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1.
Food Chem ; 301: 125240, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387040

RESUMO

Cold plasma is an emerging technology increasingly applied in the agri-food industry. For fruit and vegetables, enzymatic browning is a common phenomenon, causing quality deterioration. The objective of this study was to illustrate the effect of microscale atmospheric-pressure plasma jet (µAPPJ) plasma on the horseradish peroxidase (HRP). Results showed that after plasma treatment for 10 min, the residual activity of HRP was decreased to around 17%, and modification of secondary and tertiary structures were confirmed. The atomic force microscope (AFM) analysis revealed that the aggregation of enzyme protein was enhanced with prolonging treatment time. It was concluded that the activity of HRP could be reduced with destruction of structures and deformation of microstructure induced by µAPPJ plasma. The current study attempted to provide new idea for inhibiting browning enzymes of fruit and vegetables with plasma technology through deeper understanding of the interaction mechanism of plasma active species with enzymes.


Assuntos
Temperatura Baixa , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Gases em Plasma/farmacologia , Pressão Atmosférica
2.
Analyst ; 144(17): 5108-5116, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31373337

RESUMO

We report here the influence of antibody immobilization strategy for protein immunosensors on screen printed carbon electrode arrays in terms of antibody binding activity, analytical sensitivity, limit of detection, and stability. Horseradish peroxidase (HRP) was the model analyte with anti-HRP immobilized on the sensors, and HRP activity was used for detection. Covalently immobilized anti-HRP antibodies on electrodes coated with chitosan, electrochemically reduced graphene oxide (rGO), and dense gold nanoparticle (AuNP) films had only 20-30% of the total immobilized antibodies active for binding. Active antibodies increased to 60% with passively adsorbed antibodies on bare electrodes, to 85% with oriented antibodies using protein A covalently immobilized on AuNP-coated carbon electrode, and to 98% when attached to protein A passively adsorbed onto bare electrodes. Passively adsorbed antibodies on bare electrodes lost activity in 1-2 days, but antibodies immobilized using other strategies remained relatively stable after 5 days. Covalent immobilization gave limits of detection (LOD) of 40 fg mL-1, while passively adsorbed antibodies or protein A on carbon electrodes had LODs 4-8 fg mL-1, but were unstable. Sensitivity was highest for antibodies covalently attached to AuNP electrodes (2.40 nA per log pg per mL) that also had highest antibody coverage, and decreased slightly when protein A on AuNP was used to orient antibodies. Passively adsorbed antibodies and oriented antibodies on protein A gave slightly lower sensitivities. Immobilization strategy or antibody orientation did not have a significant effect on LOD, but dynamic range increased as the number of active antibodies on sensor surfaces increased.


Assuntos
Anticorpos Imobilizados/química , Carbono/química , Técnicas Biossensoriais/métodos , Quitosana/química , Técnicas Eletroquímicas/métodos , Eletrodos , Grafite/química , Peroxidase do Rábano Silvestre/química , Imunoensaio/métodos , Limite de Detecção , Oxirredução , Propriedades de Superfície
3.
Chemistry ; 25(54): 12576-12582, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31314132

RESUMO

Nature has evolved enzymes with exquisite active sites that catalyze biotransformations with high efficiency. However, the exploitation of natural enzymes is often hampered by poor stability, and natural enzyme production and purification are costly. Supramolecular self-assembly allows the construction of biomimetic active sites, although it is challenging to produce such artificial enzymes with catalytic activity and stability that rival those of natural enzymes. We report herein a strategy to produce a horseradish peroxidase (HRP) mimic based on the assembly of chitosan with a G-quadruplex DNA (G-DNA)/hemin complex. A network-like morphology of the assembled nanomaterial was observed together with a remarkable enhancement of peroxidase activity induced by the chitosan and G-DNA components. The turnover frequency and catalytic efficiency of the enzyme-mimicking material reached or even surpassed those of HRP. Moreover, the catalytic complex exhibited higher tolerance than HRP to harsh environments, such as extremely low pH or high temperatures. In accord with the experimental and simulated results, it is concluded that the spatial distribution of the G-DNA and chitosan components and the exposure of the catalytic center may facilitate the coordination of substrates by the hemin iron, leading to the superior activity of the material. Our work provides a simple and affordable avenue to produce highly active and robust enzyme-mimicking catalytic nanomaterials.


