RESUMEN
Synergistic dual-mode optical platforms are up-and-coming detection tools in the diagnosis and management of infectious diseases. Here, novel dual-modality fluorescence (FL) and surface-enhanced Raman scattering (SERS) techniques have been integrated into a single probe for the rapid and ultrasensitive detection of norovirus (NoV). The developed FL-SER-based biosensor relies on the dual-signal enhancements of newly synthesized sulfur-doped agar-derived carbon dots (S-agCDs). The antigen-antibody immunoreaction results in forming a core-satellite immunocomplex between anti-NoV antibody-conjugated S-agCDs and polydopamine-functionalized magnetic silver nanocubes [poly (dop)-MNPs-Ag NCs]. By deploying an immunomagnetic enrichment protocol and performing the SERS modality on a single-layer graphene substrate, norovirus-like particles (NoV-LPs) were detected across a wide range of 1 fg mL-1 - 10 ng mL-1 with an excellent limit of detection of 0.1 fg mL-1. The combined advantage of the dual-signaling properties of the biosensor was demonstrated using FL confocal imaging for "hotspots" tracking prior to SERS detection of clinical NoV in fecal specimen down to â10 RNA copies mL-1. The proposed dual-modality biosensor's performance increases the prospect of a rapid and low-cost sensitive NoV detection and surveillance option for public health.
Asunto(s)
Técnicas Biosensibles , Nanopartículas del Metal , Norovirus , Carbono , Indoles , Límite de Detección , Fenómenos Magnéticos , Polímeros , Plata , Espectrometría Raman , AzufreRESUMEN
The use of nanogels (NGs) to modulate surface-enhanced Raman scattering (SERS) activities is introduced as an innovative strategy to address certain critical issues with SERS-based immunoassays. This includes the chemical deformation of SERS nanotags, as well as their nonspecific interactions and effective "hotspots" formation. Herein, the polymeric cocoon and stimuli-responsive properties of NGs were used to encapsulate SERS nanotags containing plasmonic molybdenum trioxide quantum dots (MoO3-QDs). The pH-controlled release of the encapsulated nanotags and their subsequent localization by maleimide-functionalized magnetic nanoparticles facilitated the creation of "hotspots" regions with catalyzed SERS activities. This approach resulted in developing a biosensing platform for the ultrasensitive immunoassays of hepatitis E virus (HEV) or norovirus (NoV). The immunoassays were optimized using the corresponding virus-like particles to attain limits of detection of 6.5 and 8.2 fg/mL for HEV-LPs and NoV-LPs, respectively. The SERS-based technique achieved a signal enhancement factor of up to â¼108 due to the combined electromagnetic and chemical mechanisms of the employed dual-SERS substrate of MoO3-QDs/2D hexagonal boron nitride nanosheets. The highlight and validation of the developed SERS-based immunoassays was the detection of NoV in infected patients' fecal specimen and clinical HEV G7 subtype. Importantly, this system can be used to maintain the stability of SERS nanotags and improve their reliability in immunoassays.
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Virus de la Hepatitis E/aislamiento & purificación , Molibdeno/química , Nanogeles/química , Norovirus/aislamiento & purificación , Óxidos/química , Puntos Cuánticos/química , Anticuerpos Inmovilizados/inmunología , Técnicas Biosensibles/métodos , Virus de la Hepatitis E/inmunología , Humanos , Concentración de Iones de Hidrógeno , Inmunoensayo , Límite de Detección , Fenómenos Magnéticos , Nanopartículas/química , Norovirus/inmunología , Reproducibilidad de los Resultados , Espectrometría RamanRESUMEN
Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1-V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.
