Detection of a specific biomarker for Epstein-Barr virus using a polymer-based genosensor

• Published in: International journal of molecular sciences 2014-05, Vol.15 (5), p.9051-9066
• Main Authors: Balvedi, Renata P A, Castro, Ana C H, Madurro, João M, Brito-Madurro, Ana G
• Format: Article
• Language: English
• Subjects: Aniline Compounds - chemistry; Biomarkers; Biomarkers - analysis; Biosensing Techniques; Coordination Complexes - chemistry; DNA, Viral - metabolism; Electrochemical Techniques; Electrodes; Epstein-Barr virus; Ethidium - chemistry; Herpesvirus 4, Human - genetics; Herpesvirus 4, Human - metabolism; Humans; Nucleic Acid Hybridization; Oligonucleotides - chemistry; Oligonucleotides - metabolism; Polymers; Polymers - chemistry; Sensors; Sulfhydryl Compounds - chemistry; Viruses
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms15059051

This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 µmol·L⁻¹. In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L⁻¹ (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix (alanine, glucose, uric acid, ascorbic acid, bovine serum albumin (BSA), glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy.