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1.
Biosens Bioelectron ; 169: 112643, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007615

RESUMO

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/sangue , Coronavirus/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Influenza Humana/sangue , Pneumonia Viral/sangue , Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Células HEK293 , Humanos , Influenza Humana/diagnóstico , Influenza Humana/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Vírus da SARS/isolamento & purificação , Sensibilidade e Especificidade
2.
Anal Chem ; 92(19): 13396-13404, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32867467

RESUMO

Rapid, accurate, reliable, and risk-free tracking of pathogenic microorganisms at the single-cell level is critical to achieve efficient source control and prevent outbreaks of microbial infectious diseases. For the first time, we report a promising approach for integrating the concepts of a remarkably large Stokes shift and dual-recognition into a single matrix to develop a pathogenic microorganism stimuli-responsive ratiometric fluorescent nanoprobe with speed, cost efficiency, stability, ultrahigh specificity, and sensitivity. As a proof-of-concept, we selected the Gram-positive bacterium Staphylococcus aureus (S. aureus) as the target analyte model, which easily bound to its recognition aptamer and the broad-spectrum glycopeptide antibiotic vancomycin (Van). To improve the specificity and short sample-to-answer time, we employed classic noncovalent π-π stacking interactions as a driving force to trigger the binding of Van and aptamer dual-functionalized near-infrared (NIR) fluorescent Apt-Van-QDs to the surface of an unreported blue fluorescent π-rich electronic carbon nanoparticles (CNPs), achieving S. aureus stimuli-responsive ratiometric nanoprobe Apt-Van-QDs@CNPs. In the assembly of Apt-Van-QDs@CNPs, the blue CNPs (energy donor) and NIR Apt-Van-QDs (energy acceptor) became close to allow the fluorescence resonance energy transfer (FRET) process, leading to a remarkable blue fluorescence quenching for the CNPs at ∼465 nm and a clear NIR fluorescence enhancement for Apt-Van-QDs at ∼725 nm. In the presence of S. aureus, the FRET process from CNPs to Apt-Van-QDs was disrupted, causing the nanoprobe Apt-Van-QDs@CNPs to display a ratiometric fluorescent response to S. aureus, which exhibited a large Stokes shift of ∼260 nm and rapid sample-to-answer detection time (∼30.0 min). As expected, the nanoprobe Apt-Van-QDs@CNPs showed an ultrahigh specificity for ratiometric fluorescence detection of S. aureus with a good detection limit of 1.0 CFU/mL, allowing the assay at single-cell level. Moreover, we also carried out the precise analysis of S. aureus in actual samples with acceptable results. We believe that this work offers new insight into the rational design of efficient ratiometric nanoprobes for rapid on-site accurate screening of pathogenic microorganisms at the single-cell level in the early stages, especially during the worldwide spread of COVID-19 today.


Assuntos
Bactérias/química , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/síntese química , Nanotecnologia/métodos , Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/microbiologia , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Microbiologia de Alimentos/métodos , Humanos , Nanopartículas , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/microbiologia , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Vancomicina/farmacologia
3.
Nat Commun ; 11(1): 4851, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978386

RESUMO

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Ligantes , Engenharia Metabólica/métodos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Cristalografia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Lisina/metabolismo , Técnicas Analíticas Microfluídicas , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Termodinâmica
4.
Nat Commun ; 11(1): 4482, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901011

RESUMO

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.


Assuntos
DNA/metabolismo , Sondas Moleculares/química , Nanopartículas/metabolismo , Proteínas/metabolismo , Animais , Transporte Biológico Ativo , Técnicas Biossensoriais/métodos , Química Click , Citometria de Fluxo , Corantes Fluorescentes , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Técnicas de Sonda Molecular , Sondas Moleculares/metabolismo , Células NIH 3T3
5.
Nat Commun ; 11(1): 4757, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958771

RESUMO

Local tissue mechanics play a critical role in cell function, but measuring these properties at cellular length scales in living 3D tissues can present considerable challenges. Here we present thermoresponsive, smart material microgels that can be dispersed or injected into tissues and optically assayed to measure residual tissue elasticity after creep over several weeks. We first develop and characterize the sensors, and demonstrate that internal mechanical profiles of live multicellular spheroids can be mapped at high resolutions to reveal broad ranges of rigidity within the tissues, which vary with subtle differences in spheroid aggregation method. We then show that small sites of unexpectedly high rigidity develop in invasive breast cancer spheroids, and in an in vivo mouse model of breast cancer progression. These focal sites of increased intratumoral rigidity suggest new possibilities for how early mechanical cues that drive cancer cells towards invasion might arise within the evolving tumor microenvironment.