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Proteínas Bacterianas/genética , Biodiversidad , ADN Bacteriano/genética , Ehrlichia/fisiología , Ehrlichiosis/diagnóstico , Garrapatas/microbiología , Animales , ADN Bacteriano/análisis , Ehrlichiosis/genética , Ehrlichiosis/parasitología , Humanos , Japón , FilogeniaRESUMEN
Non-pathogenic Rickettsia species LON strains closely related to an agent of Japanese spotted fever (JSF), R. japonica, were isolated in Japan from Haemaphysalis longicornis ticks in 2001. However, the biological properties of LONs in mammalian host cells are poorly understood. In this study, microscopic analysis showed that LONs in a mouse-derived L929 host cell line were rod shaped with sizes of 0.3-0.5 × 0.5-2.0 µm. Molecular analysis revealed the existence of a LON-specific disrupted open reading frame in R. japonica-related group-specific DNA regions. Growth kinetics of LON-2 and LON-13 strains analyzed by a quantitative real-time PCR showed 100-fold or more increment of LONs cultured in L929 host cells at 30°C and slightly less increment at 33°C, and 25-fold increment in human-derived THP-1 host cells at 35°C on day 7 (168 h) post infection. The generation times of the two LON strains cultured in L929 and THP-1 were estimated to be 9.4-12.9 h and 9.6-10.9 h, respectively. To our knowledge, this is the first report on the biological characteristics of Rickettsia sp. LON strains in mammalian cells, which may provide significant information for the experimental approaches for other rickettsiae.
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Rickettsia/genética , Rickettsiosis Exantemáticas/microbiología , Garrapatas/microbiología , Animales , Línea Celular , ADN Bacteriano/aislamiento & purificación , Humanos , Ixodidae/microbiología , Japón , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Rickettsia/aislamiento & purificación , Células THP-1RESUMEN
In this report, we have examined the distance- and size-dependent localized surface plasmon resonance (LSPR) between fluorescent quantum dots (QDs) and adjacent gold nanoparticles (AuNPs) to provide a comprehensive evaluation, aiming for practical application in biosensing platform. A series of peptides with different chain lengths, connected between QDs and AuNPs is initially applied to prepare various CdSe QDs-peptide-AuNP systems to optimize LSPR signal. Separation distance between two nanoparticles of these systems before and after conjugation is also confirmed by quantum mechanical modeling and corroborated with their LSPR influenced fluorescence variations. After detailed optimizations, it can be noted that larger sized AuNPs make strong quenching of QDs, which gradually shows enhancement of fluorescence with the increment of distance and the smaller sized AuNPs. Depending on the requirement, it is possible to tune the optimized structure of the CdSe QD-peptide-AuNP nanostructures for the application. In this work, two different structural designs with different peptide chain length are chosen to construct two biosensor systems, observing their fluorescence enhancement and quenching effects, respectively. Using different structural orientation of these biosensors, two nanoconjugates has applied for detection of norovirus and influenza virus, respectively to confirm their application in sensing.
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Técnicas Biosensibles , Nanopartículas del Metal , Oro , Nanoconjugados , Resonancia por Plasmón de SuperficieRESUMEN
Combination of magnetic nanomaterials with multifunctionality is an emerging class of materials that exhibit tremendous potential in advanced applications. Synthesizing such novel nanocomposites without compromising magnetic behavior and introducing added functional properties is proven challenging. In this study, an optically active quantum dot (QD) (core) encapsulated inside iron oxide (hollow shell) is prepared as the first electrochemical/fluorescence dual-modality probe. Presence of magnetic layer on the surface enables excellent magnetic property and the encapsulating of QDs on the hollow shell structure maintains the fluorescence with minimal quenching effect, endowing for potential application with fluorescence modality readout. We successfully demonstrate dual-modality sensing utilizing of QD-encapsulated magnetic hollow sphere nanoparticles (QD@MHS NPs) with magnetic separation ability and highly integrated multimodal sensing for the detection of various viruses including hepatitis E virus (HEV), HEV-like particles (HEV-LPs), norovirus-like particles (NoV-LPs), and norovirus (NoV) from clinical specimens. Most importantly, fecal samples of HEV-infected monkey are successfully diagnosed with sensitivity similar to gold standard real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). This well-defined QD@MHS NPs-based nanoplatform intelligently integrates dual-modality sensing and magnetic bio-separation, which open a gateway to provide an efficient point-of care testing for virus diagnostics.
