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1.
Proc Natl Acad Sci U S A ; 117(39): 24450-24458, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32900935

RESUMO

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Técnicas de Laboratório Clínico/economia , Colorimetria , Infecções por Coronavirus/economia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Pandemias , Pneumonia Viral/virologia , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo , Proteínas Virais/genética , Inativação de Vírus
2.
Environ Monit Assess ; 192(10): 657, 2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32968831

RESUMO

Simple, low-cost, and sensitive methods for the assessment of hexavalent chromium as an important environmental pollutant are highly desirable, especially under resource-limited settings. Therefore, herein we propose an original approach for the simple, low-cost, selective, and extremely sensitive assessment of Cr(VI) utilizing its catalysis of the micellar sensitized o-dianisidine (DA)-hydrogen peroxide reaction. The initial rate of the amended reaction is monitored by tracing the oxidation product, either by a digital camera video recording or spectrophotometrically at 440 nm, for 120 s from mixing the reactants. The optimized reaction conditions were 5 mmol L-1 DA, 0.6 mol L-1 H2O2, 2.0 v/v% Tween 20, and 10 mmol L-1 chloroacetate buffer (pH 4.5 ± 0.1), at 30 °C. The linear calibration graph extends to 90.0 ng mL-1 Cr(VI) with detection limits (3Sb) of 0.8 and 1.0 ng mL-1, for the video recording and spectrophotometric procedures, respectively. The amended method was successfully applied to the assessment of Cr(IV) in natural and polluted industrial wastewaters. The analytical data were in excellent statistical harmony with those of the standard ETAAS method. The proposed method is two orders of magnitude more sensitive than the diphenylcarbazide standard spectrophotometric method.Graphical abstract.


Assuntos
Colorimetria , Peróxido de Hidrogênio , Cromo/análise , Monitoramento Ambiental
3.
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
4.
Exp Parasitol ; 217: 107962, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763249

RESUMO

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.


Assuntos
FMN Redutase/metabolismo , Ferro/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Membrana Celular/enzimologia , Doença de Chagas/parasitologia , Colorimetria , FMN Redutase/análise , FMN Redutase/química , Imunofluorescência , Humanos , Filogenia , Distribuição de Poisson , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Trypanosoma cruzi/classificação , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Regulação para Cima
5.
Ecotoxicol Environ Saf ; 204: 111004, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32768745

RESUMO

Consumption of seafood contaminated with ciguatoxins (CTXs) leads to a foodborne disease known as ciguatera. Primary producers of CTXs are epibenthic dinoflagellates of the genera Gambierdiscus and Fukuyoa. In this study, thirteen Gambierdiscus and Fukuyoa strains were cultured, harvested at exponential phase, and CTXs were extracted with an implemented rapid protocol. Microalgal extracts were obtained from pellets with a low cell abundance (20,000 cell/mL) and were then analyzed with magnetic bead (MB)-based immunosensing tools (colorimetric immunoassay and electrochemical immunosensor). It is the first time that these approaches are used to screen Gambierdiscus and Fukuyoa strains, providing not only a global indication of the presence of CTXs, but also the ability to discriminate between two series of congeners (CTX1B and CTX3C). Analysis of the microalgal extracts revealed the presence of CTXs in 11 out of 13 strains and provided new information about Gambierdiscus and Fukuyoa toxin profiles. The use of immunosensing tools in the analysis of microalgal extracts facilitates the elucidation of further knowledge regarding these dinoflagellate genera and can contribute to improved ciguatera risk assessment and management.


Assuntos
Ciguatoxinas/isolamento & purificação , Colorimetria/métodos , Dinoflagelados/química , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Ciguatoxinas/classificação , Especificidade da Espécie
6.
Sensors (Basel) ; 20(17)2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842601

