Foam fractionation applications

Biotechnological downstream processing faces several challenges, such as dilute product streams and contained target products which are sensitive to heat, oxidation, other chemicals, etc. State-of-the-art separation methods, e.g. chromatography, are not always the best option due to variable yield losses and high costs. Foam fractionation appears as a promising alternative unit operation in biotechnological downstream processing. From its applications in metal industry and on fish farms, it was developed further towards the recovery of phytonutrients, metabolites and proteins. However, no large scale applications of foam fractionation in biotechnological downstream processing exist yet. This is due to the complexity of various biotechnological media, which makes a universalized approach for systematic process design of protein separations difficult. Ongoing research in the fields of process engineering, surface chemistry and protein chemistry can help to close this gap. Although many different substances, such as detergents, have been separated or recovered using foam fractionation, this review focuses mainly on biotechnological applications, more specifically on protein separation.     

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.     

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 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.     

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.     

Selective elution and purification of living Trichomonas vaginalis using gravitational field-flow fractionation

Gravitational field-flow fractionation is one of the simplest separation methods for biological materials. Its potential in parasitology is demonstrated for Trichomonas vaginalis, a parasite responsible for one of the most widespread sexually transmitted diseases. It was observed that this unicellular parasite can be purified in a culture medium with a recovery of 85% for the living trophozoites. The parasite retention characteristics were different when motile living and non-motile dead cells were eluted, motile cells being less retained than the non-motile cells.     

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.     

Micropreparation of hemopoietic stem cells from the mouse bone marrow suspension by gravitational field-flow fractionation

Gravitational field-flow fractionation is a relatively simple experimental technique. This method was used for the characterization of stem cells from mouse bone marrow. Because these cells are bigger than the other cells in bone marrow, it is possible to separate them from the mixture. The fractions collected after passing through the separation channel were characterized using a Coulter Counter and used for transplantation into irradiated mice.     

Flow field-flow fractionation: a pre-analytical method for proteomics

Proteome analysis requires a comprehensive approach including high-performance separation methods, mass spectrometric analysis, and bioinformatics. While recent advances in mass spectrometry (MS) have led to remarkable improvements in the ability to characterize complex mixtures of biomolecules in proteomics, a proper pre-MS separation step of proteins/peptides is still required. The need of high-performance separation and/or isolation/purification techniques of proteins is increasing, due to the importance of proteins expressed at extremely low levels in proteome samples. In this review, flow field-flow fractionation (F4) is introduced as a complementary pre-analytical separation method for protein separation/isolation, which can be effectively utilized for proteomic research. F4 is a set of elution-based techniques that are capable of separating macromolecules by differences in diffusion coefficient and, therefore, in hydrodynamic size. F4 provides protein separation without surface interaction of the analyte with packing or gel media. Separation is carried out in an open channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly-applied, secondary flow of the fluid. Therefore, biological analytes such as proteins can be kept under a bio-friendly environment without losing their original structural configuration. Moreover, proteins fractionated on a size/shape basis can be readily collected for further characterization or proteomic analysis by MS using, for instance, either on-line or off-line methods based on electrospray ionization (ESI) or matrix-assisted laser desorption-ionization (MALDI). This review focuses on the advantages of F4 compared to most-assessed separation/isolation techniques for proteomics, and on selected applications based on size-dependent proteome separation. New method developments based on the hyphenation of F4 with on-line or off-line MS, and with other separation methods such as capillary isoelectric focusing (CIEF) are also described.     

Modification of glass channel walls for separation of biological particles by gravitational field-flow fractionation

In the gravitational field-flow fractionation of complex samples, various interaction and adsorption phenomena can occur in separation channels that influence fractionation and complicate the explanation of resulting fractograms. To overcome these problems, the glass surface was modified to create charge-free, non-adsorbing hydrophilic media for the mild treatment of hydrophilic biological particles. The modification was carried out in two steps: (1) by a simple lacquering of the glass surface with polystyrene diluted in toluene and (2) subsequent adsorption of a detergent layer on polystyrene. Essential suppression of ionic interactions between soluble low-molecular-mass compounds and the channel wall and decreased adsorption effects were demonstrated in separations of blood samples by gravitational field-flow fractionation.     

