Micro-thermal focusing field-flow fractionation

Focusing mechanism was effectively exploited to separate large (micrometer-size) particles by using new micro-thermal field-flow fractionation (micro-TFFF). It has been shown that the retention order of micrometer-size particles at high field strength can be explained by the mechanism of steric exclusion only at lowest flow rates of the carrier liquid. A simplistic, purely mechanical model of steric exclusion is not accurate to describe the retention at higher flow rates where the focusing phenomenon appears. Despite the fact that the thickness of the channel for micro-FFF cannot be reduced without taking into account a possible deterioration of the separation due to the contribution of "steric exclusion" mechanism, this paper demonstrates, in agreement with our previous results, that if the operational conditions were conveniently chosen, namely a low flow rate, a reasonable fit of the experimental retention data with the theory of steric exclusion mechanism in FFF was found and the separation of micron-size particles can be accomplished. However, high selectivity and resolution and high-speed separation were achieved if the focusing effect has clearly dominated the FFF mechanism. As a result, it seems that the micro-TFFF is the most universal technique which can be applied for the separation of the synthetic and natural macromolecules within an extended range of molar masses up to ultra-high molar masses and for the particles of various chemical nature and origin in a nano-size range as well as for large (micrometers) particles. Until nowadays, only sedimentation and flow field-flow fractionation techniques in so called "steric" modes were applied for the separations of large size particles. This application of micro-TFFF in focusing mode for the separation of large size particles is the first one described in the literature.     

Separation of human and animal cells by steric field-flow fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.     

Separation of human and animal cells by steric field-flow fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.     

The use of sedimentation field-flow fractionation to study suspended particulate matter

The current and potential uses of sedimentation field-flow fractionation (SdFFF) for characterizing suspended particulate matter (SPM) from natural waters is reviewed. Suitable sample preparation methods and run conditions are given which enable the particle size distribution of aquatic SPM to be determined. Samples collected from different natural waters display quite distinct differences in the shape of their particle size distribution.One of the major advantages of this high resolution separation technique is that fractions of specific size ranges can be collected for characterization by other analytical methods. This has been illustrated in this work with scanning electron microscopy and EDAX elemental analysis data. The potential to extend this approach using other characterization techniques such as inductively coupled-plasma mass spectrometry and X-ray diffraction is discussed.A method has been developed for combining adsorption experiments with SdFFF separations that enables the distributions of both the amount adsorbed and the surface adsorption density across the SPM size range to be determined with good resolution. This approach is illustrated by the adsorption of the herbicide glyphosate to two estuarine SPM samples.     

Physical Characterization and Quantification of Bacteria by Sedimentation Field-Flow Fractionation

Studies in microbial ecology require accurate measures of cell number and biomass. Although epifluorescence microscopy is an accepted and dependable method for determining cell numbers, current methods of converting biovolume to biomass are error prone, tedious, and labor-intensive. This paper describes a technique with sedimentation field-flow fractionation to enumerate bacteria and determine their density, size, and mass. Using cultured cells of different shapes and sizes, we determined optimum values for separation run parameters and sample-handling procedures. The technique described can separate and detect 4′, 6-diamidino-2-phenylindole-stained cells and generate a fractogram from which cell numbers and their size or mass distribution can be calculated. A direct method for estimating bacterial biomass (dry organic matter content) which offers distinct advantages over present methods for calculating biomass has been developed.     

Isolation of microfilariae from blood by gravitational field-flow fractionation

Over 100 million persons suffer from diseases caused by filariae infestation, and one billion are at risk. A simple isolation method for both analytical and preparative separation is presented. Based on the simplest field-flow fractionation technique, the gravitational one, effective isolation of microfilariae is achieved. Microfilariae are eluted in the void volume of the channel without pollution by red blood cells. The red blood cell elution peak shows a total absence of microfilariae, as demonstrated after fraction collection and microscopic investigation. The elution mode of microfilariae and red blood cells appears to be a steric one, as confirmed by a reinjection experiment. The simplicity, low cost and the relatively short time required for this separation (10 min) indicate that gravitational field-flow fractionation could become a new separation tool for screening of microfilariae. With both live and dead microfilariae, the high recovery (66–80%) allows preparative fractionation for diagnostic purposes or fundamental research.     

Separation and characterization of red blood cells with different membrane deformability using steric field-flow fractionation

Human red blood cells were treated in different ways to alter their membrane deformability, and the hydrodynamic behavior of these altered cells was studied using the steric field-flow fractionation (FFF) technique. The relationships between cell retention in the FFF channel, flow-rate of the carrier fluid and the applied field strength were studied for normal and glutaraldehyde-fixed human red cells, and separation conditions were optimized. The effect of flow-induced hydrodynamic lift forces on red cell retention in the steric FFF channel was studied, and the results suggest that the membrane deformability of the red cell is an important factor contributing to the lift force, besides other previously described effects due to density and flow velocity. Using steric FFF, a mixture of normal and glutaraldehyde-fixed human red cells was completely separated with a resolution twice that found in published d ata from gel permeation, another hydrodynamic separation technique. Partial loss of membrane deformability, induced by different degrees of glutaraldehyde-fixation, by diamide, or by a thermal treatment, has also been studied. Steric FFF is thus shown to have potential for rapid separation and differentiation of red cells with different density and membrane deformability, conditions known to be associated with, e.g., cell senescence and certain hematological diseases.     

