Spatially resolved (semi)quantitative determination of iron (Fe) in plants by means of synchrotron micro X-ray fluorescence

Iron (Fe) is an essential element for plant growth and development; hence determining Fe distribution and concentration inside plant organs at the microscopic level is of great relevance to better understand its metabolism and bioavailability through the food chain. Among the available microanalytical techniques, synchrotron μ-XRF methods can provide a powerful and versatile array of analytical tools to study Fe distribution within plant samples. In the last years, the implementation of new algorithms and detection technologies has opened the way to more accurate (semi)quantitative analyses of complex matrices like plant materials. In this paper, for the first time the distribution of Fe within tomato roots has been imaged and quantified by means of confocal μ-XRF and exploiting a recently developed fundamental parameter-based algorithm. With this approach, Fe concentrations ranging from few hundreds of ppb to several hundreds of ppm can be determined at the microscopic level without cutting sections. Furthermore, Fe (semi)quantitative distribution maps were obtained for the first time by using two opposing detectors to collect simultaneously the XRF radiation emerging from both sides of an intact cucumber leaf.

Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S–S bonds is often used in “bottom up” sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the “hidden” C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S–S bond rupture does not occur in this case.

Non-destructive depth profile reconstruction of bio-engineered surfaces by parallel-angle-resolved X-ray photoelectron spectroscopy

In the present, contribution angle-resolved X-ray photoelectron spectroscopy (AR-XPS) was proposed as a useful tool to address the challenge of probing the near-surface region of bio-active sensor surfaces. A model bio-functionalised surface was characterised by parallel AR-XPS and commercially available Thermo Avantage-ARProcess software was used to generate non-destructive concentration depth profiles of protein-functionalised silicon oxide substrates. At each step of the functionalisation procedure, the surface composition, the overlayer thickness, the in-depth organisation and the in-plane homogeneity were evaluated. The critical discussion of the generated profiles highlighted the relevance of the information provided by PAR-XPS technique.

Lanthanoid complexes with thenoyltrifluoroacetone and 4-tert-butylcalix[4]arene-tetraacetic acid tetraethyl ester: synergistic extraction and separation

The solvent extraction of fourteen lanthanoid ions with thenoyltrifluoroacetone (HTTA) in combination with tetraethyl 4-tert-butylcalix[4]arene-tetraacetic acid tetraethyl ester (S) from a perchlorate medium at constant ionic strength was investigated. The extracted species were identified as the Ln(TTA)₃·S complexes by slope analysis. Equilibrium constants, parameters for extraction, and the synergistic and separation factors between two adjacent Ln(III) ions were determined.

Nanoscale surface analysis on second generation advanced high strength steel after hot dip galvanizing

Second generation advanced high strength steel is one promising material of choice for modern automotive structural parts because of its outstanding maximal elongation and tensile strength. Nonetheless there is still a lack of corrosion protection for this material due to the fact that cost efficient hot dip galvanizing cannot be applied. The reason for the insufficient coatability with zinc is found in the segregation of manganese to the surface during annealing and the formation of manganese oxides prior coating. This work analyses the structure and chemical composition of the surface oxides on so called nano-TWIP (twinning induced plasticity) steel on the nanoscopic scale after hot dip galvanizing in a simulator with employed analytical methods comprising scanning Auger electron spectroscopy (SAES), energy dispersive X-ray spectroscopy (EDX), and focused ion beam (FIB) for cross section preparation. By the combination of these methods, it was possible to obtain detailed chemical images serving a better understanding which processes exactly occur on the surface of this novel kind of steel and how to promote in the future for this material system galvanic protection.

