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

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.

On the Challenges for the Diagnosis of SARS-CoV-2 Based on a Review of Current Methodologies

“…Similar to RT-PCR, genosensing is based on nucleic acid testing via hybridization between a probe, usually a single strand oligonucleotide, and a complementary DNA or RNA. 101 Upon hybridization of the probe immobilized on the sensor surface, a fluorescent, electrical, or electrochemical signal is produced, thus allowing the biorecognition of the target RNA. In contrast to RT-PCR, the genosensing approach is free from amplification and separation steps, making it simple and easy to handle.…”

“…Genosensors are a useful, cost-effective alternative to RT-PCR for detecting viral RNA fragments of specific sequences. Similar to RT-PCR, genosensing is based on nucleic acid testing via hybridization between a probe, usually a single strand oligonucleotide, and a complementary DNA or RNA . Upon hybridization of the probe immobilized on the sensor surface, a fluorescent, electrical, or electrochemical signal is produced, thus allowing the biorecognition of the target RNA.…”

“…In contrast to RT-PCR, the genosensing approach is free from amplification and separation steps, making it simple and easy to handle. It has been used in food analysis and environmental control, but only recently in POC diagnosis. ,, Indeed, there is a demand for further developments of commercial products for mass testing, which may explain why genosensors for SARS-CoV-2 have not been reported yet. Because genosensors usually involve immobilization of specific probes for hybridization, these devices are of significant selectivity and rarely affected by interferents.…”

Redox-Active Monolayers Self-Assembled on Gold Electrodes—Effect of Their Structures on Electrochemical Parameters and DNA Sensing Ability

“…Redox-active layers involving dipyrromethene-M 2+ -dipyrromethene or (phenantroline) 3 -Fe 2+ complexes have been successfully applied for the sensitive and selective determination of DNA and RNA targets [6][7][8]. The main goal of the research presented is the elaboration of the relationship between the chemical structure of the redox-active layer and the amount of generated analytical signal.…”