Study of bi-enzyme immobilization onto layered double hydroxides nanomaterials for histamine biosensor application.

In this work, we present the development of a hybrid biomembrane based on the immobilization of diamine oxidase (DAO) into LDH thin films for histamine detection. The LDHs preselected as host matrixes are: hydrotalcites (Mg2Al(CO3)0.5(OH)6), lowaite (Mg4Fe(OH)10Cl) and hydrocalumite (Ca2Al(OH)6Cl). The immobilized probes were characterized by atomic force microscopy (AFM) and attenuated total reflection infrared spectroscopy (IR-ATR mode). The analysis of these results shows that the immobilization of DAO occurs with all type of selected LDH and is stable after a 7 day-immersion in phosphate buffer solution. The LDH incorporating magnesium or calcium divalent cations present high-quality surface topology for DAO immobilization and the ability to keep the enzyme in a well conformation for biogenic amines catabolism and histamine detection.

A fully integrated passive microfluidic Lab-on-a-Chip for real-time electrochemical detection of ammonium: Sewage applications.

The present work reports on the development of a new generation of Lab-on-a-chip (LOC) to perform in-situ and real-time potentiometric measurements in flowing water. The device consisted of two differentiated parts: a poly (dimethylsiloxane) (PDMS) microfluidic structure obtained by soft lithography and a fully integrated chemical sensing platform including four working microelectrodes, two reference microelectrodes and one counter microelectrode for detecting ammonium in a continuous mode. The performance of the device was evaluated following its potentiometric response when analyzing ammonium containing samples. As a key parameter, its time of response was compared to that of a commercially available electrical conductivity sensor used as reference sensor during tests in laboratory using flowing tap water and technical scale using flowing wastewater. As a result, the LOC showed a slope of 55 mV/decade, a limit of detection of 4·10-5 M and a time of full response between 10 and 12 s. It was demonstrated that the device can provide fast and reliable data at real time when immersed in a laminar flow of water. Moreover, the test of robustness showed that it was still functional after immersion in sewage for at least 15 min. Besides, the LOC reported here can be helpful for a wide variety of flowing-water applications such as aqua culture outlets control, in-situ and continuous analysis of rivers effluents and sea waters monitoring among others.

Characterization of Langmuir and Langmuir-Blodgett films of a thiomacrocyclic ionophore by surface pressure and AFM.

The new synthesized thiomacrocyclic ionophore 4-phenyl-4-sulfide-11-(1-oxodecyl)-1,7-dithia-11-aza-4-phosphacyclotetradecane has proven to form Langmuir and Langmuir-Blodgett (LB) films. This ionophore shows a large affinity for copper(II) ions. Thus, the influence of the subphase composition on the surface pressure-area isotherms has been studied. The LB films have been observed by AFM and the effect of the subphase composition and the deposition surface pressure on the LB films is discussed. AFM image morphology has been correlated to the ionophore molecular structure. Surface pressure-area isotherms and AFM images show that the presence of copper(II) ions has an important role in the film structure.

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.

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.

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).

Dielectrophoresis as a tool to characterize and differentiate isogenic mutants of Escherichia coli.

In this study we report on an experimental method based on dielectrophoretic analysis to identify changes in four Escherichia coli isogenic strains that differed exclusively in one mutant allele. The dielectrophoretic properties of wild-type cells were compared to those of hns, hha, and hha hns mutant derivatives. The hns and hha genes code respectively for the global regulators Hha and H-NS. The Hha and H-NS proteins modulate gene expression in Escherichia coli and other Gram negative bacteria. Mutations in either hha or hns genes result in a pleiotropic phenotype. A two-shell prolate ellipsoidal model has been used to fit the experimental data, obtained from dielectrophoresis measurements, and to study the differences in the dielectric properties of the bacterial strains. The experimental results show that the mutant genotype can be predicted from the dielectrophoretic analysis of the corresponding cultures, opening the way to the development of microdevices for specific identification. Therefore, this study shows that dielectrophoresis can be a valuable tool to study bacterial populations which, although apparently homogeneous, may present phenotypic variability.

Aggregation profile characterisation in dielectrophoretic structures using bacteria and submicron latex particles.

A novel quantitative characterisation method for the measurement of anomalous low frequency aggregation processes on dielectrophoresis electrodes has been developed. Experimental evidence is provided for the relationship between the aggregation effect and AC electro-osmotical fluid motion theory. The aggregation profile dependence for E.coli bacteria, as a function of frequency and applied field, has been quantitatively examined. Additional experimental observations of the aggregation profiles of latex particles with dimensions of hundreds of nanometres, also confirm the relationship between this aggregation effect and the mentioned fluid motion theory.