The Design of Experiment as a Tool to Model Plant Trace-Metal Bioindication Abilities.

Bioindicator plants are species that have the capacity to linearly uptake some elements (metal and metalloids) from the growing substrate, thus reflecting their concentration in the soil. Many factors can influence the uptake of these elements by plants, among which is the simultaneous presence of several metals, a common situation in contaminated or natural soils. A novel approach that can be used to validate the bioindication ability of a species growing on a polymetallic substrate is the design of experiment (DoE) approach. The aim of the present study was to apply the DoE in full factorial mode to model the Cu, Cd, Pb, Zn, and Cr bioindication capacity of Polygonum aviculare, used as the model plant. The results showed that P. aviculare has the ability to bioindicate Cd and Cr with a linear uptake (from 0.35 to 6.66, and 0.1 to 3.4 mg kg-1, respectively) unaffected by the presence of other metals. Conversely, the uptake of Pb, Cu, and Zn is strongly influenced by the presence of all the studied metals, making their concentration in the plant shoot not proportional to that of the soil. In conclusion, these preliminary results confirmed that the DoE can be used to predict the bioindicator abilities of a plant for several elements at the same time and to evaluate the interactions that can be established between variables in the growing medium and in the plant itself. However, more studies including other plant species are needed to confirm the effectiveness of this method.

Production of Antioxidant Molecules in Polygonum aviculare (L.) and Senecio vulgaris (L.) under Metal Stress: A Possible Tool in the Evaluation of Plant Metal Tolerance.

Plants growing on heavy metal (HM)-polluted soils show toxicity symptoms, such as chlorosis and growth reduction, and undergo oxidative stress due to the formation of reactive oxygen species (ROS). Plants overcome oxidative stress by producing a wide range of antioxidant molecules, such as polyphenols and flavonoids. The aim of the present work was to study the accumulation of these molecules in response to increasing concentrations of Cd, Cr, Cu, Ni, Pb and Zn and to assess whether they can be used as a tool in assessing metal-related stress in Polygonum aviculare and Senecio vulgaris. On average, P. aviculare shoots accumulated lower amounts of metals than S. vulgaris shoots. The uptake of all six elements was correlated and proportional to their concentration in the nutrient solution (ρ > 0.9), with the bioaccumulation factor (BAF) being >1 for most of them. The present research demonstrated that 82% of the samples showed a good correlation (|ρ| > 0.5) between the level of polyphenols, flavonoids and antioxidant activity and the metal concentration in plant shoots, confirming that the metal stress level and production of phenolic compounds having antioxidant activity were strictly connected. Nonetheless, the mere quantification of these molecules cannot identify the type of metal that caused the oxidative stress, neither determine the concentration of the stressors. The five tested populations of each species did not show any specific adaptation to the environment of origin.

Heavy Metals Bioindication Potential of the Common Weeds Senecio vulgaris L., Polygonum aviculare L. and Poa annua L.

In recent years, heavy metals (HMs) levels in soil and vegetation have increased considerably due to traffic pollution. These pollutants can be taken up from the soil through the root system. The ability of plants to accumulate HMs into their tissues can therefore be used to monitor soil pollution. The aim of this study was to test the ruderal species Senecio vulgaris L., Polygonum aviculare L., and Poa annua L., as possible candidates for biomonitoring Cu, Zn, Cd, Cr, Ni and Pb in multiple environments. The soils analyzed in this work came from three different environments (urban, woodland, and ultramafic), and therefore deeply differed for their metal content, texture, pH, and organic matter (OM) content. All urban soils were characterized by high OM content and presence of anthropogenic metals like Pb, Zn, Cd, and Cu. Woodland soils were sandy and characterized by low metal content and low OM content, and ultramafic soils had high Ni and Cr content. This soil variability affected the bioindication properties of the three studied species, leading to the exclusion of most metals (Zn, Cu, Cr, Cd, and Pb) and one species (P. aviculare) due to the lack of linear relations between metal in soil and metal in plants. Senecio vulgaris and Poa annua, conversely, appeared to be good indicators of Ni in all the soils tested. A high linear correlation between total Ni in soil and Ni concentration in P. annua shoots (R2 = 0.78) was found and similar results were achieved for S. vulgaris (R2 = 0.88).

Cylindrical Layered Bone Scaffolds with Anisotropic Mechanical Properties as Potential Drug Delivery Systems.

