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A3-IL: CHOLINESTERASE BIOSENSORS
FOR THE DETECTION OF PESTICIDES AND AFLATOXIN B1
Danila Moscone, Fabiana Arduini
Dipartimento di Scienze e Tecnologie Chimiche,
Università di Roma Tor Vergata,
Via della ricerca scientifica, 00133 Roma, Italy; danila.moscone@uniroma2.it
The enzymatic methods for environmental and food contaminants detection can be successfully adopted as an alternative to classical methods (GC, HPLC) for a faster and simpler analysis. The use of cholinesterase enzymes (ChEs) for inhibition-based determination of organophosphorous and carbammic pesticides at ppb levels has shown great promise for environmental screening analysis. The interference of heavy metals occurring in the detection of pesticides based on free Acetylcholinesterase (AChE) inhibition has been investigated. The study of the reaction between thiocholine and heavy metals has been accomplished to demonstrate that some heavy metal cations, usually present in samples, could have a strong influence in the measurement of pesticides, limiting the reaction of thiocholine with DTNB (Ellman’s Reagent) and thus overestimating the pesticide concentration. This demonstrates that in the case of any method based on the use of AChE in solution with final measurement of thiocholine for pesticide detection, a separation between pesticide and heavy metals is necessary. Thus, a novel method for the detection of pesticides by inhibition of AChE has been developed using two immiscible phases in which the pesticide (organic phase) and the enzyme (aqueous phase) are solubilised. The enzyme (AChE) is then able to extract the pesticide at the aqueous/organic interface by an irreversible binding reaction. This results in an inhibition of the enzyme, which can then be related to pesticide concentration. An inhibition of 50% has been observed with 9×10-7 M of paraoxon and 1×10-7 M of carbofuran.
An alternative method to avoid the reversible inhibitor (heavy metals) during the detection of organophosphorous and carbammic pesticides (irreversible inhibitors) are the development a biosensor in which the enzyme is immobilised onto the transducer coupled with “medium exchange method” of analysis.
We proposed the screen-printed electrodes (SPEs) modified with Prussian Blue as platform for the successive immobilisation of two different ChE enzymes (AChE from electric eel and BChE from horse serum) to obtain the biosensors for organophosphorous and carbammic pesticides detection. The analysis is performed in two steps (“medium exchange” method) demonstrating that this procedure avoids the effect of any interfering compound eventually present in real samples, such as SDS (sodium dodecyl sulphate). To increase the sensitivity of ChE towards organothiophosphate, a simple and fast electrochemical method of oxidation of organothiophospate to organophosphate was carried out. The ChE and BChE biosensors demonstrated good reproducibility (RSD 9%) and low detection limit (chlorpyriphos methyl 0.5 ppb, diazinon 0.5 ppb, paraoxon 5 ppb, aldicarb 24 ppb) even when challenged with waste water in Acea Laboratory (Municipal company for drinking and waste water). For paraoxon detection another electrochemical biosensor was also developed, based on self-assembled monolayer of AChE by means of cysteamine and glutaraldeyde onto gold SPEs. The AChE activity was measured using ferricyanide as electrochemical mediator. The biosensor is characterised by a linear range up to 40 ppb. The biosensor was challenged with drinking water and river water obtaining a good accuracy.
The storage stability of biosensors developed was also investigated. The biosensor that showed the best storage stability was adopted for nerve agent detection at TNO, chemical warfare specialised center in Holland. The biosensor was challenged against the Sarin gas (0.5 and 0.1 mg/m3) at different incubation times. A 30 sec time incubation was necessary to detect 0,1 mg/m3 of Sarin gas, showing an inhibition of 30% with, however, 10 min of total analysis time. In order to decrease this time, a kinetic approach was used supported by a new version of Palm Sens® instrument software. In this way, the time of analysis was lower than 2 min, allowing to detect 0.1 mg/m3 of Sarin gas within IDLH limit (Immediately Dangerous to Life or Health= 0.2 mg/m3 for 5 min).
Recently it was demonstrated the ability of aflatoxin B1 (AFB1) to inhibit the AChE of mouse brain at ppm level [1]. We present a novel method for AFB1 determination based on AChE inhibition and the AChE residual activity is determined using the colorimetric method (Ellman’s method). ChEs from various sources were tested using AFB1 as reference aflatoxin. AChE from electric eel has shown the highest sensitivity to AFB1 and it was chosen for the rest of the work. To select and optimize the analytical procedures, the investigation on type of AChE inhibition by AFB1 was carried out. The AChE degree of inhibition by AFB1 was independent of the incubation time and the enzyme concentrations, showing the reversibility of the inhibition. This reversibility of the inhibition permits a rapid analysis of AFB1; in fact, only 3 minutes of analysis are required characterized by a linear range of 10-60 ppb. The suitability of the assay for AFB1 quantification at 100, 120 and 150 ppb in barley was also evaluated succesively, in order to reduce the detection limit in sample. An electrochemical bioassay was developed in which the acetylcholinesterase was used in solution and the residual activity was monitored using an amperometric choline oxidase biosensor. The choline oxidase was immobilized by cross-linking onto screen-printed electrodes modified with Prussian Blue, able to detect the H2O2 at low potential (-0.05 V versus screen printed internal silver pseudoreference electrode). For the development of the AFB1 assay, various parameters such as AChE and substrate concentration, methanol effect and pH study were evaluated and optimized. The linear working range was assessed to be 10-60 ppb. Finally, the matrix effect and recovery studies have been performed using commercially available olive oils. The suitability of the developed method for the directly analysis of AFB1 in olive oil samples was demonstrated, reaching a detection limit of 10 ppb of AFB1 in real samples.
References
- M.F. Cometa et al., Toxicology, 2005, 206,125-135.
Dr. Danila Moscone is a Full Professor of Analytical Chemistry at the Chemistry and Technology Department, University of Rome Tor Vergata. She is an expert in electrochemical sensor and biosensor development, their analytical evaluation and application to real samples. She has been involved in the field of biosensors for more than twenty-five years. In recent years, she is involved (within the framework of the projects funded by the European Community) in the development of new analytical methods concerning immunosensors and interference-free sensors and biosensors, based on screen-printed electrodes (SPEs), in the field of environmental, clinical and food analysis.
