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Summary Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD., We defined urinary stem cell (USC) RNA-seq signatures and provided evidence that they are a non-invasive, affordable cell source to study Duchenne muscular dystrophy (DMD), an X-linked disease, due to dystrophin gene mutations. Based on USCs’ RNA profiling and whole-genome sequencing (WGS), we identified three undetected atypical DMD mutations.

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices. Graphical abstract

Colloids are an important component of wines, but their study is challenging due to their instability. Asymmetrical Flow-Field Flow Fractionation (AF4)-multidetection is here proposed as a suitable approach to isolate and characterize red wine colloids in native state. AF4 provided size-separation and enabled quantification of the colloidal content of two wines. The gyration radius of colloids was determined by multi-angle light scattering, and ranged between 25 and 50 nm. Analysis of the collected AF4-fractions showed that proteins, polysaccharides and phenolics were present in different proportions among fractions. The composition of AF4-fractions differed between wines. SDS-PAGE analysis of AF4-fractions indicated the presence of protein-phenolics sub-aggregates only in the fractions containing colloids with small radius. The results allowed proposing a model for red wine colloids structure, which comprises two coexisting entities, one made of covalently linked proteins-phenolics sub-aggregates interacting by non-covalent forces with polysaccharides, and a second in which only polysaccharides and phenolics are present. The proposed model is consistent with the reported relative stability of red wine proteins, a fact that can be due to the stabilizing activity of polysaccharides. Given that enological practices affect the extraction of proteins, polysaccharides and phenolics, AF4-technique represents a very promising tool to investigate the effects of these practices on wine colloidal aggregation and behavior.

Background New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. Objectives To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. Methods Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector,® an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. Results Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates, and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages. Conclusions Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing. Level of Evidence: 5

Clofazimine (CLZ) is an antibiotic with a promising behavior against Gram-positive bacteria; however, the drug is completely insoluble in water and accumulates in fat tissues. We explored nanocarriers, labeled and not labeled with rhodamine, consisting of negatively charged sulfobutylether-beta-cyclodextrins for CLZ loading. A new oligomeric carrier was obtained cross-linking beta CyD with epichlorohydrin followed by sulfonation in a strongly alkaline aqueous medium. The oligomeric carrier has a MW of 53 kDa and forms small nanoparticles of a few tens of nm. With aqueous solutions containing a 25 mg/mL oligomeric carrier, we loaded up to 0.5 mg/mL of drug. The oligomers exhibited a 10-fold better loading capacity compared to monomers and formed nanoparticles with a size in the 20-60 nm range after drug loading. Circular dichroism confirmed encapsulation of the CLZ in the nanocarriers. All carriers with or without CLZ are not cytotoxic up to 1 mu M, while CLZ alone is highly cytotoxic at the same concentration. The drug has IC50 values below 100 nM against S. epidermidis. The same holds true also for clinical isolates of S. epidermidis, some displaying MDR So, the selectivity index significantly increased for CLZ/carrier systems compared to the drug alone. Taken all together, our results open new avenues for the clinical application of this antibiotic.

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector&reg, (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector&reg, profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

