Electrochemical detection of disease-relevant RNA biomarkers is on-demand to develop simple, label-free, sensitive, and low-cost biosensors. However, sequence type and length of target may result in complex secondary structure formation, which may limit the discrimination of a normal from an abnormal sequence. Here, we attempted to employ 18-mer nucleic acid probes, dubbed as microprobes, to discriminate the neurodegenerative RNA repeat sequences prone to complex secondary structure formation due to having many folds larger length than the probe. The detection strategy is simple and label-free involving surface immobilization of microprobes on gold surface and measuring charge transfer resistance (RCT) of the sensing interface by electrochemical impedance spectroscopy (EIS) before and after capturing the target RNA. Specifically, we immobilized the PNA and DNA probes with six CTG repeats—complementary to CAG repeats associated with Huntington’s disease—on polished gold electrodes (2 mm diameter) via Au-S linkage and subsequent filling layer of 6-mercaptohexanol. Then, two different RNA lengths (CAG17 and CAG68) were tested from 100 aM to 100 nM concentration range. The interface was characterized by EIS using 1 mM redox probe Fe(CN)63-/4- in phosphate buffer at pH 7.4. Open circuit potential (OCP) was obtained for each measurement and EIS measurement was performed in frequency range from 100 kHz to 100 mHz. The EIS data was fitted into Randle’s equivalent circuit using Zview software to extract the RCT values. It was observed that PNA microprobe is better than DNA microprobe in discriminating the two targets at a wide range of concentrations. The RCT signals decreased followed by the binding of the RNA targets, which may be due to the presence of high cationic strength (20 mM Mg2+) in the target environment. Interestingly, despite the large length CAG68 show lower RCT than the RCT of CAG17. The effect of the length reversed when the interface was washed with EDTA after hybridization, which confirms the effect of the cationic strength on the resistance signaling. We studied different control experiments including positive control, structural control, sequence composition control and non-targeting control.

Nucleic acid microprobes to distinguish sequence- and length-dependent RNA structures by electrochemical impedance spectroscopy

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