RESUMO
Impedance spectroscopy approaches combined with the immunosensor technology have been used for the determination of trace amounts of ciprofloxacin antibiotic belonging to the fluoroquinolone family. The sensor electrode was based on the immobilization of anti-ciprofloxacin antibodies by chemical binding onto a poly(pyrrole-NHS) film electrogenerated on a solid gold substrate. The electrode surface was modified by electropolymerization of pyrrole-NHS, antibody grafting and ciprofloxacin immunoreaction. The sensitive steps of surface modification, cyclic voltammetry (CV) and atomic force microscopy (AFM) imaging have been used for electrode surface characterization. The immunoreaction of ciprofloxacin on the grafted anti-ciprofloxacin antibody directly triggers a signal via impedance spectroscopy measurements which allows the detection of extremely low concentration of 10 pg/ml ciprofloxacin.
Assuntos
Antibacterianos/análise , Técnicas Biossensoriais/métodos , Ciprofloxacina/análise , Antibacterianos/química , Técnicas Biossensoriais/instrumentação , Ciprofloxacina/química , Eletroquímica/instrumentação , Eletroquímica/métodos , Imunoensaio/instrumentação , Imunoensaio/métodos , Microscopia de Força Atômica , Estrutura Molecular , Polímeros/química , Pirróis/química , Reprodutibilidade dos TestesRESUMO
Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.
Assuntos
Técnicas Biossensoriais/métodos , Materiais Revestidos Biocompatíveis/análise , Materiais Revestidos Biocompatíveis/química , Eletroquímica/métodos , Rodopsina/análise , Rodopsina/química , Análise Espectral/métodos , Adsorção , Técnicas Biossensoriais/instrumentação , Cristalização/métodos , Impedância Elétrica , Membranas Artificiais , Ligação Proteica , Rodopsina/ultraestrutura , Propriedades de SuperfícieRESUMO
Langmuir-Blodgett (LB) technique appears to be quite suitable for generating biospecific surfaces and it has potential application for fabricating biosensors. In this work, mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile have been transferred onto hydrophobic silver surface previously modified by 1-octadecanethiol (ODT) SAMs. In order to obtain stable LB films, the influences of different parameters - type of amphiphile, surface pressure and pH - on the properties of mixed IgG/amphiphile monolayer, were investigated. Electrochemical properties of the engineered immunosensor have been measured by impedimetric spectroscopy. The immunosensor obtained exhibits a high sensitivity and a good specificity in a linear dynamic range from 200 to 1000 ng ml(-1).