Assuntos
Materiais Biomiméticos/química , Quitosana/química , Quadruplex G , Hemina/química , Peroxidase do Rábano Silvestre/química , Nanoestruturas/química , Catálise , Domínio Catalítico , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Dinâmica Molecular , Oxirredução , Conformação Proteica , Temperatura Ambiente , Termodinâmica
4.
Anal Chim Acta ; 1074: 80-88, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159942

RESUMO

A rapid and sensitive electrochemical biosensor was constructed to detect Salmonella using invA gene biosensor. The biosensing was based on polyrrole-reduced graphene oxide (PPy-rGO) nanocomposite modified glassy carbon electrode (GCE) and signal amplification with horseradish peroxidase-streptavidin biofunctionalized gold nanoparticles (AuNPs-HRP-SA). PPy-rGO was prepared at 60 °C by chemical reduction of PPy-functionalized graphene oxide (PPy-GO) that was synthesized by in situ polymerization at room temperature. The detection signal was amplified via enzymatic reduction of H2O2 in the presence of hydroquinone (HQ) using AuNPs-HRP-SA as nanotag. Under optimal conditions, the differential pulse voltametric (DPV) signal from the biosensor was linearly related to the logarithm of target invA gene concentrations from 1.0 × 10-16 to 1.0 × 10-10 M, and the limit of detection (LOD) was 4.7 × 10-17 M. The biosensor can also detect Salmonella in the range of 9.6 to 9.6 × 104 CFU mL-1, with LOD of 8.07 CFU mL-1. The biosensor showed good regeneration ability, acceptable selectivity, repeatability and stability, which bode well as an alternative method for Salmonella screening.


Assuntos
Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Grafite/química , Nanopartículas Metálicas/química , Polímeros/química , Pirróis/química , Salmonella/isolamento & purificação , Técnicas Biossensoriais/métodos , Carbono , DNA Bacteriano/genética , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Enzimas Imobilizadas/química , Ouro/química , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Hidroquinonas/química , Limite de Detecção , Nanocompostos/química , Hibridização de Ácido Nucleico , Oxirredução , Salmonella/genética , Estreptavidina/química
5.
Anal Chim Acta ; 1076: 91-99, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203968

RESUMO

The development of an automated miniaturized analytical system that allows for the rapid monitoring of carbamazepine (CBZ) levels in serum and wastewater is proposed. Molecular recognition of CBZ was achieved through its selective interaction with microbeads carrying anti-CBZ antibodies. The proposed method combines the advantages of the micro-bead injection spectroscopy and of the flow-based platform lab-on-valve for implementation of automatic immunosorbent renewal, rendering a new recognition surface for each sample. The sequential (or simultaneous) perfusion of CBZ and the horseradish peroxidase-labelled CBZ through the microbeads is followed by real-time on-column monitoring of substrate (3,3',5,5'-tetramethylbenzidine) oxidation by colorimetry. The evaluation of the initial oxidation rate and also the absorbance value at a fixed time point provided a linear response versus the logarithm of the CBZ concentration. Under the selected assay conditions, a single analysis was completed after only 11 min, with a quantification range between 1.0 and 50 µg L-1. Detection of CBZ levels in undiluted wastewater samples was feasible after a simple filtration step while good recoveries were attained for spiked certified human serum, analyzed without sample clean-up.


