Direct Amperometric Sensing of Fish Nodavirus RNA Using Gold Nanoparticle/DNA-Based Bioconjugates

Abstract

We describe the design of a simple and highly sensitive electrochemical bioanalytical method enabling the direct detection of a conserved RNA region within the capsid protein gene of a fish nodavirus, making use of nanostructured disposable electrodes. To achieve this goal, we select a conserved region within the nodavirus RNA2 segment to design a DNA probe that is tethered to the surface of nanostructured disposable screen-printed electrodes. In a proof-of-principle test, a synthetic RNA sequence is detected based on competitive hybridization between two oligonucleotides (biotinylated reporter DNA and target RNA) complimentary to a thiolated DNA capture probe. The method is further validated using extracted RNA samples obtained from healthy carrier Sparus aurata and clinically infected Dicentrarchus labrax fish specimens. In parallel, the sensitivity of the newly described biosensor is compared with a new real-time RT-PCR protocol. The current differences measured in the negative control and in presence of each concentration of target RNA are used to determine the dynamic range of the assay. We obtain a linear response (R2 = 0.995) over a range of RNA concentrations from 0.1 to 25 pM with a detection limit of 20 fM. The results are in good agreement with the results found by the RT-qPCR. This method provides a promising approach toward a more effective diagnosis and risk assessment of viral diseases in aquaculture.

Micro- and nanosystems for the detection of hemorrhagic fever viruses

“…258 The electrochemical reaction results in the generation of electrons, thereby providing an electrical difference through an electrode surface. 259 To date, based on the electron generation mechanisms, different types of electrochemical sensors have been developed, including amperometric sensors, 260,261 impedance sensors, 262,263 and photoelectrochemical sensors. 264,265 The performance of an electrochemical sensing platform is determined both by the chosen electrode material and recognition elements (e.g., antibody, aptamer, phage), which are capable of selectively reacting with target analytes.…”

A Review of Advanced Impedance Biosensors with Microfluidic Chips for Single-Cell Analysis

“…Electrical biosensors are subdivided into potentiometric, amperometric, and impedance sensors, depending on which electrical information is measured to obtain biological information [ 2 , 3 ]. These electrical biosensing techniques have been employed in devices and systems that detect and monitor various biological materials in a range from tissues to small biomolecules such as deoxyribonucleic acid (DNA) [ 4 , 5 , 6 , 7 , 8 ]. Specially, impedance biosensors have been used to detect biological components and molecules by measuring impedance changes without requiring special labeling processing, including cells, DNA, and proteins.…”