An aptasensor for ochratoxin A based on grafting of polyethylene glycol on a boron-doped diamond microcell.

A novel strategy for the fabrication of an electrochemical label-free aptasensor for small-size molecules is proposed and demonstrated as an aptasensor for ochratoxin A (OTA). A long spacer chain of polyethylene glycol (PEG) was immobilized on a boron-doped diamond (BDD) microcell via electrochemical oxidation of its terminal amino groups. The amino-aptamer was then covalently linked to the carboxyl end of the immobilized PEG as a two-piece macromolecule, autoassembled at the BDD surface, forming a dense layer. Due to a change in conformation of the aptamer on the target analyte binding, a decrease of the electron transfer rate of the redox [Fe(CN)6](4-/3-) probe was observed. To quantify the amount of OTA, the decrease of the square wave voltammetry (SWV) peak maximum of this probe was monitored. The plot of the peak maximum against the logarithm of OTA concentration was linear along the range from 0.01 to 13.2 ng/L, with a detection limit of 0.01 ng/L. This concept was validated on spiked real samples of rice.

Qualitative assessment of the emotional and behavioural responses of haemophilia A carriers to negative experiences in their medical care.

Previous discussions with haemophilia A (HA) carriers suggested that carriers may experience inappropriate care, resulting in poor relationships with healthcare providers (HCPs; principally physicians and nurses), and unfortunate and extreme emotional and behavioural responses. This was a qualitative study to explore medical experiences of HA carriers and their emotional and behavioural responses. Eleven HA carriers and five Haemophilia Treatment Centre nurses were interviewed. Themes were identified using QSR NVivo 8.0. Carriers and nurses reported HA-related bleeding symptoms in carriers, including life-threatening haemorrhage following injury or medical intervention. Menorrhagia was common and distressing. Negative carrier experiences were related in the determination of genotypic and phenotypic status, management, precautions and HCP attitude, including dismissing carriers' symptoms, concerns or requests for care. Carriers responded with mistrust, lost confidence, disappointment, fear, anxiety, doubt of self or child, discussing experiences, avoidance of healthcare and self-treatment. Dismissive HCP attitudes, ignorance about bleeding disorders in women and unique aspects of the carrier population appear to make errors more likely. This study indicates that carriers experience inappropriate care and encounter dismissive attitudes, and respond emotionally and behaviourally. Our model suggests that systematic medical errors aggravate a negative feedback loop leading to negative emotional and behavioural responses and worsening carrier care. Improved carrier care policies and increased awareness of women's bleeding disorders may improve this situation. Further research is needed to determine whether the themes identified in this study accurately reflect the experiences of carriers in general.

Experimental study of thermodynamic surface characteristics and pH sensitivity of silicon dioxide and silicon nitride.

In this report, we have introduced a revision of the chemical treatment influence on the surface thermodynamic properties of silicon dioxide (SiO(2)) and silicon nitride (Si(3)N(4)) solid thin layers. Some characterization techniques might be used to quantify the thermodynamic properties of solid surface and predict its ability in the adhesion phenomenon. In this work, we have used static and dynamic contact angle (CA) measurements to characterize both dioxide solid surfaces being treated by using the two procedures of cleaning and chemical activation. Qualitative and quantitative concepts of analysis, using the Van Oss approach, are based on the determination of dioxide surface hydrophilic and hydrophobic features and the thermodynamic parameters such as free energy, acid, base, and Lewis acid-base surface tension components. Electrochemical capacitance-potential measurements were carried out to study the reactivity of both silicon dioxide and silicon nitride surfaces for pH variation. Furthermore, the surface roughness of these insulators was examined by using the contact angle hysteresis (CAH) measurements and atomic force microscopy (AFM). It was concluded that CA technique can be used as a suitable and base method for the understanding of surface wettability and for the control of surface wetting behavior.

Effects of active and passive land use management after cropland abandonment on water and vegetation dynamics in the Central Spanish Pyrenees.

The Mediterranean mountains have been subject to significant land abandonment process during the second half of the 20th century. The subsequent natural revegetation following abandonment in rural areas has been widely documented to have substantial implications on the hydrological cycle and the vegetation. The Spanish Pyrenees are one of the most affected areas by these land transformations which could threaten their importance for water supply and agricultural activities in the downstream lowland areas. Land managers as well as scientists around the world have taken different positions on how to deal with these land use changes. Some are in favor of active management (AM) (i.e. density reduction) while others are supporting passive management (PM) (letting the process of revegetation continue). This study aims to investigate the implication of AM and PM on hydrological and vegetation dynamics under different climate trajectories in a representative abandoned cropland catchment in the Central Spanish Pyrenees. A coupled ecohydrologic model is used to estimate the post management response of streamflow (STR), evapotranspiration (ET), soil saturation deficit (SD) and plant carbon (PC) following shrub clearing. Clearing increased annual STR by 16%, while ET and SD decreased by around -9% and -6% respectively during the first year after management with changes to monthly flows. These changes to water regimes may be even higher in wetter years. Over a 10-years period of vegetation recovery annual STR increased between 7.1% and 24.2%, while annual ET and SD decreased between -2.6% to -8.7% and -2.7% to -6% respectively due to shrub clearing, with the highest changes occurring in the first three years of AM. On the effect of climate change, our results show that a 2 °C increase in temperature could reduce AM effects on water regimes and accelerate the recovery of PC given averaged rainfall conditions.

