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1.
Anal Bioanal Chem ; 412(7): 1509-1520, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32002580

RESUMO

Highly specific enrichment of N-linked glycopeptides from complex biological samples is crucial prior to mass spectrometric analysis. In this work, a hydrophilic metal-organic framework composite is prepared by the growth of UiO-66-NH2 on graphene sheets, followed by its post-synthetic modification to attach boronic acid to form GO@UiO-66-PBA. The fabrication of graphene oxide-MOF composite results in enhanced surface area with improved thermal and chemical stability. The synthesized MOF nanocomposite is characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and BET. A crystalline structure with high porosity offering large surface area and good hydrophilicity of the nanocomposite assists as an enrichment tool in glycoproteomics. The GO@UiO-66-PBA nanocomposite selectively enriches N-linked glycopeptides from tryptic digests of horseradish peroxidase (HRP) and immunoglobulin (IgG). GO@UiO-66-PBA nanoparticles show a low detection limit (1 fmol) and good specificity (1:200), reusability and reproducibility for N-linked glycopeptide enrichment from IgG digest. The binding capacity of GO@UiO-66-PBA is 84 mg/g for protein concentration, with a good recovery of 86.5%. A total of 372 N-linked glycopeptides corresponding to different glycoproteins are identified from only 1 µL of human serum digest. Thus, the presented research work can be an efficient separation platform for N-linked glycopeptide enrichment from complex samples, which can be extended to cost-effective routine analysis. Graphical abstract.


Assuntos
Ácidos Borônicos/química , Glicopeptídeos/química , Estruturas Metalorgânicas/química , Grafite/química , Peroxidase do Rábano Silvestre/química , Humanos , Microscopia Eletrônica de Varredura , Reprodutibilidade dos Testes
2.
Chem Commun (Camb) ; 56(10): 1549-1552, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31930244

RESUMO

In accordance with the rapid increase in demand for selective and spatial chemical tagging, and accurate detection of proteins of interest, we develop a sensitive protein detection method, termed "Supra-blot" capitalizing on high-affinity host-guest interaction between cucurbit[7]uril (CB[7]) and adamantylammonium (AdA). The method can directly detect chemically tagged proteins without false-positive signals caused by endogenous biomolecules. Not only a single specific protein, but also spatially localized proteins in cells were labeled with AdA, and selectively detected by a host molecule-enzyme hybrid, CB[7]-conjugated horseradish peroxidase (CB[7]-HRP) generating amplified chemiluminescence signals. This study shows the great potential of Supra-blot for accurate and reliable detection of proteins of interest in cells.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Peroxidase do Rábano Silvestre/química , Imidazóis/química , Medições Luminescentes/métodos , Amantadina/química , Compostos de Amônio/química , Células HEK293 , Histonas/química , Histonas/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos
3.
Nat Commun ; 11(1): 41, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900396

RESUMO

The development of programmable microscale materials with cell-like functions, dynamics and collective behaviour is an important milestone in systems chemistry, soft matter bioengineering and synthetic protobiology. Here, polymer/nucleotide coacervate micro-droplets are reconfigured into membrane-bounded polyoxometalate coacervate vesicles (PCVs) in the presence of a bio-inspired Ru-based polyoxometalate catalyst to produce synzyme protocells (Ru4PCVs) with catalase-like activity. We exploit the synthetic protocells for the implementation of multi-compartmentalized cell-like models capable of collective synzyme-mediated buoyancy, parallel catalytic processing in individual horseradish peroxidase-containing Ru4PCVs, and chemical signalling in distributed or encapsulated multi-catalytic protocell communities. Our results highlight a new type of catalytic micro-compartment with multi-functional activity and provide a step towards the development of protocell reaction networks.