Assuntos
Carbamazepina/sangue , Colorimetria/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Monoclonais/imunologia , Armoracia/enzimologia , Benzidinas/química , Carbamazepina/imunologia , Peroxidase do Rábano Silvestre/química , Humanos , Microesferas , Oxirredução , Sefarose/análogos & derivados , Sefarose/química , Águas Residuárias/análise
6.
Anal Bioanal Chem ; 411(17): 3801-3810, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31172237

RESUMO

The convenience of colorimetric sensors is useful for practical applications. In this work, we constructed a novel colorimetric sensor with magnetic separation ability that can be operated in nearly neutral conditions and achieve one-step detection of metabolites. Magnetic Cu doped Fe3O4@FeOOH magnetic nanocomposite (Cu/Fe3O4@FeOOH) with an oxygen vacancy was prepared by a one-step self-assembly hydrothermal method, and fully characterized by different methods. The oxygen vacancy generated by the incorporation of Cu2+ cations into the Fe3O4@FeOOH structure was confirmed to be a vital reactive site for enhancing the catalytic activity, which opens up a new way of designing highly efficient enzyme mimics. Benefiting from its inherent horseradish-peroxidase-like activity, a simple and selective enzyme-based colorimetric sensor was developed for one-step detection of H2O2 and cholesterol, and 3,3',5,5'-tetramethylbenzidine was catalyzed by H2O2 to generate a colored product of oxidized 3,3',5,5'-tetramethylbenzidine for signaling. H2O2 and cholesterol can be linearly detected in the same range from 0.01 to 0.4 mmol L-1 with detection limits of 0.0075 mmol L-1 and 0.0082 mmol L-1, respectively. The proposed colorimetric sensor has satisfactory reusability, accuracy, and practicability in human serum samples, indicating its potential application for the detection of different metabolites in the fields of life science and analytical science. Graphical abstract.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , Cobre/química , Óxido Ferroso-Férrico/química , Peroxidase do Rábano Silvestre/química , Concentração de Íons de Hidrogênio , Magnetismo , Nanopartículas Metálicas/química , Benzidinas/química , Colesterol/sangue , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/análise , Microscopia Eletrônica de Transmissão , Oxirredução , Espectroscopia Fotoeletrônica , Reprodutibilidade dos Testes , Análise Espectral Raman , Difração de Raios X
7.
Talanta ; 200: 186-192, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036172

RESUMO

In this work, different paper surface modification strategies were compared to obtain an amine functionalized SBA-15 (N-SBA-15) composite for paper-based device development. The synthesized N-SBA-15 was characterized by N2 adsorption-desorption isotherm, and infrared spectroscopy (FTIR), and it was incorporated to different polymer matrices (κ-carrageenan (CA), polyvinyl alcohol (PVA) and polyethylenimine (PEI)) for the development of the composite modified paper-based device. The retention, interactions, and morphology of the obtained composites were investigated by absorbance measurement, FTIR and scanning electron microscopy (SEM), respectively. To demonstrate the applicability of the modified paper-based device, ascorbic acid (AA) quantification was carried out. Horseradish peroxidase (HRP) was immobilized onto the modified paper surface. HRP in the presence of H2O2 catalyzes the oxidation of 10-acetyl-3,7-dyhidroxyphenoxazine (ADHP) to highly fluorescent resorufin, which was measured by LIF detector. Thus, when AA was added to the solution, it decreases the relative fluorescence signal proportionally to the AA concentration. The linear range from 50 nmol L-1 to 1500 nmol L-1 and a detection limit of 15 nmol L-1 were obtained for AA quantitation. The obtained results allowed us to conclude that N-SBA-15/PEI composite could be considered an excellent choice for the paper-based device modification procedure due to its inherent simplicity, low cost, and sensitivity.


Assuntos
Ácido Ascórbico/análise , Papel , Polímeros/química , Dióxido de Silício/química , Adsorção , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/química , Nitrogênio/química , Tamanho da Partícula , Dióxido de Silício/síntese química , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
8.
Talanta ; 201: 52-57, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122460

RESUMO

More and more attention about food safety leads to a research hotspot to develop new detection methods for food contaminant. To address the problems of serious interference and low sensitivity, a chemiluminescent aptasensor for the detection of aflatoxin B1(AFB1) in food was developed in this paper. It is based on horseradish peroxidase (HRP) catalyze the luminol chemiluminescence reaction. The hybridization chain reaction (HCR) signal amplification strategy has been used to improve the detection sensitivity. Magnetic separation could further reduce background signal obviously at the same time. AFB1 as a model of analyte to test the capability of our developed assay system. Under the optimal experimental conditions, CL intensity showed a good linear correlation with the concentrations of AFB1 ranging from 0.5 to 40 ng mL-1. The limit of detection was estimated 0.2 ng mL-1 based on 3 times of the signal-to-noise ratio which is lower than those of the previously reported sensors. It could be used to detect AFB1 content in real samples, such as peanuts and milk which were purchased in local supermarket. The results proved that the sensing system has good anti-interference and selectivity. In all, it has potential for practical application in food safety field.


