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Doctoral Thesis
DOI
10.11606/T.88.2011.tde-21042011-155326
Document
Author
Full name
Juliana Coatrini Soares
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2011
Supervisor
Committee
Gonçalves, Débora (President)
Cavalheiro, Carla Cristina Schmitt
Moraes, Marli Leite de
Motheo, Artur de Jesus
Nogueira, Ana Rita de Araujo
Title in Portuguese
Biossensores eletroquímicos fabricados a partir da imobilização da urease em filmes de polipirrol
Keywords in Portuguese
Biossensor
Eletroquímica
Polipirrol
Urease
Abstract in Portuguese
A urease (Canavalia ensiformis DC.) foi fisicamente imobilizada em matrizes de polipirrol (PPI) com o objetivo de se detectar uréia em amostras padrão. A eletropolimerização do pirrol foi realizada por voltametria cíclica em uma faixa de potencial de -1,0 a 1,0 V vs. ECS em um meio aquoso contendo 0,2 mol/L de 'LI'CL'O IND.4' e 0,1 mol/L de pirrol. Este procedimento permitiu também a imobilização da enzima na matriz polimérica em suas formas, urease purificada (comercial) e como extrato bruto obtido a partir do feijão de porco (Jack Bean), após a adição de 300 'mü'g/mL de urease purificada ou 100 'mü'L de extrato bruto de feijão de porco. A urease purificada possui 34.375 U/g de sólido e o extrato bruto, 13.000 UA/mL, valores obtidos por titrimetria. A presença da enzima imobilizada nos filmes de PPI foi verificada por voltametria cíclica, FTIR, microscopia eletrônica de varredura (MEV), microscopia de força atômica (AFM) e por uma microbalança de cristal de quartzo (MCQE). A atividade da enzima após a imobilização nos filmes de PPI foi confirmada pela presença de íons amônio em solução, que são formados como produtos da reação de hidrólise da uréia catalisada pela enzima. Como o transdutor influencia a eficiência e a sensibilidade do biossensor, dois métodos de transdução foram estudados: cronoamperometria, aplicando-se um potencial de -0,28 V durante 120 s em tampão fosfato pH 7,0 e a cronopotenciometria, aplicando-se uma corrente de 1,0 mA durante 120 s em tampão fosfato pH 7,0 variando-se a concentração de uréia. O principal objetivo deste trabalho foi avaliar a eficiência do biossensores para a detecção de uréia por meio de transdutores potenciométricos e amperométricos e depois comparar as eficiências dos filmes de PPI/urease purificada e PPI/extrato bruto como biosensores.
Title in English
Electrochemical biosensors fabricated by the immobilization of urease in polypyrrole films
Keywords in English
Biosensor
Electrochemistry
Polypyrrole
Urease
Abstract in English
Urease (Canavalia ensiformis DC.) was physically immobilized on polypyrrole (PPy) films aiming at detecting urea in standard samples. The electropolymerization of pyrrole was performed by cyclic voltammetry at a potential range from -1.0 to 1.0 V vs SCE in an aqueous medium containing 'LI'CL'O IND.4' 0.2 mol/L and 0.1 mol/L pyrrole. This procedure also allowed us to immobilize the enzyme into the PPy matrix in forms, commercially purified and crude extract of urease obtained from Jack Bean (Canavalia ensiformis) after adding into the electropolymerization media 300 'mü'g/mL of purified urease or 100 'mü'L of crude extract. The urease solutions had units of active enzyme of 34.375 U/g (purified) and 13.000 UA/mL (crude extract), and the crude extract was obtained from Jack beans by titrimétric methods. The presence of urease immobilized into the PPy film was verified by cyclic voltammetry, FTIR, scanning electron microscopy (SEM), atomic force microscopy (AFM), and by electrochemical quartz crystal microbalance (EQCM) The activity of the enzyme after immobilizing into the PPy films was confirmed by the presence of ammonium ions in solution, since they are formed as catalytic products by urea hydrolysis reaction catalyzed enzyme. The transducer element influences the efficiency and sensitivity of the biosensor, and two transducer methods were studied: chronoamperometry, by applying a potential of -0.28 V during 120 s in buffer phosphate at pH 7.0 and chronopotentiometry, by applying a current of 1.0 mA during 120 s in buffer phosphate at pH 7.0 both after varying the urea concentration. Our main purpose was to evaluate the efficiency of the biosensors for detecting urea by means of potentiometric and amperometric transducers and then compare the efficiencies of PPY/purified urease e PPY/crude extract as biosensors.
 
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Publishing Date
2011-07-25
 
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