Development of an “electrically wired” amperometric immunosensor for the determination of biotin based on a non-diffusional redox osmium polymer film containing an antibody to the enzyme label horseradish peroxidase

An amperometric immunosensor was developed based on a non-diffusional redox polymer used for transferring electrons between the electrode surface and the antigen (horseradish peroxidase; HRP) bound to the anti-HRP antibody on the sensing surface. The redox polymer [Os(bpy)2(PVP)10Cl]Cl (bpy = bipyridyl, PVP = poly-4-vinylpyridine) was co-immobilised with the antibody on the electrode surface by crosslinking the antibody with glutaraldehyde to form a combined sensing and electron transfer system. The resulting sensing film exhibits the classical features of a kinetically fast redox couple strongly bound to an electrode surface. The properties of the sensor depend on the content of osmium polymer, the extent of crosslinking and the loading of the antibody in the film. The detection limit for HRP of the sensor was found to be 0.01 μg ml−1, which is one order of magnitude lower than that for a traditional ELISA experiment. By employing an antibiotin antibody with an immuno-competitive approach, the sensor exhibited excellent performance for the detection of the hapten biotin.