Abstract: A biosensor for the measurement of glycerol in FIA was constructed using covalently immobilized glycerokinase and glycerol-3-phosphate oxidase in conjunction with a Pt based hydrogen peroxide probe. Different immobilization strategies have been studied including random and asymmetric immobilization onto a polymeric support and immobilization onto two different membranes. The latter resulted in the best configuration for batch measurement. The most effective configuration for measurement in FIA was the immobilization of glycerokinase in a glass beads reactor coupled with glycerol-3-phosphate oxidase on a preactivated Immobilon AV membrane kept at the electrode surface. Using a 250 µL injection loop, 3 mmol ATP(Mg+2) in 0.1 M borate buffer pH 8.5 and a flow rate of 0.5 ml/min, a linear response in the 2 x 10^-6/10^-3 mol/l range and a detection limit of 5 x 10^-7 mol/l were obtained for glycerol. Lifetime of the glycerol-3-phosphate membrane was extended up to 1 month by storage in the working buffer containing 1% DEAE-dextran and 5% lactitol. More than 350 samples can be assayed with this system. The biosensor was used to monitor off-line glycerol production during alcoholic fermentations carried out at different pHs and temperatures.

Abstract: Diamine oxidase from Lens culinaris has been used for the amperometric detection of biogenic amines in cheese. The enzyme has been immobilized onto polymeric membranes or glass beads and coupled with a Pt based hydrogen peroxide electrode. The resulting enzyme electrode and reactor have been optimized For the response to the six biogenic amines more frequently found in cheese: histamine, tyramine, putrescine, cadaverine, phenylethylamine and tryptamine. Detection limits were Found to be in the micromolar range with a linearity up to 3 orders of magnitude. Rejection of the electrochemically active compounds present in cheese was effective by the electropolymerization of 1,4-diaminebenzene onto the Pt surface. The best performances in terms of sensitivity and selectivity were obtained using the enzyme reactor.

Abstract: The inhibition effect of heavy metals ions such as Hg(II), Cu(II), V(V), and Ni(II) on the activity of oxidase enzymes such as alcohol oxidase, glycerol 3-P oxidase, and sarcosine oxidase has been studied and used for the construction of calibration curves in flow injection analysis. A platinum-based H2O2 probe was used to measure the enzymatic activity of the selected oxidase enzymes. The most sensitive detection system was obtained using the glycerol 3-P oxidase/Hg(II) couple. A calibration curve was obtained in the 0.05-0.4 ppm range, with a detection limit of 0.05 ppm, and a 50% of inhibition (I-50) Of 0.2 ppm. The enzyme alcohol oxidase was used to construct calibration curves for Cu(II) and V(V). The detection limits were 2 and 0.5 ppm, respectively, with I50 ppm for cu(II) and 2.7 ppm for V(V). Ni(II) was detected in the 1-6 ppm range, using the enzyme sarcosine oxidase; I-50 was 3.8 ppm. Relative standard deviations were less than or equal to5% for each enzyme/ion metal couple. The analytical behavior was comparable to measurements in batch analysis. The analysis time was in the range of 10^-15 minutes, which makes the system suitable for fast analysis of heavy metals in the 0.1-10 ppm range. The method could be usefully adopted as on-line control or screening field-test for contaminated water samples (typically industrial effluents) according to the detection limits. This will reduce the number of samples to he analyzed by standard methods based on AAS or ICP/MS detection.

Abstract: An electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DTT), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), 1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane (p,p'-DDD) and o,p-DDT was developed. Optimization of the ELISA competition conditions, led to similar response for the p,p'-isomers. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3',5,5'-tetramethylbenzidine as substrate. The use of purified antiserum for p,p'-DDT resulted in a sensitive assay with a detection limit of 40 pg mL-1 and RSD ranging from 1 to 3% intra-day and 3 to 6% inter-day. No matrix effect for waste water samples of different origin has been evidenced. The ELISA was used to detect DDTs in 20 samples after extraction in diethylether. This method appears suitable for routine screening of DDTs without sample pre-treatment other than dilution in PBS or after organic solvent extraction if high sensitivity is required.