A new optical platform for biosensing based on fluorescence anisotropy

A novel fluorescence-based optical platform for the interrogation of an optical biochip was designed and developed. The optical biochip is made of polymethylmethacrylate (PMMA) formed by two pieces of PMMA appropriately shaped in order to obtain four microchannels that are 500 ¼m wide and 400 ¼m high. The lower part includes the micro-channels and the inlet and outlet for the fluidic, while the sensing biolayer is immobilized on the upper part. The optical signal is constituted by the fluorescence emitted by the biolayer, which is anisotropically coupled to the PMMA cover and suitably guided by the PMMA chip. The potentiality of the optical chip as a biosensor was investigated by means of a direct IgG/anti-IgG interaction carried out inside the flow channels. The mouse-IgG was covalently immobilized on the internal wall of the PMMA cover and the Cy5-labeled anti-mouse IgG was used for the specific interaction. Several chemical treatments of the PMMA surface were investigated (Poly L-lactic acid, Eudragit L100, NaOH) in order to obtain the most effective distribution of carboxylic groups useful for the covalent immobilisation of the mouse-IgG. The treatment with Eudragit L100 was found to be the most successful. Limit of detection and quantification of 0.05 ¼g/ml and 0.2 ¼g/ml respectively were obtained with the configuration described.