Assuntos
Células Artificiais/química , Catalase/química , Rutênio/química , Compostos de Tungstênio/química , Catalase/síntese química , Catálise , Peroxidase do Rábano Silvestre/química
4.
Sci Total Environ ; 704: 135246, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31787307

RESUMO

Acetaminophen (AAP) is one of the most commonly prescribed over-the-counter drugs with wide distribution in surface water, which has attracted great attention around the world. Photodegradation and enzymatic transformation are important processes for the removal of AAP in water. The influence of natural organic matter (NOM) from different sources on the transformation of AAP by horseradish peroxidase (HRP) in sunlit water was systematically studied. NOM can effectively promote the combined degradation rate of AAP in photo-enzyme coupling system (PECS), and the promotion extents of different NOMs were dependent on their aromaticity and average molecular weights. NOM with low aromaticity and low average molecular weight is more effective. In order to disclose the underlying effects of NOM clearly, the reaction mechanism was clarified through the determination of the photodegradation constant and enzymatic reaction constant. The effect of NOM structure on the photo-enzymatic transformation of AAP was quantified, which showed significant positive correlation with the SUVA254 and E2/E3 of NOM. Further investigation revealed that the amount of H2O2 generated by NOM from different sources was also closely related to SUVA254 and E2/E3 of NOM. The findings will facilitate understanding the environmental fate of AAP and other pharmaceutical products in natural water.


Assuntos
Acetaminofen/química , Modelos Químicos , Processos Fotoquímicos , Poluentes Químicos da Água/química , Peroxidase do Rábano Silvestre , Peróxido de Hidrogênio , Fotólise
5.
Chemosphere ; 239: 124716, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31521938

RESUMO

During the past several years, abundant progresses has been made in the development of immobilized oxidative enzymes with focus on finding new support materials, improving the immobilization methods and their applications. Nowadays, immobilized oxidative enzymes are broadly accepted as a green way to face the challenge of high amounts of micropollutants in nature. Among all oxidative enzymes, laccases and horseradish peroxidase were used frequently in recent years as they are general oxidative enzymes with ability to oxidize various types of compounds. Immobilized laccase or horseradish peroxidase are showed better stability, and reusability as well as easy separation from reaction mixture that make them more favorable and economic in compared to free enzymes. However, additional improvements are still essential such as: development of the new materials for immobilization with higher capacity, easy preparation, and cheaper price. Moreover, immobilization methods are still need improving to become more efficient and avoid enzyme wasting during immobilization and enzyme leakage through working cycles.


Assuntos
Biodegradação Ambiental , Enzimas Imobilizadas/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Lacase/metabolismo , Oxirredução
6.
Talanta ; 206: 120211, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514873

RESUMO

Urinary glucose determination using a glucose test strip is simple and convenient in daily self-monitoring of diabetes. However, diabetic patients exhibit acquired impaired color vision (ICV), which results in the inability to discriminate between hues. Even with the assistance of a color chart, it is still not easy for these patients to read the urinary glucose results with the naked eye. In this study, a smartphone camera using an image-based colorimetric detection method was successfully developed for quantitative analysis of urine glucose. A horseradish peroxidase-hydrogen peroxide-3,3'5,5'-tetramethylbenzidine (HRP-H2O2-TMB) system was optimized for a reliable and gradual color fading process via a glucose oxidase (GOD) catalyzed oxidation reaction. The color changes of the peroxidase-H2O2 enzymatic reactions in the 96-well microplate were captured by a smartphone RGB camera with subsequent detection of red, green, and blue (RGB) intensities decreasing at each image pixel. The highly quantitative relationships between the glucose concentrations and the color characteristic values of the blue channel of the captured images were successfully established. The high accuracy of this method was demonstrated in urine glucose measurements with a linear response over the 0.039 mg mL-1 to 10.000 mg mL-1 glucose concentration range and a 0.009 mg mL-1 detection limit. The method has great potential as a point-of-need platform for diabetic patients with defective color vision and features high accuracy and low cost.


Assuntos
Diabetes Mellitus/urina , Glucose/análise , Glicosúria/diagnóstico , Smartphone , Armoracia/enzimologia , Benzidinas/química , Compostos Cromogênicos/química , Colorimetria/métodos , Glucose/química , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Fotografação/instrumentação , Testes Imediatos
7.
Analyst ; 144(21): 6321-6326, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31552921

RESUMO

Mass spectrometry (MS)-based analysis of glycoproteins and glycopeptides requires selective separation strategies to eliminate interferences from more abundant non-glycosylated biomolecules. In this work, we describe a two-phase liquid-liquid extraction method using supramolecular polymeric nanoassemblies that can selectively and efficiently enrich glycopeptides for enhanced MS detection. The polymeric nanoassemblies are made selective for glycopeptides via the incorporation of hydrazide functional groups that covalently bind to glycans. The enrichment efficiency is further enhanced via the incorporation of acidic functional groups that lead to a proximity-assisted catalysis of the hydrazide-glycan conjugation reaction. Our results further demonstrate the value of designer supramolecular nanomaterials for the selective enrichment of modified peptides from complicated mixtures.