Megakaryocyte cell sorting from diosgenin-differentiated human erythroleukemia cells by sedimentation field-flow fractionation

Anticancer differentiation therapy could be one strategy to stop cancer cell proliferation. Human erythroleukemia (HEL) cell line, incubated with 10 microM diosgenin, underwent megakaryocytic differentiation. Thus, the association diosgenin/HEL could be used as a model of chemically induced cellular differentiation and anticancer treatment. The goal of this work was to determine the capacity of sedimentation field-flow fractionation (SdFFF) to sort megakaryocytic differentiated cells. SdFFF cell sorting was associated with cellular characterization methods to calibrate specific elution profiles. As demonstrated by cell size measurement methods, cellular morphology, ploidy, and phenotype, we obtained an enriched, sterile, viable, and functional fraction of megakaryocytic cells. Thus, SdFFF is proposed as a routine method to prepare differentiated cells that will be further used to better understand the megakaryocytic differentiation process.     

Size characterization of inclusion bodies by sedimentation field-flow fractionation

Sedimentation field-flow fractionation (sedFFF) was evaluated to characterize the size of Delta(4-23)TEM-beta-lactamase inclusion bodies (IBs) overexpressed in fed-batch cultivations of Escherichia coli. Heterologous Delta(4-23)TEM-beta-lactamase protein formed different sizes of IBs, depending upon the induction conditions. In the early phases of recombinant protein expression, induced with low concentrations of IPTG (isopropyl-beta-d-thiogalactoside), IB masses were larger than expected and showed heterogeneous size distributions. During cultivation, IB sizes showed a Gaussian distribution and reached a broad range by the end of the fed-batch cultivations. The obtained result proved the aptitude of sedFFF to rapidly assess the size distribution of IBs in a culture.     

Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.     

Flow field-flow fractionation: new method for separating, purifying, and characterizing the diffusivity of viruses.

The nature and theory of flow field-flow fractionation is described, and its potential applicability to virus-like particles is discussed. Different virus types are shown to be retained at different levels. Retention can be controlled by variation of the experimental parameters, in good agreement with theory. However, a mild adsorption effect is indicated and requires the development of alternate strategies for measuring diffusion coefficients. For Qbeta, our value agrees well within 10% of literature values; the values obtained for other viruses, using Abeta as an internal standard, are untested. Finally, it is demonstrated that flow field-flow fractionation can cleanly fractionate two viruses from one another and from an albumin impurity, that samples as large as several milligrams in size can be analyzed, and that the method has potential utility in the quantitative and qualitative analysis of virus systems.     

Diosgenin dose-dependent apoptosis and differentiation induction in human erythroleukemia cell line and sedimentation field-flow fractionation monitoring

To limit or stop cancer spreading, one of the most prevalent strategies is to induce cancer cell death. Differentiation therapy and apoptosis induction are two ways to achieve this goal. Sedimentation field-flow fractionation (SdFFF) has been described as an effective tool for cell separation, respecting integrity and viability. Because SdFFF takes advantage of intrinsic properties of eluted cells (size, density, shape), we studied the capacity of SdFFF to monitor specific biophysical modifications that occurred during cellular apoptosis or differentiation induction. Then, we used, as an in vitro cellular model of apoptosis and differentiation, diosgenin dose-dependent induction in the polyvalent human erythroleukemia cell line. Two other chemicals were used: phorbol myristate acetate (differentiation inducer) and staurosporine (apoptosis inducer). Our results demonstrated a correlation between SdFFF elution profile changes and induction of effective biological processes. Thus, after acquisition of a reference profile, SdFFF could be used alone to follow chemically induced biological events, suggesting many different applications such as testing series of molecules, evaluation of new cellular/biological models used in different life science fields, or sorting purified populations with the aim of better understanding mechanisms of induced cellular events.     

Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field-flow fractionation

The evaluation of the translation capacity of cells that produce recombinant proteins can be made by monitoring their ribosomal composition. In a previous use of asymmetrical flow field-flow fractionation (AsFlFFF) for this purpose the overall analysis time was more than 1 h and 40 min, based on a standard protocol for cell harvest, washing, cell disruption, and the final 8-min AsFlFFF determination of ribosome and subunits. In the present work the overall analysis time was reduced to 16 min. The washing step was deleted and a time-consuming freeze-thaw cycle. Cell disruption was obtained by a time-minimized lysozyme and detergent treatment for 1.5 min, respectively. The ribosomal material was finally fractionated and quantified in only 6 min, without previous centrifugation, using AsFlFFF. The great time reduction will enable the future use of AsFlFFF at-line to a growing cell cultivation, continuously monitoring the change in ribosomal composition or in other applications requiring high sample throughput. To demonstrate the high efficiency of the method the ribosome and tRNA composition in an Escherichia coli cultivation was monitored every half an hour, giving 18 measurements across the complete growth curve, a frequency of data enough to make decisions about induction or termination of the cultivation.     