Assuntos
Fenômenos Biomecânicos , Técnicas Biossensoriais/métodos , Hidrogéis/química , Animais , Técnicas Biossensoriais/instrumentação , Linhagem Celular , Elasticidade , Humanos , Camundongos , Modelos Biológicos , Neoplasias Experimentais/patologia , Esferoides Celulares/patologia , Esferoides Celulares/fisiologia , Temperatura
6.
Anal Chem ; 92(19): 13396-13404, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: covidwho-738932

RESUMO

Rapid, accurate, reliable, and risk-free tracking of pathogenic microorganisms at the single-cell level is critical to achieve efficient source control and prevent outbreaks of microbial infectious diseases. For the first time, we report a promising approach for integrating the concepts of a remarkably large Stokes shift and dual-recognition into a single matrix to develop a pathogenic microorganism stimuli-responsive ratiometric fluorescent nanoprobe with speed, cost efficiency, stability, ultrahigh specificity, and sensitivity. As a proof-of-concept, we selected the Gram-positive bacterium Staphylococcus aureus (S. aureus) as the target analyte model, which easily bound to its recognition aptamer and the broad-spectrum glycopeptide antibiotic vancomycin (Van). To improve the specificity and short sample-to-answer time, we employed classic noncovalent π-π stacking interactions as a driving force to trigger the binding of Van and aptamer dual-functionalized near-infrared (NIR) fluorescent Apt-Van-QDs to the surface of an unreported blue fluorescent π-rich electronic carbon nanoparticles (CNPs), achieving S. aureus stimuli-responsive ratiometric nanoprobe Apt-Van-QDs@CNPs. In the assembly of Apt-Van-QDs@CNPs, the blue CNPs (energy donor) and NIR Apt-Van-QDs (energy acceptor) became close to allow the fluorescence resonance energy transfer (FRET) process, leading to a remarkable blue fluorescence quenching for the CNPs at ∼465 nm and a clear NIR fluorescence enhancement for Apt-Van-QDs at ∼725 nm. In the presence of S. aureus, the FRET process from CNPs to Apt-Van-QDs was disrupted, causing the nanoprobe Apt-Van-QDs@CNPs to display a ratiometric fluorescent response to S. aureus, which exhibited a large Stokes shift of ∼260 nm and rapid sample-to-answer detection time (∼30.0 min). As expected, the nanoprobe Apt-Van-QDs@CNPs showed an ultrahigh specificity for ratiometric fluorescence detection of S. aureus with a good detection limit of 1.0 CFU/mL, allowing the assay at single-cell level. Moreover, we also carried out the precise analysis of S. aureus in actual samples with acceptable results. We believe that this work offers new insight into the rational design of efficient ratiometric nanoprobes for rapid on-site accurate screening of pathogenic microorganisms at the single-cell level in the early stages, especially during the worldwide spread of COVID-19 today.


Assuntos
Bactérias/química , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/síntese química , Nanotecnologia/métodos , Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/microbiologia , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Microbiologia de Alimentos/métodos , Humanos , Nanopartículas , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/microbiologia , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Vancomicina/farmacologia
7.
Biosens Bioelectron ; 169: 112642, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979593

RESUMO

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/genética , Técnicas Biossensoriais/instrumentação , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/virologia , Desenho de Equipamento , Fluorescência , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Smartphone
8.
Biosens Bioelectron ; 169: 112604, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32980805

RESUMO

Virus severely endangers human life and health, and the detection of viruses is essential for the prevention and treatment of associated diseases. Metal-organic framework (MOF), a novel hybrid porous material which is bridged by the metal clusters and organic linkers, has become a promising biosensor platform for virus detection due to its outstanding properties including high surface area, adjustable pore size, easy modification, etc. However, the MOF-based sensing platforms for virus detection are rarely summarized. This review systematically divided the detection platforms into nucleic acid and immunological (antigen and antibody) detection, and the underlying sensing mechanisms were interpreted. The nucleic acid sensing was discussed based on the properties of MOF (such as metal ion, functional group, geometry structure, size, porosity, stability, etc.), revealing the relationship between the sensing performance and properties of MOF. Moreover, antibodies sensing based on the fluorescence detection and antigens sensing based on molecular imprinting or electrochemical immunoassay were highlighted. Furthermore, the remaining challenges and future development of MOF for virus detection were further discussed and proposed. This review will provide valuable references for the construction of sophisticated sensing platform for the detection of viruses, especially the 2019 coronavirus.


