Use of uncertainty estimates as reported by participants in proficiency testing for the evaluation of their results: pros and cons

The aim of this paper is to study the measurement uncertainties reported in proficiency tests (PTs) using examples from PTs in the environmental sector and to compare the obtained measurement uncertainty estimates using different approaches. In addition, the paper focusses on the differences between the z-score and the zeta score. Since the year 2000, the Finnish Environment Institute has asked participants to report analytical methods as well as measurement uncertainties in connection with PT results. The measurement uncertainties of the assigned value have also been evaluated. On the basis of the results, the measurement uncertainties reported by the participants varied greatly. Participants often reported underestimated measurement uncertainties, but overestimated uncertainties were also reported. At the moment, it seems as if performance assessment should be based on the z-score because of a number of significant over- and underestimated measurement uncertainties. The zeta score should be used for information and educational purposes mainly.     

New aspects of buffering with multivalent weak acids in capillary zone electrophoresis: pros and cons of the phosphate buffer

Phosphate buffer is frequently used as background electrolyte in capillary electrophoresis. It can cover a broad range of pH due to the three dissociation constants (pK1 = 2.0, pK2 = 7.2, and pK3 = 11.0) of phosphoric acid and because it is UV-light transparent. This contribution brings a theoretical study of the analytical separation performance (sample window, regions of peak symmetry, regions of fronting and tailing peaks) of phosphate buffer, serving as a model of buffering with multivalent weak acids. The study is based on the use of peak shape diagrams and covers the pH range 2.55-11.43. New important general knowledge has been revealed that single multivalent weak acids mimic the performance of background electrolytes with multiple coanionic species for anionic analyses. It is shown that simple phosphate buffers prepared by mixing phosphoric acid and potassium hydroxide may exhibit up to two regions of symmetry, of fronting as well as of tailing zones, on the mobility scale inside the sample window. Moreover they may exhibit a "schizophrenic" region of enormous electromigration dispersion.     

The use of supercritical fluid extraction for the determination of steroids in animal tissues.

A multi-analyte, multi-matrix method was developed for the routine determination of steroids in animal tissues (skin, meat and fat). After addition of internal standards and sample pre-treatment, the analytes of interest were extracted from the matrix with unmodified supercritical CO2 and trapped directly on an alumina sorbent placed in the extraction vessel (in-line trapping under supercritical conditions). After extraction, alkaline hydrolysis was performed and the analytes were derivatised. The samples were then analysed by gas chromatography-mass spectrometry. The limit of detection for the different matrix-analyte combinations was 2 micrograms kg-1 (for melengestrol acetate 5 micrograms kg-1), the repeatability ranged from 4 to 42% (n = 9) and the reproducibility ranged from 2 to 39% (n = 3).     

The use of fluorescence polarization assays for the detection of infectious diseases

Fluorescence Polarization Assays (FPAs) have been shown to have great utility in the detection of infectious diseases. Examples are presented of the use of O-polysaccharides (OPSs) for the detection of antibodies in serum, whole milk and whole blood to gram negative organisms (Brucella spp., Salmonella spp.). The use of proteins and peptides are also described for the detection of Mycobacterium bovis and Equine Infectious Anemia Virus. Fluorescence Polarization Inhibition Assays (FPIAs) are discussed for the specific and sensitive detection and quantitation of Salmonella spp. cells from culture. An example of the detection of enterohemorrhagic E. coli (EHECS) by Strand Displacement Amplification (SDA), coupled with FP, down to the single cell level, within thirty minutes, is described.     

Preliminary study of the use of dansyl chloride to determine cyclic-3',5'-AMP in tissues.

A highly sensitive method for the determination of cyclic-3',5'-AMP has been developed which involves the reaction of the substance with dansyl chloride and the subsequent separation of the dansyl-cyclic-3',5'-AMP derivative by thin-layer chromatography. Experiments with standard solutions of 3H-cyclic-3',5'-AMP have shown that there is a direct relationship between the amount of dansyl-3H-cyclic-3',5'-AMP recovered and that dansylated. The procedure is exceedingly sensitive, allowing milligram quantities of material to be analysed for its endogeneous cyclic-3',5'-AMP content. With the use of 14C-adenine as substrate, this method permits the separation of 14C-cyclic-3',5'-AMP formed from the substrate and other 14C-containing compounds, thus allowing the turn-over of cyclic-3'-5'-AMP to be studied. The usefullness of the method is demonstrated by analysing the turn-over and endogenous content of cyclic-3'-5'-AMP in rat nervous tissue.     