Assuntos
Aflatoxina B1/análise , Aptâmeros de Nucleotídeos/química , Arachis/química , Técnicas Biossensoriais/métodos , Contaminação de Alimentos/análise , Leite/química , Animais , Aptâmeros de Nucleotídeos/genética , Armoracia/enzimologia , DNA/química , DNA/genética , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Limite de Detecção , Luminescência , Medições Luminescentes , Luminol/química , Hibridização de Ácido Nucleico , Oxirredução
9.
Talanta ; 200: 293-299, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036187

RESUMO

The co-immobilization of two enzymes onto single support commonly exhibits low efficiency due to the competition against limited sites. Water-stable metal-organic frameworks (MOFs) [i.e., PCN-333(Al)] with a high surface area and ultra-large cavities were employed to efficiently adsorb cholesterol oxidase (ChOx) and encapsulate horseradish peroxidase (HRP), respectively. The prepared PCN-333/ChOx&HRP was characterized through SEM, XRD, confocal microscopy, N2 adsorption isotherms, and thermal gravity analysis (TGA). The high surface area and high concentration of mesoporous cages resulted in the high loadings of both ChOx and HRP. The absorbed ChOx and the encapsulated HRP presented excellent activities without additional chemical modification. The immobilized enzymes were stable against protease digestion, organic solvents, temperature changes, and pH variation. Thus, a colorimetric biosensor for cholesterol detection was fabricated depending on cascade catalytic reactions of the immobilized bi-enzymes. An extended linear range from 0.0 to 40.0 µM with a low detection limit of 0.6 µM was obtained using the biosensor. The co-immobilization of the enzymes onto the surface and into the mesopores of MOFs provided a new and excellent platform for the development of highly stable and sensitive colorimetric biosensors.


Assuntos
Técnicas Biossensoriais , Colesterol Oxidase/química , Colesterol/análise , Colorimetria , Peroxidase do Rábano Silvestre/química , Estruturas Metalorgânicas/química , Adsorção , Colesterol Oxidase/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Tamanho da Partícula , Propriedades de Superfície
10.
Anal Chim Acta ; 1071: 53-58, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31128755

RESUMO

An ultrasensitively multicolor colorimetric assay on the basis of enzyme-catalyzed reaction mediated etching of gold nanobipyramids (Au NBPs) is developed for detection of blood glucose with naked eye. This assay depends on the catalytic oxidation of H2O2 (generated from glucose oxidation) by horseradish peroxidase (HRP) to generate hydroxyl radicals (•OH) with strong oxidizability, which can accelerate the Au NBPs etching and accompany with vivid color changes and localized surface plasmon resonance (LSPR) blue shift. On the basis of this, the proposed colorimetric glucose assay accomplishes a dynamic range of 0.05-90 µM with detection limit of 0.02 µM, which is almost three orders of magnitude lower than that (10 µM) based on gold nanorods (Au NRs). Moreover, the approximate glucose levels can be intuitively and conveniently judged by the color changes with naked eye. Most importantly, visually distinguish between healthy people and diabetic patients by naked eye dispense with any sophisticated instrument is the most important highlight of this colorimetric assay, indicating the available potential for diagnosis. To the best of our knowledge, this is the first report of colorimetric assay using Au NBPs as etching substrates for visually analysis of glucose.