RESUMO

The global burden of coronavirus disease 2019 (COVID-19) to public health and global economy has stressed the need for rapid and simple diagnostic methods. From this perspective, plasmonic-based biosensing can manage the threat of infectious diseases by providing timely virus monitoring. In recent years, many plasmonics' platforms have embraced the challenge of offering on-site strategies to complement traditional diagnostic methods relying on the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). This review compiled recent progress on the development of novel plasmonic sensing schemes for the effective control of virus-related diseases. A special focus was set on the utilization of plasmonic nanostructures in combination with other detection formats involving colorimetric, fluorescence, luminescence, or Raman scattering enhancement. The quantification of different viruses (e.g., hepatitis virus, influenza virus, norovirus, dengue virus, Ebola virus, Zika virus) with particular attention to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was reviewed from the perspective of the biomarker and the biological receptor immobilized on the sensor chip. Technological limitations including selectivity, stability, and monitoring in biological matrices were also reviewed for different plasmonic-sensing approaches.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Ressonância de Plasmônio de Superfície/métodos , Betacoronavirus/patogenicidade , Colorimetria , Infecções por Coronavirus/virologia , Fluorescência , Humanos , Nanoestruturas/química , Pandemias , Pneumonia Viral/virologia , Análise Espectral Raman
7.
Virus Res ; 288: 198129, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32822689

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.


Assuntos
Betacoronavirus/genética , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/métodos , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Técnicas de Laboratório Clínico/instrumentação , Colorimetria/instrumentação , Infecções por Coronavirus/virologia , Endodesoxirribonucleases/química , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Reologia , Sensibilidade e Especificidade
8.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751106

RESUMO

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.


Assuntos
Betacoronavirus/genética , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/economia , Pneumonia Viral/diagnóstico , RNA Viral/análise , Betacoronavirus/isolamento & purificação , Colorimetria/economia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/metabolismo , Carga Viral
9.
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
10.
PLoS One ; 15(7): e0235532, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614905

RESUMO

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.


Assuntos
Centrômero/genética , Colorimetria/métodos , Pichia/genética , Plasmídeos/análise , DNA Fúngico/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
11.
J Environ Radioact ; 220-221: 106299, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32658643

RESUMO

Based on the fact that uranyl ions (UO22+) adsorbed on GO can enhanced the peroxidase-like activity of graphene oxide (GO), a novel colorimetric strategy for visualizing quantitative determination of uranyl ions was established. The peroxidase-like activity of GO-UO22+ nanocomposites was assessed by catalyzing H2O2 oxidation of TMB to produce a distinct color reaction. A good linearity between the UO22+ concentration and absorption at 652 nm was acquired in the range of 5.90 × 10-6 to 9.43 × 10-4 M with a detection limit of 4.70 µM. This strategy was also successfully applied to determination of uranyl ions in environmental water samples.


Assuntos
Colorimetria , Adsorção , Grafite , Peróxido de Hidrogênio , Íons , Limite de Detecção , Peroxidase , Peroxidases , Monitoramento de Radiação
12.
Sci Transl Med ; 12(556)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32719001

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Colorimetria/métodos , Colorimetria/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Proteínas do Nucleocapsídeo/genética , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , RNA-Seq , Sensibilidade e Especificidade , Pesquisa Médica Translacional
13.
Food Chem ; 333: 127343, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32663746

RESUMO

Leuconostoc spp. are generally utilized as kimchi starters because of their beneficial effects on kimchi fermentation and sensory characteristics. We developed a DNAzyme-based colorimetric method for measuring the abundance of the kimchi starter Leuconostoc mesenteroides WiKim32. A primer set for loop-mediated isothermal amplification and target-specific DNAzyme was designed based on the WiKim32 nucleotide sequence. In the presence of the target amplicon, DNAzyme bound to it, resulting in negligible amounts of green product. In contrast, with the addition of hemin and in the absence of the target amplicon, DNAzyme fragments not bound to the target amplicon formed G-quadruplex-hemin conjugates, generating a visible green product by oxidizing 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt. There was no cross-reaction with other strains. The method had high detection sensitivity and quantitative capacity in kimchi samples without a requirement for DNA isolation. This strategy provides a rapid, sensitive, and simple detection method with possible industrial applications.


