This study presents a new strategy and level of mechanistic understanding for ultrasensitive detection of short, non-coding RNAs without target amplification or chemical modification using electrochemical biosensors. Electrochemical impedance spectroscopy (EIS) has been used for probe target interaction detection because of its high utility for sensitive and label-free measurements of the nucleic acid targets as a result of hybridisation. EIS measurements of different probe target combinations in a range of spatial orientations and sequence overlaps showed that bringing the target overhangs closer to the nanometer proximity of the electrode surface improved the EIS signal significantly. Systematic investigations using different lengths of overhangs towards the electrode surface revealed proportionally higher EIS signals with increasing lengths of the overhangs. Our observations could be explained using the Poisson-Boltzmann and Gouy-Chapman model and followed our experimental modelling. In conclusion, the optimized arrangements of our EIS biosensor system enabled us to detect microRNA-122, a known biomarker for liver injury, as well as three common isoforms to a 1 nM (equivalent to 80 fmole) detection limit. This will enable us to develop solutions for the detection of this important blood biomarker at point of care.
Keywords: Analytical methods; Biosensors; Electrochemical impedance spectroscopy; Probe-target complex; microRNA detection.
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