Acetylcholinesterase pesticides detection

Absorbance- and reflectance-based measurements are widespread, as there are many enzymatic reaction products or intermediates that are colored or if not, can react with the appropriate indicator. Sensors using acetylcholinesterase for carbamate pesticides detection are an example of indirect optical fiber biosensors. This enzyme catalyses the hydrolysis of acetylcholine with concomitant decrease in pH41 ... 

Acetylcholinesterase (AChE) isolated from various organisms has been used in the majority of pesticide biosensors. In the early 1950s potentiometric detection was adopted for pesticide detection. In the middle of the 1980s it was used for the construction of the first integrated biosensors for detection of pesticides based on inhibition of AChE. Later rapid changes in science and technology introduced novel genetically... 

A. Vakurov, C.E. Simpson, C.L. Daly, T.D. Gibson, and P.A. Millner, Acetylcholinesterase-based biosensor electrodes for organophosphate pesticide detection I. Modification of carbon surface for immobilization of acetylcholinesterase. Biosens. Bioelectron. 20, 1118-1125 (2004). 

P.R.B. de O Marques, G.S. Nunes, T.C.R. dos Santos, S. Andreescu, and J.L. Marty, Comparative investigation between acetylcholinesterase obtained from commercial sources and genetically modified Drosophila melanogaster application in amperometric biosensors for methamidophos pesticide detection. Biosens. Bioelectron. 20, 825-832 (2004). 

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

Moreover, the use of PB will not only be limited to the detection of H202 and its use in conjunction with oxidase enzymes. A recent disclosure of the electrocatalytic activity of PB towards the oxidation of thiols will also be discussed and an application with acetylcholinesterase enzymes for pesticide detection reported. 

Scheme 1. Schematic representation of the system adopted for glucose and pesticide detection. In the upper part of the scheme is shown the reaction chain for the detection of acetylthiocholine giving a measure of acetylcholinesterase (AChE) activity which can be related to pesticide content. In the lower part of the scheme is shown the classic reaction utilised in the case of an oxidase enzyme (glucose oxidase—GOx) for the detection of glucose. In the first case, the final product is thiocholine and in the second, H202, both are measured at the Prussian blue modified electrode at an applied potential of 0.2 V vs. Ag/AgCl and —0.05 V vs. Ag/AgCl, respectively.

Vakurov A., Simpson C. E., Daly C. L., Gibson T. D., and Millner R. A., Acetylecholinesterase-based biosensor electrodes for organophosphate pesticide detection II. Immobilization and stabilization of acetylcholinesterase. Biosens. Bioelectron., 20(1), 2324-2329, 2005. 

In the following section an example of the use of disposable graphite sensor based for food analysis will be described. In particular, the use of these sensors to develop enzymatic biosensors for pesticide detection based on AChE (acetylcholinesterase) enzyme inhibition will be described. 

On a different setup, Du et al. [31] cast sol-gel silica/gold nanoparticle nanocomposite films on a glassy carbon electrode, which was then impregnated with an acetylcholinesterase (AChE) enzyme (Figure 46.10). The construct was used as a biosensor for organophosphorus pesticide detection. In this system, gold nanoparticles not only offered a biocompatible microenvironment to retain the activity of adsorbed enzyme molecules but also acted as a wire to enhance the direct electron transfer rate between the enzyme active centers and the electrode surface, which otherwise would be blocked by the thick protein shell of the enzyme chemical structure. 

Wu S, Lan XQ, Zhao W, Li YP, Zhang LH, Wang HN, Han M, Tao SY (2011) Controlled immobilization of acetylcholinesterase on improved hydrophobic gold nanoparticle/Prassian blue modified surface for ultra-trace organophosphate pesticide detection. Biosens Bioelectron 27(l) 82-87. doi 10.1016/j.bios.2011.06.020... 

Ivanov AN, Younusov RR, Evtugyn GA, Arduini F, Moscone D, Palleschi G (2011) Acetylcholinesterase biosensor based on single-walled carbon nanotubes-Co phtalocyanine for organophosphoms pesticides detection. Talanta 85(1) 216-221. doi 10.1016/j.talanta. 2011.03.045... 

Du D, Ding J, Tao Y, Chen X (2008) Application of chemisorption/desorption process of thiocholine for pesticide detection based on acetylcholinesterase biosensor. Sensor Actuat B 134 908-912... 

Su S, He Y, Zhang M, Yang K, Song S, Zhang X, Fan C, Lee S (2008) High-sensitivity pesticide detection via silicon nanowires-supported acetylcholinesterase-based electrochemical sensors. Appl Phys Lett 93 023113... 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

In this system, choline formed by acetylcholinesterase is oxidized by choline oxidase and the hydrogen peroxide produced is determined using the luminol/peroxidase CL reaction. The sensor has been used for the analysis of Paraoxon and Aldicarb pesticides, with detection limits of 0.75 pg/L and 4 pg/ L, respectively. Recoveries in the range of 81-108% in contaminated samples of soils and vegetables were obtained. 

The detection of organophosphate and other pesticides based on the inhibition of the enzyme acetylcholinesterase by these compounds has received considerable attention primarily due to high specificity and sensitivity [1,7-16]. Cholinesterases, such as acetylcholinesterase,... 

N. Mionetto, J.-L. Marty and I. Karube, Acetylcholinesterase in organic solvents for the detection of pesticides biosensor application, Biosens. Bioelectron, 9 (1994) 463-470. 

S. Andreescu, L. Barthelmebs and J.-L. Marty, Immobilisation of acetylcholinesterase on screen-printed electrodes comparative study between three immobilisation methods and applications to the detection of organophosphorus pesticides, Anal. Chim. Acta, 464 (2002) 171-180. 

A. Crew, J.P. Hart, R. Wedge and J.-L. Marty, A screen-printed, am-perometric, biosensor array for the detection of organophosphate pesticides based on inhibition of wild type, and mutant acetylcholinesterases, from Drosophila melanogaster, Anal. Lett., 37 (2004) 1601-1610. 

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... 

Liu, G., Lin, Y. (2006). Biosensor based on self-assembling acetylcholinesterase on carbon nanotubes for flow injection/ amperometric detection of organophosphate pesticides and nerve agents. Anal. Chem. 78 835 3. 

Acetylcholinesterase - Nanomaterials Hybrid Sensors for the Detection of Organophosphorous and Carbamate Pesticides... 

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