Assuntos
DNA Catalítico/metabolismo , Fermentação , Alimentos e Bebidas Fermentados/microbiologia , Leuconostoc mesenteroides/isolamento & purificação , Leuconostoc mesenteroides/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Colorimetria , DNA Catalítico/química , Quadruplex G , Hemina/química , Leuconostoc mesenteroides/genética , Especificidade da Espécie
14.
Food Chem ; 332: 127431, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32645668

RESUMO

Illegal usage of ß-agonists as the animal growth promoters can lead to multiple harmful impacts to public health, thus detection of ß-agonists at trace level in complex sample matrixes is of great importance. In recent years, emergence of advanced nanomaterials greatly facilitates the advancement of sensors in terms of sensitivity, specificity and robustness. Plenty of nanoparticles-based sensors have been developed for ß-agonists determination. In this review, we comprehensively summarized the construction of emerging nanoparticles-based sensors (including colorimetric sensors, fluorescent sensors, chemiluminescent sensors, electrochemical sensors, electrochemiluminescent sensors, surface enhanced Raman scattering sensors, surface plasmon resonance sensors, quartz crystal microbalance sensors, etc.), and nanomaterial-based enzyme-linked immunosorbent assay (nano-ELISA). Impressively, the applications of nanoparticles-based sensors and nano-ELISAs in the detection of ß-agonists have also been summarized and discussed. In the end, future opportunities and challenges in the design construction of nanoparticles (NPs)-based sensors and their applications in ß-agonist assay are tentatively proposed.


Assuntos
Agonistas Adrenérgicos/análise , Nanoestruturas/química , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Colorimetria/instrumentação , Colorimetria/métodos , Humanos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos
15.
Biotechniques ; 69(3): 178-185, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32635743

RESUMO

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Guanidina , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Laboratório Clínico/normas , Colorimetria , Infecções por Coronavirus/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Fenolsulfonaftaleína
16.
Food Chem ; 332: 127392, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32623126

RESUMO

The present work describes a novel and rapid approach for evaluating total phenolic compounds (TPCs) in tea and fruits using colorimetric spots and the digital image-based (DIB) method. Colorimetric spots were formed by reacting diazotized aminobenzenes namely sulfanilic acid, sulfanilamide, or aniline with TPCs in the extract to form an azo dye. The limit of detection (LOD) was 6.5, 5.5, or 5.1 mg GAE (gallic acid equivalent) L-1 and the analytical range was 25-500, 20-500, or 18-200 mg GAE L-1, respectively. Correlation with the Folin-Ciocalteu assay was significant (Pearson coefficient, R = 0.970-0.991) while the antioxidant activity assay was moderate to high (R = 0.737-0.977). The method developed was successfully applied to the analysis of tea and fruits and showed RSD (n = 3) not exceeding 9.6, 8.5, and 9.7%, respectively. Ecologically, the DIB method developed could determine the variation of TPCs within cultivars and was found to be strongly dependent on the growing environment.


Assuntos
Benzeno/química , Colorimetria/métodos , Análise de Alimentos/métodos , Frutas/química , Fenóis/análise , Chá/química , Antioxidantes/análise , Limite de Detecção
17.
PLoS Negl Trop Dis ; 14(6): e0008364, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32492018

RESUMO

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera.


Assuntos
Colorimetria/métodos , Testes Diagnósticos de Rotina/métodos , Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/diagnóstico , Febre Hemorrágica da Crimeia/virologia , Replicação Viral , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Humanos , Imunoglobulina G/sangue , Modelos Moleculares , Nucleoproteínas/química , Nucleoproteínas/genética , Conformação Proteica
18.
Food Chem ; 328: 127099, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32474238

RESUMO

In this study, we developed a competitive colorimetric immunoassay for qualitative detection of DAN based on oxidation of iron (Ⅱ) (Fe2+) in the presence of glucose oxidase (GOx) and color change induced by Fe2+-phenanthroline (Phen) chromogenic system. Streptavidin (SA) acted as a linker between biotinylated anti-DAN-monoantibody (bio-mAb) and biotinylated GOx (bio-GOx) to form the immunocomplexes bio-mAb-SA-bio-GOx. In the absence of DAN, the immunocomplexes bio-mAb-SA-bio-GOx combining with coated DAN-ovalbumin (DAN-OVA) will be immobilized and catalyze glucose to produce H2O2. Fe2+ is oxidized to Fe3+ by H2O2, giving rise to a colorless result. In the presence of DAN, Fe2+ produces a chelation reaction with Phen, leading to orange-red color. Under optimal conditions, the detection limit (LOD) by naked eyes was 2.5 ng mL-1 in milk, chicken, beef, and pork samples. Low LOD, no matrix effect, and no signal reader requirement make it possibly applied to quickly screen DAN on site.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , Fluoroquinolonas/análise , Glucose Oxidase/metabolismo , Imunoensaio/métodos , Quelantes de Ferro/química , Fenantrolinas/química , Biocatálise , Catálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fluoroquinolonas/química , Glucose/química , Glucose Oxidase/química , Peróxido de Hidrogênio/química , Limite de Detecção
19.
Exp Parasitol ; 216: 107932, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32535113

