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A laccase biosensor, in which the enzyme was immobilized on N-succinimidyl-3-thiopropionate (NSTP)-modified gold electrodes, is reported. Two different approaches for the preparation of N-succinimidyl-terminated monolayers were evaluated: a) activation of a preformed 3-mercaptopropionic acid (MPA) SAM by reaction with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS); b) assembling of dithiobisuccinimidyl propionate (DTSP). NSTP-modified electrodes were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Biosensors prepared by covalent binding of the enzyme and by cross-linking with glutaraldehyde atop NSTP-modified electrodes were compared in terms of sensitivity and operational range for caffeic acid. A much better analytical performance was found using the latter approach. Variables affecting the amperometric detection (enzyme loading, pH and applied potential) were optimized. The operational stability and characteristics of functioning of the laccase biosensor in terms of repeatability of the amperometric measurements, reproducibility with different biosensors and useful lifetime, were evaluated. The kinetic parameters of the enzyme reactions and the analytical characteristics of the corresponding calibration plots were calculated for eight phenolic compounds. Limits of detection of 0.07 mM, 0.05 mM and 0.09 mM were obtained for caffeic acid, catechol and 3,4-dihydroxyphenylacetic acid (DOPAC), respectively. The practical usefulness of the developed biosensor was evaluated by estimating the "pool" of phenolic compounds in olive oil mill wastewaters (OMW).

Gold nanocrystal-modified glassy carbon electrodes (nAu-GCE) were prepared and used for the determination of histamine by flow injection and high performance liquid chromatography using pulsed amperometric detection (PAD) as the detection mode. Experimental variables involved in the electrodeposition process of gold from a HAuCl 4 solution were optimized. A catalytic enhancement of the histamine voltammetric response was observed at the nAu-GCE when compared with that obtained at a conventional Au disk electrode, as a consequence of the microdispersion of gold nanocrystals on the GC substrate. The morphological and electrochemical characteristics of the nAu-GCE were evaluated by SEM and cyclic voltammetry. PAD using a very simple potential waveform consisting of an anodic potential (þ 700 mV for 500 ms) and a cathodic potential (À 300 mV for 30 ms), was used to avoid the electrode surface fouling when histamine was detected under flowing conditions. Flow injection amperometric responses showed much higher I p values and signal-to-noise ratios at the nAu-GCE than at a conventional gold disk electrode. A limit of detection of 6 Â 10 À7 mol L À1 histamine was obtained. HPLC-PAD at the nAu-GCE was used for the determination of histamine in the presence of other biogenic amines and indole. Histamine was determined in sardine samples spiked at a 50 mg g À1 concentration level, with good results. Furthermore, the chromatographic PAD method was also used for monitoring the formation of histamine during the decomposition process of sardine samples.

A novel amperometric immunosensor for the determination of progesterone, based on direct attachment of antiprogesterone on a gold nanoparticles-modified graphite-Teflon composite electrode is reported. Competitive assay involving progesterone labeled with alkaline phosphatase (AP), and 1-naphtyl phosphate as the enzyme substrate, was employed. The product of the enzyme reaction, 1-naphtol was detected amperometrically at þ 0.3 V vs. Ag/AgCl. Variables involved in the immunosensor preparation such as the colloidal gold loading and the amount of anti-Prog immobilized, as well as in the immunosensing event (pH, Prog/Prog-AP ratio and volume) were optimized. A calibration plot was obtained for progesterone with a linear range between 0 and 30 ng mL À1 , and a slope value, À 1.2 nA ng À1 mL, which is remarkably higher than that reported for other progesterone biosensors. A good repeatability of the amperometric measurements, RSD ¼ 3.5% (n ¼ 10), and an acceptable fabrication reproducicility, RSD ¼ 8.2% (n ¼ 3), were obtained. The developed anti-Prog-Au coll -graphite-Teflon immunosensor was applied to the progesterone determination in milk. Recovery at the 3.5 ng mL À1 progesterone concentration level was of 101 AE 6% (n ¼ 7).

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