In the course of their life span, cells release a multitude of different vesicles in the extracellular matrix (EVs), constitutively and/or upon stimulation, carrying signals either inside or on their membrane for intercellular communication. As a natural delivery tool, EVs present many desirable advantages, such as biocompatibility and low toxicity. However, due to the complex biogenesis of EVs and their high heterogeneity in size distribution and composition, the characterization and quantification of EVs and their subpopulations still represents an enticing analytical challenge. Centrifugation methods allow to obtain different subpopulations in an easy way from cell culture conditioned medium and biological fluids including plasma, amniotic fluid and urine, but they still present some drawbacks and limitations. An unsatisfactory isolation can limit their downstream analysis and lead to wrong conclusions regarding biological activities. Isolation and characterization of biologically relevant nanoparticles like EVs is crucial to investigate specific molecular and signaling patterns and requires new combined approaches. Our work was focused on HF5 (miniaturized, hollow-fiber flow field-flow fractionation), and its hyphenation to ultracentrifugation techniques, which are the most assessed techniques for vesicle isolation. We exploited model samples obtained from culture medium of murine myoblasts (C2C12), known to release different subsets of membrane-derived vesicles. Large and small EVs (LEVs and SEVs) were isolated by differential ultracentrifugation (UC). Through an HF5 method employing UV, fluorescence and multi-angle laser scattering as detectors, we characterized these subpopulations in terms of size, abundance and DNA/protein content; moreover, we showed that microvesicles tend to hyper-aggregate and partially release nucleic matter. The quali-quantitative information we obtained from the fractographic profiles was improved with respect to Nano Tracking Analysis (NTA) estimation. The SEV population was then further separated using density gradient centrifugation (DGC), and four fractions were submitted again to HF5-multidetection. This technique is based on a fully orthogonal principle, since F4 does not separate by density, and provided uncorrelated information for each of the fractions processed. The "second dimension" achieved with HF5 showed good promise in sorting particles with both different size and content, and allowed to identify the presence of fibrilloid nucleic matter. This analytical bidimensional approach proved to be effective for the characterization of highly complex biological samples such as mixtures of EVs and could provide purified fractions for further biological characterization.

The development of plant-based protein polymers to employ in biofilm production represents the promising intersection between material science and sustainability, and allows to obtain biodegradable materials that also possess excellent physicochemical properties. A possible candidate for protein biopolymer production is phaseolin, a storage protein highly abundant in P Vulgaris beans. We previously showed that transformed tobacco chloroplasts could be employed to express a mutated phaseolin carrying a signal peptide (directing it into the thylakoids) also enriched of a cysteine residue added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds, as we previously demonstrated, and should promote polymerization. To verify the effect of the peptide modification and to quantify polymer formation, we employed hollow-fiber flow field-flow fractionation coupled to UV and multi-angle laser scattering detection (HF5-UV-MALS): HF5 allows for the selective size-based separation of phaseolin species, whereas MALS calculates molar mass and conformation state of each population. With the use of two different HF5 separation methods we first observed the native state of P.Vulgaris phaseolin, mainly assembled into trimers, and compared it to mutated phaseolin (P*) which instead resulted highly aggregated. Then we further characterized P* using a second separation method, discriminating between two and distinct high-molecular weight (HMW) species, one averaging 0.8 × 106 Da and the second reaching the tens of million Da. Insight on the conformation of these HMW species was offered from their conformation plots, which confirmed the positive impact of the Cys modification on polymerization.

Most of lateral flow immunoassay (LFIA) devices rely on gold nanoparticles (GNP) labeled antibodies or other biospecific proteins, to achieve reagent-less color-based detection. GNP size, GNP-protein conjugation level and its stability are crucial points for the development of precise and accurate methods. In addition, the purification of the GNP-protein conjugates from unreacted protein and GNP, is necessary for adequate analytical performance of the assay. To assist the synthesis and production process of GNP and their protein conjugates, we use for the first time a non-destructive, particle separation-multi-detection approach based on miniaturized flow field flow fractionation (HF5). A separation method was developed to baseline size-separate GNP, GNP-protein, protein and GNP including BSA used as a surface coater in less than 30 minutes. Freshly synthesized GNP were first characterized and then conjugated with two different model antibodies: a mouse immunoglobulin (IgG) and a fluorescein-labeled mouse immunoglobulin (FITC-IgG). The IgG-GNP complexes were fractionated using the HF5 apparatus, able to separate IgG-GNP from free proteins by their hydrodynamic size, allowing purification of the conjugation product. Both IgG-GNPs and GNPs were characterized according to their size by the MALS detector, and according to their Surface Plasmon Resonance and spectrum by UV-Vis detection, improving the results obtained via batch characterization. This simple non-invasive approach is very useful for the LFIA development and optimization: the use of HF5-mutidetection offers a unique tool for this purpose facilitating the industrialization of the process and the relate optimization and standardization.

