Application of a composite consisting of polypyrrole (PPy) and gold nanoparticles (Au Nps) to the construction of sensing layer in DNA biosensors led to the enhancement of the sensitivity due to the increase in the number of probe DNA strands in the layer by at least two orders of magnitude compared to the regular thiol intermediate layers. The probe DNA strands attached to the Au nanoparticles in the composite layer were fully available for the hybridization process. The examination of the composites containing a constant amount of Au nanoparticles and varying in PPy-layer thickness was done using electrochemical impedance spectroscopy, voltammetry and scanning electron microscopy. The optimal PPy layer thickness was determined to be in the range 10-20 nm. Such thickness of the polymer layer led to the elimination of aggregation of metal nanoparticles electrodeposited at the PPy surface. The sensor response (Delta R-ct) increased linearly with logarithm of concentration of target DNA in the range 2.10(-13)-2.10(-6) M. The obtained detection limit of target DNA in the sample was circa 8.4.10(-13) M. This limit is equivalent to the detection of circa 3.5.10(6) copies of DNA in a 7-mu l droplet or circa 5.10(11) DNA copies in one-liter sample. (C) 2014 Elsevier Ltd. All rights reserved.