Biological and chemical investigation of Allium cepa L. response to selenium inorganic compounds

The aim of this study was to evaluate the biological and chemical response of Allium cepa L. exposed to inorganic selenium compounds. Besides the investigation of the total content of selenium as well as its chemical speciation, the Allium test was used to evaluate the growth of onion roots and mitotic activity in the roots’ meristem. The total content of selenium was determined by inductively coupled plasma mass spectrometry (ICP MS). High-performance liquid chromatography (HPLC), coupled to ICP MS, was used for the selenium chemical speciation. Results indicated that A. cepa plants are able to biotransform inorganic selenium compounds into their organic derivatives, e.g., Se-methylselenocysteine from the Se(IV) inorganic precursor. Although the differences in the biotransformation of selenium are due mainly to the oxidation state of selenium, the experiment has also shown a fine effect of counter ions (H⁺, Na⁺, NH₄ ⁺) on the response of plants and on the specific metabolism of selenium.

Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis

The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50?% of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.

One-pot solvothermal synthesis of a Cu2O/Graphene nanocomposite and its application in an electrochemical sensor for dopamine

Nanocomposites composed of cuprous oxide (Cu₂O) and graphene were synthesized via reduction of copper(II) in ethylene glycol. This material possesses the specific features of both Cu₂O and graphene. Its morphology was characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. Cyclic voltammetry was used to evaluate the electrochemical response of a glass carbon electrode (GCE) modified with the nanocomposite towards dopamine (DA). Compared to the bare GCE, the Cu₂O nanoparticles modified electrode and the graphene modified electrode, the nanocomposites modified electrode displays high electrocatalytic activity in giving an oxidation peak current that is proportional to the concentration of DA in the range from 0.1 to 10???M,with a detection limit of 10?nM (S/N?=?3). The modified electrode shows excellent selectivity and sensitivity even in the presence of high concentration of uric acid and can be applied to determine DA in real samples with satisfactory results.

Profiling of secondary metabolites in tissues from Rheum palmatum L. using laser microdissection and liquid chromatography mass spectrometry

Evaluating the quality of herbal medicines by morphological features is a convenient, quick, and practical method compared with other methods that mostly depend on modern instruments. Here, laser microdissection and ultra-performance liquid chromatography are combined with mass spectrometry to map the distribution of secondary metabolites in cells or tissues of a herb itself for correlating its bioactive components and morphological features. The root and rhizome of Rheum palmatum L. were taken as research target, which is the Chinese medicine, Radix et Rhizoma Rhei. According to fluorescent microscopic characteristics, 12 herbal cells or tissues of Radix et Rhizoma Rhei were separated by laser microdissection. Thirty-eight compounds were identified or tentatively characterized in the microdissected tissues. (+)-Catechin, 1-O-galloyl-2-O-cinnamoyl-β-d-glucose, and emodin were found to be the major components in most of the tissues. The brown ergastic substances found in rays of normal and anomalous vascular bundles as well as the parenchymatous cells of rhizome pith and the parenchymatous cells of root xylem contained higher than average amounts of these three components and more kinds of secondary metabolites. Overall, results suggest that Radix et Rhizoma Rhei of larger size and with conspicuous “brocaded patterns” and star spots are of higher quality as they tend to have greater contents of bioactive components. The study provides quantitative and specific criteria by which the quality of Radix et Rhizoma Rhei can be judged. This research also established a new, reliable, and practical method for direct profiling and imaging of secondary metabolites in any herbal tissue.

Structural and electronic characterization of self-assembled molecular nanoarchitectures by X-ray photoelectron spectroscopy

Molecular monolayers and similar nanoarchitectures are indicative of the promising future of nanotechnology. Therefore, many scientists recently devoted their efforts to the synthesis, characterization, and properties of mono- and multilayer-based systems. In this context, X-ray photoelectron spectroscopy is an important technique for the in-depth chemical and structural characterization of nanoscopic systems. In fact, it is a surface technique suitable for probing thicknesses of the same order of the photoelectron inelastic mean free paths (a few tens of ångströms) and allows one to immediately obtain qualitative and quantitative data, film thickness, surface coverage, molecule footprint, oxidation states, and presence of functional groups. Nevertheless, other techniques are important in obtaining a complete spectroscopic characterization of the investigated systems. Therefore, in the present review we report on X-ray photoelectron spectroscopy of self-assembled molecular mono- and multilayer materials including some examples on which other characterization techniques produced important results.

