Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.187
Filtrar
1.
J Chem Phys ; 151(14): 144712, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615232

RESUMO

Electrogenerated chemiluminescence (ECL) is a promising technique for low concentration molecular detection. To improve the detection limit, plasmonic nanoparticles have been proposed as signal boosting antennas to amplify ECL. Previous ensemble studies have hinted that spectral overlap between the nanoparticle antenna and the ECL emitter may play a role in signal enhancement. Ensemble spectroscopy, however, cannot resolve heterogeneities arising from colloidal nanoparticle size and shape distributions, leading to an incomplete picture of the impact of spectral overlap. Here, we isolate the effect of nanoparticle-emitter spectral overlap for a model ECL system, coreaction of tris(2,2'-bipyridyl)dichlororuthenium(ii) hexahydrate and tripropylamine, at the single-particle level while minimizing other factors influencing ECL intensities. We found a 10-fold enhancement of ECL among 952 gold nanoparticles. This signal enhancement is attributed exclusively to spectral overlap between the nanoparticle and the emitter. Our study provides new mechanistic insight into plasmonic enhancement of ECL, creating opportunities for low concentration ECL sensing.


Assuntos
Nanopartículas Metálicas/química , Compostos Organometálicos/química , Propilaminas/química , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Ouro/efeitos da radiação , Luz , Luminescência , Medições Luminescentes/métodos , Nanopartículas Metálicas/efeitos da radiação , Compostos Organometálicos/efeitos da radiação
2.
Anal Bioanal Chem ; 411(23): 6067-6080, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273413

RESUMO

Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 µm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17-1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens. Graphical abstract.


Assuntos
Contaminação de Alimentos/análise , Medições Luminescentes/métodos , Leite/microbiologia , Aves Domésticas/microbiologia , Salmonella/isolamento & purificação , Animais , Galinhas/microbiologia , Contaminação de Alimentos/economia , Inocuidade dos Alimentos/métodos , Imunoensaio/economia , Imunoensaio/métodos , Separação Imunomagnética/economia , Separação Imunomagnética/métodos , Limite de Detecção , Medições Luminescentes/economia , Infecções por Salmonella/microbiologia , Fatores de Tempo
3.
Anal Bioanal Chem ; 411(23): 6049-6056, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31280477

RESUMO

As an important glycoprotein of the lectin family, soybean agglutinin (SBA) is an anti-nutritional factor with considerable toxic and side effects and plays a significant role in tumor analysis. In order to achieve the sensitive detection of SBA, a sandwich-structured electrochemiluminescence (ECL) biosensor was constructed using carboxylated carbon nitride (C-g-C3N4) as luminophore and D-galactosamine (galM) as a recognition element. A glassy carbon electrode (GCE) was modified with Au nanoparticles (Au NPs) for capturing the galM via Au-N bond, and further capturing the target SBA by specific recognition between galM and SBA. In the presence of SBA, the composite C-g-C3N4-galM was immobilized onto the electrode. With the increase in the concentration of SBA, the ECL signal from C-g-C3N4 increased, thus achieving a signal-on detection of SBA. The linear range of the biosensor was 1.0 ng/mL~10 µg/mL and detection limit for SBA was as low as 0.33 ng/mL. In this construction strategy, C-g-C3N4 not only acted as an excellent signal probe, but also as an immobilization matrix to easily achieve a high loading of the small molecule recognition element galM. This strategy provides a simple alternative SBA detection platform. Graphical abstract.


Assuntos
Galactosamina/química , Grafite/química , Substâncias Luminescentes/química , Nitrilos/química , Lectinas de Plantas/análise , Proteínas de Soja/análise , Técnicas Biossensoriais/métodos , Ácidos Carboxílicos/química , Técnicas Eletroquímicas/métodos , Ouro/química , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Nanopartículas Metálicas/química , Lectinas de Plantas/sangue , Proteínas de Soja/sangue
4.
BMC Infect Dis ; 19(1): 646, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324234

