Cholinesterase electrode

Durand P, Nicaud JM, Mallevialle J. 1984. Detection of organophosphorus pesticides with an immobilized cholinesterase electrode. J Anal Toxicol 8 112-117. 

Finally, it is possible to determine enzyme inhibitors instead of substrates. Inhibitors affect the rates of enzymatic reactions, and hence the intensity of the signal obtained. One example is the cholinesterase electrode which determines inhibittm, especially pesticides. 

The inhibition and regeneration of the cholinesterase electrode exhibit different variations with changing pH (Figure 4.27), as was the case for heavy metals. The inhilntor action of paraoxon is optimal for a pH of about 7.4 and so the serum environment favors organophosphate intoxication. Regeneration of the enzyme is favored for more basic pH values [134]. 

One example of analysis using enzymatic inhibition is the determination of pesticides by the inhibition of a cholinesterase electrode. A simplified flow-chart representing the automation of this biosensor is given in Figure 8.9. The calibration and measuring steps are followed by the test for inhibition and then regeneration. This is characteristic of measurement by inhibition. 

The cholinesterase electrode was constructed on this basis for the total determination of organophosphates and carbamates [32]. This electrode is also sensitive to other toxic compounds with very low detection limits (paraquat (3 ppm) trichlorophenol (20 ppm) methylazinphos (150 ppb) lindane (15 ppm) and mercury (4 ppm)) [135]. This type of biosensor uses enzymatic inhibition and can be incorporated in an automated system for the quality control of water, and, notably, waste water [291]. 

Biosensors ai e widely used to the detection of hazardous contaminants in foodstuffs, soil and fresh waters. Due to high sensitivity, simple design, low cost and real-time measurement mode biosensors ai e considered as an alternative to conventional analytical techniques, e.g. GC or HPLC. Although the sensitivity and selectivity of contaminant detection is mainly determined by a biological component, i.e. enzyme or antibodies, the biosensor performance can be efficiently controlled by the optimization of its assembly and working conditions. In this report, the prospects to the improvement of pesticide detection with cholinesterase sensors based on modified screen-printed electrodes are summarized. The following opportunities for the controlled improvement of analytical characteristics of anticholinesterase pesticides ai e discussed ... 

The aim of our investigation was the development of the amperometric enzyme immunosensor for the determination of Klebsiella pneumoniae bacterial antigen (Ag), causes the different inflammatory diseases. The biosensing pail of the sensors consisted of the enzyme (cholinesterase) and antibodies (Ab) immobilized on the working surface of the screen-printed electrode. Bovine seiaim albumin was used as a matrix component. 

Chemically modified electrodes, 39, 118 Chemometrics, 197 Chemoreceptor, 187 Chip, 194, 195 Chloramphenicol, 70 Chloride electrode, 159 Chlorpromazine, 34 Cholesterol, 182 Cholinesterase, 182 Chromium, 85, 86 Chronoahsorptometry, 42 Chronoamperometry, 21, 60, 130, 135, 132, 177... 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

E.V. Gogol, G.A. Evtugyn, J.L. Marty, H.C. Budnikov, and V.G. Winter, Amperometric biosensors based on Nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors. Talanta 53, 379-389 (2000). 

A. Ivanov, G. Evtugyn, L.V. Lukachova, E.E. Karyakina, HC. Budnikov, S.G. Kiseleva, A.V. Orlov, G.P. Karpacheva, and A.A. Karyakin, Cholinesterase potentiometric sensor based on graphite screen-printed electrode modified with processed polyaniline. IEEE Sensors J. 3, 333-340 (2003). 

A. Ivanov, G. Evtugyn, H. Budnikov, F. Ricci, D. Moscone, and G. Palleschi, Cholinesterase sensors based on screen-printed electrodes for detection of organophosphorus and carbamic pesticides. Anal. Bioanal. Chem. 377, 624-631 (2003). 

Frolich and colleagues (1998) analyzed ACh in human CSF by different methods, which included thermospray/mass spectroscopy, HPLC/mass spectroscopy, HPLC-EC Pt electrode and gas chromatogra-phy/mass spectroscopy (GC/MS). An SPE extraction was used for cleanup and concentration. Samples were run with and without the IMER to rule out any interference by physostigmine, a cholinesterase inhibitor, in the HPLC-EC assay. HPLC-EC and GC-MS gave data correlations with similar sensitivities, but the HPLC-EC values were 39% lower. Analysis using thermospray/mass spectroscopy and HPLC/ mass spectroscopy did not provide adequate sensitivity and the data obtained were inconsistent. 

G.A. Evtyugin, I.I. Stoikov, C.K. Budnikov and E.E. Stoikova, A cholinesterase sensor based on a graphite electrode modified with 1,3-disub-stituted calixarenes, J. Anal. Chem., 58 (2003) 1151-1156. 

P. Skladal and M. Mascini, Sensitive detection of pesticides using am-perometric sensors based on cobalt phthalocyanine-modified composite electrodes and immobilised cholinesterase, Biosens. Bioelectron, 7 (1992) 335-343. 

