RESUMEN
We report a novel in-situ electrochemical synthesis approach for the formation of functionalized graphene-graphene oxide (fG-GO) nanocomposite on screen-printed electrodes (SPE). Electrochemically controlled nanocomposite film formation was studied by transmission electron microscopy (TEM) and Raman spectroscopy. Further insight into the nanocomposite has been accomplished by the Fourier transformed infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) spectroscopy. Configured as a highly responsive screen-printed immunosensor, the fG-GO nanocomposite on SPE exhibits electrical and chemical synergies of the nano-hybrid functional construct by combining good electronic properties of functionalized graphene (fG) and the facile chemical functionality of graphene oxide (GO) for compatible bio-interface development using specific anti-diuron antibody. The enhanced electrical properties of nanocomposite biofilm demonstrated a significant increase in electrochemical signal response in a competitive inhibition immunoassay format for diuron detection, promising its potential applicability for ultra-sensitive detection of range of target analytes.
Asunto(s)
Diurona/análisis , Técnicas Electroquímicas/instrumentación , Grafito/química , Herbicidas/análisis , Inmunoensayo/instrumentación , Nanocompuestos/química , Óxidos/química , Anticuerpos Monoclonales/inmunología , Técnicas Biosensibles/instrumentación , Diurona/inmunología , Herbicidas/inmunología , Nanocompuestos/ultraestructura , Sensibilidad y EspecificidadRESUMEN
A disposable electrochemical immunosensor has been developed for the determination of phenyl urea herbicide-diuron using a low cost laser ablated gold electrodes (LC-LAGE) fabricated on polystyrene substrate. The electrodes were electrochemically deposited with prussian blue-gold nanoparticle (PB-GNP) film, and a competitive inhibition immunoassay was performed on LC-LAGE by using a specific hapten-protein conjugate. The binding of available diuron specific antibody on conjugate coated electrode was detected using alkaline phosphatase rabbit anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of electrochemically active product, 1-naphthol, which was monitored using square wave voltammetry technique. The assay exhibited an excellent sensitivity and specificity showing the dynamic response range between 1 ppt and 10 ppm for diuron with detection limit around 1 ppt. This study provides insight into development of a rapid and high-throughput screening of pesticides in environmental samples at a very low cost.
Asunto(s)
Técnicas Biosensibles/métodos , Diurona/análisis , Herbicidas/análisis , Animales , Anticuerpos Inmovilizados , Técnicas Biosensibles/estadística & datos numéricos , Diurona/inmunología , Técnicas Electroquímicas , Monitoreo del Ambiente/métodos , Oro , Haptenos , Herbicidas/inmunología , Inmunoensayo/métodos , Inmunoensayo/estadística & datos numéricos , Nanopartículas del Metal , Conejos , Sensibilidad y EspecificidadRESUMEN
Two monoclonal anti-diuron antibodies were generated that bind to diuron with an extremely low equilibrium dissociation constant. The antibodies prevented and restored in vitro and in vivo the diuron-dependent inhibition of photosynthesis. In isolated thylakoids prepared from spinach leaves (Spinacia oleracea L.) the diuron-inhibited Hill reaction was reconstituted immediately after the addition of the monoclonal antibodies. The antibodies also restored the diuron-dependent inhibition of the photosynthetic oxygen evolution of the cell wall-deficient mutant cw15 of the green alga Chlamydomonas reinhardtii Dangeard.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Chlamydomonas/efectos de los fármacos , Diurona/antagonistas & inhibidores , Fotosíntesis/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Chlamydomonas/genética , Chlamydomonas/metabolismo , Diurona/inmunología , Diurona/farmacología , Herbicidas/antagonistas & inhibidores , Herbicidas/inmunología , Herbicidas/farmacología , Mutación , Fotosíntesis/fisiologíaRESUMEN
A potato virus X (PVX) vector was used to express a single chain antibody fragment (scFv) against the herbicide diuron, as a fusion to the viral coat protein. The modified virus accumulated in inoculated Nicotiana clevelandii plants and assembled to give virus particles carrying the antibody fragment. Electron microscopy was used to show that virus particles from infected leaf sap were specifically trapped on grids coated with a diuron-BSA conjugate. The results demonstrate that the PVX vector can be used as a presentation system for functional scFv.
Asunto(s)
Fragmentos de Inmunoglobulinas/genética , Potexvirus/genética , Clonación Molecular , Diurona/inmunología , Herbicidas/inmunología , Microscopía Electrónica , Plantas Tóxicas , Proteínas Recombinantes/genética , Nicotiana/virologíaRESUMEN
The cDNAs from a hybridoma (mAb 481.1) specific for diuron, a widely used phenylurea herbicide, were cloned into the phage display vector pComb8. Antigen-binding clones were selected by panning on diuron-hapten-BSA conjugates. The nucleotide and deduced amino-acid sequences encoding the Fab regions of the light (kappa) and heavy (gamma) chains were determined. The light chain was from mouse kappa chain subgroup III and the heavy chain was a member of the mouse H chain subgroup III(d).