Enzyme Immobilization Notes

1. Pesticide solutions prepared by dilution with distilled water are very unstable. Working solutions can be also prepared by diluting the stock ones in PBS. These working solutions must be used and discharged at the end of the day.

2. Phosphate buffer may be prepared by using Sorensen's tables, but it is important to add the KCl (concentration: 0.05-1.0 M) in order to facilitate the electroconductivity of the solution between reference and working electrode.

3. AChE stock solutions are more stable in 0.9% NaCl. This solution must be kept at -4°C during storage. Use buffer solution only to dilute it before performing electrochemical assays.

4. AChEs from other sources (e.g., bovine erythrocytes, human erythrocytes) and butyrylcholinesterases (e.g., from horse and human serums) can also be used in this study. Also, genetically modified cholinesterases have proved to be very sensitive to several pesticide inhibitors (10,11).

5. The Ellman method (9,10) has to be applied in order to determine the specific activity (in activity unit [U]/mL) of the AChE working solution. This must be performed prior to enzyme immobilization in order to calculate the exact amount of the enzyme solution to be added to the PVA-SbQ in order to obtain an enzymatic charge of 1 U/electrode after immobilization

6. Chronoamperometry is performed by applying a potential of 100 mV vs silver/ silver chloride electrode, and the current is recorded with time. Current stabilization takes less than 5 min with this system. If the stabilization takes an extremely long time and results in poor current (CV > 10 %) then the obtained electrode should be changed. A new immobilization procedure, with careful homogeniza-tion of the enzymatic paste, will need to be performed.

7. The current intensity obtained after signal stabilization means the biosensor capacity. This is the practical baseline for the followed measurements.

8. This initial current value is adequate to measure and detect lower pesticide contents. Higher values may decrease the biosensor sensitivity because minor variations in current signal may be factored into the noise scale.

9. A curve of type number of determinations vs current intensity (nA) can be constructed and the coefficient of variation (%) must be determined.

10. From the complexity of the mechanisms and kinetics of AChE inhibition it is evident that the calibration curves will not have a simple linear correlation for some pesticides, but acceptable linear correlations can be obtained in a restricted region of the curve. This can be considered the practical working calibration range of the biosensor.

References

1. Chibata, I. (1995) Immobilized Enzyme. Kodansha Ltd., Halsted Press, Tokyo, Japan, 1978.

2. Mesing, R. Immobilized Enzymes for Industrial Reactors. Academic Press, New York, NY.

3. Taylor, F. R. (1991) Protein Immobilization: Fundamentals and Applications (Taylor, F. R., ed.) Marcel Dekker, New York, NY.

4. Wang, B. and Dong, S. (2000) Organic phase enzyme electrode for phenolic determination based on a functionalised sol-gel composite. J. Electroanal. Chem. 478, 45-50.

5. Wallace, G. Smyth, M. and Zhao, H. (1999) Conducting electroactive polymer-based biosensors. Trends Anal. Chem. 18, 245-251.

6. Wang, J., Nascimento, V. B., Kane, S. L., Rogers, K., Smyth, M. R. and Agnes, L. (1996) Screen-printed tyrosinase-containing electrodes for biosensing of enzyme inhibitors. Talanta 43, 1903-1907.

7. Mello, L. D. and Kubota, L.T. (2002) Analytical, nutritional and clinical methods. Review of the use of biosensors as analytical tools in the food and drink industries. Food Chem. 77(2), 237-256.

8. Ferretti, S., Paynter, S., Russel, D. A., Sapsford, K. E., and Richardson, D. J.

(2000) Self-assembled monolayers: a versatile tool for the formulation of the biosurfaces. Trends Anal. Chem. 19, 530-540.

9. Ellman, G. L., Courtney, K. P., Andres, V., and Fearstherstone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-90.

10. Nunes, G. S., Montesinos, T., Marques, P .B. O., Fournier, D., and Marty, J.-L.

(2001). Acetylcholine enzyme sensor for determining methamidophos insecticide. Evaluation of some genetically modified acetylcholinesterases from Drosophila melanogaster. Anal. Chim. Acta 434, 1-8.

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

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