3D cylindrical layered scaffolds with anisotropic mechanical properties were prepared according to a new and simple method, which involves gelatin foaming, deposition of foamed strips, in situ crosslinking, strip rolling and lyophilization. Different genipin concentrations were tested in order to obtain strips with different crosslinking degrees and a tunable stability in biological environment. Before lyophilization, the strips were curled in a concentric structure to generate anisotropic spiral-cylindrical scaffolds. The scaffolds displayed significantly higher values of stress at break and of the Young modulus in compression along the longitudinal than the transverse direction. Further improvement of the mechanical properties was achieved by adding strontium-substituted hydroxyapatite (Sr-HA) to the scaffold composition and by increasing genipin concentration. Moreover, composition modulated also water uptake ability and degradation behavior. The scaffolds showed a sustained strontium release, suggesting possible applications for the local treatment of abnormally high bone resorption. This study demonstrates that assembly of layers of different composition can be used as a tool to obtain scaffolds with modulated properties, which can be loaded with drugs or biologically active molecules providing properties tailored upon the needs.

New polluting metals. Quantification in herbal medicines by voltammetric and spectroscopic analytical methods.

A new application of voltammetric techniques in stripping mode to the quantitative determination of metals belonging to the platinum group (PGM) in herbal medicines, at the ultra-trace level, is reported. Pd (II), Pt (II) and Rh (III) are determined by means of square-wave adsorption voltammetry (SWAdSV); Os (VIII) and Ru (III) are determined through square-wave voltammetry in catalytic mode (SWCV); Ir (III) is determined through the application of square-wave catalytic voltammetry in adsorption mode (SWAdCSV). In all the applied methods, the voltammetric cell has the conventional setting with three-electrodes, which sees the presence of a suspended mercury-drop electrode (HMDE) or a glassy carbon electrode (GCE) as working electrodes for the determination of Ir (III). The auxiliary electrode was a platinum electrode, and an Ag∣AgCl∣KClsatd. electrode was employed as reference electrode. Validation of the analytical procedure here proposed has been achieved using reference standards: NIST-SRM 1570a (Spinach Leaves) and NIST-SRM 1573a (Tomato Leaves), both added with pure metal standards, obtaining satisfying precision values, better than the limits set for the validation of quantitative methods. Following the verification of the validity of the here presented procedure, commercially available herbal medicines, based on Eucalyptus globulus, Harpagophytum procumbens DC and Taraxacun officinale weber, were analyzed. Standard samples were also analyzed by atomic absorption spectroscopy, in order to have a reference technique for validating the entire procedure.

Synthesis Monitoring, Characterization and Cleanup of Ag-Polydopamine Nanoparticles Used as Antibacterial Agents with Field-Flow Fractionation.

Advances in nanotechnology have opened up new horizons in nanomedicine through the synthesis of new composite nanomaterials able to tackle the growing drug resistance in bacterial strains. Among these, nanosilver antimicrobials sow promise for use in the treatment of bacterial infections. The use of polydopamine (PDA) as a biocompatible carrier for nanosilver is appealing; however, the synthesis and functionalization steps used to obtain Ag-PDA nanoparticles (NPs) are complex and require time-consuming cleanup processes. Post-synthesis treatment can also hinder the stability and applicability of the material, and dry, offline characterization is time-consuming and unrepresentative of real conditions. The optimization of Ag-PDA preparation and purification together with well-defined characterization are fundamental goals for the safe development of these new nanomaterials. In this paper, we show the use of field-flow fractionation with multi-angle light scattering and spectrophotometric detection to improve the synthesis and quality control of the production of Ag-PDA NPs. An ad hoc method was able to monitor particle growth in a TLC-like fashion; characterize the species obtained; and provide purified, isolated Ag-PDA nanoparticles, which proved to be biologically active as antibacterial agents, while achieving a short analysis time and being based on the use of green, cost-effective carriers such as water.

Hybrid gravitational field-flow fractionation using immunofunctionalized walls for integrated bioanalytical devices.

