Abstract

Altered serum and plasma microRNA (miRNA) expression profiles have been observed in numerous human diseases, with a number of studies describing circulating miRNA biomarkers for cancer diagnosis, prognosis and response to treatment, and recruitment to clinical trials for miRNA-based drug therapy already underway. Electrochemical detection of biomarkers in urine has several significant advantages over circulating biomarker analysis including safety, cost, speed and ease of conversion to the point of care environment. Consequently, much current research is underway to identify urinary miRNA biomarkers for a variety of pathologies including prostate and bladder malignancies, and renal disorders. We describe here a robust method capable of electrochemical detection of human urinary miRNAs at femtomolar concentrations using a complementary DNA-modified glassy carbon electrode. A miR-21-specific DNA hybridisation probe was immobilised onto a glassy carbon electrode modified by sulfonic acid deposition and subsequent chlorination. In our pilot system, the presence of synthetic mature miR-21 oligonucleotides increased resistance at the probe surface to electron transfer from the ferricyanide/ferrocyanide electrolyte. Response was linear for 10nM–10 fM miR-21, with a limit of detection of 20 fM, and detection discriminated between miR-21, three point-mutated miR-21 sequences, and miR-16. We then demonstrated similar sensitivity and reproducibility of miR-21 detection in urine samples from 5 human control subjects. Our protocol provides a platform for future high-throughput screening of miRNA biomarkers in liquid biopsies.

Highlights

  • IntroductionMicroRNAs (miRNAs) are single-stranded RNA transcripts, typically of 19–23 nucleotides in length

  • MicroRNAs are single-stranded RNA transcripts, typically of 19–23 nucleotides in length

  • We investigated miR-21 detection using our biosensor in urine mimic aqueous solutions containing salt, urea and protein

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Summary

Introduction

MicroRNAs (miRNAs) are single-stranded RNA transcripts, typically of 19–23 nucleotides in length. Posttranscriptional regulators of gene expression, miRNAs are currently estimated to target over 60% of the protein coding messenger (mRNAs) encoded by the human genome. Aberrant circulating miRNA expression profiles are associated with cardiovascular disease and numerous types of malignancy, leading to the identification of novel miRNA biomarkers and miRNA-based therapeutic approaches, with recruitment to

Current address
Reagents and chemicals
Instruments
Electrode preparation and modification
Electrochemistry
Sodium chloride matrix effects
Protein matrix effects
Urine samples
Initial sensitivity testing of the sensor
Specificity of the sensor
Conclusions
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