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Técnicas Biosensibles , Nanopartículas , Puntos Cuánticos , Colorantes Fluorescentes , MagnetismoRESUMEN
A novel biosensing system based on graphene-mediated surface-enhanced Raman scattering (G-SERS) using plasmonic/magnetic molybdenum trioxide nanocubes (mag-MoO3 NCs) has been designed to detect norovirus (NoV) via a dual SERS nanotag/substrate platform. A novel magnetic derivative of MoO3 NCs served as the SERS nanotag and the immunomagnetic separation material of the biosensor. Single-layer graphene oxide (SLGO) was adopted as the 2D SERS substrate/capture platform and acted as the signal reporter, with the ability to accommodate an additional Raman molecule as a coreporter. The developed SERS-based immunoassay achieved a signal amplification of up to â¼109-fold resulting from the combined electromagnetic and chemical mechanisms of the dual SERS nanotag/substrate system. The developed biosensor was employed for the detection of NoV in human fecal samples collected from infected patients by capturing the virus with the aid of NoV-specific antibody-functionalized magnetic MoO3 NCs. This approach enabled rapid signal amplification for NoV detection with this biosensing technology. The biosensor was tested and optimized using NoV-like particles within a broad linear range from 10 fg/mL to 100 ng/mL and a limit of detection (LOD) of â¼5.2 fg/mL. The practical applicability of the developed biosensor to detect clinical NoV subtypes in human fecal samples was demonstrated by effective detection with an LOD of â¼60 RNA copies/mL, which is â¼103-fold lower than that of a commercial enzyme-linked immunosorbent assay kit for NoV.
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Técnicas Biosensibles , Grafito/química , Molibdeno/química , Nanopartículas/química , Norovirus/aislamiento & purificación , Óxidos/química , Tamaño de la Partícula , Espectrometría Raman , Propiedades de SuperficieRESUMEN
Sensitive and accurate detection methods for infectious viruses are the pressing need for effective disease diagnosis and treatment. Herein, based on V2O5 nanoparticles-encapsulated liposomes (VONP-LPs) we demonstrate a dual-modality sensing platform for ultrasensitive detection of the virus. The sensing performance relies on intrinsic peroxidase and electrochemical redox property of V2O5 nanoparticles (V2O5 NPs). The target-specific antibody-conjugated VONP-LPs and magnetic nanoparticles (MNPs) enrich the virus by magnetic separation and the separated VONP-LPs bound viruses are hydrolyzed to release the encapsulated V2O5 NPs. These released nanoparticles from captured liposomes act as peroxidase mimics and electrochemical redox indicator resulting in noticeable colorimetric and robust electrochemical dual-signal. Utilizing the superiority of dual-modality sensor with two quantitative analysis forms, norovirus like particles (NoV-LPs) can be detected by electrochemical signals with a wide linear range and low detection limit. To verify the applicability in real samples, norovirus (NoV) collected from actual clinical samples are effectively-identified with excellent accuracy. This proposed detection method can be a promising next-generation bioassay platform for early-stage diagnosis of virus disease and surveillance for public health.
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Técnicas Biosensibles/métodos , Liposomas/química , Norovirus/aislamiento & purificación , Compuestos de Vanadio/química , Infecciones por Caliciviridae/diagnóstico , Infecciones por Caliciviridae/virología , Colorimetría/métodos , Técnicas Electroquímicas/métodos , Humanos , Límite de Detección , Nanopartículas/química , Oxidación-ReducciónRESUMEN
Viral diseases are one of the most life-threatening diseases as they can erupt unpredictably and spread rapidly in any medium with a very small number of particles. Therefore, the key for lethal virus detection should be highly sensitive in the early-stage detection, which can help increase the chance of survival. Amplification of the detecting signal is one of the most promising mechanisms for the detection of low-concentration analytes. A proper amplification can develop such a kind of system where a small number of particles can produce intense signals for a prominent detection. Keeping this in mind, in this report, we have presented a fluorometric method to detect norovirus (NoV) by a newly developed fluorophore-labeled liposome and a magnetically modified Fe3O4 combined system. Homogeneously distributed amine-functionalized liposomes have been constructed filled with a strong fluorophore of calcein. Simultaneously, (3-aminopropyl)-triethoxysilane (APTES)-functionalized Fe3O4 nanoparticles are also synthesized by the standard silanization process, and these two separately synthesized nanoparticles were functionalized with an antibody to achieve specificity. The Fe3O4 and calcein-liposome system has been applied for NoV detection, which was magnetically separated from the analyte medium and then externally burst to release the fluorophores from the core of the liposome. The easiness, rapidity, and sensitivity in a wide linear range can offer a huge potential of this method in point-of-care diagnostics.
