Separation of polystyrene microbeads using dielectrophoretic/gravitational field-flow-fractionation.

The characterization of a dielectrophoretic/gravitational field-flow-fractionation (DEP/G-FFF) system using model polystyrene (PS) microbeads is presented. Separations of PS beads of different surface functionalization (COOH and none) and different sizes (6, 10, and 15 microm in diameter) are demonstrated. To investigate the factors influencing separation performance, particle elution times were determined as a function of particle suspension conductivity, fluid flow rate, and applied field frequency and voltage. Experimental data were analyzed using a previously reported theoretical model and good agreement between theory and experiment was found. It was shown that separation of PS beads was based on the differences in their effective dielectric properties. Particles possessing different dielectric properties were positioned at different heights in a fluid-flow profile in a thin chamber by the balance of DEP and gravitational forces, transported at different velocities under the influence of the fluid flow, and thereby separated. To explore hydrodynamic (HD) lift effects, velocities of PS beads were determined as a function of fluid flow rate in the separation chamber when no DEP field was applied. In this case, particle equilibrium height positions were governed solely by the balance of HD lift and gravitational forces. It was concluded that under the experimental conditions reported here, the DEP force was the dominant factor in controlling particle equilibrium height and that HD lift force played little role in DEP/G-FFF operation. Finally, the influence of various experimental parameters on separation performance was discussed for the optimization of DEP/G-FFF.     

Determination of particle sedimentation rate by ultrasonic interferometry: Role of particle size,density and volume fraction

The sedimentation rate (SR) of non-aggregated spherical particles in suspension was determined using an ultrasonic interferometry technique (Echo-Cell); this method is based on A-mode echography and measures the rate of formation of a sediment on a solid plate during settling. The particle accumulation rate, which is related to SR, is obtained from the interference of two waves reflected by two interfaces: one between the plate and the sediment and the other between the sediment and the suspension. Studies were carried out at 25°C using latex spheres of different diameters (7 to 20 μm) and densities (1.062 to 1.190 g/cm³) suspended in distilled water at various volume fractions (1% to 5%). As anticipated by the Stokes model, linear relations were found between SR and both particle density and the square of particle radius. Experimental SR values decreased with increasing suspension particle concentration; these concentration effects were in good agreement with those predicted by the Steinour model. Our results thus serve to validate the theoretical aspects of the Echo-Cell method and suggest its usefulness as a tool for studies of RBC interaction and RBC aggregation.     

MyDEP: A New Computational Tool for Dielectric Modeling of Particles and Cells

Dielectrophoresis (DEP) and electrorotation (ROT) are two electrokinetic phenomena exploiting nonuniform electric fields to exert a force or torque on biological particles suspended in liquid media. They are widely used in lab-on-chip devices for the manipulation, trapping, separation, and characterization of cells, microorganisms, and other particles. The DEP force and ROT torque depend on the respective polarizabilities of the particle and medium, which in turn depend on their dielectric properties and on the field frequency. In this work, we present a new software, MyDEP, which implements several particle models based on concentric shells with adjustable dielectric properties. This tool enables the study of the variation in DEP and ROT spectra according to different parameters, such as the field frequency and medium conductivity. Such predictions of particle behavior are very useful for choosing appropriate parameters in DEP experiments. The software also enables the study of the homogenized properties of spherical or ellipsoidal multishell particles and provides a database containing published cell properties. Equivalent electrical conductivity and relative permittivity of the cell alone and in suspension can be calculated. The software also offers the ability to create graphs of the evolution of the crossover frequencies with the electric field frequency. These graphs can be directly exported from the software.     

Blood modeling using polystyrene microspheres

The steady flow viscosity at shear rates 0 to 120 sec-1 and dynamic viscoelasticity at frequencies 0.02 to 0.8 Hz were determined for aqueous suspensions of uniform polystyrene microspheres of 1.0 micron diameter. Rheological properties of the microsphere suspensions were Newtonian for particle concentrations up to 32%. By introducing dextran and calcium chloride into the particle suspensions, non-Newtonian behavior was produced similar to that observed for human blood. The cooperative effects of dextran and calcium ions promoted aggregation of particles at a concentration as low as 12%. Thus, a suspension of uniform sized spherical polystyrene particles in aqueous solution of dextran may be made to mimic blood by controlling the surface charge on the polystyrene spheres using addition of calcium ions to the medium.     

Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.     