Assuntos
Glicopeptídeos/análise , Extração Líquido-Líquido/métodos , Nanoestruturas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Armoracia/enzimologia , Bovinos , Glicopeptídeos/química , Peroxidase do Rábano Silvestre/análise , Peroxidase do Rábano Silvestre/química , Hidrazinas/química , Imunoglobulina G/análise , Imunoglobulina G/química , Oxirredução , Fragmentos de Peptídeos/análise , Poliestirenos/química , Proteólise , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Tripsina/química
8.
Eur J Histochem ; 63(3)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31505925

RESUMO

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during post-natal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase-conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, at one-, four- and eight-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in one- and four-day-old animals and adult animals. These results were not so manifest in eight-day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in O- and N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.


Assuntos
Acetilgalactosamina/metabolismo , Brônquios/citologia , Células Epiteliais/metabolismo , Galactose/metabolismo , Alvéolos Pulmonares/citologia , Animais , Brônquios/crescimento & desenvolvimento , Feminino , Histocitoquímica/métodos , Peroxidase do Rábano Silvestre/química , Neuraminidase/química , Lectinas de Plantas/química , Gravidez , Alvéolos Pulmonares/crescimento & desenvolvimento , Ratos Wistar , Proteínas de Soja/química
9.
Mater Sci Eng C Mater Biol Appl ; 104: 109931, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499978

RESUMO

Fibrin gels are of interest as biomaterials for regenerative medicine but present poor mechanical properties, undergo fast degradation and strongly contract in presence of cells. To face these drawbacks, a fibrin network can be associated with another polymer network, in an Interpenetrating Polymer Network (IPN) architecture. In this study, we report the properties of an IPN comprising a fibrin (Fb) network and a silk fibroin (SF) network. This IPN is synthesized through the action of 2 enzymes, each one being specific of one protein gelation, i.e. thrombin (Tb) for Fb gelation, and horseradish peroxidase (HRP) for SF gelation. The effective formation of both Fb and SF networks in an IPN architecture was first verified at qualitative and quantitative levels. The resulting IPN was easily manipulable, displayed high viscoelastic properties and showed homogeneous macro- and micro-structure. Then the degradability of the IPN by two proteases, thermolysin (TL) and trypsin (TRY), obeying different mechanisms was presented. Finally, two-dimensional culture of human fibroblasts on the IPN surface induced little material contraction, while fibroblasts showed healthy morphology, displayed high viability and produced mature extracellular matrix (ECM) proteins. Taken together, the results suggest that this new IPN have a strong potential for tissue engineering and regenerative medicine.


Assuntos
Fibrina/farmacologia , Fibroínas/farmacologia , Peroxidase do Rábano Silvestre/metabolismo , Polímeros/farmacologia , Trombina/metabolismo , Proliferação de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/biossíntese , Fibronectinas/biossíntese , Humanos , Recém-Nascido , Masculino
10.
Mikrochim Acta ; 186(10): 683, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31529296