Validation procedures of sedimentation field-flow fractionation techniques for biological applications

Sedimentation field-flow fractionation (SdFFF) offers great potential for the separation of submicrometer and micrometer-sized species. The availability of commercial instrumentation and the versatility of this method originated its success. At this stage of development, SdFFF techniques are mature enough for use in analytical research, development and even routine work. However, prior to their use, these techniques like any other methodologies, have to be validated. As the application of SdFFF techniques to cell separation is being constantly developed, we have investigated separation performance according to validation rules classically defined for separation methods (chromatography) in the case of cellular materials.     

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.     

Micro-thermal field-flow fractionation of bacteria

The retention of Staphylococcus epidermidis bacteria cells, achieved with the use of micro-thermal field-flow fractionation and described in this paper, represents the first experimental proof that the separation and characterization of the bio-macromolecules and biological particles is possible by exploiting Ludwig-Soret effect of thermal diffusion. The experiments were carried out under gentle experimental conditions preventing the denaturation of the bacteria. Lift forces, appearing at high linear velocities of the carrier liquid, generated the focusing mechanism of the retention which resulted in high-speed and high-performance separation performed in less than 10 min.     

蛋白质纯化技术研究进展

蛋白质组学研究的基础就是蛋白质的分离。对于天然蛋白来说,可能需要一系列的纯化步骤才能获得纯度满足研究要求的蛋白质,但是蛋白质在分离过程中常常由于溶液环境变化或外力作用造成构象变化而引起失活。本文首先介绍了常用的蛋白质分离纯化技术及其研究进展,包括膜分离技术、沉淀分离技术、电泳分离技术以及层析分离技术等常用的蛋白质纯化技术,总结了现有技术存在的问题,并对近年来发展的新型蛋白质分离技术--非对称流场流分离技术进行了介绍和展望。     

Molecular mass sorting of proteome using hollow fiber flow field-flow fractionation for proteomics

Hollow fiber flow field-flow fractionation (HF FlFFF) has been demonstrated as a tool for pre-fractionating proteomes by differences in molecular mass (Mr), where the resulting protein fractions are subsequently digested and analyzed by shotgun proteomics using two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-LC-ESI-MS/MS). HF FlFFF is a separation device capable of fractionating proteins or cells by hydrodynamic radius, and protein fraction can be readily collected as intact conditions in aqueous buffer solutions. In this study, HF FlFFF was applied to fractionate the proteome of Corynebacterium glutamicum, a well known soil bacterium that has been widely used in bioindustry due to its remarkable ability to secrete high amounts of glutamic acid. The collected HF FlFFF fractions of different MW intervals were enzymatically digested for protein identification by 2D-LC-ESI-MS/MS. Experiments showed improvements in protein identification when HF FlFFF pre-fractionation was applied, due to decreases in the ionization suppression effect and the MS exclusion effect by spectral congestion. Pre-fractionation of C. glutamicum proteome allowed us to find 90 additional proteins by 2D-LC-ESI-MS/MS that were not found by a direct shotgun analysis without pre-fractionation. A total of 415 proteins were found overall with 203 proteins commonly found from experiments with and without pre-fractionation.     

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.     

Two dimensional (pI & ds) separation of phosphorylated proteins by isoelectric focusing/asymmetrical flow field-flow fractionation: application to prostatic cancer cell line

This study demonstrates the use of on-line isoelectric focusing/asymmetrical flow field-flow fractionation (IEF-AF4), a non-gel based high speed two dimensional (isoelectric point and hydrodynamic diameter) protein separation device used for the isolation/separation of phosphoproteins. IEF-AF4 performance was evaluated by first fractionating α-casein molecules at different pIs and sizes. Collected proteins were analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS(n)) to determine various isoforms of the phosphopeptides as well as the relative ratio of phosphorylated and unmodified peptides. A narrow pH cut (ΔpH=0.5) of carrier ampholyte was used in IEF-AF4 to finely resolve phosphoproteins by pI. When the channel lane of multilane AF4 became acidic, the relative ratio of phosphorylated to unmodified or less phosphorylated peptides increased. The current method was applied to prostate cancer cell lysates to demonstrate that IEF-AF4 can examine the relative abundances of specific phosphoproteins, known as biomarkers, in prostate cancer. While affinity-based enrichment methods remove unmodified peptides, IEF-AF4 offers intact phosphoprotein separation at the protein level without removing unmodified proteins. IEF-AF4 enables quantitative analysis without isotope labeling.     

Rapid protein separation and diffusion coefficient measurement by frit inlet flow field-flow fractionation.

In this study three flow field-flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4-20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and gamma-globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3-4%) is found. High-speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.     

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.