A NOVEL BIOSENSOR SENSITIVE TO BETA CASEIN USING AFM AND IMPEDANCE SPECTROSCOPY

The aim of this work is to give a short survey on possibilities of application of biosensors, electrochemical sensors and measuring methods in dairy farming. AFM and Electrochemical Impedance Spectroscopy (EIS) have been used for the characterization of the immobilization of ?-casein proteins over an antibody functionalized Si3N4 substrate and to study the antibody–antigen interaction. After cleaning, the substrate electrodes were functionalized with 10mM/l concentration of 10- (Carbomethoxy)decyldimethylchloro-silane in 1:3 CCl4:Heptane solution overnight. Then, we immersed them into HCl for 12 hours to convert the methyl esther group into an acid. They were activated by immersing them for 15 minutes into a 0.1 M ethanol solution of a mixture of N-(3-Dimethylaminopropyl)- N’ -ethyl-carbodiimidehydro-chloride (EDC), 2,3,4,5,6-Pentafluorophenol 99 % C6F5OH (PFP), and N,N-Diidopropylethylamine 99 % (EDIPA). Later, the samples were cleaned with ethanol and dried again. Finally, a drop with the antibody solution was deposited over the samples and they were kept into the fridge for one hour. Then, they were thoroughly rinsed with PBS and dried under a N2 flow. The presence of immobilized antibodies on the surface was visualized by AFM. The impedance analysis was performed with the “VMP multichannel potentiostat” in the frequency range (0.1–100) kHz, using a modulation voltage of 28 mV. During measurements the potential was kept at zero volts. The technique based on EIS is capable of distinguishing between different concentrations of ?-casein (2x10-3 mg/mL to 1.78x10-3mg/mL) , responsible for milk degradation. ACKNOWLEDGEMENTS The authors wish to acknowledge the financial support from projects MINAHE 2 (TEC2005-07996-C02-01) and AECI A/2675/05 and A/7177/06. El CIBER de Bioingeniería, Biomateriales y Nanomedicina es una iniciativa del ISCIII.

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