Pierluigi Reschiglian

Field-flow fractionation in bioanalysis: A review of recent trends

Field-flow fractionation (FFF) is a mature technique in bioanalysis, and the number of applications to proteins and protein complexes, viruses, derivatized nano- and micronsized beads, sub-cellular units, and whole cell separation is constantly increasing. This can be ascribed to the non-invasivity of FFF when directly applied to biosamples. FFF 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 field. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media and without using degrading mobile phases. The fractionation device can be also easily sterilized, and analytes can be maintained under a bio-friendly environment. This allows to maintain native conditions of the sample in solution. In this review, FFF principles are briefly described, and some pioneering developments and applications in the bioanalytical field are tabled before detailed report of most recent FFF applications obtained also with the hyphenation of FFF with highly specific, sensitive characterization methods. Special focus is finally given to the emerging use of FFF as a pre-analytical step for mass-based identification and characterization of proteins and protein complexes in proteomics.

Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection

AbstractFoodborne 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 recent advancements 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. FigureNew analytical formats are under development to allow multiplexed detection of foodborne pathogens, thus cutting assay times and costs and enabling large scale screening procedures. A variety of analytical strategies are being explored to reach this goal. This review covers the recent progresses, limitations and future challenges of these approaches

Energy transfer from silica core-surfactant shell nanoparticles to hosted molecular fluorophores.

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.

Asymmetrical flow field-flow fractionation with multi-angle light scattering detection for the analysis of structured nanoparticles

Synthesis and applications of new functional nanoparticles are topics of increasing interest in many fields of nanotechnology. Chemical modifications of inorganic nanoparticles are often necessary to improve their features as spectroscopic tracers or chemical sensors, and to increase water solubility and biocompatibility for applications in nano-biotechnology. Analysis and characterization of structured nanoparticles are then key steps for their synthesis optimization and final quality control. Many properties of structured nanoparticles are size-dependent. Particle size distribution analysis then provides fundamental analytical information. Asymmetrical flow field-flow fractionation (AF4) with multi-angle light scattering (MALS) detection is able to size-separate and to characterize nanosized analytes in dispersion. In this work we focus on the central role of AF4-MALS to analyze and characterize different types of structured nanoparticles that are finding increasing applications in nano-biotechnology and nanomedicine: polymer-coated gold nanoparticles, fluorescent silica nanoparticles, and quantum dots. AF4 not only size-fractionated these nanoparticles and measured their hydrodynamic radius (r(h)) distribution but it also separated them from the unbound, relatively low-M(r) components of the nanoparticle structures which were still present in the sample solution. On-line MALS detection on real-time gave the gyration radius (r(g)) distribution of the fractionated nanoparticles. Additional information on nanoparticle morphology was then obtained from the r(h)/r(g) index. Stability of the nanoparticle dispersions was finally investigated. Aggregation of the fluorescent silica nanoparticles was found to depend on the concentration at which they were dispersed. Partial release of the polymeric coating from water-soluble QDs was found when shear stress was induced by increasing flowrates during fractionation.

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.

Hyperlayer hollow-fiber flow field-flow fractionation of cells.

Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing. Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FIFFF channel design. The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage. which is particularly appealing for analytical bio-applications. Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FIFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae). Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FIFFF of cells, at superior size-based selectivity and reduced analysis time.

Host–Guest Interactions in Fe(III)-Trimesate MOF Nanoparticles Loaded with Doxorubicin

Doxorubicin (DOX) entrapment in porous Fe(III)-trimesate metal organic frameworks (MIL-100(Fe)) nanoparticles was investigated in neutral Tris buffer via UV-vis absorption, circular dichroism (CD), and fluorescence. The binding constants and the absolute spectra of the DOX-MIL-100(Fe) complexes were determined via absorption and fluorescence titrations. A binding model where DOX associates as monomer to the dehydrated Fe3O (OH)(H2O)2 [(C6H3)(CO2)3]2 structural unit in 1:1 stoichiometry, with apparent association constant of (1.1 to 1.8) × 10(4) M(-1), was found to reasonably fit the experimental data. Spectroscopic data indicate that DOX binding occurs via the formation of highly stable coordination bonds between one or both deprotonated hydroxyl groups of the aglycone moiety and coordinatively unsaturated Fe(III) centers. Complete quenching of the DOX fluorescence and remarkable thermal and photochemical stability were observed for DOX incorporated in the MIL-100(Fe) framework.

Separation of mitochondria by flow field-flow fractionation for proteomic analysis

Flow field-flow fractionation (FlFFF) has been utilized for size-based separation of rat liver mitochondria. Collected fractions of mitochondria of various sizes were examined by confocal microscopy, and mitochondria of each fraction were lysed and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the comparison of protein patterns in differently sized mitochondria by densitometric measurements, and for protein characterization of some gel spots with nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). FlFFF fractions of the mitochondria were also tryptically digested for shotgun proteomic characterization of mitochondrial proteins/peptides by nLC-ESI-MS-MS. Peak area (integrated ion counts) of some peptides extracted from LC-MS chromatograms were examined at different fractions for the quantitative comparison. Among 130 proteins, 105 unique proteins were found to be mitochodrial from the off-line combination of FlFFF and nLC-ESI-MS-MS analysis. It also showed that 23 proteins were found in all fractions but some proteins were found exclusively in certain fractions. Among 25 proteins listed from other subcellular species, seven proteins were known to exist in mitochondria as well as in other subcellular locations, which may support the possible translocation or multiple localizations of proteins among organelles. This study demonstrated effective use of FlFFF for the isolation and/or enrichment of intact mitochondria isolated from cells, as well as its potential use for the fractionation of other subcellular components in the framework of subcellular functional proteomics.

Flow field-flow fractionation for the analysis of nanoparticles used in drug delivery

Structured nanoparticles (NPs) with controlled size distribution and novel physicochemical features present fundamental advantages as drug delivery systems with respect to bulk drugs. NPs can transport and release drugs to target sites with high efficiency and limited side effects. Regulatory institutions such as the US Food and Drug Administration (FDA) and the European Commission have pointed out that major limitations to the real application of current nanotechnology lie in the lack of homogeneous, pure and well-characterized NPs, also because of the lack of well-assessed, robust routine methods for their quality control and characterization. Many properties of NPs are size-dependent, thus the particle size distribution (PSD) plays a fundamental role in determining the NP properties. At present, scanning and transmission electron microscopy (SEM, TEM) are among the most used techniques to size characterize NPs. Size-exclusion chromatography (SEC) is also applied to the size separation of complex NP samples. SEC selectivity is, however, quite limited for very large molar mass analytes such as NPs, and interactions with the stationary phase can alter NP morphology. Flow field-flow fractionation (F4) is increasingly used as a mature separation method to size sort and characterize NPs in native conditions. Moreover, the hyphenation with light scattering (LS) methods can enhance the accuracy of size analysis of complex samples. In this paper, the applications of F4-LS to NP analysis used as drug delivery systems for their size analysis, and the study of stability and drug release effects are reviewed.

Gravitational field-flow fractionation for the characterisation of active dry wine yeast.

Gravitational field-flow fractionation (GrFFF) is applied to the fractionation of active dry wine yeast. An experimental approach to the analysis of the effects that field variation by changing mobile phase composition and flow-rate have on the fractionation process of standard particles (polystyrene) was first developed to further obtain effective fractionation of wine yeast by GrFFF. Scanning electron microscopy and Coulter counter particle size measurements were used to monitor the fractionation extent and capabilities of GrFFF to describe the distribution of yeast cells populations.