The rapid development of nanoproducts in pharmaceutical field highlights the need for robust analytical methods to ensure their quality and stability. Nanoparticles of different nature and composition (NPs) are employed for many purposes, such as the improvement of drug solubility/bioavailability and the controlled delivery of drugs. Among NPs features, particle size distribution (PSD) plays a fundamental role in determining NPs properties. Nevertheless the high development of different NPs, authorities such as the FDA and the European Union highlight the lack of robust characterization methods and quality control for nanomaterials. Among the techniques for the size-characterization of particles, Field-Flow Fractionation (FFF) represents a competitive choice: due to the absence of a stationary phase, the separation mechanism is gentle with total maintenance of the native properties of the analytes. In this paper the microcolumn variant of FFF, the Hollow-Fiber Flow FFF (HF5), is coupled on-line with Multi-Angle Light Scattering (MALS) for the development of methods for the characterization of NPs as quality control in new pharmaceutical field. The HF5-MALS was applied to the size c haracterization in different preservation conditions of silver polyvinylpyrrolidone-stabilized NPs (AgPVP) used for their antimicrobial activity. The ratio of gyration and hydrodynamic radii of AgPVP was evaluated for fresh and aged NPs, suggesting in this case aggregation rearrangement. The influence of different PVP coating and dilution factor was also studied. Finally the metal ion release was determined in relationship to these shape modifications. The HF5-MALS method is robust and reproducible and it can be considered as an important tool for the development of analytical platform for quality control of NPs.

The use of stem cells for regenerative applications and immunomodulatory effect is increasing. Amniotic epithelial cells (AECs) possess embryonic-like proliferation ability and multipotent differentiation potential. Despite the simple isolation procedure, inter-individual variability and different isolation steps can cause differences in isolation yield and cell proliferation ability, compromising reproducibility observations among centers and further applications. We investigated the use of a new technology as a diagnostic tool for quality control on stem cell isolation. The instrument label-free separates cells based on their physical characteristics and, thanks to a micro-camera, generates a live fractogram, the fingerprint of the sample. Eight amniotic membranes were processed by trypsin enzymatic treatment and immediately analysed. Two types of profile were generated: a monomodal and a bimodal curve. The first one represented the unsuccessful isolation with all recovered cell not attaching to the plate, while for the second type, the isolation process was successful, but we discovered that only cells in the second peak were alive and resulted adherent. We optimized a Quality Control (QC) method to define the success of AEC isolation using the fractogram generated. This predictive outcome is an interesting tool for laboratories and cell banks that isolate and cryopreserve fetal annex stem cells for research and future clinical applications.

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Clofazimine (CLZ) is an antibiotic with a promising behavior against Gram-positive bacteria; however, the drug is completely insoluble in water and accumulates in fat tissues. We explored nanocarriers, labeled and not labeled with rhodamine, consisting of negatively charged sulfobutylether-β-cyclodextrins for CLZ loading. A new oligomeric carrier was obtained cross-linking βCyD with epichlorohydrin followed by sulfonation in a strongly alkaline aqueous medium. The oligomeric carrier has a MW of 53 kDa and forms small nanoparticles of a few tens of nm. With aqueous solutions containing a 25 mg/mL oligomeric carrier, we loaded up to 0.5 mg/mL of drug. The oligomers exhibited a 10-fold better loading capacity compared to monomers and formed nanoparticles with a size in the 20-60 nm range after drug loading. Circular dichroism confirmed encapsulation of the CLZ in the nanocarriers. All carriers with or without CLZ are not cytotoxic up to 1 μM, while CLZ alone is highly cytotoxic at the same concentration. The drug has IC

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal–organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delivery applications and gave fundamental data on their size distribution and stability. [Figure not available: see fulltext.].