Characterization of reaerosolization from impingers in an effort to improve airborne virus sampling

Aims:  To assess the impact of reaerosolization from liquid impingement methods on airborne virus sampling.
Methods and Results:  An AGI-30 impinger containing particles [MS2 bacteriophage or 30-nm polystyrene latex (PSL)] of known concentration was operated with sterile air. Reaerosolized particles as a function of sampling flow rate and particle concentration in the impinger collection liquid were characterized using a scanning mobility particle sizer. Reaerosolization from the impinger was also compared to that from a BioSampler. Results show that reaerosolization increases as flow rate increases. While the increased particle concentration in the impinger collection liquid leads to an increase in the reaerosolization of PSL particles, it does not necessarily lead to an increase in the reaerosolization of virus particles. Reaerosolization of virus particles begins to decrease as the particle concentration in the impinger collection liquid rises above 10⁶ PFU ml⁻¹. This phenomenon results from aggregation of viral particles at high concentrations. Compared with micron-sized particles, nanosized virus particles are easier to aerosolize because of reduced inertia. Reaerosolization from the BioSampler is demonstrated to be significantly less than that from the impinger.
Conclusions:  Reaerosolization from impingement sampling methods is a mode of loss in airborne virus sampling, although it is not as significant a limitation as the primary particle size of the aerosol. Utilizing a BioSampler coupled with short sampling periods to prevent high accumulative concentrations can minimize the impact of reaerosolization.
Significance and Impact of the Study:  This study confirms reaerosolization of virus particles to be a mode of loss in impingement sampling and identifies methods to minimize the loss.     

The latex particle adherence (LPA) assay for detection of leukocytes with adhesive surface properties.

A new, simple, and rapid in vitro assay has been developed for identification of adherent and nonadherent leukocytes. The assay is based on adherence of latex (polystyrene) particles to the cell surface. Using the latex particle adherence (LPA) assay, the percentage of adhesive leukocytes has been determined in human peripheral blood mononuclear preparations and in the lymph nodes, thymus, bursa of Fabricius, spleen, and bone marrow of mouse, chicken, and rat origin. The highest proportion of LPA-positive cells was found in peritoneal exudate, bone marrow, and spleen, the lowest proportion, in thymus and bursa of Fabricius. LPA-Positive cells in human peripheral blood mononuclear preparations were identified as surface immunoglobulin-positive lymphocytes nonrosetting with sheep red blood cells. LPA-Positive cells in peritoneal exudate were identified as macrophages. Incubation of leukocyte suspensions on polystyrene petri dishes or nylon wool columns reduces substantially the percentage of LPA-positive cells in the nonadherent fraction. The LPA assay seems to be a method of choice for establishing the relationship between adhesiveness of the cell surface and other cell membrane markers on a single-cell level.     

Sedimentation volume, redispersibility and appearance of a polystyrene latex suspension in the presence of nonionic surface active agents

The sedimentation volume, redispersibility and appearance of a polystyrene latex suspension in the presence of nonionic surface active agent of polyoxyethylene glycol monoethers of n-hexadecanol have been investigated. At low concentrations of nonionic surface agents, hard caked suspensions were found. This is probably due to the attraction between the uncoated areas or the mutual adsorption of the adsorbed molecules on the bare surface of the particles in the sediment. At high concentrations of nonionic surface active agents, the redispersibility of the particles was affected by the steric repulsive force.     

Multiphase electrodes for microbead control applications: integration of DEP and electrokinetics for bio-particle positioning

Advances in microfabrication have introduced new possibilities for automated, high-throughput biomedical investigations and analysis. Physical effects such as dielectrophoresis (DEP) and AC electrokinetics can be used to manipulate particles in solution to coordinate a sequence of bioanalytical processing steps. DEP is accomplished with non-uniform electric fields that can polarize particles (microbeads, cells, viruses, DNA, proteins, etc.) in suspension causing translational or rotational movement. AC electrokinetics is another phenomena involved with movement of particles in suspension with electric fields and is comprised of both electro-thermal and electro-osmotic effects. This paper investigates single layer electrodes that are effective for particle localization and clustering based on DEP and AC electrokinetic effects. We demonstrate a novel multi-electrode setup capable of clustering particles into an array of discrete bands using activated and electrically floating electrodes. These bands shift to adjacent regions on the electrode surface by altering the electrode activation scheme. The predictability of particle placement to specific locations provides new opportunities for integration and coordination with raster scanning lasers or a charge coupled device (CCD) for advanced biomedical diagnostic devices, and more sophisticated optical interrogation techniques.     

