Detection of hepatitis E virus genotype 3 in wastewater by an electrochemical genosensor.

Hepatitis E Virus (HEV) is an etiologic agent of hepatitis worldwide. HEV genotype 3 is the most prevalent in non-endemic regions, identified in humans, pigs and environmental samples. Thus, considering the zoonotic nature of HEV genotype 3, viral genome detection in wastewater concerns public health authorities. Electrochemical biosensors are promising analytical tools for viral genome detection in outside settings. This work reports on a highly specific, sensitive and portable electrochemical genosensor to detect HEV genotype 3 in wastewater samples. Based on the alignment analysis of HEV genotype 3 genome sequences available in GenBank, highly specific DNA target probes were designed to hybridize a target sequence within the ORF2/ORF3 overlapping genome region of HEV in between a biotinylated capture probe and a signal probe labeled with digoxigenin, in a sandwich-type format. An anti-Dig antibody labeled with the horseradish peroxidase (HRP) enzyme allowed electrochemical detection. The specificity of the target molecular probes of the viral genome was determined before the biosensor assembly by in silico analysis, PCR and qPCR assays demonstrating efficient amplification of two targets, i.e., nucleotides 5338-5373 and 5328-5373, but this last one of higher performance. The electrochemical response of the genosensor with synthetic HEV was target concentration-dependent in a linear range from 300 pM to 2.4 nM, with a sensitivity of 16.93 µA/nM, a LOD 1.2 pM and high reproducibility. The genosensor response was differential when interrogated with the HEV genotype 3 viral genomes from wastewater against other four viruses. Therefore, the approach offers a step forward to the epidemiologic surveillance of viruses in wastewater as an early warning system.

Electrochemical genosensor for the specific detection of SARS-CoV-2.

Infection caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the Coronavirus disease (COVID-19) and the current pandemic. Its mortality rate increases, demonstrating the imperative need for acute and rapid diagnostic tools as an alternative to current serological tests and molecular techniques. Features of electrochemical genosensor devices make them amenable for fast and accurate testing closer to the patient. This work reports on a specific electrochemical genosensor for SARS-CoV-2 detection and discrimination against homologous respiratory viruses. The electrochemical biosensor was assembled by immobilizing thiolated capture probes on top of maleimide-coated magnetic particles, followed by specific target hybridization between the capture and biotinylated signaling probes in a sandwich-type manner. The probes were rigorously designed bioinformatically and tested in vitro. Enzymatic complexes based on streptavidin-horseradish peroxidase linked the biotinylated signaling probe to render the biosensor electrochemical response. The genosensor showed to reach a sensitivity of 174.4 µA fM-1 and a limit of detection of 807 fM when using streptavidin poly-HRP20 enzymatic complex, detected SARS-CoV-2 specifically and discriminated it against homologous viruses in spiked samples and samples from SARS-CoV-2 cell cultures, a step forward to detect SARS-CoV-2 closer to the patient as a promising way for diagnosis and surveillance of COVID-19.

Differential detection of zika virus based on PCR.

Tropical countries are highly prone to infectious diseases such as the one caused by zika virus. Infection by zika is clinically and epidemiologically highly relevant. For example, when women are infected by zika during the first trimester of pregnancy, the child incurs a high risk of microcephaly and acute neurological syndromes. In adults, the virus is associated with the Guillain-Barré syndrome and other disorders. The worldwide emergency caused by zika in 2013/14 demonstrated the need for rapid and accurate diagnostic tools for the virus. Current diagnostic methods include virus isolation, serological tests, and molecular assays. However, virus isolation requires labor-intensive and time-consuming cell culture; serological detection suffers from cross-reactivity caused by previous exposure to homologous arboviruses that cause symptoms like those caused by zika, while molecular tools commonly are not designed for differential zika detection. This work reports on developing a specific molecular detection method based on phylogenetically conserved primers designed for the specific diagnosis of the zika virus. The zika primers were systematically selected through a rigorous bioinformatic analysis and demonstrated the capability to be highly specific. We tested our primers on synthetic DNA, cell cultures and samples from patients infected with zika, dengue and chikungunya and found that they detected zika with specificity high enough for differential virus diagnosis.

Gold nanoparticle/DNA-based nanobioconjugate for electrochemical detection of Zika virus.

The development of a stable nanobioconjugate based on gold nanoparticles (AuNPs) linked to single-strand DNA (ssDNA) is reported for amplification of the electrochemical signal of a Zika virus (ZIKV) genetic material-based bioassay, with high sensitivity. The genosensor was assembled either at a screen-printed gold electrode (SPAuE) or a screen-printed carbon electrode decorated with hierarchical gold nanostructures (SPCE/Au), with Ru3+ as an electrochemical reporter. The genosensor response, interrogated by differential pulse voltammetry (DPV) at the transient current density, was linear from 10 to 600 fM and from 500 fM to 10 pM of the target, with a sensitivity of 2.7 and 2.9 µA cm-2 M-1 and a limit of detection of 0.2 and 33 fM at the SPAuE and SPCE/Au, respectively. The resultant genosensor detected ZIKV genetic material in raw serum samples from infected patients, with no sample pretreatment in a polymerase chain reaction amplification-free assay. The proposed ultrasensitive nanobioconjugate-based system offers a step forward to the diagnosis of the ZIKV, closer to the patient, and holds the potential for signal amplification in biosensing of a myriad of applications.Graphical abstract.

Genosensors for differential detection of Zika virus.

Zika virus (ZIKV) is considered an emerging infectious disease of high clinical and epidemiological relevance. The epidemiological emergency generated by the virus in Latin America and Southeast Asia in 2014 evidenced the urgent need for rapid and acute diagnostic tools. The current laboratory diagnosis of ZIKV is based on molecular and serological methods. However, molecular tools need expensive and sophisticated equipment and trained personnel; and serological detection may suffer from cross-reactivity. In this context, genosensors offer an attractive alternative for field-ready, early and accurate diagnosis of ZIKV. This work reports on the development of genosensors for the differential detection of ZIKV and its discrimination from dengue (DENV) and chikungunya (CHIKV) homologous arboviruses. We designed specific capture and signal probes by bioinformatics, and prove their specificity to amplify the target genetic material by the polymerase chain reaction (PCR). The designed biotinylated capture and digoxigenin (Dig)-labeled signal probes hybridized the target in a sandwich-type format. An anti-Dig antibody labeled with the horseradish peroxidase (HRP) enzyme allowed for both optical and electrochemical detection. The genosensors detected the ZIKV genetic material in spiked serum, urine, and saliva samples and cDNA from infected patients, discriminating them from the DENV and ZIKV genetic material. The proposed system offers a step forward to the differential diagnosis of the ZIKV, closer to the patient, very promising for diagnosis and surveillance of this rapidly emerging disease.