Electrochemical detection of urinary microRNAs via sulfonamide-bound antisense hybridisation

Highlights • A modified glassy carbon electrochemical sensor for microRNAs was developed.• The electrode allowed detection of femtomolar concentrations of miR-21.• The method was applied to detection of urinary miR-21.


Electronic Supplementary Information
Contents 1. Table S1:-DNA and RNA sequences 2. Figure S2:-Regeneration of the electrode 3. Figure S3:-Electrochemical impedance spectroscopy (EIS) 4. Figure S4-5:-Example overlays of EIS and coulometric data 5. Figure S6:-EIS data for mismatched sequences 6. Figure S7:-EIS data of miR-16 target compared to miR-21 7. Figure S8:-Storage of the probe at increasing temperature for 24 hours 8. Figure S9-S10:-Results of EIS and coulometric analysis with sodium chloride matrix 9. Figure S11:-Result of analysis with urea matrix 10. Figure S12:-Effect of protein and use of proteinase K 11. Figure S13:-Proteinase K treatment of urine 12. Figure S14:-Negative control of urine treatment procedure, using competitive PNA binding 13. Figure S15:-Positive control of urine treatment procedure, using non-complementary PNA 14. Figure S16:-Negative control of urine treatment procedure using RNase A treatment 15. Figure S17:-Resulting concentrations of urine via PCR 16. Figure S18-S23:-Electrochemical measurements of urine 17. Figure S24-S25:-Comparison of direct RT and extraction of known miRNA concentrations 18. Figure S26:-Optimisation of hybridisation time 5'NH2-C6-CGC CAA TAT TTA CGT GCT GCT A Figure S2 Change in coulometric response when the electrode is regenerated through a thermal denaturation procedure to remove the hybridised miR-21 followed by another hybridisation event with an identical concentration of 10 −10 M miR-21.

Procedure
The electrode was submerged in a 1 mL Eppendorf containing the TMD (50 mM Tris-HCl, 20 mM MgCl2 and 1 mM dithiothreitol pH 8.0) buffer at 95 °C for 20 minutes to remove the RNA from the duplex. Following this, the electrode was submerged into ice cold TMD buffer for 10 minutes. Finally, the electrode was sonicated in the buffer for 2 minutes to remove any residual adsorbed RNA. The equivalent circuit model used to interpret electrical impedance data using EIS spectrum analyser.

Note
This data shows that over 50% of the response is maintained after storage of the probe for 24 hours at elevated temperatures of 40 °C and 50 °C.

Note
Protein fouls the electrode surface, however in the presence of proteinase K this effect is greatly reduced.

Note
Treatment of urine with a PNA complementary to the target miRNA resulted in negligible signal. This implies that responses obtained from proteinase K treated and filtered urine are from the miRNA target rather than protein fouling.

Note
This experiment shows that the positive control, using a PNA sequence specific to a miRNA that is not the probe target, resulted in a significant signal change being obtained upon hybridisation. A further implication is that the responses obtained from the proteinase K treated and filtered urine are from the specific miRNA target.

Note
Addition of RNase degrades RNA in the urine resulting in negligible current change. Finally, a miR-16 'spike' is used to show that the signal can be restored, and also that the RNase used previously (and likely any originally present in the urine) is removed upon filtration through the 10 kDa spin filter. Figure S19 Coulometric responses of Figure S16 converted into concentration using the calibration plot shown in figure 1 of the manuscript.

Note
Higher bar is a lower concentration.   Figure S24 The difference in CT values obtained upon PCR amplification of a range of synthetic miR-21 solutions, one set where RT was directly performed, and one where an extraction step was performed first. Figure S25 The loss of concentration observed when an extraction step is performed prior to RT-qPCR amplification of a synthetic miR-21 solution compared to one where the RT is performed using the solution directly.

Note
The difference in RT-qPCR response, between extracted and directly analysed samples, decreased with decreasing initial miRNA concentration. With an initial concentration of 10 −8 M, a decrease of approx. 7000× upon extraction is observed; this is lowered to approx. 700 at 10 −10 M and further decreased at 10 −12 and 10 −14 M. The electrochemical analyses do not require extraction and so are not susceptible to these losses at higher miRNA concentrations.