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| Author(s): |
Josiane Caetano Dragunski
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Química de São Carlos (IQSC/BT) |
| Defense date: | 2007-03-02 |
| Examining board members: |
Sergio Antonio Spinola Machado;
Emanuel Carrilho;
Eder Tadeu Gomes Cavalheiro;
Valber de Albuquerque Pedrosa;
Nelson Ramos Stradiotto
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| Advisor: | Sergio Antonio Spinola Machado |
| Abstract | |
This work describes the development, characterization and utilization of a carbon paste biosensor based in the acetylcholinesterase enzyme for carbamates determinations in foodstuff, as well as the study of rate constants for enzyme/substrate reaction in solution. Stability tests were initially performed for both the substrate and the enzyme. In these tests, the signal for UV-vis adsorption for the substrate shows no inhibition during 11 days while for the enzyme it has been demonstrated that a considerable loss of activity occurs after three days from the solution preparation. In the electrode preparation, some experimental parameters were optimized, such as the amount of enzyme and the content of cobalt ftalocyanine (CoPC) in the electrode, as well as the employed percentage of glutaraldehide. The highest analytical signals were obtained for the addition of 2.40x10-3 g enzyme, 0.90x10-3 g CoPC (related to the massa of 0,017g of carbon paste) and a 1% glutaraldehide solution. The next step was to carry out an experiment based in the inhibition of enzyme activity by the pesticide. For this, the biosensor was immersed in 5.00x10-5 e 1.00x10-4 mol L-1 carbaryl solutions. It was observed that, by increasing the carbaryl concentration, the electrochemical signal of the sensor was inhibited proportionally. This was indicative that the sensor was adequate to be used in carbaryl monitoring and analytical determinations. The analytical determinations of carbamate pesticides were performed in 0.1 mol L-1 phosphate buffer, pH 7,4, with incubation time of 8, 12 and 15 minutes for carbaryl, metomil and aldicarb, respectively. The detection (LD) and quantification (LQ) limits obtained with the biosensor were 2.00x10-6 mol L-1 (0.40 mg L-1) and 6.70 x 10-6 mol L-1 (1.30 mg L-1) for carbaryl, 1.88x10-7mol L-1 (30.45 micro g L-1) and 6.26x10-7 mol L-1 (0.10 mg / L-1) for metomil and 1.10x10-6 mol L-1 (0.20 mg L-1) and 3.60x10-6 mol L-1 (0.70 mg L-1) for aldicarb. For the commercial formulation of metomil, Lannate, LD and LQ obtained were 2.13x10-7 mol L-1 (34.50 microg L-1) and 7.09x10-7 mol L-1(0.12 mg L-1), respectively. The HPLC measurements showed LD and LQ of 1.58x10-8 mol L-1 (3.18micro g L-1) and 5.27x10-8 mol L-1 (10.60 micro g L-1) for carbaryl and 9.02x10-10 mol L-1 (0.15 micro g L-1) and 3.00x10-9 mol L-1 (48.60 micro g L-1) for metomil. Recovering tests were also done with both analytical techniques for carbaryl and Lannate. The obtained recoveries using the biosensor were in the range of 76.83 to 106.67% for carbaryl and 78.00 to 96.50% for Lannate, while using the HPLC, the recoverings were 78.00 a 108.33% for carbaryl and 57.00 to 99.50% for Lannate. The recovering of aldicarb in tomatoes, with HPLC, were 62.40 %. The study of the enzymatic reaction in solution showed that the employed methodology allows to obtain the rate constant values for the rate determining step of the AchE/AchI reaction. It was observed that these rate constant values were strongly dependent in the pesticide concentrations for fenitrothion (organofosforous) and carbaryl (carbamate). At low concentration levels of the pesticide in the electrolyte, all the rate constants showed similar values but, when the pesticide concentration was raised, fenitrothion was found to exert a more powerful inhibition action for the enzyme activity than carbaryl, thus suggesting its higher toxic character. These results showed the development of a possible analytical methodology for quantification of these pesticides, by calculating the rate constant value and its dependence to the pesticide concentration in solution. (AU) | |