In this work, the biospecific recognition antigen-antibody reaction was implemented in gravitational field-flow fractionation (GrFFF), a flow-assisted separation technique for micron-sized particles, in order to realize a hybrid immunomodulated GrFFF system in which two different principles are combined to achieve highly versatile fractionation. Micron-sized polystyrene beads coated with horseradish peroxidase (HRP) were used as a model sample, and anti-HRP antibodies were immobilized on the accumulation wall of the GrFFF channel. Ultrasensitive chemiluminescence imaging was employed to visualize the beads during elution and to optimize experimental conditions. The same principle was then applied to real biological samples composed by Yersinia enterocolitica and Escherichia coli cells. Results show the possibility to modify the elution of selected sample components and even to retain them into the channel. The hybrid immunomodulated GrFFF system is a step towards the development of a module that could be integrated in a lab-on-a-chip-based point-of-care testing device which includes sample pre-analytical cleanup and analysis.

Strontium-Substituted Hydroxyapatite-Gelatin Biomimetic Scaffolds Modulate Bone Cell Response.

Strontium has a beneficial role on bone remodeling and is proposed for the treatment of pathologies associated to excessive bone resorption, such as osteoporosis. Herein, the possibility to utilize a biomimetic scaffold as strontium delivery system is explored. Porous 3D gelatin scaffolds containing about 30% of strontium substituted hydroxyapatite (SrHA) or pure hydroxyapatite (HA) are prepared by freeze-drying. The scaffolds display a very high open porosity, with an interconnectivity of 100%. Reinforcement with further amount of gelatin provokes a modest decrease of the average pore size, without reducing interconnectivity. Moreover, reinforced scaffolds display reduced water uptake ability and increased values of mechanical parameters when compared to as-prepared scaffolds. Strontium displays a sustained release in phosphate buffered saline: the quantities released after 14 d from as-prepared and reinforced scaffolds are just 14 and 18% of the initial content, respectively. Coculture of osteoblasts and osteoclasts shows that SrHA-containing scaffolds promote osteoblast viability and activity when compared to HA-containing scaffolds. On the other hand, osteoclastogenesis and osteoclast differentiation are significantly inhibited on SrHA-containing scaffolds, suggesting that these systems could be usefully applied for local delivery of strontium in loci characterized by excessive bone resorption.

Gelatin Porous Scaffolds as Delivery Systems of Calcium Alendronate.

The systemic administration of bisphosphonates (BPs) for the treatment of metabolic diseases characterized by abnormal bone loss suffers from several adverse side effects, which can be reduced by implementation of alternative modes of administration. In this work, glutaraldehyde cross-linked gelatin scaffolds are proposed as delivery systems of calcium alendronate monohydrate (CaAL•H2 O). The 3D highly porous scaffolds display a relevant interconnected porosity (>94%), independently from CaAL•H2 O content (0, 3, and 6 wt%). At variance, pore size varies with composition. The relative increase of the number of smaller pores on increasing BP content is in agreement with the parallel significant increase of the compressive modulus and collapse strength. The scaffolds exhibit a sustained CaAL•H2 O release profile, and a significant amount of the drug is retained in the scaffolds even after 14 d. In vitro tests are carried out using cocultures of osteoblast (OB) and osteoclast (OC). The evaluation of differentiation markers is performed both on the supernatants of cell culture and by means of quantitative polymerase chain reaction. The results indicate that BP containing scaffolds support osteoblast proliferation and differentiation, whereas they inhibit osteoclast viability and activity, displaying a promising beneficial role on bone repair processes.

Hollow-fiber flow field-flow fractionation and multi-angle light scattering investigation of the size, shape and metal-release of silver nanoparticles in aqueous medium for nano-risk assessment.

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.

Field-flow fractionation of cells with chemiluminescence detection.

Field-flow fractionation is a separation technique characterized by a retention mechanism which makes it suitable for sorting cells over a short analysis time, with low sample carry-over and preserving cell viability. Thanks to its high sensitivity, chemiluminescence detection is suitable for the quantification of just a few cells expressing chemiluminescence or bioluminescence. In this work, different formats for coupling gravitational field-flow fractionation and chemiluminescence detection are explored to achieve ultra-sensitive cell detection in the framework of cell sorting. The study is carried out using human red blood cells as model sample. The best performance is obtained with the on-line coupling format, performed in post-column flow-injection mode. Red cells are isolated from diluted whole human blood in just a few minutes and detected using the liquid phase chemiluminescent reaction of luminol catalysed by the red blood cell heme. The limit of detection is a few hundred injected cells. This is lower than the limit of detection usually achieved by means of conventional colorimetric/turbidimetric methods, and it corresponds to a red blood cell concentration in the injected sample of five orders of magnitude lower than in whole blood.