Metabonomics studies of intact hepatic and renal cortical tissues from diabetic db/db mice using high-resolution magic-angle spinning 1H NMR spectroscopy

A metabonomics approach based on high-resolution magic-angle spinning (HRMAS) ¹H NMR spectroscopy was applied to investigate the metabolite composition in intact hepatic tissues and renal cortical tissues from db/db mice of 8 weeks old, an animal model of type 2 diabetes mellitus. Compared to the control group, the hepatic tissues of diabetic mice have elevated levels of triglyceride and bile acid and declined levels of trimethylamine-N-oxide, phosphocholine, glycerophosphocholine, and choline. The biochemical changes are less obvious in renal cortical tissues of diabetic mice. The WET_CPMG pulse sequence was selected for our metabonomics study after the quality and reproducibility of the spectra obtained from the NOEPR, NOEPR_CPMG, and WET_CPMG pulse sequences were analyzed together with principal component analysis. The influence of line-broadening factor of exponential window function for spectral manipulation on class separation was paid attention to for the first time, and an optimal value was obtained under our experimental conditions. These studies show the efficiency of HRMAS ¹H NMR spectroscopy for tissue metabonomics study in combination with multivariate statistical analysis, which may help to explore the etiological factor of diabetes mellitus from a new perspective.

Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry

The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca²⁺. In-depth MS and MS/MS analysis of the cross-linked products was aided by ¹⁵ ? N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca²⁺-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca²⁺-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

Voltammetric determination of bisphenol A in food package by a glassy carbon electrode modified with carboxylated multi-walled carbon nanotubes

A highly sensitive and mercury-free method for determination of bisphenol A (BPA) was established using a glassy carbon electrode that was modified with carboxylated multi-walled carbon nanotubes. A sensitive oxidation peak is found at 550?mV in linear sweep voltammograms at pH?7. Based on this finding, trace levels of bisphenol A can be determined over a concentration range that is linear from 10?nM to 10⁴?nM, the correlation coefficient being 0.9983, and the detection limit (S/N?=?3) being 5.0?nM. The method was successfully applied to the determination of BPA in food package.

Smart methanol sensor based on silver oxide-doped zinc oxide nanoparticles deposited on microchips

We have prepared calcined silver oxide-doped zinc oxide nanoparticles (NPs) by a hydrothermal method using reducing agents in alkaline medium. The doped NPs were characterized by UV/vis, FTIR, and X-ray photoelectron spectroscopy, and by X-ray powder diffraction and field-emission scanning electron microscopy. The NPs were deposited on microchips to result in a sensor that has a fast response to methanol in the liquid phase. Features include high sensitivity, low-sample volume, reliability, reproducibility, ease of integration, long-term stability, and enhanced electrochemical responses. The calibration plot is linear (r²?=?0.9981) over the 0.25 mmolL^?1 to 0.25 molL^?1 methanol concentration range. The sensitivity is ~7.917 μA cm^?2 mmolL^?2, and the detection limit is 71.0?±?0.5 μmolL^?1 at a signal-to-noise-ratio of 3.

Micro-/nanofluidic device for tunable generation of a concentration gradient: application to Caenorhabditis elegans chemotaxis

In this study, we propose a novel micro-/nanofluidic device that can generate a chemical concentration gradient using a parallel nanochannel as gradient generator. This device is easy to fabricate, showing high reproducibility. Its main feature is the multiple-nanochannel-based gradient generator, which permits the diffusion of small molecules and tunably generates concentration gradients. The nanopattern for the nanochannels can be rapidly and easily fabricated by wrinkling a diamond-like carbon thin film which is deposited on a polydimethylsiloxane substrate; the generation of the concentration gradient can be adjusted by controlling the dimensions of the nanochannels. The developed gradient generator is embedded into a microfluidic device to study chemotaxis in the nematode Caenorhabditis elegans, which has a highly developed chemosensory system and can detect a wide variety of chemical molecules. This device shows good performance for rapid analysis of C. elegans chemotaxis under sodium chloride stimuli.

