Highly Selective Detection of Single-Nucleotide Polymorphisms Using a Quartz Crystal Microbalance Biosensor Based on the Toehold-Mediated Strand Displacement Reaction

Abstract

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.

Toehold-mediated DNA displacement-based surface-enhanced Raman scattering DNA sensor utilizing an Au–Ag bimetallic nanodendrite substrate

“…The intensity decrease continued up to 60 min and no meaningful intensity change occurred afterward. This observation is analogous with those reported in previous publications [12,19] in which a double-strand was also pre-immobilized on a substrate surface before measurements; while, the displacement was completed approximately in 10 min when a double-strand was in a solution without immobilization on the surface [7,11,28]. The Raman spectrum acquired at 60 min of the displacement from the 200 pM sample (black) is also shown in Fig.…”

“…Larkey et al [11] demonstrated a miRNA fluorescence sensor utilizing fluorescence quenching that occurs when two different dyes tagged at both ends of a freed reporter DNA became close by forming a hairpin structure (limit of detection (LOD): 10 nM). A quartz crystal microbalance (QCM)-based sensing scheme was also studied [12]. A hairpin capture probe became an open-loop structure by hybridization with a target DNA and then a streptavidin coupled reporter strand was allowed to bind on the opened probe for mass amplification (LOD: 0.3 nM).…”

Electrochemical DNA biosensor based on gold nanorods for detecting hepatitis B virus

“…Numerous techniques, including quartz-crystal microbalance [4], electrochemical [5], optic [6], and surface plasmon resonance [7] methods, have been widely studied for DNA detection. Among them, electrochemical biosensors have become very popular because of their great advantages, including simplicity, rapidity, portability, low cost, and high sensitivity [8].…”

Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

“…Such detection limit is comparable to those reported methods coupled with complicated signal amplification means or even more sensitive than other common label-free approaches for microRNA detection (Table 1). In addition, the relative standard deviation (n ¼ 6) of the sensor for These comparisons indicate the high selectivity of the method, which is associated with the highly specific sequence-dependence of the TSDR [34].…”