Interest in stem cell separation and purification from easily accessible clinical specimens is booming due to the increase of cell therapy applications. The recovery of pluripotent or multipotent stem cells in human sources different from the embryo requires the use of effective methods of cell sorting/enrichment. Among these sources, perinatal tissues retain cells with pivotal stem cell features such as high self-renewal ability, wide differentiation potential and immunomodulatory properties. In this perspective, methods exploiting cell biophysical differences in a less dependent process of identification based on specific markers are therefore promising. These methods allow cell isolation irrespective of the broad and diversified surface antigenic panel that usually limits the ability to easily distinguish cells as in the case of mesenchymal stromal/stem cell separation. In addition, the use of non- or minimally invasive tag-less techniques might be a way to preserve stem cell features of the selected product and reduce regulatory issues related to their use in regenerative applications. In this review, non-invasive cell sorting techniques based on microfluidic systems and relevant patents are described. In particular applications of emerging separation approach, Field-Flow Fractionation (FFF), for perinatal stem cell sorting are cited. Protocols and applications based on FFF-derived techniques are detailed

Due to the increased use of silver nanoparticles in industrial scale manufacturing, consumer products and nanomedicine reliable measurements of properties such as the size, shape and distribution of these nano particles in aqueous medium is critical. These properties indeed affect both functional properties and biological impacts especially in quantifying associated risks and identifying suitable risk-mediation strategies. The feasibility of on-line coupling of a fractionation technique such as hollow-fiber flow field flow fractionation (HF5) with a light scattering technique such as MALS (multi-angle light scattering) is investigated here for this purpose. Data obtained from such a fractionation technique and its combination thereof with MALS have been compared with those from more conventional but often complementary techniques e.g. transmission electron microscopy, dynamic light scattering, atomic absorption spectroscopy, and X-ray fluorescence. The combination of fractionation and multi angle light scattering techniques have been found to offer an ideal, hyphenated methodology for a simultaneous size-separation and characterization of silver nanoparticles. The hydrodynamic radii determined by fractionation techniques can be conveniently correlated to the mean average diameters determined by multi angle light scattering and reliable information on particle morphology in aqueous dispersion has been obtained. The ability to separate silver (Ag(+)) ions from silver nanoparticles (AgNPs) via membrane filtration during size analysis is an added advantage in obtaining quantitative insights to its risk potential. Most importantly, the methodology developed in this article can potentially be extended to similar characterization of metal-based nanoparticles when studying their functional effectiveness and hazard potential.

Monoclonal antibodies (mAbs) are promising reagents both for the manufacture of drug substances and for their employment as a drug themselves, but to be approved for utilization, according to FDA recommendations and WHO guidelines, they have to undergo verifications regarding their purity, stability and percentage of aggregates. Moreover, stability tests of lots have to be performed in order to verify molecular size distribution over time and lot-to-lot consistency. Recent works in literature have highlighted the need for suitable, sensitive and reliable complementary analytical techniques for the characterization of mAbs and quantification of aggregates. Size-exclusion chromatography (SEC) is the reference technique in the biopharmaceutical industry for its robustness, high performance and simple use; however it presents some limitations especially toward the separation and detection of aggregates with high molecular weight. On the other hand, flow field-flow fractionation (F4) in its miniaturized version (hollow fiber flow field-flow fractionation, HF5) shows comparable performances with interesting additional advantages: a broad size range, gentle separation mechanism with low dilution factor and higher sensitivity. To propose HF5 as a complementary technique for evaluating aggregates' content in mAbs samples, a comparative study of both SEC and HF5 performances has been made. In this work, SEC and HF5 were coupled with UV and multi-angle light scattering detection and employed first in separating standard samples of proteins mixture used as a sample model. Then, a screening of mobile phases and an evaluation of separation performances was performed on a therapeutic mAbs formulation, demonstrating the complementarities between SEC and HF5 and their possible use as a separative platform approach for the characterization and quality control of protein drugs. © 2014 Elsevier B.V.