Spatial and temporal distribution of inorganic species in water and sediments of the Tronto River ecosystem.

Analytical results are reported for the determination of inorganic species in water and sediments sampled in the Tronto river ecosystem. The species determined are Cd, Co, Cr, Cu, Fe, Mn, Ni, Zn, Hg, F, NO3-, SO4-, Na, K, Ca, Mg, NH4+ in integrated water, and Cd, Co, Cr, Cu, Fe, Mn, Ni, Zn, Hg in sediments. For all the elements, in addition to detection limits, precision and accuracy are given: the former, expressed as relative standard deviation (Sr), and the latter, expressed as relative error (e), were good, being in all cases lower than 5%.

Working without accumulation membrane in flow field-flow fractionation. Effect of sample loading on retention.

Membraneless hyperlayer flow field-flow fractionation (Hyp FIFFF) has shown improved performance with respect to Hyp FIFFF with membrane. The conditions for high recovery and recovery independent of sample loading in membraneless Hyp FIFFF have been previously determined. The effect of sample loading should be also investigated in order to optimize the form of the peaks for real samples. The effect of sample loading on peak retention parameters is of prime importance in applications such as the conversion of peaks into particle size distributions. In this paper, a systematic experimental work is performed in order to study the effect of sample loading on retention parameters. A procedure to regenerate the frit operating as accumulation wall is described. High reproducibility is obtained with low system conditioning time.

Size characterization of barley starch granules by gravitational field-flow fractionation: a rapid, low-cost method to assess the brewing capability of different strains.

Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch granules are more susceptible to enzymatic hydrolysis. When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred. Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time. In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented. For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF. As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.

Tandem hollow-fiber flow field-flow fractionation.

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 HF5 (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.

Biocompatible channels for field-flow fractionation of biological samples: correlation between surface composition and operating performance.

Biocompatible methods capable of rapid purification and fractionation of analytes from complex natural matrices are increasingly in demand, particularly at the forefront of biotechnological applications. Field-flow fractionation is a separation technique suitable for nano-sized and micro-sized analytes among which bioanalytes are an important family. The objective of this preliminary study is to start a more general approach to field-flow fractionation for bio-samples by investigation of the correlation between channel surface composition and biosample adhesion. For the first time we report on the use of X-ray photoelectron spectroscopy (XPS) to study the surface properties of channels of known performance. By XPS, a polar hydrophobic environment was found on PVC material commonly used as accumulation wall in gravitational field-flow fractionation (GrFFF), which explains the low recovery obtained when GrFFF was used to fractionate a biological sample such as Staphylococcus aureus. An increase in separation performance was obtained first by conditioning the accumulation wall with bovine serum albumin and then by using the ion-beam sputtering technique to cover the GrFFF channel surface with a controlled inert film. XPS analysis was also employed to determine the composition of membranes used in hollow-fiber flow field-flow fractionation (HF FlFFF). The results obtained revealed homogeneous composition along the HF FlFFF channel both before and after its use for fractionation of an intact protein such as ferritin.

Bacteria sorting by field-flow fractionation. Application to whole-cell Escherichia coil vaccine strains.

Sorting and quantification of deactivated bacteria is an important way of quality control for whole-cell bacterial vaccines. In general, surface features of deactivated bacteria used for whole-cell bacterial vaccines affect the immunoresponse to bacteria-associated antigens. Enumeration of bacteria is also an important process development parameter for these vaccines. Field-flow fractionation (FFF) was previously applied to the separation of bacteria. For the first time, FFF is used for sorting bacteria strains of the same species on the basis of differences in bacterial membrane characteristics. Two FFF techniques, gravitational FFF (GrFFF) and asymmetrical flow FFF (AsFIFFF), are shown to be able to fractionate, distinguish, and quantify different deactivated Escherichia coli strains used for vaccines. E. coli can differ in the presence of fimbriae on the bacterial membrane. Fimbriae affect E. coli pathology and thus the use of E. coli for vaccines. GrFFF and AsFIFFF are able to fractionate fimbriated/ nonfimbriated cells in mixtures of different strains. While GrFFF is characterized by low cost and simplicity, As-FIFFF shows a higher performance in size fractionation with a high-speed separation. Coupled, on-line UV/visible turbidimetry yields the relative numbers of fractionated cells and sample recovery. Scanning electron microscopy and quasi-elastic light scattering are employed as uncorrelated techniques for size and morphology analysis of the E. coli strains.