RESUMO

Neglected tropical diseases, such as Chagas disease caused by the protozoa Trypanosoma cruzi, affect millions of people worldwide but lack effective treatments that are accessible to the entire population, especially patients with the debilitating chronic phase. The recognition of host cells, invasion and its intracellular replicative success are essential stages for progression of the parasite life cycle and the development of Chagas disease. It is predicted that programmed cell death pathways (apoptosis) would be activated in infected cells, either via autocrine secretion or mediated by cytotoxic immune cells. This process should play a key role in resolving infections by hindering the evolutionary success of the parasite. In this research, we performed assays to investigate the role of the lectin galectin-3 (Gal3) in parasite-host signaling pathways. Using cells with endogenous levels of Gal3 compared to Gal3-deficient cells (induced by RNA interference), we demonstrated that T. cruzi mediated the survival pathways and the subverted apoptosis through Gal3 promoting a pro-survival state in infected cells. Infected Gal3-depleted cells showed increased activation of caspase 3 and pro-apoptotic targets, such as poly (ADP-ribose) polymerase (PARP), and lower accumulation of anti-apoptotic proteins, such as c-IAP1, survivin and XIAP. During the early stages of infection, Gal3 translocates from the cytoplasm to the nucleus and must act in survival pathways. In a murine model of experimental infection, Gal3 knockout macrophages showed lower infectivity and viability. In vivo infection revealed a lower parasitemia and longer survival and an increased spleen cellularity in Gal3 knockout mice with consequences on the percentage of T lymphocytes (CD4+ CD11b+) and macrophages. In addition, cytokines such as IL-2, IL-4, IL-6 and TNF-α are increased in Gal3 knockout mice when compared to wild type genotype. These data demonstrate a Gal3-mediated complex interplay in the host cell, keeping infected cells alive long enough for infection and intracellular proliferation of new parasites. However, a continuous knowledge of these signaling pathways should contribute to a better understanding the mechanisms of cell death subversion that are promoted by protozoans in the pathophysiology of neglected diseases such as Chagas disease.


Assuntos
Apoptose/fisiologia , Doença de Chagas/parasitologia , Galectina 3/fisiologia , Trypanosoma cruzi/fisiologia , Análise de Variância , Animais , Western Blotting , Caspase 3/análise , Sobrevivência Celular , Doença de Chagas/mortalidade , Chlorocebus aethiops , Colorimetria , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Galectina 3/análise , Galectina 3/genética , Células HeLa , Humanos , Imunofenotipagem , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parasitemia/mortalidade , Parasitemia/parasitologia , Fenótipo , Baço/patologia , Células Vero
20.
Food Chem ; 329: 127224, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32516716

RESUMO

To overcome the drawbacks of antibody labeling dependence and single-readout system in the conventional lateral flow immunoassays (LFIAs) as well as the non-targeted combination of new capture agents reported recently for pathogen detection, in this work, a multi-readout and label-free LFIA was proposed for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) based on a nanozyme-bacteria-antibody sandwich pattern. A type of functional nanozyme-mannose modified Prussian blue (man-PB), was introduced as the recognition agent as well as signal indicator. Apart from original signal intensity on the T-line, the peroxidase-like catalytic activity-driven generation of colorimetric signal could be used as another format of quantitation. Importantly, such LFIA could exhibit excellent performance for target pathogens detection separately with a quantitative range of 102-108 cfu·mL-1 and a low detection limit of 102 cfu·mL-1 based on different readout formats, indicating the application potential of the proposed LFIA in real samples.


Assuntos
Escherichia coli O157/isolamento & purificação , Imunoensaio , Nanopartículas/química , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Catálise , Colorimetria , Escherichia coli O157/imunologia , Ferrocianetos/química , Microbiologia de Alimentos , Limite de Detecção , Manose/química , Nanopartículas/metabolismo
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