778]. Batch fabrication time for the device presented here was 1.5 h including channel coating time to suppress electroosmotic flow. Devices were constructed out of poly-isobornyl acrylate and glass. A standard microscope with a UV source was used for sample detection. Separations were demonstrated using Promega BenchTop 100 bp ladder in hydroxyl ethyl cellulose (HEC) and oligonucleotides of 91 and 118 bp were used to characterize sample injection and extraction of DNA bands. The end result was an inexpensive micro-capillary electrophoresis device that uses tools (e.g. micropipette, electrophoretic power supplies, and microscopes) already present in most labs for sample manipulation and detection, making it more accessible for potential end users.

Double focus fluorescence correlation spectroscopy (dfFCS) was used to determine electrophoretic mobilities of short double-stranded DNA (dsDNA)-fragments (75 base pairs (bp) -1019 bp) in microfluidic channels. The electrokinetic flow profile across a microchannel was measured with 1 microm spatial resolution and separated in electroosmotic and electrophoretic contributions. Experiments show that the free solution mobility is independent of DNA length. The diffusion constant is additionally determined by FCS and follows a length dependent rod-diffusion model. We interpret the electrophoretic mobilities using a modified Nernst Einstein relation, which additionally takes Manning condensation and counterion induced hydrodynamic retardation forces into account. In 3% w/v polyethylene oxide (PEO)-network (M(r) 3 .10(5) Dalton) the electrophoretic velocities become size-dependent with a power-law exponent be-tween 0.28 and 0.31. Mixtures of dsDNA-fragments exhibit distinguishable peaks in the dfFCS cross-correlation function. The potential of dfFCS for realtime micro-analysis in terms of speed and spatial resolution is discussed.

We present a microelectrophoresis chip that is simple to fabricate using the microfluidic tectonics (microFT) platform (Beebe, D. J. et al., Proc. Natl. Acad. Sci. USA 2000, 97, 13488-13493; Agarwal, A. K. et al.,. J. Micromech. Microeng. 2006, 16, 332-340). The device contains a removable capillary insert (RCI) for easy sample collection after separation (Atencia, J. et al.,. Lab Chip 2006, DOI: 10. 1039/b514068d). Device construction is accomplished in less than 20 min without specialized equipment traditionally associated with microelectrophoresis chip construction. microFT was used to build a PAGE device utilizing two orthogonal microchannels. One channel performs standard separations, while the second channel serves as an access point to remove bands of interest from the chip via the RCI. The RCI contains an integrated electrode that facilitates the removal of bands using electrokinetic techniques. The device was characterized using prestained proteins (Pierce BlueRanger and TriChromRanger). Samples were loaded into the microelectrophoresis device via a standard micropipette. An electrical field of 40 V/cm was used to separate and collect the proteins. The microPAGE device is simple to fabricate, benefits from microscale analysis, and includes an on-chip collection scheme that interfaces the macroworld with the microworld.

We demonstrate a versatile microfabricated electrophoresis platform, incorporating arrays of integrated on-chip electrodes, heaters, and temperature sensors. This design allows a range of different sieving gels to be used within the same device to perform separations involving both single- and double-stranded DNA over distances on the order of 1 cm. We use this device to compare linear and cross-linked polyacrylamide, agarose, and thermo-reversible Pluronic-F127 gels on the basis of gel casting ease, reusability, and overall separation performance using a 100 base pair double-stranded DNA ladder as a standard sample. While cross-linked polyacrylamide matrices provide consistently high-quality separations in our system over a wide range of DNA fragment sizes, Pluronic gels also offer compelling advantages in terms of the ability to remove and reload the gel. Agarose gels offer good separation performance, however, additional care must be exercised to ensure consistent gel properties as a consequence of the need for elevated gel loading temperatures. We also demonstrate the use of denaturing cross-linked polyacrylamide gels at concentrations up to 19% to separate single-stranded DNA fragments ranging in size from 18 to 400 bases in length. Primers differing by 4 bases at a read length of 30 bases can be separated with a resolution of 0.9-1.0 in under 20 min. This level of performance is sufficient to conduct a variety of genotyping assays including the rapid detection of single nucleotide polymorphisms (SNPs) in a microfabricated platform. The ability to use a single microelectrophoresis system to satisfy a wide range of separation applications offers molecular biologists an unprecedented level of flexibility in a portable and inexpensive format.