RESUMO

BACKGROUND: The aim of this study was to establish a set of assessment methods suitable for evaluating the complex indoor environment of hospital wards and to ascertain the composition of bacteria and microbial ecology of hospital wards. METHODS: Colony-forming units (CFUs), PM2.5 detection, real-time PCR, and adenosine triphosphate (ATP) bioluminescence assay were employed to evaluate the complexity of indoor air in 18 wards of nine departments in a hospital and two student dormitories in a university. Subsequently, the microbial samples were quantified and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Although the studied indices were relatively independent, the PM2.5 content was correlated with bacterial CFUs determined by passive sedimentation method, bacterial and fungal counts measured by real-time PCR, and ATP bioluminescence assay. The composition of microorganisms in the air of hospital wards differed from that in the air of student dormitories. The dominant genera in hospital wards were Staphylococcus (39.4%), Micrococcus (21.9%), Corynebacterium (11.7%), Kocuria (4.4%), Bacillus (2.9%), Streptococcus (1.6%), Moraxella (1.6%), and Enterococcus (1.3%), and the microbial ecology differed between Respiration Dept. III and other hospital departments. Additionally, 11.1 and 27.3% of bacteria in hospital wards and student dormitories were not identified, respectively. CONCLUSIONS: Assessment of environmental quality of hospital wards should be based on comprehensive analysis with multiple indicators. There may be imbalances in the microbial diversity in the hospital wards, therefore, monitoring of the environmental quality of hospitals is important in the prevention of nosocomial infections.


Assuntos
Microbiologia do Ar , Poluição do Ar em Ambientes Fechados/análise , Hospitais , Medições Luminescentes/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trifosfato de Adenosina/análise , Carga Bacteriana/métodos , China , Contagem de Colônia Microbiana/métodos , Humanos , Material Particulado/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus
5.
Food Chem ; 298: 125066, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31261009

RESUMO

A ultrasensitive chemiluminescence (CL) method for the determination of fluoroquinolones (FQs) in milk was proposed based on the enhancement effect of FQs on the CL reaction between cerium(IV) and methoxylated Cypridina luciferin analogs (MCLA). Prior to the CL determination, molecularly imprinted polymers (MIPs) with superior recognition performances were adopted to the clean-up and extraction of the family of FQs. Then the developed CL system coupling with MIPs (MIP-CL) was applied to the selective determination of FQs in milk. Under the optimized conditions, the method was validated with respect to linearity, precision, accuracy, limits of detection and quantification. The relative standard deviation (RSD) on intra-day assay was below 5.1%, and detection limit was as low as 0.10 nmol/L. The consistency of this method and HPLC method was compared and validated by the paired t-test. The results indicated that the proposed method allowed class-specific determination of FQs in complex matrices.


Assuntos
Fluoroquinolonas/análise , Medições Luminescentes/métodos , Impressão Molecular/métodos , Polímeros/química , Animais , Cério/química , Fluoroquinolonas/isolamento & purificação , Limite de Detecção , Leite/química , Extração em Fase Sólida
6.
Int J Nanomedicine ; 14: 4293-4307, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354261

RESUMO

Purpose: Antibodies are key reagents in the development of immunoassay. We attempted to develop high-performance CPP immunoassays using high-affinity monoclonal antibodies prepared via cytokine-assisted immunization. Methods: We used fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to assist traditional subcutaneous immunization of preparing high-affinity monoclonal antibodies, and further to develop high-performance immunoassay methods for CPP. Results: This novel immune strategy significantly enhanced immune response against CPP. Six anti-CPP monoclonal antibodies (mAbs) with high affinity were successfully screened and selected for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect CPP in the range of 1.2-1250 pmol L-1 with a detection limit of 6.25 pmol L-1. Significantly, the whole incubation process can be completed in 30 min as compared to about 4.5 hr for the control ELISA kit. Furthermore, this assay exhibited high sensitivity and specificity, low intra-assay and inter-assay coefficients of variation (CVs < 15%). The developed assay was applied in the detection of CPP in 115 random serum samples and results showed a high correlation with data obtained using a commercially available ELISA kit (correlation coefficient, 0.9737). Conclusion: Our assay could be applied in the point-of-care testing of CPP in the serum samples, and also the method developed in this study could be adopted to explore the detection and diagnosis of other biomarkers for various diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Citocinas/metabolismo , Glicopeptídeos/sangue , Imunização , Imunoensaio/métodos , Medições Luminescentes/métodos , Testes Imediatos , Animais , Anticorpos Monoclonais/biossíntese , Feminino , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Nanopartículas de Magnetita/química , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade
7.
Food Chem ; 297: 124930, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253279