Assuntos
Glicemia/análise , Colorimetria/métodos , Ouro/química , Nanopartículas Metálicas/química , Armoracia/enzimologia , Glicemia/química , Cor , Diabetes Mellitus/sangue , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Oxirredução , Propriedades de Superfície
11.
Anal Bioanal Chem ; 411(16): 3581-3589, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31089784

RESUMO

The use of aptamers in various analytical applications as molecular recognition elements and alternative to antibodies has led to the development of various platforms that facilitate the sensitive and specific detection of targets ranging from small molecules and proteins to whole cells. The goal of this work was to design a universal and adaptable sandwich-type aptasensor exploiting the unique properties of DNA binding proteins. Specifically, two different enzyme-DNA binding protein conjugates, GOx-dHP and HRP-scCro, were used for the direct detection of a protein using two aptamers for target capture and detection. The specific dsDNA binding sequence for each DNA binding protein tag was incorporated in the form of a hairpin at one end of each aptamer sequence during the synthesis step. Detection was accomplished by an enzymatic (GOx/HRP) cascade reaction after the binding of each enzyme conjugate to its corresponding binding sequence on each aptamer. The proposed sandwich-type aptasensor was validated for the detection of thrombin, which is one of the most commonly used model targets with known dual aptamers. The limit of detection accomplished was 0.92 nM which is comparable with other colorimetric platforms reported in the literature. The sensitivity of the aptasensor was easily modulated by changing the number of dsDNA binding sites incorporated in the aptamer sequences, thus controlling the enzyme stoichiometry. Finally, the potential use of the proposed sensing approach for real sample testing was demonstrated using spiked human plasma and no significant matrix effects were observed when up to 2% plasma was used.


Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas de Ligação a DNA/química , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Sequência de Bases , Sítios de Ligação , Técnicas Biossensoriais , Proteínas Sanguíneas/análise , Colorimetria/métodos , Técnicas Eletroquímicas/métodos , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Limite de Detecção , Reprodutibilidade dos Testes
12.
Anal Chim Acta ; 1064: 33-39, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-30982515

RESUMO

The detection of sequence-specific microRNAs (miRNAs) is an important factor to the diseases diagnosis. Herein, a triple signal amplification electrochemical biosensor for highly sensitive detection of miRNA-21 was developed based on a duplex-specific nuclease (DSN)-assisted target recycling combined with gold nanoparticles (NPs), horseradish peroxidase (HRP) enzymatic signal amplification. The electrochemical biosensor generated significantly amplified amperometric current changes (Δi) for the detection of miRNA-21 down to 43.3 aM, and Δi was proportional to the logarithm of the concentration of miRNA-21 within the range of 0.1 fM to 100 pM. Meanwhile, the inherent selectivity of the term hairpin capture probe endowed the biosensor with high differentiation of similar miRNAs. The good feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in A549 lysates, which indicates its promising potential in biomedical research and clinical analysis.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Ouro/química , Peroxidase do Rábano Silvestre/metabolismo , Nanopartículas Metálicas/química , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico , Células A549 , Espectroscopia Dielétrica , Peroxidase do Rábano Silvestre/química , Humanos , MicroRNAs/metabolismo , Células Tumorais Cultivadas
13.
Anal Chim Acta ; 1065: 12-20, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31005144

RESUMO

We are reporting an innovative building-block for the development of biosensors based on the non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with avidin (MWCNTs-avidin). In this work, at variance with previous reports, avidin has the double role of simultaneously being the exfoliating agent of MWCNTs and the platform for anchoring different biotinylated biomolecules. The optimum dispersion was obtained by sonicating for 5.0 min 0.50 mgmL-1 MWCNTs with 1.00 mgmL-1 avidin solution prepared in 50:50 v/v ethanol/water. As proof-of-concept, we immobilized biotinylated horseradish peroxidase (b-HRP) at glassy carbon electrodes (GCE) modified with MWCNTs-avidin to develop a hydrogen peroxide biosensor using hydroquinone as redox mediator. Surface plasmon resonance, electrochemical impedance spectroscopy, cyclic voltammetry and amperometry demonstrated that, even after the partial denaturation of avidin due to the drastic conditions used to functionalize the MWCNTs, it preserves the biorecognition properties and efficiently interacts with biotinylated horseradish peroxidase (b-HRP). The analytical characteristics of the resulting hydrogen peroxide biosensor are the following: linear range between 1.0 × 10-6 M and 1.4 × 10-5 M, sensitivity of (1.37 ±â€¯0.04) x 105 µAM-1, detection limit of 24 nM and reproducibility of 2.9%. The sensor was challenged with different samples, a mouthwash solution, human blood serum and milk, with very good performance.


