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).

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.

E-Nose and e-Tongue combination for improved recognition of fruit juice samples.

There are many important challenges related to food security analysis by application of chemical and electrochemical sensors. One critical parameter is the development of reliable tools, capable of performing an overall sensory analysis. In these systems, as much information as possible is required in relation to smell, taste and colour. Here, we investigated the possibility of using a multisensor data fusion approach, which combines an e-Nose and an e-Tongue, adept in generating combined aroma and taste profiles. In order to shed light on this concept, classification of various Tunisian fruit juices using a low-level of abstraction data fusion technique was attempted. Five tin oxide-based Taguchi Gas Sensors were applied in the e-Nose instrument and the e-Tongue was designed using six potentiometric sensors. Four different commercial brands along with eleven fruit juice varieties were characterised using the e-Nose and the e-Tongue as individual techniques, followed by a combination of the two together. Applying Principal Component Analysis (PCA) separately on the respective e-Nose and e-Tongue data, only few distinct groups were discriminated. However, by employing the low-level of abstraction data fusion technique, very impressive findings were achieved. The Fuzzy ARTMAP neural network reached a 100% success rate in the recognition of the eleven-fruit juices. Therefore, data fusion approach can successfully merge individual data from multiple origins to draw the right conclusions that are more fruitful when compared to the original single data. Hence, this work has demonstrated that data fusion strategy used to combine e-Nose and e-Tongue signals led to a system of complementary and comprehensive information of the fruit juices which outperformed the performance of each instrument when applied separately.

Evidence of ammonium ion-exchange properties of natural bentonite and application to ammonium detection.

Ammonium exchange with hybrid PVC-bentonite (mineral montmorillonite clay) thin film was revealed using FTIR spectroscopy, EDX, cyclic voltammetry and electrochemical impedance spectroscopy. The effect of ammonium exchange on the charge transfer resistance of PVC-bentonite hybrid thin film was attributed to a modification of the intersheet distance and hydration of bentonite crystals. The obtained impedimetric ammonium sensor shows a linear range of detection from 10(-4)M to 1M and a detection limit around 10(-6)M.

Cadmium-sensitive electrode based on tetracetone derivatives of p-tert-butylcalix[8]arene.

The performance of a cadmium-sensitive electrode based on the tetracetone derivatives of p-tert butylcalix[8]arene was investigated. The ion-sensitivity of the calix[8]arene was examined via cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectrometry, UV/Vis spectroscopy and FT-IR spectroscopy. The sensitive membrane containing the active ionophore was cast onto the surface of a gold electrode. The electrode exhibited a linear relationship between the charge transfer resistance (Rct) and the logarithm of the detected ion concentration. The cathodic peak at a potential of 0.56 V increased linearly as the Cd(2+) ion concentration increased. The detection limit of the device reached 10(-7) M with high sensitivity toward cadmium.

Development and optimization of a novel conductometric bi-enzyme biosensor for L-arginine determination.

A highly sensitive conductometric biosensor for l-arginine determination was developed by exploiting the unique biorecognition capacities of two enzymes of urea cycle - arginase (E.C. 3.5.3.1) and urease (E.C. 3.5.1.5). The enzymes were co-immobilized in a single bioselective membrane on the working sensor, while a lysine rich bovine serum albumin (BSA) membrane was immobilized on the reference sensor, allowing differential measurements. The optimum percentage ratio of arginase and urease within the bioselective membrane was determined when the biosensor sensitivity to l-arginine and urea was optimum. Analytical characteristics of the conductometric biosensor for l-arginine determination were compared for two types of enzyme immobilization (cross-linking with glutaraldehyde (GA) and entrapment in the polymeric membrane). The optimum features in terms of the sensitivity, the linear range, and the detection limit (4.2 µS/mM, 0.01-4mM, and 5.0 × 10(-7)M, respectively) were found for l-arginine biosensor based on enzyme cross-linking with GA. A quantitative determination of l-arginine in the real sample (a drinkable solution "Arginine Veyron") gave a satisfactory result compared to the data provided by the producer (a relative error was 4.6%). The developed biosensor showed high operational and storage stability.

