Development of Innovative and Versatile Polythiol Probes for Use on ELOSA or Electrochemical Biosensors: Application in Hepatitis C Virus Genotyping

Abstract

The aim of this study was to develop versatile diagnostic tools based on the use of innovative polythiolated probes for the detection of multiple viruses. This approach is compatible with optical enzyme-linked oligosorbent assay (ELOSA) or electrochemical (biosensors) detection methods. The application targeted here concerns the rapid genotyping of Hepatitis C virus (HCV). HCV genotyping is one of the predictive parameters currently used to define the antiviral treatment strategy and is based on the sequencing of the viral NS5b region. Generic and specific NS5b amplicons were produced by real-time polymease chain reaction (RT-PCR) on HCV(+) human plasma. Original NS5b probes were designed for genotypes 1a/1b, 2a/2b/2c, 3a, and 4a/4d. Robust polythiolated probes were anchored with good efficacy on maleimide-activated microplates (MAM) and gold electrodes. Their grafting on MAM greatly increased the sensitivity of the ELOSA test which was able to detect HCV amplicons with good sensitivity (10 nM) and specificity. Moreover, the direct and real-time electrochemical detection by differential pulse voltammetry enabled a detection limit of 10 fM to be reached with good reproducibility. These innovative polythiolated probes have allowed us to envisage developing flexible, highly sensitive, and easy-to-handle platforms dedicated to the rapid screening and genotyping of a wide range of viral agents.

Nucleic acid electrochemical and electromechanical biosensors: a review of techniques and developments

“…Some of the more striking innovations in DNA detection have occurred in the field of virus detection, especially with respect to probe design. Researchers who examined sensing of viruses investigate various capture probes that are tagged with redox species (Lereau et al 2013, Grabowska et al 2014a. In one approach, polythiolated DNA probe tagged with ferrocene was evaluated (Lereau et al 2013).…”

“…Researchers who examined sensing of viruses investigate various capture probes that are tagged with redox species (Lereau et al 2013, Grabowska et al 2014a. In one approach, polythiolated DNA probe tagged with ferrocene was evaluated (Lereau et al 2013). When the capture probe was not hybridized, the ferrocene molecule on the flexible ssDNA is more mobile and migrates to the electrode surface.…”

“…Therefore, PCR was necessary for the detection of the parasite in a real water sample, especially because the infective dose is ∼30 oocysts. The study by Lereau et al must be noted for its novel probe design to achieve high sensitivity (Lereau et al 2013). The researchers utilized a novel polythiolated 15-mer ssDNA probe with three ferrocene residue at the end of the strand to achieve a 10-fM limit detection for a 105-mer oligonucleotide corresponding to the 3a hepatitis C virus genotype (Lereau et al 2013).…”

“…The study by Lereau et al must be noted for its novel probe design to achieve high sensitivity (Lereau et al 2013). The researchers utilized a novel polythiolated 15-mer ssDNA probe with three ferrocene residue at the end of the strand to achieve a 10-fM limit detection for a 105-mer oligonucleotide corresponding to the 3a hepatitis C virus genotype (Lereau et al 2013). The polythiol modification permitted greater signal stability as the probe was less likely to degrade and detach from the gold sensor surface.…”

“…Furthermore, nucleotides are more resilient at high temperatures over a range of pH values. DNA sensors can also genotype a virus for tailoring treatment, as in the case of HPV infection (Lereau et al 2013). Hence, this review is limited to nucleic acid-based detection of pathogens.…”