Assuntos
Avidina/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/análise , Nanotubos de Carbono/química , Animais , Espectroscopia Dielétrica , Eletrodos , Peroxidase do Rábano Silvestre/química , Humanos , Leite/química , Ressonância de Plasmônio de Superfície
14.
Anal Bioanal Chem ; 411(19): 4951-4961, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30982928

RESUMO

Immunomagnetic separation (IMS) was combined with flow-based chemiluminescence sandwich immunoassays (CL-SIA) for the quantification of Staphylococcal enterotoxin B in milk. Therefore, iron oxide-shell silica-core magnetic nanocomposites were conjugated to biotinylated anti-SEB antibodies (MNC-IgGs). MNC-IgGs were applied successfully for (i) capturing SEB in milk samples by an affinity reaction, (ii) magnetophoretic collection on antibody spots in a channel of a flow-based immunochip, and (iii) sensitive enzymatic chemiluminescence detection of biotin labels by poly(horseradish peroxidase)-streptavidin. IMS was performed in 0.6 mL and 100 mL milk samples resulting in detection limits of 50 ng L-1 and 0.39 ng L-1, respectively, for the combined analytical method. It was shown that the assay sensitivity was dramatically improved by the combination of IMS with flow-based CL-SIA compared to CL-SIA directly applied with milk samples (detection limit 130 ng L-1). The IMS-CL-SIA has a time-to-result of 2-3 h. The reported combined analytical method can be used for a rapid control of SEB in complex food matrices such as milk. In future, even the monitoring of multiple contaminants in food or water may be performed by IMS-CL-SIA. Graphical abstract.


Assuntos
Enterotoxinas/análise , Imunoensaio/métodos , Separação Imunomagnética/instrumentação , Luminescência , Magnetismo , Leite/química , Nanocompostos/química , Staphylococcus aureus/química , Superantígenos/análise , Animais , Automação , Biotina/análise , Microbiologia de Alimentos/métodos , Peroxidase do Rábano Silvestre/química , Limite de Detecção , Estreptavidina/química
15.
J Nanobiotechnology ; 17(1): 35, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823927

RESUMO

BACKGROUND: Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera. RESULTS: In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R2 = 0.914) was found between the percent competitive inhibition values of cELISA and HI titers. CONCLUSIONS: A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.


Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática , Vírus da Doença de Newcastle/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Anticorpos Antivirais/isolamento & purificação , Camelus , Galinhas , Escherichia coli , Biblioteca Gênica , Células HEK293 , Peroxidase do Rábano Silvestre/química , Humanos , Proteínas Recombinantes/química , Sensibilidade e Especificidade
16.
Anal Chim Acta ; 1058: 1-8, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-30851843

RESUMO

Most of the photoelectrochemical (PEC) bioassays need to immobilize biomolecules on electrodes, which lead to tedious modification processes, damaged biomolecules, as well as crippled sensitivity/accuracy and low throughput of the performances. To overcome these drawbacks, we now introduce an exquisitely split-mode (which separates the bioreaction (performed in microplates) from the PEC detection (conducted in PEC cell)) cathodic photoelectrochemistry for probing versatile biocatalytic events with high throughput. Specifically, the enzymatically in situ generated 1,2-bezoquinone was covalently attached onto the PbSe quantum dots (QDs) modified indium tin oxide (ITO) (ITO/PbSe) photocathode through the connector of chitosan (CS). And the attached 1,2-bezoquinone acted as an efficient electron acceptor to promote the cathodic photocurrent of the ITO/PbSe electrode, enabling us to probe quinones-generating oxidoreductase (by taking horseradish peroxidase (HRP) as a model) coupled biocatalytic cascades including the alkaline phosphatase (ALP)/HRP and the glucose oxidase (GOx)/HRP cascades. Quantitative probing for ALP activity in a wide linear range of 5.0 × 10-3 to 10 U/L with the detection limit of 1.2 × 10-3 U/L was realized. While a wide linear range of 5.0 × 10-8 to 1.0 × 10-4 moL/L with a quite low detection limit of 1.0 × 10-8 moL/L was obtained for the glucose assay. In addition, this testing protocol was also extended to an immunoassay (taking carcinoembryonic antigen (CEA) as an example) using HRP as a catalytic tracer. The developed bioassays show high sensitivity and good selectivity for CEA detection in the linear range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.02 pg/mL. Moreover, the proposed detection has distinctive merits because it not only avoids the adverse effects of the surface confined biomolecules for crippling the signal transduction, but also it has enhanced throughput.