RESUMO

A boronate-modified magnetic affinity sorbent was prepared by adopting hyperbranched polyethyleneimine as the scaffold to amplify initiator sites. 3-Acrylamidophenylboronic acid was employed as monomer to proceed in situ free-radical polymerization on magnetite (Fe3O4) nanoparticles. Due to the improved density of boronic acid polymers, the adsorbent exhibited high adsorption capacity, typically (134 ± 8) µg mg-1 for dopamine, (92 ± 7) µg mg-1 for catechol, (363 ± 14) µg mg-1 for ovalbumin and (464 ± 22) µg mg-1 for horseradish peroxidase. These capacities are much higher than those of other adsorbents. The sorbent was applied to the enrichment of catecholamines from spiked human urine. Owing to the high adsorption capacity, only 1.0 mg of adsorbent was sufficient to eliminate the interferences and enrich the targets (dopamine, norepinephrine and epinephrine) within 5 min. They were quantified by HPLC. The recoveries from spiked samples range between 83.5% ~106%, with relative standard deviations of 3.2 ~ 9.4% (n = 5). The separation of glycoproteins from egg white was also accomplished prior to their analysis by PAGE. In the authors' perception, this approach is promising in that the density of functional groups on the adsorbent is strongly increased. Graphical abstract The preparation routine of boronate affinity magnetic adsorbent (Fe3O4@HpAAPBA). The adsorbent is used for the magnetic solid phase extraction of cis-dol compounds from real sample.


Assuntos
Ácidos Borônicos/química , Catecolaminas/urina , Nanopartículas de Magnetita/química , Polietilenoimina/química , Catecóis/química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Dopamina/química , Clara de Ovo/química , Epinefrina/química , Glicoproteínas/isolamento & purificação , Peroxidase do Rábano Silvestre/química , Humanos , Norepinefrina/química , Ovalbumina/química , Extração em Fase Sólida
11.
ACS Appl Mater Interfaces ; 11(40): 37313-37321, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517474

RESUMO

A simple process is developed for the one-step preparation of dual-compartment alginate microcapsules with controlled size and structure from microfluid-generated water-in-water-in-oil (W/W/O) emulsion droplet. Unlike other methods that rely on transient W/W/O emulsion droplet, we introduce an aqueous two-phase system (ATPS) to form a stable W/W/O emulsion droplet as a template for preparing dual-compartment alginate microcapsules. Two different bioactive molecules are able to be spatially confined encapsulated in the shell and core of alginate microcapsules due to the partitioning effect of ATPS and the high viscosity of alginate solution. Moreover, an enzyme cascade reaction with a spatial confined glucose oxidase and horseradish peroxidase in the shell and core of alginate microcapsules confirms its excellent biocompatibility and high activity. This method provides a green platform for enzyme-catalyzed tandem reactions and controlled sequential release of multiple drugs based on alginate microcapsules.


Assuntos
Alginatos/química , Cápsulas/química , Emulsões/química , Microfluídica , Óleos/química , Água/química , Biocatálise , Fluoresceína-5-Isotiocianato/análogos & derivados , Glucose Oxidase/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Microfluídica/instrumentação , Tamanho da Partícula , Soroalbumina Bovina
12.
Sensors (Basel) ; 19(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500124

RESUMO

This study explores the use of a butyrylcholinesterase (BChE)-based, reversible reaction biosensor using screen-printed electrodes (SPEs) having a smaller working surface area than the single-use electrodes previously studied. Previous research demonstrated the prospective application of a single-use biosensor fabricated with an acetylcholinesterase (AChE) enzyme encapsulated in peptide nanotubes (PNTs) and enhanced with horseradish peroxidase (HRP) to detect organophosphorus compounds (OPCs) in aqueous and gas phases. In the current study, potential improvements to the biosensor are investigated. BChE-based biosensors were fabricated using PNTs, HRP, and Nafion in combination to increase the reactive surface area, enhance sensitivity, and maintain enzyme stability. Cyclic voltammetry (CV) was used along with the new modified sensor to measure malathion concentration in the gas phase. The results show that a BChE-based biosensor could reliably measure gas phase malathion concentrations between 6-25 ppbv by CV with the extent of inhibition linearly proportional to the malathion concentration (R2 = 0.941). This research demonstrated that fabricated BChE-based biosensors could be stored without cold storage requirement for up to six weeks with minimal performance degradation. Moreover, the sensor electrodes were each reused several times, and were still useable at the conclusion of the research. This research demonstrates the potential of fabricating a reusable, inexpensive biosensor that is capable of OPC detection with high sensitivity and a low detection limit without a long-term cold storage requirement.


