Acetylcholinesterase biosensors

Biosensors may provide the basis for in-field analyses and real-time process analysis. However, biosensors are generally limited to the determination of a limited range of analytes in defined matrices. Enzyme-based biosensors, principally acetylcholinesterase (AChE) inhibition, have been successfully used in environmental analysis for residues of dichlorvos and paraoxon, " carbaryl " and carbofuran. " Immunochemically based biosensors may be the basis for the determination of pesticide residues in liquid samples, principally water and environmental samples, but also fruit juices. The sensors can be linked to transducers, for example based on a piezo-... 

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... 

C. Cremisini, A.D. Sario, J. Mela, R. Pilloton, and G. Paleshci, Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor. Anal. Chim. Acta 311, 273—280 (1995). 

F.N. Kok and V. Hasirci, Determination of binary pesticide mixtures by an acetylcholinesterase-choline oxidase biosensor. Biosens. Bioelectron. 19, 661-665 (2004). 

C. la Rosa, F. Pariente, L. Hernandez, and E. Lorenzo, Determination of organophosphorus and car-bamic pesticides with an acetylcholinesterase amperometric biosensor using 4-aminophenyl acetate as substrate. Anal. Chim. Acta 295, 273-282 (1993). 

D.P. Nikolelisa, M.G. Simantirakia, C.G. Siontoroua, and K. Tothb, Flow injection analysis of carbo-furan in foods using air stable lipid film based acetylcholinesterase biosensor. Anal. Chim. Acta 537, 169-177 (2005). 

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). 

R.A. Doong and H.C. Tsai, Immobilization and characterization of sol-gel-encapsulated acetylcholinesterase fiber-optic biosensor. Anal. Chim. Acta 434, 239-246 (2001). 

K. Anitha, S.V. Mohan, SJ. Reddy, Development of acetylcholinesterase silica sol-gel immobilized biosensor — an application towards oxydemeton methyl detection. Biosens. Bioelectron. 20, 848—856 (2004). 

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. 

H. Schulze, S. Vorlova, F. Vilatte, T.T. Bachmann and R.D. Schmid, Design of acetylcholinesterases for biosensor applications, Biosens. Bioelectron, 18 (2003) 201-209. 

S. Sotiropoulou, D. Fournier and N.A. Chaniotakis, Genetically engineered acetylcholinesterase-based biosensor for attomolar detection of dichlorvos, Biosens. Bioelectron, 20 (2005) 2347-2352. 

S. Sotiropoulou and N.A. Chaniotakis, Lowering the detection limit of the acetylcholinesterase biosensor using a nanoporous carbon matrix, Anal. Chim. Acta, 530 (2004) 199-204. 

Our research group is working on the development of electrochemical biosensors for the detection of microcystin and anatoxin-a(s), based on the inhibition of protein phosphatase and acetylcholinesterase, respectively. These enzyme biosensors represent useful bioanalytical tools, suitable to be used as screening techniques for the preliminary yes/no detection of the toxicity of a sample. Additionally, due to the versatility of the electrochemical approach, the strategy can be applied to the detection of other cyanobacterial toxins. 

Acetylcholinesterase-based biosensor for electrochemical anatoxin-a(s) detection... 

The main drawback of acetylcholinesterase-based biosensors is the lack of selectivity because, as we mentioned, this enzyme is inhibited not only by anatoxin-a(s) but also by insecticides such as organ-ophosphorates and carbamates. This problem can be overcome by the choice of specific mutant enzymes. The combined use of mutants highly sensitive to anatoxin-a(s) and resistant to most insecticides and vice versa allows us to unambiguously discriminate between the cyanobacterial toxin and insecticides. 

Acetylcholinesterase was immobilised by entrapment into a PVA-SbQ matrix (see experimental details in Refs. [88,95]). The need of polymer hydration slightly increases the response times, when compared to other immobilisation techniques. Nevertheless, the entrapment presents the advantage of providing biosensors with longer lifetimes due to the protective effect of the polymer matrix. 

The developed biosensor was applied to the analysis of cyanobacterial bloom samples from freshwater lakes of Spain, Greece, France, Scotland and Denmark. Two samples from Scotland and one from Denmark irreversibly inhibit the acetylcholinesterase. The estimated concentrations were between 1.5 and 30nmol/g of dry weight, values extremely high when compared to the intraperitoneal 50% lethal dose of anatoxin-a(s) in mice (121 nmol/kg). 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

E. Devic, D. Li, A. Dauta, P. Henriksen, G.A. Codd, J.-L. Marty and D. Fournier, Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases, Appl. Environ. Microbiol., 68 (2002) 4102 4106. 

F. Villate, H. Schulze, R.D. Schmid and T.T. Bachmann, A disposable acetylcholinesterase-based electrode biosensor to detect anatoxin-a(s) in water,Anal. Bioanal. Chem., 372 (2002) 322-326. 

B. Bucur, S. Andreescu and J.-L. Marty, Affinity methods to immobilize acetylcholinesterases for manufacturing biosensors, Anal. Lett., 37 (2004) 1571-1588. 

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... 

L. Doretti, D. Ferrara, S. Lora, F. Schiavon and F.M. Veronese, Acetylcholine biosensor involving entrapment of acetylcholinesterase and poly (ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane, Enzyme Microb. Technol., 27 (2000) 279-285. 

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. 

Antibodies to OP-tyrosine will be made. These antibodies will be used to diagnose OP exposure in a biosensor assay with saliva, sweat, or urine. New biomarkers of OP exposure will be identified using mass spectrometry and the new OP-tyrosine antibodies. The identification of new biomarkers for low-dose OP exposme is expected to lead to an understanding of how neurotoxicity is caused by OP doses that are too low to inhibit acetylcholinesterase. For example, it is possible that disruption of microtubule polymerization by OP-adduct formation may explain cognitive impairment from OP exposure. 

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