Assuntos
Técnicas Biossensoriais/métodos , Estruturas Metalorgânicas/química , Viroses/virologia , Vírus/isolamento & purificação , Animais , Anticorpos Antivirais/análise , Antígenos Virais/análise , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Modelos Moleculares , Impressão Molecular/instrumentação , Impressão Molecular/métodos , Ácidos Nucleicos/análise , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Viroses/diagnóstico
9.
Biosens Bioelectron ; 167: 112479, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32763826

RESUMO

COVID-19 pandemic outbreak is the most astounding scene ever experienced in the 21st century. It has been determined to be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the global pandemic, the lack of efficient rapid and accurate molecular diagnostic testing tools has hindered the public opportunely response to the emerging viral threat. Herein, a DNA nanoscaffold hybrid chain reaction (DNHCR)-based nucleic acid assay strategy is reported for rapid detection of SARS-CoV-2 RNA. In this method, the DNA nanoscaffolds have been first constructed by the self-assembly of long DNA strands and self-quenching probes (H1). Then, the SARS-CoV-2 RNA will initiate the hybridization of H1 and free H2 DNA probes along the nanoscaffold, and an illuminated DNA nanostring is instantly obtained. By taking advantages of the localization design of the H1 probes and the temperature tolerance of the isothermal amplification, the proposed DNHCR method can detect target at short responding time (within 10 min) and mild condition (15 °C-35 °C). Moreover, the reliability of DNHCR method in serum and saliva samples have also been validated. Therefore, DNHCR-based method is expected to provide a simple and faster alternative to the traditional SARS-CoV-2 qRT-PCR assay.


Assuntos
Betacoronavirus , Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , DNA/síntese química , DNA/química , DNA/genética , Estudos de Viabilidade , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Nanoestruturas/química , Nanotecnologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
10.
Biosens Bioelectron ; 167: 112494, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32791468

RESUMO

G-quadruplex is a non-canonical nucleic acid structure formed by the folding of guanine rich DNA or RNA. The conformation and function of G-quadruplex are determined by a number of factors, including the number and polarity of nucleotide strands, the type of cations and the binding targets. Recent studies led to the discovery of additional advantageous attributes of G-quadruplex with the potential to be used in novel biosensors, such as improved ligand binding and unique folding properties. G-quadruplex based biosensor can detect various substances, such as metal ions, organic macromolecules, proteins and nucleic acids with improved affinity and specificity compared to standard biosensors. The recently developed G-quadruplex based biosensors include electrochemical and optical biosensors. A novel G-quadruplex based biosensors also show better performance and broader applications in the detection of a wide spectrum of pathogens, including SARS-CoV-2, the causative agent of COVID-19 disease. This review highlights the latest developments in the field of G-quadruplex based biosensors, with particular focus on the G-quadruplex sequences and recent applications and the potential of G-quadruplex based biosensors in SARS-CoV-2 detection.


Assuntos
Betacoronavirus , Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Quadruplex G , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/tendências , Técnicas de Laboratório Clínico/tendências , Colorimetria , Técnicas Eletroquímicas , Corantes Fluorescentes , Humanos , Pandemias
11.
Biosensors (Basel) ; 10(9)2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847008

RESUMO

Cytokines are a family of proteins which play a major role in the regulation of the immune system and the development of several diseases, from rheumatoid arthritis to cancer and, more recently, COVID-19. Therefore, many efforts are currently being developed to improve therapy and diagnosis, as well as to produce inhibitory drugs and biosensors for a rapid, minimally invasive, and effective detection. In this regard, even more efficient cytokine receptors are under investigation. In this paper we analyze a set of IL-6 cytokine receptors, investigating their topological features by means of a theoretical approach. Our results suggest a topological indicator that may help in the identification of those receptors having the highest complementarity with the protein, a feature expected to ensure a stable binding. Furthermore, we propose and discuss the use of these receptors in an idealized experimental setup.