RESUMO

A new microfluidic chemiluminescence (MF-CL) method for rapidly assaying the total antioxidant capacity (TAC) of apple and pomegranate juices and honey samples was developed. The method exploited the NaHCO3-H2O2-Co2+ CL reaction. It was found that gallic acid (GA), catechin, caffeic acid, ferulic acid and rutin, as selected phenolic antioxidants, could suppress the CL reaction. The linear range and limit of detection of the method for the antioxidants were as follows: 0.5-3 mg L-1 and 0.27 mg L-1 for GA, 0.2-5.0 mg L-1 and 0.17 mg L-1 for catechin, 0.03-2.0 mg L-1 and 0.03 mg L-1 for caffeic acid, 0.3-2.0 mg L-1 and 0.23 mg L-1 for ferulic acid and 0.3-4.0 mg L-1 and 0.15 mg L-1 for rutin. GA was used as the standard, and the TAC of the fruit juices and honey samples as presented as GA equivalents (GAE). MF-CL was compared with DPPH and Folin-Ciocalteau (FC) methods.


Assuntos
Antioxidantes/química , Sucos de Frutas e Vegetais/análise , Mel/análise , Dispositivos Lab-On-A-Chip , Medições Luminescentes/métodos , Cobalto/química , Ácido Gálico/química , Peróxido de Hidrogênio/química , Medições Luminescentes/instrumentação , Bicarbonato de Sódio/química
8.
Anal Chim Acta ; 1075: 137-143, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196419

RESUMO

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.


Assuntos
Sondas de DNA/química , DNA de Cadeia Simples/análise , Proteína BRCA2/genética , Sondas de DNA/genética , DNA de Cadeia Simples/genética , Desoxirribonuclease IV (Fago T4-Induzido)/química , Feminino , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Neoplasias Ovarianas/genética , Mutação Puntual , Telomerase/genética
9.
Dokl Biochem Biophys ; 485(1): 107-110, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201626

RESUMO

In this study, we formulated the principles of designing bioluminescent enzyme tests for assessing the quality of complex media, which consist in providing the maximum sensitivity to potentially toxic chemicals at a minimal impact of uncontaminated complex media. The developed principles served as a basis for designing a new bioluminescent method for an integrated rapid assessment of chemical safety of fruits and vegetables, which is based on using the luminous bacteria enzymes (NAD(P)H:FMN oxidoreductase and luciferase) as a test system.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , FMN Redutase/química , Análise de Alimentos/métodos , Luciferases/química , Medições Luminescentes/métodos
11.
Chem Commun (Camb) ; 55(52): 7458-7461, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31184643

RESUMO

The development of a sensitive and reliable method for the detection of bioaccumulated heavy metal toxins is highly desirable for biotoxicity evaluation. However, the conventional biotoxicity evaluation method based on luminescent bacteria suffers from only being able to detect the overall toxicity without selectivity in light-off detection mode. Although various synthetic fluorescent probes have been developed for the selective detection of heavy metal ions, they usually suffer from aggregation-caused quenching after local accumulation in biological systems. To tackle these challenges, we herein develop a dual detection strategy for bioaccumulated Hg2+ based on turn-off of the bioluminescence of P. phosphoreum bacteria by disrupting the quorum sensing system and turn-on of the photoluminescence of an aggregation-induced emission (AIE) probe by forming aggregates with Hg2+ inside the bacteria. It is expected that the dual detection strategy would find broad applications in the evaluation of bioaccumulated toxins.


