Detection acetylcholinesterase

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

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. 

Of the known cyanobacterial toxins only anatoxin-fl(s) is detected and is, therefore, able to be screened for by acetylcholinesterase inhibition. 

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). 

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... 

V.B. Kandimalla and H.X. Ju, Binding of acetylcholinesterase to multiwall carbon nanotube-cross-linked chitosan composite for flow-injection amperometric detection of an organophosphorous insecticide. Chem. Eur. J. 12, 1074—1080 (2006). 

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). 

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

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). 

A.K. Singh, A.W. Flounders, J.V. Volponi, C.S. Ashley, K. Wally, and J.S. Schoeniger, Development of sensors for direct detection of organophosphates. Part I immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports. Biosens. Bioelectron. 14, 703-713 (1999). 

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). 

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. 

Increased levels of urinary catecholamines may also be associated with accumulation of acetylcholine that resulted from acetylcholinesterase inhibition by disulfoton. No human data were located to support this, but limited animal data provide some evidence. Disulfoton exposure caused a 173% and 313% increase in urinary noradrenaline and adrenaline levels in rats, respectively, within 72 hours (Brzezinski 1969). The major metabolite of catecholamine metabolism, HMMA, was also detected in the urine from rats given acute doses of disulfoton (Wysocka-Paruszewska 1971). 

The only known change in neurotransmitter metabolism so far detected is a deficiency of acetylcholine in the brain. This has been shown in post-mortem studies on the brains of patients with Alzheimer s disease. Some success in reducing the symptoms of the disease has been obtained with drugs that inhibit the activity of acetylcholinesterase leading to an increase in the acetylcholine concentration, but the improvement is minimal so that its use is controversial. 

J. van der Greef, R. Verpoorte High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products. J Chromatogr A 2000, 872, 61-73. 

Zhang C., and Malhotra, S.V., Increased paraoxon detection by acetylcholinesterase inactivation with ionic liquid additives, Talanta, 67, 560-565, 2005. 

Numerous postcolumn enzymatic reactors have been designed for LCEC. Enzymes can be used to produce an electroactive compound from the analyte of interest or, alternatively, to generate an electroactive species that is proportional to analyte concentration. An example of the latter is the detection of acetylcholine [46]. In this case, acetylcholinesterase is used to convert acetylcholine to choline. The resulting choline is reacted with choline oxidase to produce hydrogen peroxide. The amount of hydrogen peroxide produced is directly proportional to the initial concentration of acetylcholine. Detection limits are in the 100 femto-mole range. 

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,... 

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,... 

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

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