Assuntos
Benzoquinonas/química , Antígeno Carcinoembrionário/análise , Técnicas Eletroquímicas/métodos , Chumbo/química , Fotoquímica/métodos , Pontos Quânticos/química , Compostos de Selênio/química , Fosfatase Alcalina/química , Anticorpos/imunologia , Armoracia/enzimologia , Benzoquinonas/síntese química , Antígeno Carcinoembrionário/imunologia , Quitosana/química , Enzimas Imobilizadas/química , Glucose Oxidase/química , Ouro/química , Peroxidase do Rábano Silvestre/química , Imunoensaio/métodos , Raios Infravermelhos , Chumbo/efeitos da radiação , Limite de Detecção , Nanopartículas Metálicas/química , Pontos Quânticos/efeitos da radiação , Reprodutibilidade dos Testes , Compostos de Selênio/efeitos da radiação
17.
Analyst ; 144(9): 3038-3044, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30907399

RESUMO

We herein report a facile approach for the preparation of horseradish peroxidase (HRP)-mimic glucose oxidase-conjugated graphene oxide/MnO2 (GOD-GO/MnO2) as new nanozyme to detect the glucose concentration in whole blood. The nano-sized of MnO2 nanoparticles embedded in bovine serum albumin (BSA)-coated GO by in situ growth were evaluated focusing on the principle of HRP-mimic activity catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. Furthermore, we constructed dual sensing platforms based on the combination of a plasma separation pad and GOD-GO/MnO2 for direct detection of glucose concentration in whole blood by colorimetric assay without blood sample pretreatment. As a proof-of-concept, a limit of detection of 3.1 mg dL-1 for glucose was obtained with a wide linear quantification range from 25 mg dL-1 to 300 mg dL-1 through visual observation and quantitative analysis, suggesting potential clinical applications in blood glucose monitoring for diabetic patients.


Assuntos
Glicemia/análise , Colorimetria/métodos , Glucose Oxidase/química , Grafite/química , Compostos de Manganês/química , Nanopartículas/química , Óxidos/química , Animais , Benzidinas/química , Materiais Biomiméticos/química , Técnicas Biossensoriais/métodos , Análise Química do Sangue/métodos , Bovinos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Limite de Detecção , Oxirredução , Ratos , Soroalbumina Bovina/química , Estreptozocina
18.
Analyst ; 144(9): 2936-2941, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30920552

RESUMO

Imidacloprid (IMD) is one of the most used pesticides worldwide as a systemic insecticide as well as for pest control and seed treatment. The toxic and potential carcinogenic character of IMD makes its monitoring of great relevance in the field of agriculture and environment, so sensitive methodologies for in field analysis are strongly required. In this context, we have developed a competitive immunoassay for the determination of IMD using specific monoclonal antibodies followed by electrochemical detection on screen-printed carbon electrodes (SPCE). The optimized immunosensor exhibited a good reproducibility (RSD of 9%) and a logarithmic response in the range 50-10 000 pM of IMD, with an estimated detection limit (LOD) of 24 pM, which was below the maximum levels allowed by the legislation. High-Performance Liquid Chromatography-Mass Spectrometry-Mass Spectrometry (HPLC-MSMS) and Enzyme-Linked Immunosorbent Assay (ELISA) analysis were also performed for comparison purposes, where the electrochemical immunosensor exhibited a wider range of response and a lower detection limit. Matrix effects below 6.5% were obtained using tap water samples. All these characteristics make our electrochemical immunosensor a valid and advantageous tool for the in field determination of IMD.