RESUMEN
Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum. Indirect immunofluorescence assay (IFA) is generally used for HGA serodiagnosis. A. phagocytophilum immunodominant P44 major outer membrane proteins are encoded by p44/msp2 multigene family, responsible for IFA reactivity. However, because multiple P44-related proteins may involve immunoreactivity in IFA, the available diagnostic antigens remain obscure. In this study, we identified 12 B-cell epitopes on triple P44-related proteins using peptide array that reacted with 4 HGA patients' sera. Then, peptide spot immunoassay using 14 synthetic peptides derived from those 12 epitopes as antigens was applied for the detection of antibody to A. phagocytophilum from patients with fever of unknown origin. The sensitivities and diagnostic efficiencies of this immunoassay were higher than those of Western blot analysis using 3 recombinant proteins previously developed. Thus, the immunoassay using our epitope-derived antigens, which has higher diagnostic performances, may have significant benefit for HGA serodiagnosis.
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Anaplasma phagocytophilum/inmunología , Anaplasmosis/diagnóstico , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Epítopos de Linfocito B/inmunología , Inmunoensayo/métodos , Secuencia de Aminoácidos , Anaplasma phagocytophilum/aislamiento & purificación , Anaplasmosis/sangre , Anaplasmosis/microbiología , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Epítopos de Linfocito B/química , Humanos , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
Anaplasma phagocytophilum is an obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease. This bacterium expresses various 44-kDa major outer membrane proteins encoded by the p44/msp2 multigene family to avoid the host immune system. We previously detected A. phagocytophilum p44/msp2 from the tick Haemaphysalis longicornis in Mie Prefecture, Japan in 2008. In this study, we further investigated a total of 483 H. longicornis ticks (220 adults and 263 nymphs) collected from the Mie Prefecture by PCR targeting p44/msp2 to characterize the p44/msp2 multigene family of A. phagocytophilum. Six of the 483 ticks tested were PCR-positive for A. phagocytophilum p44/msp2, and these positive individuals were at the nymph stage of the tick life cycle. Cloning, sequencing, and phylogenetic analyses of the amplicons revealed that the 11 p44/msp2 clones obtained from the positive ticks shared a 54.9%-99.3% amino acid sequence similarity with the 27 previously identified clones from HGA patients in Japan. In particular, 6 p44/msp2 clones displayed the highest similarities (97.2%-99.3%) with 3 previously identified clones (FJ417343, FJ417345, FJ417357). Thus, the data from this study provide important public health information regarding A. phagocytophilum infection transmitted by H. longicornis ticks, especially at the nymph stage.
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Anaplasma phagocytophilum/genética , Garrapatas/microbiología , Secuencia de Aminoácidos , Anaplasma phagocytophilum/aislamiento & purificación , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Variación Genética , Familia de Multigenes , Receptor 2 Gatillante de la Citotoxidad Natural , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
Norovirus (NoV) is a leading cause of acute gastroenteritis. The low infectious dose and environmental stability of NoV facilitate its effective transmission through a variety of modes such as food, water and person-to-person. The available enzyme-linked immunosorbent assay (ELISA) for NoV detection has low sensitivity due to the low catalytic activity of the peroxidase used, and thus, a reliable ultrasensitive bioassay is needed. In this study, we apply the enhanced peroxidase-like activity of silver ion-incorporated gold nanoparticles (Au/Ag NPs) in a colorimetric bioassay for NoV detection. NoV was captured by anti-NoV genogroup II antibodies, which were immobilized on the surface of a 96-well microtiter plate and formed a sandwich structure among anti-NoV Ab, NoV and Ab-Au NP. Then, Ag ion-containing hydroquinone was added to form Au/Ag core/shell NPs. When H2O2/3,3',5,5'-tetramethylbenzidine (TMB) solution was added to the wells, Ag ions were liberated from the surface of Au/Ag NPs and enhanced the oxidation of TMB. These reactions enhanced the oxidation of TMB and developed an intense blue color. The Au/Ag NPs were shown to exhibit higher affinity and catalytic efficiency for H2O2 and higher catalytic velocity based on the kcat of up to 7-fold compared with Au NPs. The bioassay was then optimized to detect clinically isolated NoV using NoV-like particles (NoV-LPs). NoV-LPs were detected with a limit of detection of 10.8â¯pg/mL, corresponding to 1000- and 100-fold higher sensitivity compared to the gold-immunoassay and horseradish peroxidase-based ELISA, respectively. Clinically isolated NoV GII.4 and NoV GII.3 were detected in the range of 102-106 copies of viral RNA/mL fecal solution with a detection limit of 13.2 copies/mL fecal solution for NoV GII.4, equivalent to 132 copies of viral RNA/g feces and indicating significantly higher sensitivity compared to commercial immunoassay kits. This bioassay represents a workable detection assay for low concentrations of NoV that is applicable for early-stage diagnosis for public hygiene.
