Sabine M. Gautier

Luminescence Fiber-Optic Biosensor

Abstract A novel type of biosensors involving immobilized bioluminescence enzymes and a fiber-optic probe has been developed. The enzymes were immobilized on preactivated Nylon membranes placed in close contact with the tip of a bundle of optical glass fibers. The fiber-optic sensor was immersed in a stirred and thermostated cell protected from ambient light by a PVC jacket. The light emitted by the luminescence reactions was conducted through the fiber bundle to the photomultiplier tube of a luminometer. With immobilized firefly luciferase from Photinus pyralis, light emission could be linearly related to ATP concentration in the range 2.8 × 10−10 − 1.4 × 10−6 M. When co-immobilizing bacterial luciferase and oxidoreductase from Vibrio fischeri, NADH measurements could be performed from 3 × 10−10 M to 3 × 10−6 M. The luminol chemiluminescence reaction catalyzed by immobilized horseradish peroxidase has also been used for hydrogen peroxide determination. The standard curve was linear from 2 × 10−8 M to 2 ×...

Processing, microstructure and toughness of Al2O3 platelet-reinforced hydroxyapatite

Abstract Experimental design has been used to analyse the processing of alumina platelet-reinforced hydroxyapatite ceramics. Composite materials were developed by slip casting and hot pressing. The most appropriate slurry composition allowing production of homogeneous composites was 65 wt% of powder and 3.1 wt% of deflocculant. HAP slurries containing up to 20 vol% of platelets had a quasi-Newtonian behaviour and a shear thinning flow was found at 30 vol% of alumina. A preferred orientation of platelets within the matrix was observed which led to anisotropic mechanical characteristics. Platelet incorporation induced strong toughening of the matrix. In comparison with the monolithic HAP (KlC⊥ = 0.75 (± 0.15) MPa √m; KlC// = 0.65 (± 0.20) MPa √m), the toughness increased up to 20 vol% of alumina content and reached maximum values of KlC⊥ = 2.95 (± 0.45) MPa ⊥m and KlC// = 1.95 (± 0.35) MPa ⊥m.

CHARACTERIZATION OF HOT PRESSED AL2O3-PLATELET REINFORCED HYDROXYAPATITE COMPOSITES

Hydroxyapatite reinforced with monocrystalline Al2O3 platelets was densified by hot pressing. The effect of volume fraction and size of platelets on the microstructure, strength and toughness was investigated. It was demonstrated that no phase degradation occured during thermal treatments. A better homogeneity of composite mixtures was achieved when large platelets had been used. In return, the incorporation of small platelets appeared more favourable to increase the mechanical characteristics although limiting effect induced by microstructural defects. The flexural strength can reach 140 MPa with an associated fracture toughness of 2.5 MPa % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaaOaaaeaaca% qGTbaaleqaaaaa!36F8!\[\sqrt {\text{m}} \] compared to 137 MPa and 1.2 MPa % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaaOaaaeaaca% qGTbaaleqaaaaa!36F8!\[\sqrt {\text{m}} \] for dense monolithic HAP. The observation of crack propagation allowed us to point out the mechanisms responsible of this toughening. Crack deflection on disk faces should be considered as the initiating phenomenon leading to platelet debonding, crack bridging or branching. Crack deflection and branching may also result in the formation of unbroken ligaments of material which bridge the crack.

Multi-function fibre-optic sensor for the bioluminescent flow determination of ATP or NADH

Abstract A multi-function biosensor for the determination of either ATP or NADH using a single bioluminescence-based fibre-optic probe is described. This was made possible by co-immobilizing the firefly luciferase from Photinus pyralis for ATP analysis with the bacterial/oxidoreductase system from Vibrio harveyi for NADH analysis, on the same preactivated polyamide membrane. Compatible analytical conditions with regard to the activity and stability of each bioluminescent system were selected, enabling them to attain their highest performances. It was possible to perform continuous-flow measurements of ATP and NADH over a wide (log-log) linear calibration range with a relative standard deviation of 4.0–4.5% and detection limits of 0.25 pmol ATP and 5 pmol NADH.

Fibre-optic sensor with Co-immobilised bacterial bioluminescence enzymes

A fibre-optic bioluminescent sensor for the microdetermination of NADH is described. Measurements can be performed in the linear range 1 x 10(-9) M-3 x 10(-6) M with a detection limit of 3 x 10(-10) M using the bacterial luciferase and NAD(P)H:FMN oxidoreductase co-immobilised on a preactivated polyamide membrane. The relative standard deviation was 4.8-5.5% at 4 x 10(-8) M NADH and the steady-state response time was 2 min. When stored at -20 degrees C with 20% glycerol, the activity of the bioactive membranes was higher than that measured prior to freezing and then remained stable for more than four months.