A.L. Hart, W.A. Collier and D. Janssen, The response of screenprinted enzyme electrodes containing cholinesterases to organophosphates in solution and from commercial formulations, Biosens. Bioelectron, 12 (1997) 645-654. 

B. Bucur, A.F. Danet and J.-L. Marty, Cholinesterase immobilisation on the surface of screen-printed electrodes based on Concanavalin A affinity, Anal. Chim. Acta, 530 (2005) 1-6. 

With regard to assaying the inhibitory activity of extracts electro-chemically, one of the problems of assays using sensors based on cholinesterase was that considerable time, e.g. 30-45 min [46,47], could be needed for the activity of the enzyme electrode to fall below control levels. The time increased as the level of inhibition decreased. Such lengthy assays make any number of serial assays impractical. In previous work [48,49], it had been noted that if sensors were exposed to solution containing inhibitors and then allowed to dry, they could be... 

Fig. 28.3. Reproduced from Fig. 3 of Ref. [45], with permission from Elsevier. Detection of organo-phosphates extracted from wool, (a) Extracts containing either chlorfenvinphos (C) or diazinon (D) numbers are X in 10-XM. (b) Extracts containing mixtures of the two total concentration of organo-phosphates was 10-5M. Electrodes were exposed to the extracts, dried overnight and the currents generated in the presence of butyrylthiocholine measured. White columns, electric eel acetylcholinesterase grey, bovine erythrocyte acetylcholinesterase and black, horse serum butyryl cholinesterase.

A.L. Hart and W.A. Collier, Stability and function of screen printed electrodes, based on cholinesterase, stabilised by a co-polymer/sugar alcohol mixture, Sens. Actuators B, 53 (1998) 111-115. 

The pesticides are detected electrochemically by measuring the degree of inhibition of cholinesterase on the screen-printed electrodes. The degree of inhibition can be thought of as the ratio of the response of electrodes (to substrate) exposed to pesticide (standard or wool extract) to that of electrodes not exposed to pesticide or exposed to a blank (extract from wool which has not been exposed to pesticide). An efficient and convenient way of doing this is to expose the SPCEs to standards and wool extracts prior to measurement of inhibition in the electrochemical cell. 

This method of pesticide detection is based on the inhibition of cholinesterase activity. This is a non-specific measurement and as such cannot determine which pesticide the enzyme electrode had been exposed to. This protocol has described a method for extraction and detection of diazinon, but other organophosphate pesticides can be substituted (e.g. chlorfenvinphos) bearing in mind that the enzyme has different inhibition constants for other pesticides and quantities may need to be altered. 

It may be possible to use an array of electrodes containing various enzymes in combination with multivariate statistical analyses (principal component analysis, discriminant analysis, partial least-squares regression) to determine which pesticide(s) the SPCE has been exposed to and possibly even how much, provided sufficient training sets of standards have been measured. The construction methods for such arrays would be the same as described in this protocol, with variations in the amounts of enzyme depending on the inhibition constants of other cholinesterases for the various pesticides of interest. 

Enzyme electrodes are essentially immobilized enzyme systems. Crochet and Montalvo (C15) developed a technique for cholinesterase based on coupling a small pH electrode to a thin polymer membrane. At the electrode surface, cholinesterase interacted with acetylcholine to produce acetic acid, which was detected by the pH electrode. Excellent sensitivity was achieved by the use of a very thin film of enzyme solution, with extremely low strength of buffer containing the enzyme and almost complete suppression of spontaneous hydrolysis of the substrate. 

B8. Baum, G., Ward, F. B., and Yaverbaum, S., Kinetic analysis of cholinesterases using a choline ester sensitive electrode. Clin. Chim. Acta 36, 405-408 (1972). 

C15. Crochet, K. L., and Montalvo, J. G., Enzyme electrode system for assay of serum cholinesterase. Atuil. Chim. Acta 66, 259-269 (1973). 

Organophosphorus compounds are irreversible inhibitors of acetylcholine esterase and butyrylcholine esterase (BuChe, EC 3.1.1.8) because the phosphate group is irreversibly bound by the enzyme. Therefore, organophosphorus pesticides can be detected by using the free enzyme. Since the activity of cholinesterases (ChE) in normal serum is rather large (800 UA), untreated serum pools may be employed for inhibitor determination. Gruss and Scheller (1987) have shown that the hydrolysis of butyrylthiocholine iodide can be directly indicated at a membrane-covered platinum electrode polarized to +470 mV. Twenty seconds after sample addition a steady value proportional to the enzyme activity was obtained in the differentiated current-time curve. Injection of an inhibitor decreased the rate of thiocholine formation, so that the residual activity could be evaluated after 30 s (Fig. 115). 

Needle electrodes were placed in intercostal muscles in 10 subjects who received 70-150 pg of sarin by the single-breath technique of vapor administration (MOD13). Although eight noted chest tightness, none had changes in whole blood cholinesterase, and there were no abnormalities in the electrical pattern from the muscles. It was concluded that muscular abnormalities did not contribute to the sensation of dyspnea. 

Electrocatalytic oxidation of thiocholine is achieved at lower potentials using a modified graphite electrode featuring a redox mediator. However, TCNQ exhibits the most suitable characteristics to be used in cholinesterase biosensors. 

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