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Técnicas Biosensibles , Colorimetría , Inmunoensayo , Norovirus/aislamiento & purificación , Catálisis , Oro/química , Peroxidasa de Rábano Silvestre/química , Peróxido de Hidrógeno/química , Límite de Detección , Nanopartículas del Metal/química , Norovirus/patogenicidad , Plata/químicaRESUMEN
A new method of label free sensing approach with superior selectivity and sensitivity towards virlabel-freeon is presented here, employing the localized surface plasmon resonance (LSPR) behavior of gold nanoparticles (AuNPs) and fluorescent CdSeTeS quantum dots (QDs). Inorganic quaternary alloyed CdSeTeS QDs were capped with L-cysteine via a ligand exchange reaction. Alternatively, citrate stabilized AuNPs were functionalized with 11-mercaptoundecanoic acid to generate carboxylic group on the gold surface. The carboxylic group on the AuNPs was subjected to bind covalently with the amine group of L-cysteine capped CdSeTeS QDs to form CdSeTeS QDs/AuNPs nanocomposites. The fluorescence of CdSeTeS QDs/AuNPs nanocomposite shows quenched spectrum of CdSeTeS QDs at 640â¯nm due to the close interaction with AuNPs. However, after successive addition of norovirus-like particles (NoV-LPs), steric hindrance-induced LSPR signal from the adjacent AuNPs triggered the fluorescence enhancement of QDs in proportion to the concentration of the target NoV-LPs. A linear range of 10-14 to 10-9 gâ¯mL-1 NoV-LPs with a detection limit of 12.1â¯×â¯10-15 gâ¯mL-1 was obtained. This method was further applied on clinically isolated norovirus detection, in the range of 102-105 copies mL-1 with a detection limit of 95.0 copies mL-1, which is 100-fold higher than commercial ELISA kit. The superiority of the proposed sensor over other conventional sensors is found in its ultrasensitive detectability at low virus concentration even in clinically isolated samples. This proposed detection method can pave an avenue for the development of high performance and robust sensing probes for detection of virus in biomedical applications.
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Compuestos de Cadmio/química , Infecciones por Caliciviridae/diagnóstico , Oro/química , Nanopartículas del Metal/química , Norovirus/aislamiento & purificación , Puntos Cuánticos/química , Compuestos de Selenio/química , Resonancia por Plasmón de Superficie/métodos , Infecciones por Caliciviridae/virología , Heces/virología , Humanos , Límite de Detección , Resonancia por Plasmón de Superficie/economía , Telurio/químicaRESUMEN
Japanese encephalitis (JE) is an acute viral disease caused by the Japanese encephalitis virus (JEV). JEV strains are classified into 5 genotypes (I-V). JEV genotype V strains have never been detected in Japan to date, but they were recently detected in South Korea. In the present analysis, we tried to determine if a JEV genotype V strain caused any JE case in Japan in 2016. Serum and cerebrospinal fluid samples were collected from 10 JE patients reported in Japan in 2016. JEV RNA was not detected in any of the samples. Although JEV is a single-serotype virus, it can be expected that the neutralizing antibody titers against JEV genotype V strains are higher than those against genotype I and III strains in the serum of patients with JE in Japan whose causative JEV was the genotype V strain. The neutralizing antibody titers against the JEV genotype V strain were not higher than those against the genotype I or III strain in any serum samples. Therefore, the evidence that the JEV genotype V strain caused any JE case in Japan in 2016 was absent.