Assuntos
Técnicas Biossensoriais/métodos , Interleucina-6/análise , Receptores de Interleucina-6/análise , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Aptâmeros de Nucleotídeos/química , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Humanos , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/imunologia , Limite de Detecção , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Receptores de Interleucina-6/imunologia
12.
Nat Commun ; 11(1): 3881, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753572

RESUMO

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.


Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Potenciais de Ação/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Técnicas Biossensoriais/instrumentação , Cálcio/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Imagem Óptica/instrumentação , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Propanolaminas/farmacologia
13.
Biosens Bioelectron ; 166: 112455, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32739797

RESUMO

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the coronavirus disease 2019 (COVID-19) worldwide pandemic. This unprecedented situation has garnered worldwide attention. An effective strategy for controlling the COVID-19 pandemic is to develop highly accurate methods for the rapid identification and isolation of SARS-CoV-2 infected patients. Many companies and institutes are therefore striving to develop effective methods for the rapid detection of SARS-CoV-2 ribonucleic acid (RNA), antibodies, antigens, and the virus. In this review, we summarize the structure of the SARS-CoV-2 virus, its genome and gene expression characteristics, and the current progression of SARS-CoV-2 RNA, antibodies, antigens, and virus detection. Further, we discuss the reasons for the observed false-negative and false-positive RNA and antibody detection results in practical clinical applications. Finally, we provide a review of the biosensors which hold promising potential for point-of-care detection of COVID-19 patients. This review thereby provides general guidelines for both scientists in the biosensing research community and for those in the biosensor industry to develop a highly sensitive and accurate point-of-care COVID-19 detection system, which would be of enormous benefit for controlling the current COVID-19 pandemic.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/isolamento & purificação , Betacoronavirus/genética , Betacoronavirus/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/tendências , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/tendências , Infecções por Coronavirus/epidemiologia , Desenho de Equipamento , Genoma Viral , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Pneumonia Viral/epidemiologia , Testes Imediatos , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírion/isolamento & purificação
14.
Biosens Bioelectron ; 166: 112436, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32750677

RESUMO

Our recent experience of the COVID-19 pandemic has highlighted the importance of easy-to-use, quick, cheap, sensitive and selective detection of virus pathogens for the efficient monitoring and treatment of virus diseases. Early detection of viruses provides essential information about possible efficient and targeted treatments, prolongs the therapeutic window and hence reduces morbidity. Graphene is a lightweight, chemically stable and conductive material that can be successfully utilized for the detection of various virus strains. The sensitivity and selectivity of graphene can be enhanced by its functionalization or combination with other materials. Introducing suitable functional groups and/or counterparts in the hybrid structure enables tuning of the optical and electrical properties, which is particularly attractive for rapid and easy-to-use virus detection. In this review, we cover all the different types of graphene-based sensors available for virus detection, including, e.g., photoluminescence and colorimetric sensors, and surface plasmon resonance biosensors. Various strategies of electrochemical detection of viruses based on, e.g., DNA hybridization or antigen-antibody interactions, are also discussed. We summarize the current state-of-the-art applications of graphene-based systems for sensing a variety of viruses, e.g., SARS-CoV-2, influenza, dengue fever, hepatitis C virus, HIV, rotavirus and Zika virus. General principles, mechanisms of action, advantages and drawbacks are presented to provide useful information for the further development and construction of advanced virus biosensors. We highlight that the unique and tunable physicochemical properties of graphene-based nanomaterials make them ideal candidates for engineering and miniaturization of biosensors.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Grafite , Pneumonia Viral/diagnóstico , Vírus/isolamento & purificação , Reações Antígeno-Anticorpo , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/tendências , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/estatística & dados numéricos , Colorimetria , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , DNA Viral/análise , DNA Viral/genética , Técnicas Eletroquímicas , Desenho de Equipamento , Grafite/química , Humanos , Luminescência , Nanoestruturas/química , Hibridização de Ácido Nucleico , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Pontos Quânticos/química , Análise Espectral Raman , Ressonância de Plasmônio de Superfície , Virologia/métodos , Vírus/genética , Vírus/patogenicidade
15.
Biosens Bioelectron ; 166: 112471, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777726

