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Abstract: FR-PO973

Detection of Urinary MicroRNA Biomarkers Using Diazo Sulfonamide-Modified Screen Printed Carbon Electrodes

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic

Authors

  • Bowen, Timothy, Cardiff University, Cardiff, United Kingdom
  • Smith, Daniel A., Cardiff University, Cardiff, United Kingdom
  • Simpson, Kate A., Cardiff University, Cardiff, United Kingdom
  • Newbury, Lucy Jade, Cardiff University, Cardiff, United Kingdom
  • Fraser, Donald, Cardiff University, Cardiff, United Kingdom
  • Redman, James Edward, Cardiff University, Cardiff, United Kingdom
Background

We have established RT-qPCR-based protocols for urinary microRNA (miRNA) quantification to determine expression profiles for diabetic kidney disease (DKD) and predict graft function following renal transplantation. In parallel we are developing electrochemical quantification methods, and have demonstrated that urinary miRNA detection by glassy carbon electrode-based biosensors is more sensitive than RT-qPCR. Here we describe development of disposable screen printed carbon electrode (SPCE)-based miRNA sensors that can discriminate between urine samples from DKD patients and controls with similar sensitivity.

Methods

Screen-printed SPCEs were modified by deposition of a diazotised naphthalene sulfonic acid derivative, 4-amino-3-hydroxy-1-napthalene sulfonic acid (ANSA). The ANSA was then transformed into a sulfonyl chloride, before a 5’-amine-tagged DNA oligonucleotide with complementary sequence to the target miRNA was attached via a sulfonamide linkage to complete the biosensor. Analysis of biosensor output was carried out via reductive and oxidative chronocoulometry, obtained by measuring negative and positive potential sweeps using a ferri/ferrocyanide electrolyte, respectively. Selected miRNA readings were compared before and after hybridization in exogenous control miRNA dilution series, and between urine samples from DKD patients and controls.

Results

We demonstrated a linear response for our SPCE sensors across physiologically relevant concentrations of exogenous miR-21, replicating the femtomolar limit of detection from our previous glassy carbon electrode-biosensor studies. Subsequently, our SPCE sensors successfully detected a DKD-associated decrease in miR-192 that we reported previously following RT-qPCR analysis. Using histochemistry and atomic force microscopy analyses throughout the biosensor fabrication process, we observed sequential deposition of sensor components at the electrode surface which demonstrated the desired biosensor composition.

Conclusion

Our disposable electrode-based biosensors have strong potential for use in rapid, highly sensitive miRNA biomarker quantification in urine and other body fluids. In parallel studies we have identified urinary miRNA expression profiles associated with renal pathologies, and are now adapting our technology for clinical testing purposes.

Funding

  • Government Support - Non-U.S.