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 μM, diosgenin induced megakaryocytic differentiation, in contrast to 40 μM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.     

Characterization of Silver Nanoparticles under Environmentally Relevant Conditions Using Asymmetrical Flow Field-Flow Fractionation (AF4)

The development of methods to monitor manufactured nanomaterials in the environment is one of the crucial areas for the assessment of their risk. More specifically, particle size analysis is a key element, because many properties of nanomaterial are size dependent. The sizing of nanomaterials in real environments is challenging due to their heterogeneity and reactivity with other environmental components. In this study, the fractionation and characterization of a mixture of polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) of three different sizes were investigated using asymmetrical flow field-flow fractionation (AF4) coupled with UV-Vis spectrophotometry. In particular, the effects of electrolyte composition and natural organic matter (NOM) on the particle size and stability were evaluated. The fractogram peaks (i.e., stability) of three different AgNPs decreased in the presence of both 10 mM NaCl and 10mM CaCl₂, while increased with increasing concentration of humic acid (HA). In addition, the hydrodynamic diameters of AgNPs in both electrolytes slightly increased with an increase of HA concentration, suggesting the adsorption (coating) of HA onto the particle surface. It is also interesting to note that an increase in the particle size depended on the types of electrolyte, which could be explained by the conformational characteristics of the adsorbed HA layers. Consistent these results, AgNPs suspended in lake water containing relatively high concentration of organic carbon (TOC) showed higher particle stability and larger particle size (i.e., by approximately 4nm) than those in river water. In conclusion, the application of AF4 coupled with highly sensitive detectors could be a powerful method to characterize nanoparticles in natural waters.     

Vesicle size distributions measured by flow field-flow fractionation coupled with multiangle light scattering.

The separation method, flow field-flow fractionation (flow FFF), is coupled on-line with multiangle laser light scattering (MALLS) for simultaneous measurement of the size and concentration of vesicles eluting continuously from the fractionator. These size and concentration data, gathered as a function of elution time, may be used to construct both number- and mass-weighted vesicle size distributions. Unlike most competing, noninvasive methods, this flow FFF/MALLS technique enables measurement of vesicle size distributions without a separate refractive index detector, calibration using particle size standards, or prior assumptions about the shape of the size distribution. Experimentally measured size distributions of vesicles formed by extrusion and detergent removal are non-Gaussian and are fit well by the Weibull distribution. Flow FFF/MALLS reveals that both the extrusion and detergent dialysis vesicle formation methods can yield nearly size monodisperse populations with standard deviations of approximately 8% about the mean diameter. In contrast to the rather low resolution of dynamic light scattering in analyzing bimodal systems, flow FFF/MALLS is shown to resolve vesicle subpopulations that differ by much less than a factor of two in mean size.     

Field-flow fractionation and biotechnology

The gentle separation mechanism has made field-flow fractionation particularly suited to samples of biotechnological interest, from proteins and nucleic acids to viruses, subcellular units and whole cells. Recent progress in field-flow fractionation technology, as well as the development of coupled techniques combining field-flow fractionation capabilities with the specificity and sensitivity of well-established analytical methods, opens up new biotechnological applications for field-flow fractionation. The most recent appealing applications include: sorting and fingerprinting of bacteria for whole-cell vaccine production; noninvasive and tagless sorting of immature and stem cells; separation of intact proteins and enzymes in top-down proteomics; and the development of flow-assisted, multianalyte immunoassays using nano- and micron-sized particles with immobilized biomolecules.     

Changes in plasma membrane and microfilaments accompanying morphologic differentiation in Chinese hamster ovary (CHO) cells.

Studies on the application of the techniques of counter-current distribution (CCD) in aqueous two-phase systems and multiple sedimentation for the fractionation of metaphase chromosomes are presented. The two-phase systems were composed of aqueous solutions of Dextran 500 and poly(ethylene)glycol 6000 (PEG). It has been found that different groups of chromosomes differ in their distribution between the two phases and that the introduction of PEG with covalently attached positively or negatively charged groups provides a means of steering the distribution of chromosomes. A rough fractionation of chromosomes on the basis of size is possible by the technique of multiple sedimentation and this, in combination with CCD, yields 10 fractions of chromosomes. Partition and CCD in aqueous two-phase system separate chromosomes according to their surface properties and may prove useful for isolation of individual chromosomes in bulk.     

Field- and flow-dependent trapping of red blood cells on polycarbonate accumulation wall in sedimentation field-flow fractionation

Sedimentation field-flow fractionation (SdFFF) instrumentation is now mature. Methodological procedure and particle separation development rules are well established even in the case of biological species. However, in some biological applications, retention properties of samples not predicted by any field-flow fractionation (FFF) elution models are observed. It is demonstrated that the trapping of cellular material in the separation system is not related to geometrical instrumentation features but to channel wall characteristics. The physicochemical particle–wall attractive interactions are different depending on the flow-rate and field intensity applied. Separation power in SdFFF for biological species is therefore limited by the intensity of these interactions. In terms of separation, a balance is to be found between external field and flow intensity to limit particle–wall interactions.