Assuntos
Mercúrio/química , Photobacterium/química , Corantes Fluorescentes/química , Íons/química , Luz , Medições Luminescentes/métodos , Mercúrio/farmacologia , Microscopia Confocal , Photobacterium/isolamento & purificação , Teoria Quântica , Percepção de Quorum/efeitos dos fármacos
12.
Analyst ; 144(10): 3250-3259, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31049499

RESUMO

The trend for improved more precise diagnostics and management of disease heavily relies on the measurement of panels of biomarkers in physiological samples of patients. Ideally, the ultimate goal would be to detect as many clinically relevant biomarkers as possible in a single drop of blood, achieving quick, sensitive, reproducible, and affordable detection in small volume physiological samples. Bioluminescent (BL) proteins provide many of the desired characteristics required for such labels, including detection at extremely low concentrations, no interference from physiological fluids leading to excellent detection limits, and compatibility with many miniaturized systems. However, to date the use of BL proteins has been restricted by their limited multiplexing capabilities. BL proteins typically exhibit a single emission profile and decay kinetics making the simultaneous detection of multiple analytes difficult. Recent progresses in this area include the use of two different engineered luminescent proteins to achieve resolved signals via one-dimensional time resolution. This approach, however, to date only lead to a dual analyte detection. Herein, we have demonstrated that using a two-dimensional approach that combines both temporal and spatial resolution, we can expand the multiplexing capabilities of bioluminescent proteins. To that end, the photoprotein aequorin (AEQ) has been employed for the simultaneous detection of three separate analytes in a single well, differentiated through the use of three discrete time/wavelength windows. Through a combination of site-specific mutations and synthetic coelenterazines "semi-synthetic" AEQ variants have been developed with altered emission profiles and decay kinetics. In this study, two AEQ mutant proteins were genetically conjugated to three pro-inflammatory cytokines (tumor necrosis factor alpha, interleukins 6 and 8) resulting in AEQ-labeled cytokines. These fusion proteins were combined with synthetic coelenterazines resulting in proteins with differing emission maxima and half-lives to allow for the simultaneous detection of all three cytokines in a single sample. The validity of the assay was demonstrated in serum by employing human physiological samples and comparing our results with commercially available individual tests for each of the three cytokines.


Assuntos
Equorina/química , Interleucina-6/sangue , Interleucina-9/sangue , Fator de Necrose Tumoral alfa/sangue , Equorina/genética , Animais , Cabras , Humanos , Hidrozoários/química , Imidazóis/química , Imunoensaio/métodos , Imunoglobulina G/imunologia , Interleucina-6/imunologia , Interleucina-9/imunologia , Limite de Detecção , Luminescência , Medições Luminescentes/métodos , Camundongos , Mutação , Pirazinas/química , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/imunologia
13.
Analyst ; 144(11): 3668-3675, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31086892

RESUMO

Exosomes are non-invasive biomarkers for cancer diagnosis. Herein, we describe an electrochemiluminescent (ECL) aptasensor for the detection of exosomes from breast tumor cells. Mercaptopropionic acid (MPA)-modified Eu3+-doped CdS nanocrystals (MPA-CdS:Eu NCs) and H2O2 were used as ECL emitters and coreactant, respectively. The exosomes are recognized and captured by the CD63 aptamer, and then form a G-quadruplex/hemin DNAzyme, which efficiently catalyzes the decomposition of H2O2, resulting in the decreased ECL signal of MPA-CdS:Eu NCs. The exosomes from breast tumor cells (MCF-7 cells) can be detected in the range of 3.4 × 105 to 1.7 × 108 particles per mL. The limit of detection (LOD) was estimated to be 7.41 × 104 particles per mL at a signal-to-noise ratio of 3. The aptasensor has been successfully used to detect exosomes in the serum.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA Catalítico/química , DNA/química , Exossomos/química , Hemina/química , Nanopartículas Metálicas/química , Ácido 3-Mercaptopropiônico/química , Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Técnicas Biossensoriais/métodos , Neoplasias da Mama/diagnóstico , Compostos de Cádmio/química , Carbono/química , DNA/metabolismo , DNA Catalítico/genética , DNA Catalítico/metabolismo , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Európio/química , Exossomos/metabolismo , Quadruplex G , Hemina/metabolismo , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Medições Luminescentes/métodos , Células MCF-7 , Sulfetos/química
14.
Analyst ; 144(12): 3773-3781, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31089613

RESUMO

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.