Novel conductometric biosensor based on three-enzyme system for selective determination of heavy metal ions.

A differential pair of planar thin-film interdigitated electrodes, deposited on a ceramic pad, was used as a conductometric transducer. The three-enzyme system (invertase, mutarotase, glucose oxidase), immobilized on the transducer surface, was used as a bioselective element. The ratio between enzymes in the membrane was found experimentally considering the highest biosensor sensitivity to substrate (sucrose) and heavy metal ions. Optimal concentration of sucrose for inhibitory analysis was 1.25 mM and incubation time in the investigated solution amounted to 10-20 min. The developed biosensor demonstrated the best sensitivity toward ions Hg(2+) and Ag(+). A principal possibility of the biosensor reactivation either by EDTA solution after inhibition with silver ions or by cysteine solution after inhibition with mercury ions was shown.

Effect of different modifications of BEA-zeolites on operational characteristics of conductometric biosensor.

Effect of different modifications of zeolite Na(+)-BEA on working characteristics of urease-based conductometric biosensor was studied. As the biosensor sensitive elements were used bioselective membranes based on urease and various zeolites immobilised with bovine serum albumin on the surface of conductometric transducers. Influence of zeolites on sensitivity of urea biosensor was investigated as well as reproducibility of biosensor signal and reproducibility of activity of the bioselective element after different variants of urease immobilisation on the surface of conductometric transducer. The biosensors based on zeolites (NH4(+)-BEA 30 and H(+)-BEA 30) were shown to be the most sensitive. Concentration of these zeolites in the bioselective membrane was optimized. Use of zeolites modified with methyl viologen and silver was ascertained to be of no prospect for urea conductometric biosensors. It was demonstrated that characteristics of urea biosensors can be regulated, varying zeolites modifications and their concentrations in bioselective membranes.

Integrity characterization of myoglobin released from poly(ε-caprolactone) microspheres using two analytical methods: UV/Vis spectrometry and conductometric bi-enzymatic biosensor.

Myoglobin (Mb)-loaded poly(ε-caprolactone) (PCL) microparticles were prepared by multiple emulsion with solvent extraction/evaporation method under more or less deleterious operating conditions. The protein integrity was monitored using both UV/Vis absorbance ratio method at specific wavelengths and a conductometric bi-enzymatic biosensor based on proteinase K and pronase. Under standard operating conditions, Mb remained in native conformation, while different degrees of protein denaturation were observed by changing the encapsulation conditions. It was shown that solvent elimination under reduced pressure and in a lower extent addition of a higher molecular weight PCL led to protein alteration. In the first case, the loss of protein integrity can be attributed to residual solvent entrapped in particles whose solidification was accelerated. In the second case, denaturation may be explained by an increase in the protein exposure time at water/organic solvent interface due to an increase in organic phase viscosity.

Electrochemical monitoring of chlorhexidine digluconate effect on polyelectrolyte immobilized bacteria and kinetic cell adhesion.

The electrochemical impedance spectroscopy (EIS) technique has been used as a sensitive method to explore the effect of antibacterial molecules on immobilized bacteria and biofilm formation. In this work, we describe the electrochemical spectroscopy as a powerful method to monitor the effect of chlorhexidine digluconate (CHX-Dg) on polyelectrolyte immobilized Escherichia coli K12 MG1655 and the kinetics of cell adhesion on gold electrodes. The experimental impedance data were modeled with a Zview program to find the best equivalent electrical circuit and analyse its parameter's properties. Polyelectrolyte multilayer formation on the electrode surface and bacteria immobilization greatly increased the electron-transfer resistance (R(et)) and reduced the constant phase element (CPE(dl)). The effect of CHX-Dg was studied in a 0.5 x 10⁻4 mmol l⁻¹ to 0.5 mmol l⁻¹ range. The relation between the evolution of R(et) and CHX-Dg concentration was found to be negatively correlated. When CHX-Dg was added, the electrochemical monitoring of the bacterial kinetic adhesion showed that the electrode's capacity (C(P)) variation remained stable, demonstrating that the addition of CHX-Dg in the broth inhibited bacterial adhesion.

