Rational engineering of synergically stabilized aptamer-cDNA duplex probes for strand displacement based electrochemical sensors

Electrochemical aptamer (EA) sensors employing electrode-bound aptamer-cDNA (cDNA: complementary DNA) duplex probes are simple and versatile sensing platforms for detection of many analytes. Target recognition in these sensors is a strand displacement process where target binding to aptamer displaces the cDNA strand. Despite many displacement-type EA sensors, a common drawback exists that sensor response rate is relatively slow (typically with target binding equilibrium time ranging from 0.5 to 3 h), which is mainly caused by a suboptimal conformation of the aptamer sequence in the conventional aptamer-cDNA duplex probe containing a relatively large number of aptamer-involved base pairs. Realizing that the key to optimizing sensor sensitivity and response rate of displacement-type EA sensors lies in engineering of a class of aptamer-cDNA probes with moderate duplex stability and a minimum amount of aptamer-involved base pairs, we explore here a unique means for rational design of this type of aptamer-cDNA duplex probes by using the concepts of synergic stabilization and base-pair regulation. We have integrated a short non-aptamer base-pair sequence and a short aptamer-involved base-pair sequence to engineer a class of synergically stabilized aptamer-cDNA duplex probe. Through further regulation of the number of aptamer-involved base pairs within the duplex we can tune not only the signal increase but also the response rate of these synergically stabilized duplex probes to achieve optimal sensor performance. The rationally engineered synergically stabilized aptamer-cDNA duplex probes are sensitive and exhibit significantly enhanced response rates (with target binding equilibrium times of 1 min for ATP and 2 min for cocaine). The concepts of synergic stabilization and base-pair regulation can be generalized to any aptamer-cDNA duplex probes and will help guide further development of displacement-type EA sensors for sensitive and quick detection of a wide variety of target analytes. A class of synergically stabilized aptamer-cDNA duplex probe (B) is engineered by integrating a short aptamer-involved (AI) base pair sequence and a short additional (SA) DNA sequence. The synergically stabilized probe possesses the advantage of less aptamer bases paired to the cDNA bases over the conventional probe (A, the duplex is made up entirely of AI base pairs), which provides significantly enhanced response rates of electrochemical aptamer sensors based on target-induced s