Ultrasensitive electrochemical genosensor for direct detection of specific RNA sequences derived from avian influenza viruses present in biological samples

Acta Biochim Pol. 2019 Aug 22;66(3):299-304. doi: 10.18388/abp.2019_2774.

Abstract

An electrochemical genosensor based on an epoxy-phenanthroline-Fe(III)-NH2-ssDNA layer for the detection of RNA derived from Avian Influenza is presented. The biosensor preparation consists of: (I) modification of gold electrodes with aminoethanethiol, (II) modification of the self-assembled monolayer of aminoethanethiol with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline using "click" chemistry, (III) a first step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (IV) a second step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (V) immobilization of the single stranded amino-DNA probe via "click" chemistry between epoxy and amino groups. The interactions between the ssDNA probe and RNA targets were explored with Osteryoung Square Wave Voltammetry. The genosensor showed a remarkable detection limit of 3 copies/µL (5 aM) for RNA extracted from A/swan/Poland/305/06 (H5N1) containing a fully complementary sequence. A linear dynamic range for this sequence was observed from 3.0×103 to 3.0×105 [copies/µl]. RNA extracted from A/mallard/Poland/446/09 (H7N7), containing a non-complementary sequence, generated a much weaker response. Moreover, the developed genosensor allows to distinguish RNA present in biological samples having 2, 3 and 4 mismatches. This biosensing approach can become a potential alternative tool for detecting RNA samples in biomedical research and early clinical diagnosis of avian influenza viruses.

MeSH terms

  • Animals
  • Base Sequence*
  • Biosensing Techniques / instrumentation
  • Biosensing Techniques / methods*
  • Chick Embryo
  • DNA, Single-Stranded / chemistry
  • Electrochemical Techniques / instrumentation
  • Electrochemical Techniques / methods*
  • Electrodes
  • Epoxy Compounds / chemistry
  • Ferric Compounds
  • Gold
  • Influenza A virus / genetics*
  • Influenza A virus / isolation & purification
  • Influenza in Birds / virology*
  • Poland
  • Poultry / virology
  • RNA, Complementary
  • RNA, Viral / chemistry
  • Sensitivity and Specificity
  • Sequence Analysis, RNA / methods*

Substances

  • DNA, Single-Stranded
  • Epoxy Compounds
  • Ferric Compounds
  • RNA, Complementary
  • RNA, Viral
  • Gold