Effect of the duplex length on the sensing performance of a displacement-based electrochemical nucleic acid sensor with an adjustable point mutation discrimination function

Abstract

One “signal on” electrochemical DNA sensing system was established based on the competitive displacement and hybridization-induced conformation change. This sensing system is composed of two probes, one linear capturing probe (CP) and one stem-loop signaling probe (SP) which are co-immobilized to the surface of Au electrode. Before adding target DNA, a 4–14 base-pair duplex formed between two probes forces the redox methylene blue (MB) far away from Au electrode surface, thereby producing low background current; after adding target DNA, the more stable duplex formed between CP and target DNA undoes the former duplex to release the SP. The released SP recovers the original stem-loop structure which drives MB close to the electrode surface, thereby producing high detection signal. The effect of the duplex length (DL) on sensing performance was systematically investigated. The research results show that the DL can pose a big effect on the signal enhancement (SE), the sensing response speed, the sensitivity and the specificity, further affecting the comprehensive performance of sensing system involved. Using the short DL, such as 4 or 6, the duplex is not rigid enough to support two strand probes well; using the long ones, such as 12 or 14, the stability of the duplex is so high that the displacement reaction is not easy to carry out. Using 10DL, the best comprehensive performance can be obtained. Under the optimized conditions, a short designated 17-base sequence could be quantified over the ranges from 0.001 to 1nM with a linear correlation of r2=0.9953 and a limit of detection (LOD) was confirmed to be ∼1pM for the proposed sensor. When used for the analysis of point mutation target, the sensor also features an excellent discrimination ability and its good adjustability.