Asymmetrical flow field-flow fractionation as a novel technique for the analysis of PS-b-PI copolymers

Asymmetrical flow field-flow fractionation (AF4) was used as a fractionation technique to investigate the molecular heterogeneity of poly(styrene-b-isoprene) diblock copolymers synthesized by either sequential living anionic polymerization or coupling of living precursor blocks. AF4 coupled to multi-angle laser light scattering (MALLS), refractive index (RI), and ultraviolet (UV) detectors was used to separate the diblock copolymers from the homopolymers and coupling products, and the molar masses of the different components were analyzed. In order to get more information about the separated block copolymers, homopolymers, and coupling products, fractions were collected directly after the AF4 channel. The collected fractions were analyzed offline by ¹H NMR to provide identification of the different species and additional information on the true chemical composition, and the microstructure of the diblock copolymer was obtained.

Metallomic study on plasma samples from Nile tilapia using SR-XRF and GFAAS after separation by 2D PAGE: initial results

An investigation was made on plasma samples obtained after protein separation. The proteome of the plasma of Nile tilapia (Oreochromis niloticus) was separated by 2D PAGE, and manganese and zinc in protein spots was qualitatively and quantitatively determined by synchrotron radiation X-ray fluorescence (SR-XRF) and graphite furnace atomic absorption spectrometry (GFAAS). Manganese and zinc are present in four and six plasma protein spots, respectively. These ions are bound to proteins with molecular weights ranging from 19 to 70?kDa and with isoelectric point (pI) ranging from 4.7 to 6.3. The concentrations of manganese and zinc bound to these proteins as determined by GFAAS following acid digestion of the spots range from 0.8 to 2.6?mg of manganese, and from 1.0 to 6.3?mg of zinc, respectively, per g of protein.

Chromatographic and mass spectrometric fingerprinting analyses of Angelica sinensis (Oliv.) Diels-derived dietary supplements

Angelica sinensis (Oliv.) Diels (“Danggui” in Chinese) is one of the most commonly used traditional Chinese medicines. It has been used to invigorate blood circulation for the treatment of anemia, hypertension, chronic bronchitis, asthma, rheumatism, and cardiovascular diseases. There are a number of A. sinensis-derived dietary supplements in the US markets. However, no study have been conducted to investigate the quality of these dietary supplements. In this paper, high-performance liquid chromatographic and flow-injection mass spectrometric fingerprints were both evaluated to assess the consistency of A. sinensis-derived dietary supplements. Similarity analysis was carried out on the high-performance liquid chromatographic (HPLC) fingerprints. Meanwhile, principal component analysis (PCA) was performed on the data obtained from flow-injection mass spectrometric (FIMS) fingerprints, which can analyze each sample in 2 min, compared with 30 min required for the chromatographic fingerprint. Both methods show significant chemical differences between samples that may be due to differences in growing locations, growing conditions, harvesting times, and/or botanical processing. The loading plots obtained from PCA singled out the discriminatory ions that were responsible for chemical differences of A. sinensis-derived dietary supplements.

Determination of carbohydrates present in Saccharomyces cerevisiae using mid-infrared spectroscopy and partial least squares regression

A fast and simple method to control variations in carbohydrate composition of Saccharomyces cerevisiae, baker's yeast, during fermentation was developed using mid-infrared (mid-IR) spectroscopy. The method allows for precise and accurate determinations with minimal or no sample preparation and reagent consumption based on mid-IR spectra and partial least squares (PLS) regression. The PLS models were developed employing the results from reference analysis of the yeast cells. The reference analyses quantify the amount of trehalose, glucose, glycogen, and mannan in S. cerevisiae. The selection and optimization of pretreatment steps of samples such as the disruption of the yeast cells and the hydrolysis of mannan and glycogen to obtain monosaccharides were carried out. Trehalose, glucose, and mannose were determined using high-performance liquid chromatography coupled with a refractive index detector and total carbohydrates were measured using the phenol–sulfuric method. Linear concentration range, accuracy, precision, LOD and LOQ were examined to check the reliability of the chromatographic method for each analyte.