Bioluminescence-based fibre-optic sensor with entrapped co-reactant: an approach for designing a self-contained biosensor

Abstract The possibility of designing a self-contained fibre-optic biosensor, i.e., that can be used without renewing reagents in the probe, was investigated. A probe specific for NADH involves immobilized bioluminescent enzymes which require the presence of two co-reactants [a flavin substrate (FMN) and an aldehyde] to catalyse light emission. The FMN was non-covalently immobilized in a synthetic film and was internally released in the vicinity of the bound enzymes, at the sensing tip of the bioprobe. Release of FMN was achieved from the two different matrices tested: a collagen film and a poly(vinyl alcohol) (PVA) network. Continuous-flow assays of NADH could be performed over a linear dynamic range from 10 pmol to 1 nmol. A PVA matrix appears to be a promising support for designing a self-contained biosensor, as 30–35 reliable measurements (R.S.D.=5%) could be achieved without a decrease in the sensor signal, compared with only 10–15 assays with a collagen film.

Alternate determination of ATP and NADH with a single bioluminescence-based fiber-optic sensor

Abstract A novel approach in the design of biosensors is reported. It consists of the use of two different enzymatic systems co-immobilized on the same support allowing the alternate detection of two different analytes without changing the sensing element of the sensor. Based on this approach, a multifunctional bioluminescence fiber-optic sensor for the microdetermination of ATP and NADH has been developed. The firefly luciferase from Photinus pyralis has been successfully co-immobilized with the bacterial bioluminescent system from Vibrio fischeri on a preactivated polyamide membrane. When using such a bioactive matrix associated with a fiber-optic sensor, the alternate measurement of ATP or NADH could be performed with the same probe in the linear range 1 × 10 −10 to 1 × 10 −6 M and 1 × 10 −9 to 1 × 10 −6 M, respectively. The relative standard deviation was around 6% at 9 × 10 −9 M ATP or at 4 × 10 −8 M NADH.

Design of bioluminescence-based fiber optic sensors for flow-injection analysis

A fiber optic sensor based on enzyme-catalyzed light-emitting reactions has been developed and integrated in a flow-injection analysis (FIA) system. The firefly luciferase, specific for ATP, and the bacterial oxidoreductase/luciferase system, specific for NADH, have been immobilized on preactivated polyamide membranes. ATP and NADH analysis could be performed in the range from 0.1 pmol to 3 nmol and from 0.5 pmol to 1 nmol, respectively. By co-immobilizing these two bioluminescence systems on the same membrane, a multi-function biosensor has been designed allowing the alternate determination of ATP or NADH with the same sensitivity as that obtained with the two different mono-functional biosensors. A partly self-contained biosensor has been also developed for the flow injection analysis of NADH. For this purpose, FMN (one of the substrates of the bacterial bienzymatic system) has been embedded in a synthetic matrix. Different supports have been tested for the non-covalent immobilization of this substrate and its release in the immediate vicinity of the bound enzymes. Using a photo-crosslinked poly(vinyl alcohol) support, 40 reliable assays (CV = 4.5%) could be performed without changing or reloading the matrix.

Dehydrogenase activity monitoring by flow-injection analysis combined with luminescence-based fibre-optic sensors

Dehydrogenase activities can be measured using a bioluminescence-based fibre-optic sensor associated with flow-injection analysis. The sensing element of the fibre-optic sensor consists of bacterial bioluminescent enzymes covalently bound on a preactivated polyamide membrane. Optimum conditions for the dehydrogenase-catalysed reaction compatible with the activity and stability of the bioluminescent system were selected with lactate dehydrogenase as an enzyme model permitting the detection of 5 IU l−1. The operational stability of the bioluminescent enzymes was not impaired by repeated contact with the substrates and the products of the dehydrogenase reaction.

Highly Stable Bioluminescence-Based Fiber-Optic Sensor Using Immobilized Enzymes from Vibrio Harveyi

Abstract The properties of the bioluminescent enzymatic systems from V. fischeri and V.harveyi immobilized on preactivated polyamide membranes, as well as the characteristics of a fiber-optic biosensor equipped with such membranes, have been studied. Particular attention has been paid to the stability of the bioactive matrices under working conditions, since this remains a key-point for designing operational biosensors of practical use. Whatever the origin of the bioluminescent system, the microdetermination of NADH could be performed at the nanomolar level with detection limits of 2 nM and 0.3 nM with the systems from V. harveyi and V. fischeri, respectively. the use of polyethyleneglycol (PEG 600) improves the operational stability of the bi-enzymatic system from V. fischeri, but a recalibration must be carried out every ten assays. Although the immobilized system from V. harveyi exhibited a lower activity than the enzymes from V. fischeri, its excellent operational stability (100 assays performed withi...