The peptide hormone angiotensin II: its new functions in tissues and organs

The peptide hormone angiotensin II is well established to play an endocrine role in the regulation of blood pressure, fluid and electrolyte homeostasis. In addition to its hemodynamic function, recent studies have shown that numerous tissues and organs contain their own locally generated angiotensin products (angiotensin II, III, IV and Ang 1-7) and they exhibit their respective activities. Such an intrinsic angiotensin-generating system renders to specific tissue function of our body, frequently via the regulatory mechanism of a paracrine, autocrine or intracrine manner. These tissues and organs include, to name but a few, the brain, bone marrow, adipose, epididymis, carotid body, liver, and pancreas. This local system has been shown to be responsive to various stimuli of physiological and pathophysiological importance. Moreover, the locally generated angiotensin peptides have multiple and novel actions including cell growth, anti-proliferation, apoptosis, reactive oxygen species generation, hormonal secretion, pro-inflammatory, and pro-fibrogenic actions, as well as vasoconstriction and vasodilatation. Notwithstanding the emerging roles of angiotensin II in various tissues and organs, the physiological significance and ultimately the clinical relevance remain largely undefined. Future target for these new functions by making use of specific renin-angiotensin system inhibitors, such as the angiotensin-converting enzyme and angiotensin II receptor blockers either in mono-therapy or in combination, could be of clinical importance. The current review is to focus on some of the new functions arising from the locally formed angiotensin II in tissues and organs, with particular attention to its emerging roles in the liver and the pancreas.     

Validation study for the determination of tetracycline residues in animal tissues.

An interlaboratory study of the liquid chromatographic (LC) determination of tetracyclines--oxytetracycline (OTC), tetracycline (TTC), and chlortetracycline (CTC)--in animal tissues was conducted. Isolation was performed with oxalic buffer followed by dechelation and deproteination with oxalic acid-acetonitrile. The extract was cleaned with a styrene-divinylbenzene cartridge. LC analysis was performed with a PLRP-S column and 0.01 M oxalic acid-acetonitrile (75 + 25, v/v) as mobile phase. Participants analyzed 2 control and 10 fortified porcine muscle and kidney samples. Additionally, porcine muscle samples containing incurred residues of tetracyclines were analyzed. Mean recoveries of fortified residues from porcine tissue ranged from 76.00 to 86.89%. Repeatabilities varied from 2.05% for OTC to 3.61% for TTC for muscle samples and from 6.75% for CTC to 8.74% for OTC for kidney samples. Reproducibilities ranged from 2.05 to 4.30% for muscle samples and from 15.77 to 18.81% for kidney samples.     

Biofunctionalization of aqueous dispersed, alumina membrane-templated polymer nanorods for use in enzymatic chemiluminescence assays

The noncovalent immobilization of alkaline phosphatase (ALP) onto aqueous dispersed nylon 6 nanorods ( approximately 310 nm mean diameter; approximately 6 microm mean length) prepared by anodic aluminum oxide (AAO) membrane templating was studied. Using multi-stacked layer-by-layer (LBL) assembly with the cationic quaternary ammonium polymer Sapphire II , the amount of ALP enzyme loaded onto the polymer nanostructures was found to be 115+/-7 microg mg(-1) nanorod. The biofunctionalized nanorods were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD . The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme are different from those of soluble ('free') ALP. While the K(m) value was measured to be 156 microM for free ALP, the apparent K(m) value determined for the LBL-immobilized ALP is approximately 20% lower (122 microM). Furthermore, despite the relatively high enzyme loading capacity of the nanorods, the specific activity of the bound ALP enzyme was found to be almost nine times lower than that measured for free ALP. Finally, additional experiments revealed that the catalytic activities of both free ALP and nanorod-conjugated ALP are affected similarly by changes in pH, with optimal performance levels occurring under conditions of pH 9.5. To the best of our knowledge, this study represents the first report examining the preparation of aqueous dispersed, AAO-templated polymer nanorods for potential application as enzyme scaffolds in chemiluminescent-based assay systems.     

The use of 1H NMR microscopy to study proton-exchange membrane fuel cells.

To understand proton-exchange membrane fuel cells (PEMFCs) better, researchers have used several techniques to visualize their internal operation. This Concept outlines the advantages of using 1H NMR microscopy, that is, magnetic resonance imaging, to monitor the distribution of water in a working PEMFC. We describe what a PEMFC is, how it operates, and why monitoring water distribution in a fuel cell is important. We will focus on our experience in constructing PEMFCs, and demonstrate how 1H NMR microscopy is used to observe the water distribution throughout an operating hydrogen PEMFC. Research in this area is briefly reviewed, followed by some comments regarding challenges and anticipated future developments.     