Assuntos
Bioensaio/métodos , Proteínas Luminescentes/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/química , Cromatografia de Afinidade/métodos , Complexos de Coordenação/química , Escherichia coli/genética , Histidina/genética , Histidina/metabolismo , Humanos , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Simulação de Acoplamento Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/química , Platina/química , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética
15.
Food Chem ; 292: 98-105, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054698

RESUMO

Accurate and early diagnosis of mycotoxin is particularly significant to the food and agricultural product safety. In the present work, a sensitive and effective monitoring method for zearalenone (ZEN) was exploited based on a novel self-enhanced electrochemiluminescence (ECL) aptasensor. The self-enhanced lumonophore was compounded by electrostatically combining amine-functionalized Ru(bpy)32+-doped silica nanoparticles (NH2-Ru@SiO2 NPs) and nitrogen doped graphene quantum dots (NGQDs) together. Since the emitter and co-reactant simultaneously existed in the same nanoparticle, shortened electron-transfer distance and decreased energy loss was obtained. Therefore, self-enhanced ECL aptasensor based on the novel complex expressed the widest linear range of 10 fg mL-1-10 ng mL-1 and the lowest detection limit of 1 fg mL-1 for ZEN detection. More importantly, ZEN produced during the mildew process of corn flour was monitored by the developed aptasensor, which exhibited superior determination and potential application in real samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Medições Luminescentes/métodos , Nanopartículas/química , Zea mays/metabolismo , Zearalenona/análise , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Farinha/análise , Grafite/química , Limite de Detecção , Compostos Organometálicos/química , Pontos Quânticos/química , Reprodutibilidade dos Testes , Dióxido de Silício/química , Zea mays/química
16.
J Agric Food Chem ; 67(20): 5711-5719, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31042038

RESUMO

Although dicamba has long been one of the most widely used selective herbicides, some U.S. states have banned the sale and use of dicamba because of farmers complaints of drift and damage to nonresistant crops. To prevent illegal use of dicamba and allow monitoring of nonresistant crops, a rapid and sensitive method for detection of dicamba is critical. In this paper, three novel dicamba haptens with an aldehyde group were synthesized, conjugated to the carrier protein via a reductive-amination procedure and an indirect competitive chemiluminescent enzyme immunoassay (CLEIA) for dicamba was developed. The assay showed an IC50 of 0.874 ng/mL which was over 15 times lower than that of the conventional enzyme immunoassay. The immunoassay was used to quantify dicamba concentrations in field samples of soil and soybean obtained from fields sprayed with dicamba. The developed CLEIA showed an excellent correlation with LC-MS analysis in spike-and-recovery studies, as well as in real samples. The recovery of dicamba ranged from 86 to 108% in plant samples and from 105 to 107% in soil samples. Thus, this assay is a rapid and simple analytical tool for detecting and quantifying dicamba levels in environmental samples and potentially a great tool for on-site crop and field monitoring.


Assuntos
Anticorpos/análise , Dicamba/química , Haptenos/química , Herbicidas/química , Técnicas Imunoenzimáticas/métodos , Medições Luminescentes/métodos , Animais , Anticorpos/imunologia , Imunização , Técnicas Imunoenzimáticas/instrumentação , Medições Luminescentes/instrumentação , Espectrometria de Massas , Estrutura Molecular , Folhas de Planta/química , Coelhos , Poluentes do Solo/química , Soja/química , Espectrometria de Massas em Tandem
17.
Virol J ; 16(1): 66, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31109347

RESUMO

BACKGROUND: Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR). METHODS: We changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3GREM-EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells. RESULTS: We successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3GREM-EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows. CONCLUSION: The new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.


Assuntos
Vírus da Leucemia Bovina/genética , Medições Luminescentes/veterinária , Mutação , Plasmídeos/genética , Elementos de Resposta , Sequências Repetidas Terminais , Animais , Bovinos , Linhagem Celular , Feminino , Genes Reporter , Glucocorticoides , Vírus da Leucemia Bovina/isolamento & purificação , Medições Luminescentes/métodos , Regiões Promotoras Genéticas , Sensibilidade e Especificidade
18.
Methods Mol Biol ; 1947: 361-376, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969428