RESUMO

The infection and spread of pathogens (e.g., COVID-19) pose an enormous threat to the safety of human beings and animals all over the world. The rapid and accurate monitoring and determination of pathogens are of great significance to clinical diagnosis, food safety and environmental evaluation. In recent years, with the evolution of nanotechnology, nano-sized graphene and graphene derivatives have been frequently introduced into the construction of biosensors due to their unique physicochemical properties and biocompatibility. The combination of biomolecules with specific recognition capabilities and graphene materials provides a promising strategy to construct more stable and sensitive biosensors for the detection of pathogens. This review tracks the development of graphene biosensors for the detection of bacterial and viral pathogens, mainly including the preparation of graphene biosensors and their working mechanism. The challenges involved in this field have been discussed, and the perspective for further development has been put forward, aiming to promote the development of pathogens sensing and the contribution to epidemic prevention.


Assuntos
Bactérias/isolamento & purificação , Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Grafite , Pandemias , Pneumonia Viral/diagnóstico , Vírus/isolamento & purificação , Animais , Bactérias/genética , Bactérias/patogenicidade , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Grafite/química , Humanos , Técnicas de Diagnóstico Molecular , Nanotecnologia , Vírus/genética , Vírus/patogenicidade
16.
Sensors (Basel) ; 20(15)2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32752043

RESUMO

Coronaviruses have received global concern since 2003, when an outbreak caused by SARS-CoV emerged in China. Later on, in 2012, the Middle-East respiratory syndrome spread in Saudi Arabia, caused by MERS-CoV. Currently, the global crisis is caused by the pandemic SARS-CoV-2, which belongs to the same lineage of SARS-CoV. In response to the urgent need of diagnostic tools, several lab-based and biosensing techniques have been proposed so far. Five main areas have been individuated and discussed in terms of their strengths and weaknesses. The cell-culture detection and the microneutralization tests are still considered highly reliable methods. The genetic screening, featuring the well-established Real-time polymerase chain reaction (RT-PCR), represents the gold standard for virus detection in nasopharyngeal swabs. On the other side, immunoassays were developed, either by screening/antigen recognition of IgM/IgG or by detecting the whole virus, in blood and sera. Next, proteomic mass-spectrometry (MS)-based methodologies have also been proposed for the analysis of swab samples. Finally, virus-biosensing devices were efficiently designed. Both electrochemical immunosensors and eye-based technologies have been described, showing detection times lower than 10 min after swab introduction. Alternative to swab-based techniques, lateral flow point-of-care immunoassays are already commercially available for the analysis of blood samples. Such biosensing devices hold the advantage of being portable for on-site testing in hospitals, airports, and hotspots, virtually without any sample treatment or complicated lab precautions.


Assuntos
Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/virologia , Humanos , Imunoensaio/métodos , Pandemias , Pneumonia Viral/virologia , Proteômica/métodos , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos
17.
ACS Sens ; 5(9): 2747-2752, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32820626

RESUMO

With the current intense need for rapid and accurate detection of viruses due to COVID-19, we report on a platform technology that is well suited for this purpose, using intact measles virus for a demonstration. Cases of infection due to the measles virus are rapidly increasing, yet current diagnostic tools used to monitor for the virus rely on slow (>1 h) technologies. Here, we demonstrate the first biosensor capable of detecting the measles virus in minutes with no preprocessing steps. The key sensing element is an electrode coated with a self-assembled monolayer containing the measles antibody, immobilized through an N-heterocyclic carbene (NHC). The intact virus is detected by changes in resistance, giving a linear response to 10-100 µg/mL of the intact measles virus without the need to label or process the sample. The limit of detection is 6 µg/mL, which is at the lower limit of concentrations that can cause infections in primates. The NHC-based biosensors are shown to be superior to thiol-based systems, producing an approximately 10× larger response and significantly greater stability toward repeated measurements and long-term storage. This NHC-based biosensor thus represents an important development for both the rapid detection of the measles virus and as a platform technology for the detection of other biological targets of interest.