Principles of separating micro-organisms from suspensions using ultrasound

C.A. MILES, M.J. MORLEY, W.R. HUDSON AND B.M. MACKEY. 1995. When particles in suspension are placed in a stationary ultrasonic field, they form bands of high concentration at half-wavelength intervals. Several theoretical papers have recently contributed to an understanding of the process but, for particles as small as bacteria, there are substantial discrepancies between the different theories, and a lack of published observations.
The threshold amplitudes required to band latex spheres (0.5-5.0 μm diameter) were measured in the frequency range 1–3 MHz and used to establish conditions, reported here for the first time, suitable for banding and moving vegetative bacterial cells in pure culture. Ultrasonic means for separating and concentrating cream and bacteria at opposite ends of a tube containing a mixture of Escherichia coli and diluted milk are also described and the breakdown of theoretical equations at low particle size is discussed.     

Determination of the enantiomerization energy barrier of some 3-hydroxy-1,4-benzodiazepine drugs by supercritical fluid chromatography

In this study, nucleotide adsorption-desorption behaviour of boronic acid-carrying uniform, porous particles was investigated. The particles were produced by a "multi-step microsuspension polymerization" in the form of poly(styrene-vinylphenyl boronic acid-divinylbenzene) terpolymer. In the first step of the production method, uniform polystyrene latex particles (6.2 microm in size) were obtained by dispersion polymerization. These particles were first swollen by a low molecular mass organic agent (i.e. dibutylphthalate, DBP) and then by a monomer mixture including styrene (S), 4-vinylphenyl boronic acid (VPBA) and divinylbenzene (DVB). The particle uniformity was protected in both swelling stages by adjusting DBP/polystyrene latex and monomer mixture/polystyrene latex ratios. Polymerization of the monomer mixture in the swollen seed particles provided boronic acid-carrying uniform, porous particles 11-12 microm in size. To have uniform particles with different porosities and boronic acid contents, the feed concentration of boronic acid-carrying monomer and the monomer/seed latex ratio were changed. The particles were tried as sorbent for the adsorption of a model nucleotide (i.e., beta-nicotinamide adenine dinucleotide, beta-NAD). In the beta-NAD adsorption experiments, the maximum equilibrium adsorption was obtained at pH 8.5 which was very close to pKa of boronic acid. The incorporation of boronic acid functionality provided a significant increase in the beta-NAD adsorption. In contrast to plain poly(styrene-co-divinylbenzene) particles, four-fold higher beta-NAD adsorption was obtained with the boronic acid functionalized particles. Beta-NAD was desorbed from the particles with the yields higher than 90% by weight.     

Feasibility of Focused Beam Reflectance Measurement (FBRM) for Analysis of Pharmaceutical Suspensions in Preclinical Development

This study examined the use of focused beam reflectance measurement (FBRM) for qualitative and quantitative analysis of pharmaceutical suspensions with particular application to toxicology supply preparations for use in preclinical studies. Aqueous suspensions of ibuprofen were used as prototype formulations. Initial experiments were conducted to examine the effects of operational conditions including FBRM probe angle, probe location, and mixing (method and rate of mixing) on the FBRM analysis. Once experimental conditions were optimized, the homogeneity and sedimentation-redispersion of particles in the suspensions were assessed. Ibuprofen suspension under continuous agitation was monitored using FBRM for 60 h to study particle size change over time. Another study was performed to determine if particle count rates obtained by FBRM could be correlated to suspension concentration. The location and the angle of the FBRM probe relative to the beaker contents, and the rate and the method of mixing the suspension were found to be sensitive parameters during FBRM analysis. FBRM was able to monitor the process of particle sedimentation in the suspension. The attrition of ibuprofen particles was detectable by FBRM during prolonged stirring with an increase in the number of smaller particles and decrease in the number of larger particles. A strong correlation was observed between particle count rate by FBRM and ibuprofen concentration in the suspension. Also, change in content uniformity in the suspension at different locations of the beaker was represented by FBRM particle count. Overall, FBRM has potential to be a useful tool for qualitative and quantitative analysis of pharmaceutical suspensions.     