An overview on enzyme-mimicking nanomaterials for use in electrochemical and optical assays

Various kinds of nanomaterials have been described in recent years that represent stable and low-cost alternatives to biomolecules (such as enzymes) for use in (bio)analytical methods. The materials typically include, metal/metal oxides, metal complexes, nanocomposites, porphyrins, phthalocyanines, smart polymers, and carbonaceous nanomaterials. Due to their biomimetic and other properties, such nano-materials may replace natural enzymes in chemical sensors, biosensors, and in various kinds of bioassays. This overview (with 252 references) highlights the analytical potential of such nanomaterials. It is divided into sections on (a) the types of nanomaterials according to their intrinsic nature, (b) non-enzymatic sensor designs (including electrochemical, colorimetric, fluorescent and chemiluminescent methods), and (c), applications of non-enzymatic sensors in the biomedical, environmental and food analysis fields. We finally address current challenges and future directions.

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Graphical abstract This review discusses different types of nanomaterials, which are explored as a potential biomimetic material to replace the natural enzyme in the field of biosensors, and have found widespread applications in biomedical, food and environmental analysis.

     

Determination of aminothiols in body fluids, cells, and tissues by capillary electrophoresis

Oxidative stress is present in cardiovascular diseases and hyperhomocysteinemia, an independent risk factor for these diseases. It may play a role by inducing production of oxygen free radicals. Reduced glutathione is the most abundant intracellular low-molecular-weight thiol and plays an essential role in protecting cells from toxic species. The thiol-containing compounds which are the most often considered in biological analysis, are homocysteine (Hcy), cysteine (Cys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), gamma-glutamyl-cysteine (gammaGlu-Cys), and their derivatives. These aminothiols are present in body fluids or cells, associated with proteins or occur free (reduced and oxidized). These free forms may play a role in the pathogenesis of disease. Because Hcy (with Cys) exhibits pro-oxidative properties and GSH (with Cys-Gly) antioxidative properties, and because there is extensive interconversion between these metabolites, their simultaneous analysis in biological samples is necessary to examine their role in human disease. Capillary electrophoresis (CE) seems to be a solution to reach this goal. No extensive review reports the analysis of aminothiols using CE. This review describes the different CE approaches which have been used to separate and assay aminothiols, and the different obtained datas.     

Synthesis of anthrose lipidic derivative as mimic of B. anthracis BclA glycoprotein for use in ELISA-like binding assays

The surfaces of Bacillus anthracis endospores expose anthrose-containing oligosaccharides, which have been considered for use as a target for specific detection of the spores. In this direction, we have developed an efficient and straightforward synthetic strategy toward anthrose lipidic derivate tetradecyl 4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-β-d-glucopyranoside 16 as a model target for B. anthracis spores. The ability of the prepared anthrose and glucose (for control purposes) lipidic derivatives to display on a multiwell plate was demonstrated by a colorimetric phenol-sulfuric acid assay and their potential utility in multiwell binding assays was assessed using fluorescein-labeled concanavalin A (ConA-FITC) and Aleuria aurantia (AAL-FITC).     

Photogenotoxicity of mammalian cells: a review of the different assays for in vitro testing

During the past several years, phototoxicity has been studied at the molecular level, and these studies have provided new insights in the field of DNA lesion characterization, DNA repair and cell response to ultraviolet (UV)-induced stress. The development of new antibiotics and antiinflammatory drugs has highlighted the necessity to develop the assessment of phototoxicity in the safety evaluation of new chemical compounds. This paper aims at reviewing the known molecular mechanisms of the cellular response to UV-induced stress, the in vitro methods that can be proposed and used to screen for toxicity of sunlight and the photosensitization process resulting from the activation of drugs by light. UV sources, biological systems and endpoints of interest in that particular objective are listed. Phototoxic effects span from the cytotoxic-apoptotic effect to the induction of primary DNA damage, DNA repair and a variety of stress genes acting on the cell cycle and the fate of the cell. Ultimately, it can lead to the induction of hereditary DNA modification. A variety of assays are proposed to specifically address all these particular consequences of UV-induced toxicity.     