Structural insight into PPARgamma ligands binding.

Peroxisome Proliferator Activated Receptors (PPARs) are a family of three related nuclear receptors first cloned in 1990. Their involvement in glucidic and lipidic homeostasis quickly made them an attractive target for the treatment of metabolic syndrome, the most prevalent mortality factor in developed countries. They therefore attracted much synthetic efforts, more particularly PPARgamma. Supported by a large number of crystallographic studies, data derived from these compounds lead to a fairly clear view of the agonist binding mode into the Ligand Binding Domain (LBD). Nearly all the compounds conform to a three-module structure, with a binder group involved in a series of hydrogen bonds in front of the ligand-dependent Activation Function (AF2), a linker mostly arranged around a phenoxyethyl and an effector end occupying the large cavity of the binding site. Following the marketing of the glitazones and the observation of the hepatotoxicity of troglitazone, variations in the binder led to the glitazars, and then pharmacomodulations have been undertaken on the two other modules, leading to a large family of highly related chemical structures. Some compounds, while still adhering to the three-module structure, diverge from the mainstream, such as the phthalates. Curiously, these plasticizers were known to elicit biological effects that led to the discovery of PPARs but were not actively studied as PPARs agonists. As the biological effects of PPARs became clearer, new compounds were also found to exert at least a part of their actions by the activation of PPARgamma.

Response of fibroblast activity and polyelectrolyte multilayer films coating titanium.

OBJECTIVES: The study of surface properties is a recent and crucial issue in the biomaterial fields applied to Odontology. The reference biomaterial in dental implantology is titanium. The principal objective is a perfect bio-integration in the oral ecosystem, both in terms of mucosal and bone tissues. The aim of this work was to optimize the tissue-titanium interface by applying polyelectrolyte multilayer films on the surface of titanium. METHODS: The experimental study was undertaken on pure titanium samples. Two types of film ending with polycations or polyanions were selected. Both film types were built with a first poly(ethyleneimine) (PEI) base layer and composed either of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) or of hyaluronic acid (HA) and poly(l-lysine) (PLL) layers. Final architectures were as follows: PEI-(PSS/PAH)(10), or PEI-(PSS/PAH)(10)-PSS, or chemically cross-linked PEI-(HA/PLL)(10) or PEI-(HA/PLL)(10)-HA. An analysis of the physicochemical characteristics of the surfaces was carried out by tensiometry measurements (dynamic contact angle, wettability, contact angle hysteresis) and atomic force microscopy. A biological study with human fibroblasts was followed over a 7-day culture period at days 0, 2, 4 and 7 to observe the cellular response in terms of morphology (scanning electron microscopy) and viability (Mosmann's test). RESULTS: The results showed that polyelectrolyte multilayer films could be successfully deposited onto titanium as previously described for glass or composite. Fibroblast adhesion and proliferation was strongly dependent on film type. SEM observations of cells on the different films agreed with the viability cell test. Furthermore, films containing PSS/PAH generated a better cellular response than films containing cross-linked HA/PLL. CONCLUSION: PSS/PAH polyelectrolyte films coating titanium could represent a new approach for oral bio-integration with great potential for clinical application in the fields of dental implantology. More particularly, the specific biofunctionalization of PSS/PAH films coating titanium could be envisioned by introducing layers of molecules that encourage the bio-integration process between the films.

[Biosensors for assay of glycoalkaloids in potato tubers].

The possibility of commercial application of biosensors based on pH-sensitive field-effect transistors and butyrylcholinesterase to glycoalkaloid analysis in potato tubers was studied. The main analytical features of the designed biosensors and measurement conditions were optimized. The biosensor was applied to quantitative analysis of glycoalkaloids in tubers of different potato varieties. The results proved to be in good agreement with those obtained by conventional protocols. Experiments on glucose assay were performed. An inverse correlation between the contents of glucose and glycoalkaloids in potato tubers was demonstrated.

A novel tool for the detection of allergic sensitization combining protein microarrays with human basophils.