Collection characteristics of a batch-type wetted wall bioaerosol sampling cyclone

The collection efficiency and sample retention of a batch-type wetted wall bioaerosol sampling cyclone (BWWC) were experimentally characterized. The BWWC is designed to sample air at 400 l/min and concentrate the particles into 12 ml of water. Aerosol is transported into a cylindrically-shaped axial flow cyclone through a tangential slot and the particles are impacted on the inner wall, which is wetted by air shear acting on a liquid pool at the base of the cyclone. The retention of collected particles and the aerosol collection efficiency of the BWWC were evaluated with polystyrene latex beads (PSL), sodium fluorescein/oleic acid droplets, and Bacillus atrophaeus (aka BG) spores. The retention of particles was determined by adding hydrosol directly into the device, running the BWWC for a pre-set period of time, and then determining the amount of particulate matter recovered relative to the initial amount. For 1-μm diameter PSL, 90% of the particles were recoverable from the cyclone body immediately after their introduction; however, only 10% were retained in the collection liquid after 8 h of operation. The aerosol sampling efficiency was determined by comparing the amount of particulate matter collected in the liquid with that collected by a reference filter. The collection efficiency was 50–60% for 1- and 3-μm polystyrene (PSL) particles, and 1.5% for 10-μm oleic acid particles. The efficiency for 3-μm oleic acid droplets was 35%. Explanations are provided for the difference between liquid and solid particle behavior, and for the low efficiency for the large liquid particles. The collection efficiency for single spore BG was slightly lower than that for 1-μm PSL.     

The Influence of Preculture Conditions and Food Quality on the Ingestion and Digestion Process of Three Species of Heterotrophic Nanoflagellates

The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated. Three species of gram-negative bacteria, one species of gram-positive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria). Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles. In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis. Digestion was found to be particle-specific. Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles. The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content. "Selective digestion" could be shown to be an important selection mechanism concerning natural food particles. The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field. In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.     

Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation

We have developed a rapid and precise electron microscope technique for the quantitation of gold particles in suspension using latex microspheres as a reference (EM latex technique). This technique allowed us to determine the specific absorption of colloidal gold at its absorption maximum (520 nm) and the average number of ligands ([125I]IgG) bound to one gold particle. On the basis of these values important binding characteristics of protein-gold complexes to cell surfaces were analyzed in a model system consisting of Staphylococcus aureus with protein A on the cell wall as a specific binding site for IgG-Au. Our observations showed that the number of binding sites represented by one IgG-gold complex depended primarily on the particle size, with one 20-nm IgG-Au corresponding to 15 and one 6-nm IgG-Au to 2.5 binding sites. Hence, the efficiency of binding of IgG-Au complexes increased with decreasing gold particle size. Saturation of binding sites, however, was not achieved. The technique also made possible the determination of the affinity between IgG-Au complexes and the cell surface; this affinity can either be regarded as a characteristic of the ligand IgG or of the gold particle. We observed that the affinity of IgG decreased with the size of the gold particles to which IgG was bound, whereas the affinity of the entire gold particle increased with particle size. The EM latex technique for quantitation of gold particles extends the general use of protein-gold complexes to the quantitative characterization of their interaction with cell surface constituents.     

Sub-micron particle behaviour and capture at an immuno-sensor surface in an ultrasonic standing wave

The capture of 200 nm biotinylated latex beads from suspensions of concentration 10(7) to 2.5 x 10(8) particle/ml on an immuno-coated surface of the acoustic reflector in an ultrasound standing wave (USW) resonator has been studied while the acoustic pathlength was less than one half wavelength (lambda/2). The particles were delivered to the reflector's surface by acoustically induced flow. The capture dependencies on suspension concentration, duration of experiments and acoustic pressure have been established at 1.09, 1.46 and 1.75 MHz. Five-fold capture increase has been obtained at 1.75 MHz in comparison to the control (no ultrasound) situation. The contrasting behaviours of 1, 0.5 and 0.2 mum fluorescent latex beads in a lambda/4 USW resonator at 1.46 MHz have been characterized. The particle movements were observed with an epi-fluorescent microscope and the velocities of the particles were measured by particle image velocimetry (PIV). The experiments showed that whereas the trajectories of 1 mum particles were mainly affected by the direct radiation force, 0.5 mum particles were influenced both by the radiation force and acoustic streaming. The 0.2 mum latex beads followed acoustic streaming in the chamber and were not detectably affected by the radiation force. The streaming-associated behaviour of the 200 nm particles has implications for enhanced immunocapture of viruses and macromolecules (both of which are also too small to experience significant acoustic radiation force).     