The rapid development of protein-based pharmaceuticals highlights the need for robust analytical methods to ensure their quality and stability. Among proteins used in pharmaceutical applications, an important and ever increasing role is represented by monoclonal antibodies and large proteins, which are often modified to enhance their activity or stability when used as drugs. The bioactivity and the stability of those proteins are closely related to the maintenance of their complex structure, which however are influenced by many external factors that can cause degradation and/or aggregation. The presence of aggregates in these drugs could reduce their bioactivity and bioavailability, and induce immunogenicity. The choice of the proper analytical method for the analysis of aggregates is fundamental to understand their (size) dimensional range, their amount, and if they are present in the sample as generated by an aggregation or as an artifact due to the method itself. Size exclusion chromatography is one of the most important techniques for the quality control of pharmaceutical proteins; however, its application is limited to relatively low molar mass aggregates. Among the techniques for the size characterization of proteins, field-flow fractionation (FFF) represents a competitive choice because of its soft mechanism due to the absence of a stationary phase and application in a broader size range, from nanometer- to micrometer-sized analytes. In this paper, the microcolumn variant of FFF, the hollow-fiber flow FFF, was online coupled with multi-angle light scattering, and a method for the characterization of aggregates with high reproducibility and low limit of detection was demonstrated employing an avidin derivate as sample model.

An integrated sensing system is presented for the first time, where a metal oxide semiconductor sensor-based electronic olfactory system (MOS array), employed for pathogen bacteria identification based on their volatile organic compound (VOC) characterisation, is assisted by a preliminary separative technique based on gravitational field-flow fractionation (GrFFF). In the integrated system, a preliminary step using GrFFF fractionation of a complex sample provided bacteria-enriched fractions readily available for subsequent MOS array analysis. The MOS array signals were then analysed employing a chemometric approach using principal components analysis (PCA) for a first-data exploration, followed by linear discriminant analysis (LDA) as a classification tool, using the PCA scores as input variables. The ability of the GrFFF-MOS system to distinguish between viable and non-viable cells of the same strain was demonstrated for the first time, yielding 100 % ability of correct prediction. The integrated system was also applied as a proof of concept for multianalyte purposes, for the detection of two bacterial strains (Escherichia coli O157:H7 and Yersinia enterocolitica) simultaneously present in artificially contaminated milk samples, obtaining a 100 % ability of correct prediction. Acquired results show that GrFFF band slicing before MOS array analysis can significantly increase reliability and reproducibility of pathogen bacteria identification based on their VOC production, simplifying the analytical procedure and largely eliminating sample matrix effects. The developed GrFFF-MOS integrated system can be considered a simple straightforward approach for pathogen bacteria identification directly from their food matrix. Graphical abstract An integrated sensing system is presented for pathogen bacteria identification in food, in which field-flow fractionation is exploited to prepare enriched cell fractions prior to their analysis by electronic olfactory system analysis.

Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

Foodborne illnesses caused by pathogenic bacteria represent a widespread and growing problem to public health, and there is an obvious need for rapid detection of food pathogens. Traditional culture-based techniques require tedious sample workup and are time-consuming. It is expected that new and more rapid methods can replace current techniques. To enable large scale screening procedures, new multiplex analytical formats are being developed, and these allow the detection and/or identification of more than one pathogen in a single analytical run, thus cutting assay times and costs. We review here recentadvancements in the field of rapid multiplex analytical methods for foodborne pathogenic bacteria. A variety of strategies, such as multiplex polymerase chain reaction assays, microarray- or multichannel-based immunoassays, biosensors, and fingerprint-based approaches (such as mass spectrometry, electronic nose, or vibrational spectroscopic analysis of whole bacterial cells), have been explored. In addition, various technological solutions have been adopted to improve detectability and to eliminate interferences, although in most cases a brief pre-enrichment step is still required. This review also covers the progress, limitations and future challenges of these approaches and emphasizes the advantages of new separative techniques to selectively fractionate bacteria, thus increasing multiplexing capabilities and simplifying sample preparation procedures.

