RESUMO
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.
Assuntos
Técnicas Biossensoriais , COVID-19 , Técnicas Biossensoriais/métodos , COVID-19/diagnóstico , Técnicas Eletroquímicas/métodos , Humanos , Pandemias , SARS-CoV-2/genética , EstreptavidinaRESUMO
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.
Assuntos
DNA de Cadeia Simples/química , Nanopartículas Metálicas/química , RNA Viral/sangue , Carga Viral/métodos , Zika virus/química , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/genética , Técnicas Eletroquímicas/métodos , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Hibridização de Ácido Nucleico , RNA Viral/genética , Rutênio/químicaRESUMO
Nanobioconjugates are hybrid materials that result from the coalescence of biomolecules and nanomaterials. They have emerged as a strategy to amplify the signal response in the biosensor field with the potential to enhance the sensitivity and detection limits of analytical assays. This critical review collects a myriad of strategies for the development of nanobioconjugates based on the conjugation of proteins, antibodies, carbohydrates, and DNA/RNA with noble metals, quantum dots, carbon- and magnetic-based nanomaterials, polymers, and complexes. It first discusses nanobioconjugates assembly and characterization to focus on the strategies to amplify a biorecognition event in biosensing, including molecular-, enzymatic-, and electroactive complex-based approaches. It provides some examples, current challenges, and future perspectives of nanobioconjugates for the amplification of signals in electrochemical biosensing.
Assuntos
Técnicas Biossensoriais/métodos , Nanoestruturas/química , Ácidos Nucleicos/química , Proteínas/química , Aptâmeros de Nucleotídeos/química , Biomarcadores/análise , Técnicas Eletroquímicas , Humanos , Polímeros/química , Toxinas Biológicas/análiseRESUMO
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.