Assuntos
Anticorpos Monoclonais/imunologia , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Inseticidas/análise , Neonicotinoides/análise , Nitrocompostos/análise , Animais , Armoracia/enzimologia , Benzidinas/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Carbono/química , Bovinos , Compostos Cromogênicos/química , Água Potável/análise , Técnicas Eletroquímicas/instrumentação , Eletrodos , Peroxidase do Rábano Silvestre/química , Inseticidas/imunologia , Limite de Detecção , Neonicotinoides/imunologia , Nitrocompostos/imunologia , Oxirredução , Coelhos , Reprodutibilidade dos Testes , Soroalbumina Bovina/química
19.
Analyst ; 144(8): 2584-2593, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30830127

RESUMO

The fast and precise detection of potential allergen-specific immunoglobulin E (sIgE) is imperative for the diagnosis and appropriate treatment of allergic diseases. In this study, we have successfully fabricated a novel paper-based immunoassay device for the detection of sIgE in allergic diseases. We used Can f 1, one of the main dog allergens, as a model allergen to detect sIgE in human sera. To achieve excellent performance, the experimental parameters were optimized. Further, we extended this device for potential applications in the clinical diagnosis of allergic diseases: worthwhile clinical performance in the detection of allergens was achieved as compared to that achieved by commercial enzyme-linked immunosorbent assay (ELISA) kit. Therefore, it was proven that this strategy has the advantages of high-throughput, rapid, sensitive, and highly accurate detection of trace amounts of sIgEs. Furthermore, by simply changing the antigen and antibody, this device could be used for the high-throughput detection of other allergens, so as to achieve multiallergen detection and appropriate desensitization therapy, thereby making it promising in the determination of allergic diseases in clinics.


Assuntos
Alérgenos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Medições Luminescentes/métodos , Papel , Alérgenos/genética , Alérgenos/isolamento & purificação , Animais , Armoracia/enzimologia , Bovinos , Ensaio de Imunoadsorção Enzimática/instrumentação , Escherichia coli/genética , Peroxidase do Rábano Silvestre/química , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina E/imunologia , Luminescência , Luminol/química , Oxirredução , Reprodutibilidade dos Testes , Soroalbumina Bovina/química , Temperatura Ambiente
20.
Luminescence ; 34(3): 368-374, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30887691

RESUMO

The occurrence of many diseases is closely related to the high expression of DNA methyltransferase 1 (DNMT1). However, most studies are focused on the detection of DNMT1 activity, a few are concerned with the detection of DNMT1 content. In this study, we developed a simple and highly sensitive chemiluminescence (CL) assay for the detection of DNMT1 content. In this method, anti-DNMT1 monoclonal antibody was coated on a polystyrene microplate to capture DNMT1. Then anti-DNMT1 polyclonal antibody and goat anti-rabbit immunoglobulin G with horseradish peroxidase (IgG-HRP) were respectively added to combine with captured DNMT1 to form a sandwich structure. Finally, the HRP could catalyze CL substrate and achieve CL signal response. Based on this novel sensitive strategy, the recovery percents were in the ranges from 71.5% to 91.0%. The precision of intra-assays and inter-assays were 5.45%-11.29% and 7.03%-11.25%, respectively. The method was successfully applied for the determination of DNMT1 in human serum. The detection results of serum samples showed that the proposed assay had a high correlation with enzyme-linked immunosorbent assay (ELISA) kit. Compared with the ELISA kit (limit of detection = 0.1 ng/mL), the method has a lower limit of detection of 0.042 ng/mL. Therefore, our method has the potential for the detection of DNMT1 content in clinical diagnosis.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Medições Luminescentes/métodos , Peroxidase do Rábano Silvestre/química , Humanos
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