Comparative study of xylanase kinetics using dinitrosalicylic,arsenomolybdate, and ion chromatographic assays

Xylanases are commonly assayed by the dinitrosalicylic acid (DNS) or the arsenomolybdate (ARS) method. However, specific activities are many times higher with DNS than with ARS. This is because the DNS assay is more reactive and the ARS assay is less reactive with xylooligosaccharides than with xylose. Xylose is often used as a standard, even though oligosaccharides are prevalent, so the DNS method overestimates and the ARS method underestimates specific activity. Ion chromatography, with pulsed amperometric detection, separates and measures all products and intermediates, but quantitation on a molar basis is difficult, because few xylooligosaccharide response factors are known. This report directly compares these three assay methods for the assay of xylanase activities.     

Bioconjugation of alkaline phosphatase to mechanically processed, aqueous suspendible electrospun polymer nanofibers for use in chemiluminescent detection assays

Aqueous suspendible polymer nanostructures were prepared by simple microtome processing of electrospun nylon 6 nanofibers and were used to immobilize calf intestinal alkaline phosphatase (ALP) by either covalent or noncovalent bioconjugation chemistries. It was found that noncovalent immobilization of ALP to the mechanically cut nanofibers (mean length approximately 4 microm; mean diameter approximately 80 nm) using a multi-stacked, layer-by-layer (LBL) approach with the cationic polymer Sapphire II resulted in the highest enzyme loading (48.1 +/- 0.4 microg . mg(-1) nanofiber) when compared to other covalent immobilization methods based on glutaraldehyde crosslinking. The biofunctionalized nanofibers were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD. The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme were influenced by the particular types of immobilization methods employed. In terms of the overall catalytic performance of the various immobilized ALP systems, a single-stacked LBL assembly approach resulted in the highest level of enzymatic activity per unit mass of nanofiber support. To the best of our knowledge, this study represents the first report examining the preparation of mechanically shortened, aqueous dispersed electrospun polymer nanofibers for potential application as enzyme scaffolds in chemiluminescent-based assay systems.     

Hypericin and hypocrellin induced apoptosis in human mucosal carcinoma cells.

Potent photosensitizers hypocrellin A (HA), hypocrellin B (HB) and hypericin (HY) are lipid-soluble perylquinone derivatives of the genus Hypericum and have a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by HA, HB and HY are still unclear. Moreover, no reports have mentioned cell apoptosis induced by HA, HB and HY in human nasopharyngeal carcinoma (NPC) and other mucosal cells. In this study, we attempt to clarify the photodynamic effects of HA, HB and HY compounds in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human NPC cells as well as human mucosal colon and bladder cells. Using these cell lines we investigated few hallmarks of apoptotic commitments in a drug dose dependent manner. Tumor cells photo-activated with HA, HB and HY showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by all tumor cell lines as evidenced by the externalization of phosphatidylserine. Under apoptotic conditions, Western blot analysis of poly(ADP-ribose) polymerase, a caspases substrate, showed the classical cleavage pattern (116 to 85 kDa) associated with apoptosis in HA, HB and HY-treated cell lysates. In addition, 85 kDa cleaved product was blocked by the tetrapepdide caspase inhibitors such as DEVD-CHO or z-VAD-fmk. Both inhibitors protect tumor cells from apoptosis. These results demonstrate that tumor cell death induced by HA, HB and HY is mediated by caspase proteases. This study also identifies HB as a more potent and promising photosensitizer for the treatment of mucosal cancer cells.     

Analytical performances of FT-IR spectrometry and imaging for concentration measurements within biological fluids, cells, and tissues

FT-IR spectrometry has proved to be a useful tool for determining a series of plasma molecular concentrations. Dedicated experiments were first performed to test the analytical performance that could be obtained by FT-IR spectrometry using a synthesized N3-peptide exhibiting a -N3 absorption centered at 2110 cm(-1), a spectral region where no organic material of biological samples absorbs. Further, we investigated whether this technology was able to allow quantification of metabolic parameters (glucose and lactic acid) within plasma, cells, and tissues as an alternative method to the "classical" biochemical approaches, which require sophisticated biological material treatment and expensive reagents. For this purpose we used a series of plasma samples to determine glucose and lactic acid concentrations, which are common markers of cancer growth. We compared the results of the main spectral data treatments commonly achieved for FT-IR data analysis, such as univariate (Beer-Lambert) or multivariate (PLS) calibrations, as well as the deconvolution of the spectral interval of interest (1200-900 cm(-1)). No significant differences were found regarding the analytical performances of these methods. Spectral deconvolution was finally undertaken on cultured and on xenografted cells (U87 glial cells implied in human gliomas) to determine glucose and lactic acid concentrations. In this case, qualification was allowed by FT-IR imaging on the cellular models since biochemical approaches are not efficient to reach metabolic concentrations at the cellular level while keeping tissue organization.