Detection of a Specific Biomarker for Epstein-Barr Virus Using a Polymer-Based Genosensor

Abstract

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.

A novel polymer‐based genosensor for the detection and quantification of Streptococcus pneumoniae in genomic DNA sample

“…The immobilization of probe on the surface of the graphite electrode without modification and modified with poly(4‐aminophenol) occurred successfully. The current response in +0.96 V and +1.23 V was associated to the oxidation peak of guanosine and adenosine, respectively, in accordance with the literature . By analyzing the guanosine oxidation peak (Fig.…”

“…In addition, the presence of free functional groups including amino and hydroxyl gives a favorable microenvironment, which may increase the retention of biomolecules . These functional groups also favor the stability and maintenance of biological activity of the device .…”

“…Electrochemical DNA sensing bears advantages as fast response time, high sensitivity, and low cost . This technique employs immobilized DNA sequences on the sensor surface as a recognition element and the sequence‐specific hybridization can be monitored and analyzed .…”

“…The hybridization signal can be either directly detected (by monitoring the DNA oxidation or reduction process) or indirectly detected (using an indicator of the hybridization) . It is known that the use of electrochemical indicators improves the system sensitivity .…”

“…The development of polymeric films and their application in biological sensors have been widely reported in literature. For the development of molecular diagnostic platforms, polymeric films are derived from hydroxyphenylacetic acid , 4‐aminophenol , 3‐aminophenol , and 4‐aminothiophenol . Electrodes modified with poly(4‐aminophenol) present two oxidizable groups (NH 2 and OH) .…”