Charge in ionic micelles determines the trends of their stability and their practical applications. Charge can be calculated from zeta potential (zeta) measurements, which, in turn, can be obtained by Doppler microelectrophoresis. In this study, the electrophoretic properties of dodecyltrimethylammonium bromide (DTAB) in KBr aqueous solution (0-6 mM) were determined by Doppler microelectrophoresis. At very low surfactant concentrations (up to 6 mM), zeta potential was quite constant and due to the ionized monomers (DTA+). Above 6 mM, zeta potential increased to a maximum at surfactant concentrations still below the critical micellar concentration (CMC). This increase could be explained by a formation of nonmicellar aggregates of DTAB. Then, above the CMC, zeta potential underwent an abrupt reduction, which was dependent qualitatively and quantitatively on KBr concentration, and which could be due to an increase of the number of counterions adsorbed on the micelle surface. Calculation of effective micellar charge from zeta potential gave the surface charge density. Comparing this value with the theoretical, obtained from geometrical considerations, a fraction of 0.29 of charged micellar headgroups was obtained when DTAB was in aqueous solution, which is consistent with the value obtained by conductivity measurements.

In this retrospect of approximately 30 years of work with micromethods, some of them developed in our own laboratory, their principles and application to different separation problems are described, such as one- and two-dimensional microelectrophoresis in capillaries and microslab gels, isoelectric focusing in capillaries or microslab gels, microchromatography, microphotometry, and microfluorometry for qualitative and quantitative evaluation of separation patterns. In addition, some useful auxiliary methods are also described, e.g., a method for quantitative protein determination in a microliter volume when neither the volume nor the protein content in that volume are known, and methods for the determination of glycoproteins, amino acids, and sugars in the picomole range.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

A set-up for casting fluorescent indicator agarose gels on ultrathin polyacrylamide microelectrophoresis gel media (Pharmacia PhastGel media) is described. The zymogram system allows a rapid and sensitive detection of deoxyribonuclease in various gel media, following isoelectric focusing, native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Red blood cell (RBC) electrophoresis measurements in polymer solutions have recently been introduced as a promising approach for investigating polymer-cell interactions near the RBC surface. A polymer-poor depletion layer near the RBC has been demonstrated: for depletion layers thicker than the double layer, viscosity within the depletion layer, rather than suspending medium viscosity, affects cell mobility. Using a well-documented model of sepsis in rats, we have induced RBC membrane damage, and then measured the electrophoretic mobility of rat RBC from control and septic animals. Mobility measurements were carried out for cells suspended in polymer-free buffer and in 0.5-2% solutions of dextran 500 (500 kDa molecular mass); RBC aggregation in autologous plasma and in dextran 500 was also studied. Our results indicate: (i) as anticipated from prior studies, the aggregation of RBC from septic animals is markedly enhanced (p

Intracellular microelectrophoresis was applied to investigate the electrokinetic properties of human buccal epithelium cell nuclei after exposure of cells to microwaves of wavelengths of 8 mm (f = 37.5 GHz) and 16 mm (f = 18.75 GHz) at a surface power density of 0.2 mW/cm(2). Irradiated or nonirradiated cells were suspended in a flat microelectrophoretic chamber and exposed to an electric field of 15 V/cm at a current flow of 0.1 mA. The cells, whose nuclei altered their intracellular location towards the anode of the externally applied electric field, were considered to have negatively charged nuclei. The percentage of cells with electrophoretically movable nuclei was determined as the value of electronegativity of cell nuclei (ENN). Microwaves induced changes of ENN during irradiation of 15-60 s. If cells of a donor had an elevated initial level of ENN, it decreased during irradiation. On the contrary, if cells of another donor had a low initial ENN level, irradiation induced ENN increase. No significant difference between the action of microwaves of wavelengths of 8 mm and 16 mm was found. However, microwave irradiation caused an increase in membrane permeability for the in vivo dye indigo Carmine in cells of all donors irrespectively of the initial levels they showed. This suggests that electrokinetic properties of nuclei in cells do not only depend on cell membrane permeability.

Micro-polyacrylamide gel electrophoresis (micro-PAGE) has been shown to be useful for molecular size separation of urinary proteins. In addition to the sodium dodecyl sulfate micro-gradient gel electrophoresis in 10 μL capillaries, which is used routinely for examination of proteinuria in our laboratory, we tried micro-gradient slab gel electrophoresis (micro-slab-PAGE) for molecular weight determination of urinary proteins. It was also employed for monitoring patients with proteinuric diseases. For detailed studied of urinary proteins, micro-two-dimensional-electrophoresis is recommended.

The electrophoretic mobility of brush border membrane vesicles purified from the rabbit ileum by chromatography on porous glass beads has been measured with a Laser-Zee microelectrophoresis instrument. The value obtained was measured in a solution containing 0.1 M NaCl, 10 mM HEPES-Tris and 50 mM mannitol at pH 7.37 and 20 °C was -1.41 μm s−1 V−1 cm. The mobility depended on both pH and ionic strength of the solution. The isoelectric point was observed to be at pH 4.25. The brush border membrane vesicle surface was found to be highly negatively charged: there are 1000–2000 negative charges on each vesicle at ionic strength 0.1. Treatments with cation had an effect on mobility and gave a reversal of surface charge.