Electrochemical genosensor based on disc and screen printed gold electrodes for detection of specific DNA and RNA sequences derived from Avian Influenza Virus H5N1

Highlights

• Miniaturized genosensor detects 1 pM of 20-mer DNA and 280-mer RNA sequences derived from H5N1.

• Genosensors discriminate between different positions of complementary sequence in RNA.

• Miniaturized genosensor has potential as device for early diagnosis of H5N1.

Abstract

The genosensors based on thiolated ssDNA probe deposited on the two types of gold electrodes: screen-printed (miniaturized) and disc electrodes destined for determination of specific sequences of DNA and RNA derived from Avian Influenza Virus H5N1 have been proposed. The working principle of genosensor is based on the ion-channel mechanism. The analytical signals generated upon hybridization processes were recorded using electrochemical technique – Osteryoung square wave voltammetry in the presence of a redox active marker [Fe(CN)₆]^3−/4− in the sample solution. The miniaturized genosensor based on screen printed gold electrodes was able to detect the 20-mer complementary DNA oligonucleotide sequence as well as ∼280-mer RNA sequences containing the complementary 20-mer sequence in various positions: at 3′-terminus, at 5′-terminus and in the middle of the RNA transcript at the 1 pM concentration. The measuring systems were selective. Non-complementary 20-mer oligonucleotide sequence as well as RNA transcript without complementary region generated weak response. The RNA transcripts were also tested with gold disc electrodes modified in the same manner. This device was able to detect ∼280-mer RNA sequences, but at higher concentration of 10 pM. The good discrimination of the position of complementary part in the ∼280-mer RNA sequences was observed with using both types of modified electrodes.

Introduction

Avian influenza virus (AIV), especially H5N1 has become nowadays a very dangerous pathogen threatening not only for poultry. The H5N1 is the virus which mainly affects the birds and usually does not spread among people. Nevertheless, highly pathogenic H5N1 virus has been registered in about 650 confirmed human infections (with approximately 60% of deaths) in 15 countries [1]. The greatest threat to mankind would be if a person who is ill with seasonal flu would also become infected with avian flu. Then there is the probability that the H5N1 virus can exchange genetic information with the human flu virus and acquire the ability for transmission from human to human. An easily human-transmissible AIV strain could have disastrous implications. Thus, the development of methods for early diagnosis, as well as for preventions is essential. Highly sensitive, accurate and rapid tests for identification of AIV infection would allow early antiviral therapy.

However, the most commonly used methods of virus detection are laborious, time-consuming, expensive, require specialized equipment and trained personnel. These include, for example: cultivation of viruses in cell culture, immunofluorescence, serological methods, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) [2], [3], [4], [5], [6], [7], reverse transcription-polymerase chain reaction (RT-PCR) [8], [9], RT-PCR with detection by ELISA [10], real-time reverse transcription-polymerase chain reaction (RRT-PCR) [11], [12] and nucleic acid sequence-based amplification (NASBA) [13], [14], [15].

Therefore, in order to minimize the social and economic costs, the development of rapid diagnostic tests is indispensable [16]. It is believed that this type of tests should meet the following conditions: high efficiency, the ability to detect multiple targets, accuracy: sensitivity and specificity, speed, ease of use, the appropriateness of the use of research in the field and affordable price. These conditions fulfil the electrochemical biosensors. The biosensors are analytical tools developed for specific and selective detection of analytes such as: nucleic acids, drugs or proteins that are crucial in the field of diagnostics.

The high sensitivity, selectivity, compatibility with modern micro-fabrication technologies, inexpensive portability, time-saving, fast data analysis and that they are label-free make electrochemical biosensors an excellent candidates for a wide variety applications in areas such as medical diagnostics, forensics, biodefense, food contamination and environmental monitoring. Thus, the area of electrochemical biosensor technology has expanded from day to day [17], [18], [19], [20], [21], [22], [23], [24], [25].

The screen-printed electrodes have been designed especially for miniaturization of electrochemical analytical systems. These disposable sensors can be easily modified in various ways. They are also suitable candidates for measuring of multiple biological samples, as they require a small sample volume [26].

Here, we report on a sensitive ion-channel mimetic miniaturized genosensor incorporated mixed monolayer of thiolated DNA probe (SH-NC3) and 6-mercaptohexanol (MCH) deposited onto screen printed gold electrode (SPGE) surfaces for electrochemical detection of specific DNA and RNA sequences derived from AIV H5N1. The electroanalytical signals generated based on the DNA–DNA and DNA–RNA duplexes formed at the electrode surface were explored using Osteryoung square wave voltammetry (OSWV) and cyclic voltammetry (CV) in the presence of [Fe(CN)₆]^3−/4− as an electroactive redox marker. As targets short (20-mer) single stranded DNA sequences and long (ca. 280-mer) RNA transcripts with different localization of the 20-mer region complementary to the probe were used. The RNA transcripts responses recorded using modified SPGEs were compared with gold disc electrodes (GDEs) modified in the same manner.