Mediated enzyme screen-printed electrode probes for clinical, environmental and food analysis

Since the appearance of the first amperometric biosensors based on the coupling of oxidase enzymes and relying on the final detection of H2O2, it was clear that one of the major problems related to this kind of configuration was due to the high overpotential needed for H2O2 oxidation (ca. 0.7V vs. Ag/AgCl). At this potential in fact, many electroactive substances (i.e. ascorbic acid, uric acid etc.), usually present in real samples, could also be oxidised to give interfering signals. The same problem was also present when other amperometric biosensors, based on different class of enzymes, were assembled. For example, the amperometric detection of nicotinamide adenine dinucleotide (NADH) has been a matter of investigation for many years in the biosensor field [1,2]. The problem associated with amperometric detection of NADH is similar to that of H2O2, with a very high overpotential required [3,4] and with electrode fouling due to the presence of radical intermediates produced during NADH oxidation that then interfere with the measurement [5]. Another class of enzymes that has found wide application in the biosensor field in the last decades is that of the cholinesterases which have been mainly used for the detection of pesticides. For the amperometric detection of cholinesterase activity, both the substrates acetylcholine and acetylthiocholine have been extensively used [6–9], the latter being preferred because this avoids the use of another enzyme, choline oxidase, which is usually coupled with acetylcholinesterase. However, the amperometric measurement of thiocholine, produced by