Electrochemical aptamer sensors using electrode-bound, redox reporter-tagged aptamer probes represent a unique biosensor platform for the selective and reagentless analysis of target analytes. Signaling in this type of biosensor is based on an electrochemical collision mechanism that target binding induces change in flexibility or conformation of the electrode-bound aptamer probe, which in turn alters the efficiency with which the probe-linked redox reporters collide with the electrode surface and exchange electrons. As such, sensor signal gain can be enhanced through introducing a displacement recognition system in which an aptamer-DNA duplex instead of the parent aptamer is used to achieve low background current and large target-induced change in flexibility or conformation of the redox reporter-tagged probe. Previously reported aptamer-DNA duplex probes, however, were commonly engineered as long, fully-matched duplex structures. They only functioned at elevated temperature and needed a long response time due to their high stability. In response, we report here a re-engineered aptamer-DNA duplex that can respond to target at room temperature with short response time. This re-engineered duplex probe is rationally designed based on a new conception of mismatch tuning of the aptamer-DNA duplex. That is, various numbers of mismatched bases are incorporated into aptamer-DNA duplex to lower its bonding strength and so as to facilitate aptamer-target interaction during the displacement recognition process. This class of mismatched duplex probe combines the advantages of low background current for high signal gain and being able to function at room temperature with short response time. The "mismatch tuning" strategy is easy to generalize and could be helpful for the further development of electrochemical aptamer sensors for sensitive and rapid quantification of many target analytes. (C) 2016 Elsevier Ltd. All rights reserved.