Electrochemical biosensing of sequence-specific nucleic acid hybridization offers unprecedented opportunities for decentralized genetic testing which requires portable, cost-effective and low-power readout devices and has received considerable attention during the past decade. To date, most of the electrochemical nucleic acid biosensors that have been developed are based on heterogeneous assay in which interaction between surface-immobilized probe and target strand is translated into an informational electrochemical signal. The achievement of high sensitivity demands laborious optimization of probe immobilization including the surface chemistry and coverage to maximize hybridization efficiency and meanwhile minimize non-specific binding/adsorption events. More importantly, conf...[ Read more ]

Electrochemical biosensing of sequence-specific nucleic acid hybridization offers unprecedented opportunities for decentralized genetic testing which requires portable, cost-effective and low-power readout devices and has received considerable attention during the past decade. To date, most of the electrochemical nucleic acid biosensors that have been developed are based on heterogeneous assay in which interaction between surface-immobilized probe and target strand is translated into an informational electrochemical signal. The achievement of high sensitivity demands laborious optimization of probe immobilization including the surface chemistry and coverage to maximize hybridization efficiency and meanwhile minimize non-specific binding/adsorption events. More importantly, configurational freedom of the surface-immobilized probe is restrained, resulting in reduced hybridization kinetics and specificity compared to fluorescent nucleic acid assays which are normally homogenous. Recently, our group developed an immobilization-free detection approach which requires no probe immobilization step and solves the fore-mentioned issues. However, the sensitivity of this method is relatively low due to its diffusion-controlled nature.

In this thesis work, we developed a group of ultrasensitive immobilization-free electrochemical DNA biosensors based on enzyme-assisted and enzyme-free signal amplification strategy. These sensors utilize the catalytic activity of different DNA enzymes and kinetically-controlled DNA assembly reaction to achieve linearly or even exponentially amplified electrochemical signal. Competitive detection limits as well as ultrahigh selectivity to distinguish even single base mismatches have been demonstrated with these presented sensors, which could be a promising alternative to the conventional electrochemical biosensors for developing portable and cost-effective DNA sensing devices.