RESUMO
A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).
Assuntos
Técnicas Biossensoriais/instrumentação , Colina/análise , Colina/farmacocinética , Eletroquímica/instrumentação , Glândula Submandibular/metabolismo , Trifosfato de Adenosina/farmacologia , Oxirredutases do Álcool/química , Animais , Técnicas Biossensoriais/métodos , Colina/química , Materiais Revestidos Biocompatíveis/síntese química , Eletroquímica/métodos , Membranas Artificiais , Fosfatidilcolinas/metabolismo , Fosfolipases/análise , Fosfolipases/metabolismo , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Glândula Submandibular/efeitos dos fármacos , TemperaturaRESUMO
The enzyme horseradish peroxidase (HRP) has been entrapped in situ by electropolymerization of pyrrole onto a platinum electrode. The latter was previously coated by a polypyrrole layer for better adhesion of the biocatalyst film and in order to avoid the enzyme folding onto the Pt electrode. The biosensor allowed the determination of hydrogen peroxide in the concentration range comprised between 4.9 x 10(-7) and 6.3 x 10(-4) M. The biosensor retained more than 90% of its original activity after 35 days of use.
