Metodi di analisi basati sull’inibizione delle colinestrerasi per applicazioni nel campo ambientale e alimentare

The increasing interest in analytical systems based on enzyme inhibition, as demonstrated by several recent reviews, shows the importance of this type of biosensings and biosensors in the field of environmental and food analysis. The advantages associated with these sensing devices, particularly the easiness in using them, combined with the low costs, make these biosensors suitable for "in situ" monitoring also by non skilled personnel. During the three years of my PhD thesis, a complete study of ChEs based biosensors for pesticides, nerve agents and aflatoxin B detection has been carried out. The interference of heavy metals occurring in the detection of pesticides based on free Acetylcholinesterase (AChE) inhibition, has been investigated. The study proposed a new approach to overcome the interferences by the interaction between thiocholine and heavy metals using two different phases with a good sensitivity (an inhibition of 50% has been observed with 9⋅10-7 M of paraoxon). An alternative method to measure pesticides avoiding interferences is the use of the biosensors using the “medium exchange method”. The screen-printed electrodes (SPEs) modified with Prussian Blue were used as substrate for the successive immobilisation of two different ChE enzymes (acetylcholinesterase, AChE and butyrylcholinesterase, BChE) to obtain the biosensors for pesticides detection. The method of analysis is achieved in two steps (“medium exchange” method) avoiding the effect of any interfering compound eventually present in real samples, such as SDS (sodium dodecyl sulfate) for example. Different pesticides were tested: AChE based biosensors have demonstrated a higher sensitivity towards aldicarb (50% inhibition with 50 ppb) and carbaryl (50% inhibition with 85 ppb) while BChE biosensors have demonstrated a higher affinity towards paraoxon (50% inhibition with 4 ppb) and chlorpyrifos-methyl oxon (50% inhibition with 1 ppb) using 30 minutes as incubation time. Real water samples were then analysed with this system demonstrating the suitability of the method. The biosensor previous reported was also optimised in order to have a faster measurement and more stable biosensor for nerve agents detection. The Sarin and VX solution were tested. We have challenged our biosensor also against the Sarin gas obtaining a degree of inhibition higher than 20% with Sarin gas at 0,05 mg/m3 in 30 sec as incubation time. The results obtained demonstrated that using the developed system it is possible to detect low concentration of Sarin in a few minutes according to the level Immediately Dangerous to Life or Health (IDLH). Furthermore, the biosensor showed a good storage stability at room temperature in dry conditions. In the last chapter the biosensing for aflatoxin B (AFB) detection has been proposed for the first time. A preliminary investigation of the kinetics and inhibitory mechanism of AFB1 on acetylcholinesterase indicated a reversibility type and mixed mechanism. The reversibility of the inhibition permits a rapid analysis of AFB1 (3 minutes) with a detection limit of 10 ppb. To evaluate the selectivity of this method, the cross reactivity was investigated showing that the assay detected AFB1 and AFB2 with similar sensitivity. The suitability of the assay for AFB1 quantification in barley was also evaluated with good recovery.