BACKGROUND: Protein microarray (PM) is a powerful alternative to costly or labour-intensive diagnostic for the large-scale detection of allergen-specific IgE. In this study, we established a proof-of-concept that coupling the diversity of protein array with the biological output of basophilic cells is a feasible proposition. METHOD: Human basophils purified from the peripheral blood of healthy donors were stripped, re-sensitized with the serum or IgE preparation to be tested, and incubated with manually spotted protein array chips (FAST slides). The basophilic cell lines KU-812 and RBL-703/21 likewise sensitized were compared with peripheral blood basophils by the same approach. Purified basophils or other basophilic cells were incubated with FAST slides for various periods of time, washed, and cell binding was visualized by light microscopy. Basophil activation, indicating the effective cross-linking of IgE by allergens, was monitored via up-regulation of basophil activation surface marker (CD 63). RESULTS: Purified stripped peripheral basophils, re-sensitized with the serum of a grass pollen-allergic patient, displayed strong binding to anti-IgE antibody and grass pollen extract with relatively low unspecific binding. Similar results were obtained with RBL-703/21, which may be a good replacement for peripheral basophils to avoid the costly, cumbersome and time-consuming basophil purification. CONCLUSION: Our data suggest that coupling the diversity of a PM approach with the potential functionality and biological activity of a cell-based test is feasible and may result in a new system to detect allergic sensitization.

Formaldehyde-sensitive sensor based on recombinant formaldehyde dehydrogenase using capacitance versus voltage measurements.

A new formaldehyde-selective biosensor was constructed using NAD(+)- and glutathione-dependent recombinant formaldehyde dehydrogenase as a bio-recognition element immobilised on the surface of Si/SiO(2)/Si(3)N(4) structure. Sensor's response to formaldehyde was evaluated by capacitance measurements. The calibration curves obtained for formaldehyde concentration range from 10 microM to 20mM showed a broad linear response with a sensitivity of 31 mV/decade and a detection limit about 10 microM. It has been shown that the output signal decreases with the increase of borate buffer concentration and the best sensitivity is observed in 2.5mM borate buffer, pH 8.40. The response of the created formaldehyde-sensitive biosensor has also been examined in 2.5mM Tris-HCl buffer, and the shift to the positive bias of the C(V) curves along with the potential axis has been observed, but the sensitivity of the biosensor in this buffer is decreased dramatically to the value of 2.4 mV/decade.

Immobilization of E. coli bacteria in three-dimensional matrices for ISFET biosensor design.

In recent years, cell-based biosensors (CBBs) have been very useful in biomedicine, food industry, environmental monitoring and pharmaceutical screening. They constitute an economical substitute for enzymatic biosensors, but cell immobilization remains a limitation in this technology. To investigate into the potential applications of cell-based biosensors, we describe an electrochemical system based on a microbial biosensor using an Escherichia coli K-12 derivative as a primary transducer to detect biologically active agents. pH variations were recorded by an ion-sensitive field effect transistor (ISFET) sensor on bacteria immobilized in agarose gels. The ISFET device was directly introduced in 100 ml of this mixture or in a miniaturized system using a dialysis membrane that contains 1 ml of the same mixture. The bacterial activity could be detected for several days. The extracellular acidification rate (ECAR) was analyzed with or without the addition of a culture medium or an antibiotic solution. At first, the microorganisms acidified their micro-environment and then they alkalinized it. These two phases were attributed to an apparent substrate preference of bacteria. Cell treatment with an inhibitor or an activator of their metabolism was then monitored and streptomycin effect was tested.

RPBS: a web resource for structural bioinformatics.

RPBS (Ressource Parisienne en Bioinformatique Structurale) is a resource dedicated primarily to structural bioinformatics. It is the result of a joint effort by several teams to set up an interface that offers original and powerful methods in the field. As an illustration, we focus here on three such methods uniquely available at RPBS: AUTOMAT for sequence databank scanning, YAKUSA for structure databank scanning and WLOOP for homology loop modelling. The RPBS server can be accessed at http://bioserv.rpbs.jussieu.fr/ and the specific services at http://bioserv.rpbs.jussieu.fr/SpecificServices.html.

Formaldehyde assay by capacitance versus voltage and impedance measurements using bi-layer bio-recognition membrane.

A novel formaldehyde sensitive biosensor based on bacterial formaldehyde dehydrogenase (FDH) as a bio-recognition element has been developed. The bio-recognition membrane had bi-layer architecture and consisted of FDH, cross-linked with albumin, and of the cofactor NAD at a high concentration level (first layer). The second layer was a negatively charged Nafion membrane, which prevented a leakage of negatively charged NAD molecules from the bio-membrane. As transducers, gold electrodes SiO(2)/Si/SiO(2)/Ti/Au and electrolyte-insulator-semiconductor Si/SiO(2) (EIS) structures have been used. Changes in capacitance and impedance properties of the bio-recognition membrane have been used for monitoring formaldehyde concentration in a bulk solution. It has been shown that formaldehyde can be detected within a concentration range from 1 microM to 20mM depending on the type of transduction used, with a detection limit of 1 and 100 microM for gold-based and EIS-based transducers, respectively.