Assuntos
Anticorpos Imobilizados/imunologia , Benzimidazóis/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Vírus do Sarampo/isolamento & purificação , Anticorpos Imobilizados/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Limite de Detecção , Vírus do Sarampo/imunologia
18.
Biosens Bioelectron ; 167: 112479, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: covidwho-684409

RESUMO

COVID-19 pandemic outbreak is the most astounding scene ever experienced in the 21st century. It has been determined to be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the global pandemic, the lack of efficient rapid and accurate molecular diagnostic testing tools has hindered the public opportunely response to the emerging viral threat. Herein, a DNA nanoscaffold hybrid chain reaction (DNHCR)-based nucleic acid assay strategy is reported for rapid detection of SARS-CoV-2 RNA. In this method, the DNA nanoscaffolds have been first constructed by the self-assembly of long DNA strands and self-quenching probes (H1). Then, the SARS-CoV-2 RNA will initiate the hybridization of H1 and free H2 DNA probes along the nanoscaffold, and an illuminated DNA nanostring is instantly obtained. By taking advantages of the localization design of the H1 probes and the temperature tolerance of the isothermal amplification, the proposed DNHCR method can detect target at short responding time (within 10 min) and mild condition (15 °C-35 °C). Moreover, the reliability of DNHCR method in serum and saliva samples have also been validated. Therefore, DNHCR-based method is expected to provide a simple and faster alternative to the traditional SARS-CoV-2 qRT-PCR assay.


Assuntos
Betacoronavirus , Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , DNA/síntese química , DNA/química , DNA/genética , Estudos de Viabilidade , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Nanoestruturas/química , Nanotecnologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
ACS Sens ; 5(9): 2747-2752, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: covidwho-740029

RESUMO

With the current intense need for rapid and accurate detection of viruses due to COVID-19, we report on a platform technology that is well suited for this purpose, using intact measles virus for a demonstration. Cases of infection due to the measles virus are rapidly increasing, yet current diagnostic tools used to monitor for the virus rely on slow (>1 h) technologies. Here, we demonstrate the first biosensor capable of detecting the measles virus in minutes with no preprocessing steps. The key sensing element is an electrode coated with a self-assembled monolayer containing the measles antibody, immobilized through an N-heterocyclic carbene (NHC). The intact virus is detected by changes in resistance, giving a linear response to 10-100 µg/mL of the intact measles virus without the need to label or process the sample. The limit of detection is 6 µg/mL, which is at the lower limit of concentrations that can cause infections in primates. The NHC-based biosensors are shown to be superior to thiol-based systems, producing an approximately 10× larger response and significantly greater stability toward repeated measurements and long-term storage. This NHC-based biosensor thus represents an important development for both the rapid detection of the measles virus and as a platform technology for the detection of other biological targets of interest.


Assuntos
Anticorpos Imobilizados/imunologia , Benzimidazóis/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Vírus do Sarampo/isolamento & purificação , Anticorpos Imobilizados/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Limite de Detecção , Vírus do Sarampo/imunologia
20.
PLoS One ; 15(8): e0237583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804936

RESUMO

Identification and quantification of plant flavonoids are critical to pharmacokinetic study and pharmaceutical quality control due to their distinct pharmacological functions. Here we report on a novel plant flavonoid electrochemical sensor for sensitive and selective detection of dihydromyricetin (DMY) based on double- layered membranes consisting of gold nanoparticles (Au) anchored on reduced graphene oxide (rGO) and molecularly imprinted polymers (MIPs) modified glassy carbon electrode (GCE). Both rGO-Au and MIPs membranes were directly formed on GCE via in-situ electrochemical reduction and polymerization processes step by step. The compositions, morphologies, and electrochemical properties of membranes were investigated with X-ray powder diffractometry (XRD), Fourier transform infrared spectrum (FTIR), Field emission scanning electron microscopy (FESEM) combined with various electrochemical methods. The fabricated electrochemical sensor labeled as GCE│rGO-Au/MIPs exhibited excellent performance in determining of DMY under optimal experimental conditions. A wide linear detection range (LDR) ranges from 2.0×10-8 to 1.0×10-4 M together with a low limit of detection (LOD) of 1.2×10-8 M (S/N = 3) were achieved. Moreover, the electrochemical sensor was employed to determine DMY in real samples with satisfactory results.


Assuntos
Carbono/química , Técnicas Eletroquímicas/instrumentação , Flavonoides/análise , Flavonóis/análise , Grafite/química , Técnicas Biossensoriais/métodos , Eletrodos , Ouro , Limite de Detecção , Nanopartículas Metálicas , Impressão Molecular , Extratos Vegetais/análise , Polímeros/síntese química , Polímeros/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
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