RESUMEN
Initially developed for the construction of DNA chips, the polypyrrole approach has been extended to other biochemical compounds (mainly proteins and oligosaccharides). This method allows one to copolymerize a pyrrole monomer with a biomolecule bearing a pyrrole group; this reaction is based on an electrochemical process allowing a very fast coupling of the biomolecule (probe) to a gold layer used as a working electrode. Fluorescence-based detection processes are classically used for evidence biorecognition on biochips; in order to avoid the labeling of the targets, we propose an alternative method--surface plasmon resonance imaging (SPRi). Surface plasmon resonance (SPR) is a typical label-free method for real-time detection of the binding of biological molecules onto functionalized surfaces. This surface-sensitive optical method is based upon evanescent wave sensing on a thin metal layer. The SPR approach described herein is performed in an imaging geometry that allows simultaneous monitoring of biorecognition reactions occurring on an array of immobilized probes (chip). In a SPRi experiment, local changes in reflectivity are recorded with a charge-coupled device (CCD) camera and are exploited to monitor up to 100 different biological reactions occurring on the molecules linked to the polypyrrole matrix. This method will be applied to protein recognition.
Asunto(s)
Polímeros/química , Análisis por Matrices de Proteínas/métodos , Pirroles/química , Resonancia por Plasmón de Superficie/métodos , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Análisis por Matrices de Proteínas/instrumentación , Estreptavidina/químicaRESUMEN
Protein microarray is a promising technology that should combine rapidity and easy use with high throughput and versatility. This article describes a method in which an electrocopolymerization process is employed to graft biological molecules on to a chip so that surface plasmon resonance imaging may be used to detect molecular interactions. Copolymerization of pyrrole-modified protein and pyrrole is an efficient grafting process which immobilizes molecules at defined positions on a gold surface. Surface plasmon resonance imaging is an optical technique that allows real-time simultaneous detection of molecular interactions on a large number of spots without labeling. This method was successfully used to analyze antibody-antigen interactions. This illustrates its high specificity and good sensitivity and demonstrates its suitability for biological studies.