This work emphasizes on the design criteria of aptamer-based biosensors for adenosine and the comparison of their performances by using both electrochemical and biochemical methods. The adenosine biosensors have been typically prepared by hybridizing a short bridging DNA (modified with a thiol for the attachment to gold electrode) to the aptamer strand that releases upon adenosine binding. We have carefully compared the two "isomeric" designs, one with the thiol-linker tethered to the 3'-hydroxyl and the other to the 5'-phosphate end of the bridging DNA. It has been demonstrated through electrochemical and gel electrophoresis experiments that the 3'-thiol bridged sensor shows much better performance than its 5'-counterpart on surface, whereas in solution phase the two sensor systems are equally sensitive. We believe that the "transient" secondary structure of the aptamer sequence restricts the freedom of the strand displacement upon analyte binding, leading to a sluggish release of the aptamer strand from the 5'-thiol-bridging DNA. (C) 2009 Elsevier B.V. All rights reserved.