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.

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.

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.

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.

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.

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.

A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs.

A novel urea biosensor based on immobilised recombinant urease as sensitive element and ion sensitive field effect transistor as transducer was developed. Recombinant urease from E. coli with an increased Km was photoimmobilised in PVA/SbQ (poly(vinyl alcohol) containing styrylpyridinium) membrane and has demonstrated quite good performance as biosensitive element. Enzymatic field effect transistors based on such a bioselective element were studied in model buffer solutions. This biosensor demonstrated an extended dynamic range up to 80 mM, a quite good reproducibility (standard deviation of the sensor responses was approximately 2.5%, n= 20 for urea concentration 10 mM) and a high stability. Such characteristics fit with the analytical requirements needed for urea control in plasma and liquids used during renal dialysis.

Use of competitive inhibition for driving sensitivity and dynamic range of urea ENFETs.

An urea biosensor based on urease-BSA (bovine serum albumin) membrane immobilised on the surface of an ion-sensitive field effect transistor (ISFET) has been studied in a mix buffer solution composed of potassium phosphate, Tris, citric acid and sodium tetraborate. In this mix buffer, the biosensor showed a dynamic larger than the one observed in a phosphate or Tris buffer. Investigation of the individual effect of each component of the buffer solution on the biosensor response has shown that tetraborate anion acts as a strong competitive inhibitor for the hydrolysis reaction of urea catalysed by urease. The biosensor response was investigated in a phosphate buffer with different concentrations of tetraborate anion. The results showed that the apparent constant of Michaelis-Menten, K(m(app)), increases from 4.3 to 79.3 mM, for experiments realised without and with 0.5 mM sodium tetraborate, respectively. The mean value, determined graphically, for the inhibition constant, K(i), was 29 microM. The graphical representation of biosensor calibration curves in semilogarithmic co-ordinates showed that the linear range of the biosensor can be extended up to three orders of magnitude, allowing an urea detection in a concentration range 0-100 mM.

Impedimetric immunosensor using avidin-biotin for antibody immobilization.

The potentialities of an electrodeposited biotinylated polypyrrole film as an immobilisation matrix for the fabrication of impedimetric immunosensors are described. Biotinylated antibody (anti-human IgG), used as a model system, was attached to free biotin groups on the electrogenerated polypyrrole film using avidin as a coupling reagent. This immobilization method allows to obtain a highly reproducible and stable device. The resulting immunosensor has a linear dynamic range of 10-80 ng ml(-1) of antigen and a detection limit of 10 pg ml(-1). Furthermore, this immunosensor exhibited minor loss in response after two regeneration steps.

A novel enzyme biosensor for steroidal glycoalkaloids detection based on pH-sensitive field effect transistors.

For the design of a biosensor sensitive to steroidal glycoalkaloids, pH-Sensitive Field Effect Transistors as transducers and immobilised butyrylcholinesterase as a biorecognition element have been used. The total potato glycoalcaloids can be measured by this biosensor in the concentration range 0.5-100 microM with detection limits of 0.5 microM for alpha-chaconine and of 2.0 microM for alpha-solanine and solanidine, respectively. The responses of the developed biosensors were reproducible with a relative standard deviation of about 1.5% and 5% for intra- and inter-sensor responses (both cases, n=10, for an alkaloid concentration of 5 microM), respectively. Moreover, due to the reversibility of the enzyme inhibition, the same sensor chip with immobilised butyrylcholinesterase can be used several times (for at least 100 measurements) after a simple washing by a buffer solution and can be stored at 4 degrees C for at least 3 months without any significant loss of the enzymatic activity.

Development of enzyme biosensor based on pH-sensitive field-effect transistors for detection of phenolic compounds.

This article describes a biosensor based on pH-sensitive field-effect transistors (pH-FETs) as transducer, and immobilised enzyme tyrosinase as biorecognition element, which was used for the determination of phenolic compounds in water solutions. The biologically active membrane was formed by cross-linking of tyrosinase with bovine serum albumin (BSA) in saturated glutaraldehyde (GA) vapours on the sensitive transducer surface. The main analytical characteristics were studied under different conditions as well as the possibility to optimise these working parameters. Different factors such as the pH of immobilisation, the enzyme loading, the time of exposition to glutaraldehyde vapours were investigated in regards to the influence on sensitivity, limit of detection, dynamic range, and operational and storage stability.

A miniaturized urea sensor based on the integration of both ammonium based urea enzyme field effect transistor and a reference field effect transistor in a single chip.

A urea biosensor prepared by covalent binding of urease directly to the surface of an ammonium-sensitive field effect transistor (FET) is described. Nonactin incorporated in carboxylated polyvinyl chloride was used to obtain the sensitive membrane of the ammonium-sensitive FET. The grafting of urease on the polyvinylchloride-COOH membrane surface was performed through carbodiimide coupling. The activity of the immobilized enzyme was spectrometrically controlled through the time-dependent disappearance of the absorbance of NADH at 340 nm. An apparent activity of 50% was found, compared with free enzyme. The sensitivity of the urea enzyme FET is 50 mV/pUrea working in a differential mode of 2 muM to 1 mM, this sensitivity being constant during 15 days. Finally, in order to test the potentialities of the urea biosensor for the environmental applications, the detection of heavy metal ions such as Cu(II) and Hg(II) in solution was performed by measuring the remaining activity of the inhibited enzyme.