Reinjection of one ore more collected fractions of eluted samples is recognized as a useful procedure in analytical separation techniques, among which field-flow fractionation (FFF), to improve the actual separation of complex samples. Hollow-fiber flow FFF (HF5) is a micro-channel subset of flow FFF (F4), which has recently reached a performance comparable to that of standard, flat-channel F4. To further improve HF5 of complex protein samples, we present a new device and method for in-line, reinjection HF5 that we call tandem H F5 (HF5/HF5). HF5 is ideally suited for tandem operation because (1) small channel volume and low operation flow rates allow reducing dilution and volume of the collected fractions, and (2) the relaxation/focusing step that takes place between the 1st and 2nd run (refocusing) allows reestablishing the volume and concentration of the sample plug before the 2nd elution. HF5/HF5 proves particularly effective in the case of oligomeric proteins since it allows collecting and reinjecting the bands that correspond to each separated oligomeric form. This provides information on the dynamic equilibria between the different oligomers. For HF5/HF5 operations, a modified, prototype HF5 instrumentation is presented which includes a "trap" constituted of a four-port, two-way valve positioned downstream the UV detector and a collection loop. The effect of refocusing conditions on HF5/HF5 performance is investigated by varying refocusing time. With a complex protein samples such as blood serum, HF5/HF5 can improve detectability of the low abundance components since overloading effects due to high-abundance components are reduced. This is shown for serum lipoproteins: while after the 1st run high density lipoproteins (HDLs) are not separated from high-abundance serum proteins, after the 2nd run it is shown possible to separate the HDL subclasses. (C) 2011 Elsevier B.V. All rights reserved.

Very monodisperse water-soluble silica core-surfactant shell nanoparticles (SCSS NPs) doped with a rhodamine B derivative were prepared using micelles of F127 as nanoreactors for the hydrolysis and condensation of the silica precursor tetraethoxysilane (TEOS). The functionalization of the rhodamines with a triethoxysilane group allowed the covalent binding of the fluorophores to the silica core: no leaking of the dye was observed when the NPs were purified either by ultrafiltration (UF) or dialysis. The diameter of the core (d(c) = 10 ± 1 nm) was determined by TEM and subtracted from the hydrodynamic diameter, measured by DLS, (d(H) = 24 nm, PdI = 0.1) to calculate the shell thickness (∼7 nm). The presence of a single population of NPs with a radius compatible with the one measured by DLS after UF was confirmed by AF4-MALS-RI measurements. The concentration of the NPs was measured by MALS-RI. This allowed us to determine the average number of rhodamine molecules per NP (10). The ability of the NPs to host hydrophobic species as cyanines in the SS was confirmed by fluorescence anisotropy measurements. Steady-state and time-resolved fluorescence measurements allowed us to observe the occurrence of a very efficient Förster resonance energy transfer process from the covalently linked rhodamines to the hosted cyanines. In particular, the analysis of the TCSPC data and steady-state measurements revealed that the adsorption of a single cyanine molecule causes an almost complete quenching of the fluorescence of the NP. Thanks to these observations, it was possible to easily determine the concentration of the NPs by fluorescence titration experiments. Results are in good agreement with the concentration values obtained by MALS-RI. Finally, the hosted cyanine molecule could be extracted with (±)-2-octanol, demonstrating the reversibility of the adsorption process.

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.

Alterations of lipoproteins (LPs) and related lipid levels in blood serum are correlated to the risk of coronary artery disease (CAD). Fast, possibly automated methods to obtain complete, multi-parametric LP profiles are therefore welcome to be developed for routine, clinical analysis practice. In this work, asymmetrical flow field-flow fractionation (AF4) with on-line, dual post-fractionation reaction detection (PFRD) is applied to develop a method for single-run, simultaneous quantification of cholesterol (CHOL) and triglycerides (TGs) in each fractionated LP class. The enzymatic reagents used for the post-fractionation reaction are available as commercial kits for certified, standard clinical protocols for the analysis of CHOL and TGs in serum. Using CHOL and glycerol as reference standards, a new procedure is applied to optimize the experimental conditions for PFRD-based, quantitative analysis. Upon optimized PFRD and AF4 conditions, results obtained for the determination of total CHOL (TC), TGs, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) in a set of serum samples from healthy donors are found in agreement with the values provided by a clinical laboratory. The intra-day and inter-day precisions of the method were found always lower than 10% (CV). When the method was applied to serum samples from patients affected by sepsis, differences in CHOL and TG profiles between patients and healthy donors were observed.