An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water

Dielectrophoresis (DEP), the motion of a particle caused by an applied electric field gradient, can concentrate microorganisms non-destructively. In insulator-based dielectrophoresis (iDEP) insulating microstructures produce non-uniform electric fields to drive DEP in microsystems. This article describes the performance of an iDEP device in removing and concentrating bacterial cells, spores and viruses while operated with a DC applied electric field and pressure gradient. Such a device can selectively trap particles when dielectrophoresis overcomes electrokinesis or advection. The dielectrophoretic trapping behavior of labeled microorganisms in a glass-etched iDEP device was observed over a wide range of DC applied electric fields. When fields higher than a particle-specific threshold are applied, particles are reversibly trapped in the device. Experiments with Bacillus subtilis spores and the Tobacco Mosaic Virus (TMV) exhibited higher trapping thresholds than those of bacterial cells. The iDEP device was characterized in terms of concentration factor and removal efficiency. Under the experimental conditions used in this study with an initial dilution of 1 x 105 cells/ml, concentration factors of the order of 3000x and removal efficiencies approaching 100% were observed with Escherichia coli cells. These results are the first characterization of an iDEP device for the concentration and removal of microbes in water.     

Electrophoresis of colloidal biological particles

Microscope electrophoresis was used to measure the electrophoretic mobility of polystyrene latex particles and bacterial, and mammalian tissue cells. The submicroscopic hydrophilic colloids (gelatin, serum albumin, and staphylococcal enterotoxin B) were adsorbed on latex carrier particles to determine their electrophoretic mobility and the effect of concentration, pH, electrolyte addition, and buffer ionic strength. Mobility curves as a function of pH were established for latex particles at 1 ppm concentration indicating an isoelectric point (IEP) at pH 3.6. The IEP for Escherichia coli B cells was measured at pH 2.8, Serratia marcescens at pH 2.6, Bacillus subtilis var. niger at pH 2.9, and L strain mouse fibroblast cells at pH 4.4. Using an adsorption technique, isoelectric points were measured for proteins: gelatin (acid form) at pH 9.4, serum albumin at pH 4.9, and staphylococcal enterotoxin B at pH 6.3. Procefures for examining electrophoretic characteristics of microscopic and submicroscopic biological particles are described in order to standardize procedures and to generate results applicable to an understanding of parameters influencing concentration and purification of colloidal biological particles.     

Retention of ingested latex particles in Peyer's patches of germfree and conventional mice

Conventional and germfree mice ingested a suspension of 2-micron latex particles in drinking water for a 15-day period. Number and distribution of intestinal Peyer's patches did not differ significantly in the two types of mice. Cleared Peyer's patches were compared with regard to size and particle content. The location of particles within Peyer's patch follicles of germfree mice was similar to that of conventional mice, but the latter had significantly larger follicles and greater accumulations of latex particles. Latex concentration varied with patch location. Proximal patches contained the majority of particles in germfree mice, whereas particles were most abundant in distal patches of conventional mice. The results show that particle uptake into Peyer's patches takes place even in the complete absence of bacteria in the gut.     

Immunodevice for simultaneous detection of two relevant tumor markers based on separation of different microparticles by dielectrophoresis

In this study, a rapid immunosensing system has been developed for simultaneous analysis of two tumor markers, alpha-fetoprotein (AFP) and prostate-specific antigen (PSA). The strategy for rapid multisensing is based on rapid immunoreactions occurring on the surface of microparticles and the spatial separation of different particles that exhibit distinct dielectrophoretic (DEP) properties. Recognition events for immunoreactions have been performed on the surfaces of two different microparticles conjugated with two different antibodies: polystyrene (PS) microparticles with an anti-AFP antibody and gold-coated (50 nm) PS microparticles with an anti-PSA antibody. The DEP devices consisted of an upper indium tin oxide (ITO) glass and a lower ITO electrode with a castellated structure. Sandwich structured immunocomplexes of AFP and PSA were created on the microparticles and then labeled with fluorescent molecules via a secondary antibody. After introducing the particles into the DEP devices, an alternating current (AC) voltage (20 V peak-to-peak voltage and 30 kHz) was applied between the upper ITO and lower electrodes to manipulate the particles with negative dielectrophoresis (n-DEP).The uncoated PS particles and the gold-coated PS particles rapidly moved and separated to form wave-like line and triangular aggregates, respectively. The measurements of the fluorescence signals from the uncoated and gold-coated PS particles directed to different regions of the DEP device permit the determination of the concentrations of AFP and PSA simultaneously. No cross-reactivity was observed for either of the immunorecognition events. Limits of detection achieved for the AFP and PSA assays were 0.18 and 1.1 ng mL(-1), respectively, which satisfy medical requirements for both antigens in human serum. The total assay time required for the simultaneous detection of the two different analytes in this study (25 min) was shortened compared to the conventional enzyme-linked immunosorbent assay.