Biotin/avidin system for the generation of fully renewable DNA sensor based on biotinylated polypyrrole film

Abstract

A DNA sensor based on electropolymerized biotinylated polypyrrole film was prepared. Biotinylated DNA probes were immobilized on the polypyrrole-biotin film via an intercalated layer of avidin creating the sensing layer, polypyrrole-biotin/avidin/DNA probe. From this sensor, gravimetric measurements performed with a quartz crystal microbalance (QCM) showed that the amount of DNA probe immobilized is controlled by the quantity of biotin units included within the polypyrrole-biotin film. The maximum coverage of DNA probes was achieved with a copolymer electrosynthesized from a pyrrole-biotin and un-biotinylated pyrrole monomer ratio of 1/5. Furthermore, the sensing layer can be re-generated several times with a minimum loss of activity. After the hybridization between immobilized DNA probes and complementary DNA strands, two ways of regeneration are possible. The complementary oligonucleotide (ODNc) strands can be selectively removed: (i) by solubilization of the avidin layer, which can be lifted off to give the polypyrrole-biotin film on which a new DNA sensor can be built (strategy 1) or (ii) by denaturation of the DNA duplex, leaving the polypyrrole-biotin/avidin/ODN probes sensor ready for use in a subsequent recognition processor (strategy 2). For both strategies, the first regeneration step lead to a loss of activity of 15–20%. Subsequent regenerations can be achieved without further loss of activity.