Background: The interest in stem cell (SC) isolation from easily accessible clinical specimens is booming. The lack of homogeneity in pluri/multipotent SC preparation blurs standardization, which however is recommended for successful applications. Multipotent mesenchymal SCs (MSCs) in fact express a broad panel of surface antigens, which limit the possibility of sorting homogeneous preparations by using an immunotag-based method. Methods: We present a tag-less, flow-assisted method to purify, distinguish, and sort pluri/multipotent SCs obtained from clinical specimens, based on differences in the biophysical properties that cells acquire when in suspension under fluidic conditions. A suspension of cells in a transport fluid is injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30 min), sorted cells are collected. Results: We obtained baseline separation between MSCs and epithelial cells, which are important contaminants of isolated MSCs. The extent of separation is evaluated by flow cytometry through detection of a specific epithelial antigen. MSCs from various human sources also prove to have different, characteristic, highly-reproducible fractionation profiles. Finally, we evaluated the dissimilar differentiation potential among cell fractions obtained from sorting a single MSC source. After differentiation induction, a fraction displayed a differentiation yield close to 100%, whereas unfractionated cells contained only 40% of responding cells. Conclusions: The results demonstrate that the method presented is able to obtain selected and well-characterized living MSCs with an increased differentiation yield. Its reduced cost, full biocompatibility, and scale-up potential could make this method an effective procedure for stem cell selection. © 2009 Clinical Cytometry Society

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.

Hollow-fiber flow field-flow fractionation is here applied to untreated, whole human blood serum. Matrix-assisted, laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) of serum fractions shows mass signals in the M r range where low-abundance, serum protein components are known to be present, though a membrane of nominal 30 000 Da cutoff was employed for the fractionation device. Using diluted sera spiked with low amounts (0.06–0.1%, w/w) of an artificial mixture constituted the human adrenocorticotropic hormone fragments 18–39 ( M r = 2465.7) and 7–38 ( M r = 3659.2), and of bovine insulin ( M r = 5734), horse cytochrome c ( M r = 12 384) and chicken lysozyme ( M r = 14 388), a hybrid fractionation/microfiltration mechanism shows to govern the separation of the low- M r components.

Films based on TiO2 nanoparticles (NPs) have been successfully used as sensing elements in chemical sensors. TiO2 colloidal suspensions can be obtained by spontaneous hydrolysis in acidic solutions of Ti(IV) compounds. The obtained TiO2 NPs can be employed to build up nanostructured films. With the purpose of preparing TiO2-based nanostructured, imprinted materials as sensing elements for piezoelectric sensors, we obtained TiO2 NP dispersions by hydrolyzing potassium titanyl oxalate in the presence of a target analyte (tyrosine). Since morphological properties of the synthesized NPs are known to influence the nanostructured film characteristics, an analytical strategy to characterize such colloidal systems can combine a size-based separation method with spectroscopic analysis to correlate the particle size distribution (PSD) with the particle-target interaction properties able to determine the sensing efficiency. In this work, we present the characterization of colloidal tyrosine-TiO2 NP systems by flow field-flow fractionation (FlFFF) with online, UV/Vis absorption detection and offline fluorescence analysis. FlFFF eliminates the possible contribution of free tyrosine to the absorption and fluorescence properties of the NPs. FlFFF also fractionates NPs on a size basis. Particle size distribution (PSD) profiles of the fractionated NPs are then obtained by conversion of the multi-wavelength UV/Vis fractograms. Size of the fractionated NPs is finally related to fluorescence properties of the collected NPs fractions. Good correlation between the fluorescence intensity, which is proportional to the tyrosine uptake, and the FlFFF-based, NP mass-size frequency distribution finally confirms the existence of tyrosine-TiO2 NP interaction.