RESUMO

Engineered G protein-coupled receptors (DREADDs, designer receptors exclusively activated by designer drugs) are convenient tools for specific activation of GPCR signaling in many cell types. DREADDs have been utilized as research tools to study numerous cellular and physiologic processes, including regulation of neuronal activity, behavior, and metabolism. Mice with random insertion transgenes and adeno-associated viruses have been widely used to express DREADDs in individual cell types. We recently created and characterized ROSA26-GsDREADD knock-in mice to allow Cre recombinase-dependent expression of a Gαs-coupled DREADD (GsD) fused to GFP in distinct cell populations in vivo. These animals also harbor a CREB-activated luciferase reporter gene for analysis of CREB activity by in vivo imaging, ex vivo imaging, or biochemical reporter assays. In this chapter, we provide detailed methods for breeding GsD animals, inducing GsD expression, stimulating GsD activity, and measuring basal and stimulated CREB reporter bioluminescence in tissues in vivo, ex vivo, and in vitro. These animals are available from our laboratory for non-profit research.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Genes Reporter , Processamento de Imagem Assistida por Computador/métodos , Medições Luminescentes/métodos , Moduladores de Transporte de Membrana/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Integrases/metabolismo , Camundongos , Especificidade de Órgãos , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais
19.
Biosens Bioelectron ; 135: 8-13, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30981028

RESUMO

In this work, the ZnO nanostars with excellent catalytic performance were firstly used as the coreaction accelerator of luminol-O2 system to construct a biosensor for ultrasensitively detecting microRNA-21 (miRNA-21) in cancer cells. Specifically, ZnO nanostars could expedite the reduction of dissolved O2, generating more reactive oxygen species (ROSs) to extremely promote electrochemiluminescence (ECL) luminous efficiency of luminol. Thus luminol-functionalized Au NPs@ZnO (L-Au NPs@ZnO) nanomaterials were employed as signal probe to fabricate sensing nano-platform for achieving significant ECL emission as "signal on" state. Moreover, upon the addition of a tiny minority of target miRNA-21, massive ferrocene (Fc) could be immobilized on the sensing interface through hybridization chain reaction (HCR) triggered-DNA dendrimers self-assembly, in which Fc consumed dissolved O2 for prominently quenching the ECL emission of signal probe and then reached a "signal off" state. As a result, the biosensor performed a good linearity in 100 aM - 100 pM and a low limit of detection (LOD) down to 18.6 aM. In general, this work utilized a new coreaction accelerator as an efficient amplification approach for ultrasensitively detecting target analyses, providing a promising approach in luminol-centric ECL bioanalysis fields.


Assuntos
Ouro/química , Substâncias Luminescentes/química , Luminol/química , MicroRNAs/análise , Óxido de Zinco/química , Técnicas Biossensoriais/métodos , DNA/química , Dendrímeros/química , Técnicas Eletroquímicas/métodos , Células HeLa , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Células MCF-7 , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Oxigênio/química
20.
Biosens Bioelectron ; 135: 95-101, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31004926

RESUMO

This work utilized ultrathin metal-organic layer (MOL) to immobilize luminophores for effectively shortening the ion/electron-transport distance and relieving the diffusional constraints of ion/electron, which greatly enhanced the ECL efficiency and intensity. Moreover, the MOL's immobilization amount of luminophores should be higher than these of bulk MOFs because MOLs possess more accessible postmodification sites for the luminophores with minimal diffusion barriers. As expected, our proof-of-concept experiment indicated that the Hf-MOL's loading number of Ru(bpy)2(mcpbpy)2+ was about 1.74 times that of a 3D mesoporous MOF (PCN-777), and the ECL efficiency and intensity of PEI@Ru-Hf-MOL were around 1.27 times and 14.5 times those of PEI@Ru-PCN-777, respectively. In view of these merits, this work utilized the prepared PEI@Ru-Hf-MOL as a highly efficient sensing platform for simple, rapid and sensitive detection of mucin 1, which exhibited a broad linearity from 1 fg/mL to 10 ng/mL and a low detection limit of 0.48 fg/mL. This work provided a practicable strategy to develop high-performance ECL materials, and therefore opened up a new avenue to design ultrasensitive ECL biosensors, which expanded the application potential of MOLs in ECL assays.


Assuntos
Complexos de Coordenação/química , Háfnio/química , Substâncias Luminescentes/química , Mucina-1/sangue , Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Humanos , Imunoensaio/métodos , Medições Luminescentes/métodos , Modelos Moleculares
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA