Gapped-duplex structure to label-free mismatch detection of pathogen DNA on solid substrate

a r t i c l e i n f o Keywords: Gapped-duplex DNA-mediated electron transfer Label-free Cryptosporidium parvum Electrochemical impedance spectroscopy A label-free platform for DNA mismatch detection, that has the potential to overcome the major drawbacks of conventional microarrays, is proposed. We have designed a gapped-duplex-based approach for achieving horizontally aligned probe-DNA on a solid substrate that offers low steric hindrance with excellent mismatch discrimination capability, enhanced association constant, and a much faster response time (less than 15 min). The electron transfer characteristics of DNA sequences immobilized by proposed method on electrode surface are compared with those from conventional immobilization method. The potential of the method in discriminating single nucleotide polymorphism variants among Cryptosporidium parvum genotypes (e.g., human and bovine) is experimentally appraised. Microarray technology has allowed researchers to confine DNA detection and synthesis reactions to such as DNA on solid substrate-based mi-croscale platforms [1–5]. Going by the convention, immobilized single-stranded DNA(ss-DNA) are called probes and the unknown DNA strands in the solution are termed target DNA (T-DNA). In a conventional DNA hybridization assay, the target DNA molecules in a solution collide with the probes that are tethered vertically to a solid substrate resulting in successful hybridization on the solid surface [6]. As the reaction proceeds, the initially formed duplex molecules forms a dense canopy that acts as a resistive layer which increases the activation energy of the further reaction. This effectively leads to a decrease in the diffusion rate of the target towards the surface [7,8]. The reaction rate of a DNA hybridization assay can be increased significantly only by making the probe-DNA more accessible. Hence, we intend to address the rate-limiting step that govern most hybridization assays. The amount of immobilized probe-DNA can be controlled by varying immobilization-based parameters such as concentration , pH, temperature, or time [6,8,11,12]. However, the most accepted immobilization step involves a 9-to 12-h long probe immobilization, which is the rate-limiting step [12]. Furthermore, the success of a DNA hybridization assay heavily depends on factors like density of probe-DNA [8], position of the mismatch [9] and length of the probe [10]. Considering these drawbacks of the existing techniques, we propose a novel gapped-duplex-based mismatch detection platform that is rapid, sensitive, label-free, and controllable. This method consists of a mixed monolayer of thiolated poly-T single-stranded 'DNA posts' and 6-mercapto-1-hexanol (MCH). Cryptosporidium parvum is a deadly waterborne protozoan parasite that causes debilitating to life-threatening …