[Foamy virus, an original retroviral model]. / Les virus foamy, un modèle rétroviral original.

Foamy viruses (FVs) or spumaviruses are complex retroviruses isolated in several mammal species like cats, cattle and horses. Highly prevalent in non-human primates they are not naturally present in humans, although several cases of simian-to-human transmissions have been described. Interestingly, the replication strategy of FVs differs in many aspects from that of other retroviruses, presenting features that are closely related to pararetroviruses, exemplified by the hepatitis B virus (HBV), but also characteristics that are closely related to yeast retrotransposons leding to the creation of the distinct Spumaretrovirina subfamily.

Insulator semiconductor structures coated with biodegradable latexes as encapsulation matrix for urease.

A new urea biosensor for clinical applications was obtained by immobilization of urease within different latex polymers functionalized by hydroxy, acetate and lactobionate groups. Responses of these biosensors based on pH-ion-selective field effect insulator-semiconductor (IS) systems to urea additions were evaluated by capacitance measurements. UV-visible spectroscopy was used to check the urease activity in various matrixes. A good retention of the catalytic urease activity in the case of the cationic polymers was observed. In addition, rotating disk electrode experiments were carried out to determine the matrix permeability characteristics. Under optimal conditions, i.e. buffer capacity corresponding to 5 mM phosphate buffer, the urea enzyme insulator semiconductor (ENIS) sensors showed a linear response for urea concentrations in the range 10(-1.5) to 10(-4)M. Furthermore, kinetic parameters for the immobilized urease were obtained from Lineweaver-Burk plot. Clearly, a fast response and a good adhesion for the urease-acetate polymer composite films, prepared without using glutaraldehyde as cross-linking agent was observed.

Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase.

Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes.

Development of novel enzyme potentiometric biosensor based on pH-sensitive field-effect transistors for aflatoxin B1 analysis in real samples.

This study aimed at the development and optimization of a potentiometric biosensor based on pH-sensitive field-effect transistors and acetylcholinesterase for aflatoxin B1 determination in real samples. Optimal conditions for bioselective elements operation were defined and analytical characteristics of the proposed biosensor were studied. The proposed biosensor characterized high operational stability and reproducibility of signal. Selectivity of acetylcholinesterase-biosensor to aflatoxins in relation to other groups of toxic substances was analyzed. The developed biosensor was applied to the determination of aflatoxin B1 in real samples (sesame, walnut and pea).

Investigating specific antigen/antibody binding with the atomic force microscope.

The aim of this work is to detect immune complexes without any kind of labelling of each of the immunological species, with a view to create a very sensitive biosensor. This is achieved by using the atomic force microscopy. We have proceeded by imaging the antibody (anti-rabbit IgG) or anti-rabbit IgG moieties adsorbed onto mica surface, before and after incubation of two kinds of antigens: a specific (rabbit IgG) and a non-specific one (sheep IgG). The analysis using the height histograms reveals many interesting features. We propose a general framework for interpreting these analysis, which enables the discrimination between specific and non-specific complexes.

Red blood cells imaging and antigen-antibody interaction measurement.

In the present study the atomic force microscope (AFM) was used to image the surface morphology of red blood cells (RBC) for the first time. The AFM yielded very reproducible images without appreciable modifications of the sample surfaces. In addition to this topographical imaging, we have developed an experimental approach to measure the binding strength between antibody (anti-A), and the RBC antigen A, when reversible bonds between specific molecules such as antigen and antibody mediate the adhesion. The experimental results suggest that the procedure established here may be used for specific antibody detection. This study has also enhanced our understanding under physiological conditions of molecular interaction in particular antigen-antibody.

Brain glucose: voltammetric determination in normal and hyperglycaemic rats using a glucose microsensor.

Pulsed voltammetry applied to glucose oxidase-coated carbon fibre electrodes (glucose sensor) was used for brain glucose determination in normal and streptozotocin-treated rats (experimental diabetes mellitus). Glucose levels increased in the frontal cortex of diabetic animals compared with the controls (+262%). Glucose levels were also increased in their CSF (+48%) and plasma (+64%), determined in ex vivo conditions. The validity of the glucose sensor determinations, as well as that of the experimental model of diabetes used, was checked using the Beckman glucose analyser and a radioimmunoassay for plasma insulin. Insulin, unlike glucose, was decreased in diabetic animals. The sensor described here ensures precise determinations and is suitable for use in experimental models where alterations in glucose metabolism occur.