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A novel label-free electrochemical method for protein detection based on redox properties of silver was developed. As recognition elements, thrombin-binding aptamers were used. Screen printed electrodes modified with silver nanoparticles (AgNP) were employed as a sensing platform for aptasensor devices. The oxidation of silver upon polarization served as a basis for analytical response. Three different thrombin binding aptamers with various surface concentrations were studied. Linear range of aptasensor response corresponded to the 10 À9 M to 10 À7 M thrombin concentration range and the detection limit was 10 À9 M.

Using two DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nt in length) to produce aptamer heterodimeric constructs results into affinity enhancement. The apparent dissociation constant (Kdapp) measured at the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (Kdapp = 0,2-0,4 nМ) which were approximately 30-fold less than for the complexes with the primary aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer used could bind to the exosite 1. The measured value of Kdapp for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5,3 and 190 nM, respectively). Upon exposure to the UV radiation at 308 nm of the equimolar mixtures of thrombin with the photoaptamer construct, the equal yield of the crosslinked complexes was observed at concentrations which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.

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