Impedimetric immunosensor based on SWCNT-COOH modified gold microelectrodes for label-free detection of deep venous thrombosis biomarker.

Measurement of D-dimer has subsequently become an essential element in the diagnostics of deep vein thrombosis and pulmonary embolism; in this context microelectrodes with an area of 9×10(-4) cm(2) were used to develop impedimetric immunosensor for detecting deep venous thrombosis biomarker (D-dimer). The biosensor is based on functionalized carbon nanotubes (SWCNT-COOH) where the antibody (anti-D-dimer) was immobilized by covalent binding. The electrical properties and the morphology of the biolayer were characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry and atomic force spectroscopy (AFM). Impedimetric microimmunosensor allows to obtain sensitivity of 40.1 kΩ µM(-1) and detection limit of 0.1 pg/mL (0.53 fM) with linear range from 0.1 pg/mL to 2 µg/mL (0.53 fM to 0.01 µM). We demonstrate that using carbon nanotubes and microelectrodes, high sensitivity and dynamic range were obtained. The biosensor exhibited a short response time of 10 min. Moreover, the studied immunosensor exhibits good reproducibility (R.S.D. 8.2%, n=4).

Electrochemical detection of D-dimer as deep vein thrombosis marker using single-chain D-dimer antibody immobilized on functionalized polypyrrole.

We describe a rapid and sensitive method for detection and quantification of d-dimer which is a biomarker present at elevated concentrations in patients with deep vein thrombosis (DVT) disorders. The method uses an immunosensor based on a single-chain antibody (ScAb) immobilized on a transducer surface and with a densely packed receptor layer. Detection is based on the redox activity of a N-alpha bis(carboxymethyl)-L-lysine (ANTA)/Cu2+ complex attached to a polypyrrole backbone. The resulting hybrid material: polypyrrole ANTA/metal complex/His-tag ScAb was characterized by AFM, surface plasmon resonance (SPR) and differential pulse voltammetry (DPV) for the optimization of the biosensor formation. The biosensor offers a promising template for antibody immobilization and for immunodetection of a specific D-dimer. The biosensor shows a remarkable variation in redox activity of the ANTA/Cu2+ complex after the D-dimer association with a binding constant Kd of 1 ng mL(-1). Electrochemical impedance spectroscopy (EIS) allows monitoring D-dimer association with a linear response between 0.1 ng mL(-1) and 500 ng mL(-1) and a detection limit of 100 pg mL(-1) in PBS is obtained. The biolayer exhibits the same sensitivity for the detection of d-dimer in human patient plasma samples. This assay method is versatile, offers enhanced performance for the evaluation of proteins association and could easily be extended to the detection of other proteins, present in serum human sample.

A rapid and sensitive alcohol oxidase/catalase conductometric biosensor for alcohol determination.

A new conductometric biosensor has been developed for the determination of short chain primary aliphatic alcohols. The biosensor assembly was prepared through immobilization of alcohol oxidase from Hansenula sp. and bovine liver catalase in a photoreticulated poly(vinyl alcohol) membrane at the surface of interdigitated microelectrodes. The local conductivity increased rapidly after alcohol addition, reaching steady-state within 10 min. The sensitivity was maximal for methanol (0.394+/-0.004 microS microM(-1), n=5) and decreased by increasing the alcohol chain length. The response was linear up to 75 microM for methanol, 70 microM for ethanol and 65 microM for 1-propanol and limits of detection were 0.5 microM, 1 microM and 3 microM, respectively (S/N=3). No significant loss of the enzyme activities was observed after 3 months of storage at 4 degrees C in a 20mM phosphate buffer solution pH 7.2 (two or three measurements per week). After 4 months, 95% of the initial signal still remained. The biosensor response to ethanol was not significantly affected by acetic, lactic, ascorbic, malic, oxalic, citric, tartaric acids or glucose. The bi-enzymatic sensor was successfully applied to the determination of ethanol in different alcoholic beverages.

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.

[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.

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.

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-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.