Assuntos
Técnicas Biossensoriais , Gases/isolamento & purificação , Malation/isolamento & purificação , Nanotubos de Peptídeos/química , Acetilcolinesterase , Butirilcolinesterase/química , Enzimas Imobilizadas/química , Gases/química , Ouro/química , Peroxidase do Rábano Silvestre/química , Limite de Detecção , Malation/química , Nanotubos de Carbono/química , Compostos Organofosforados/química , Compostos Organofosforados/isolamento & purificação , Água/química
13.
Biosens Bioelectron ; 142: 111578, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31422223

RESUMO

The sensitive and accurate detection of cardiac troponin I (cTnI) is critical for myocardial infarction diagnosis. In this work, a dual-aptamer-based electrochemical (EC) biosensor was designed for cTnI detection based on the DNA nanotetrahedron (NTH) capture probes and multifunctional hybrid nanoprobes. First, the NTH-based Tro4 aptamer probes were anchored on a screen printed gold electrode (SPGE) surface through the Au-S bond, providing an enhanced spatial dimension and accessibility for capturing cTnI. Then, the hybrid nanoprobes were fabricated by using magnetic Fe3O4 nanoparticles as nanocarriers to load a large amount of cTnI-specific Tro6 aptamer, natural horseradish peroxidase (HRP), HRP-mimicking Au@Pt nanozymes and G-quadruplex/hemin DNAzyme. This signaling nanoprobes are capable of specifically recognizing the target cTnI based on the Tro6 aptamer and amplifying the signals to improve the detection sensitivity via enzymatic processes. We found the remarkable enhanced effect of EC signal to be attributed to the co-catalysis effect of hybrid nanozymes, HRP and DNAzyme. The target cTnI was sandwiched between the two types of aptamers (Tro4 and Tro6) on the electrode interface. Finally, this EC aptasensing platform exhibited great analytical performance with a wide dynamic range of 0.01-100 ng mL-1 and a low detection limit of 7.5 pg mL-1 for cTnI. The high selectivity, sensitivity and reliability of EC aptasensor can provide great potential in the clinic disease diagnostics.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Ácidos Nucleicos Imobilizados/química , Troponina I/sangue , Catálise , Sondas de DNA/química , DNA Catalítico/química , Técnicas Eletroquímicas/métodos , Quadruplex G , Ouro/química , Hemina/química , Peroxidase do Rábano Silvestre/química , Humanos , Limite de Detecção , Platina/química , Reprodutibilidade dos Testes , Troponina I/análise
14.
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
15.
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
16.
Anal Chim Acta ; 1079: 164-170, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387707

RESUMO

Ultrathin two-dimensional (2D) metal-organic framework (MOF) nanosheets have attracted numerous attention due to their unparalleled advantages such as unique structures, large surface areas, good stability and high catalytic efficiency. In this paper, 2D Cu(bpy)2(OTf)2 nanosheets (Cu-MOF, bpy = 4,4-bipyridine, OTf = trifluoromethanesulfonate) are first utilized to achieve the fluorescent detection for H2O2 and glucose with their intrinsic peroxidase-like activity. The fluorescent biosensor Cu-MOF nanosheets were achieved by the efficient catalysis of non-fluorescent thiamine (TH) to strong fluorescent thiochrome in the presence of H2O2. With the combination of glucose oxidase, highly selective and sensitive fluorescent detection for glucose could be established with a detection limit of 0.41 µM and two separate linear ranges of 10-100 µM and 100-1000 µM, respectively. Furthermore, the developed method has been applied to human serum for the quantitative determination of glucose as a clinical diagnosis indicator of diabetes mellitus.


Assuntos
Materiais Biomiméticos/química , Glicemia/análise , Peróxido de Hidrogênio/análise , Estruturas Metalorgânicas/química , Aspergillus niger/enzimologia , Materiais Biomiméticos/síntese química , Técnicas Biossensoriais/métodos , Cobre/química , Fluorescência , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Estruturas Metalorgânicas/síntese química , Espectrometria de Fluorescência/métodos , Tiamina/análogos & derivados , Tiamina/química
17.
Anal Chim Acta ; 1079: 94-102, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387724

RESUMO

A shrimp tropomyosin (TPM) immunosensor has been developed and optimized to detect trace amounts of shrimp (in the ppm range), based on a combination of an amperometric transduction, magnetic particles and disposable screen-printed electrodes. The approach is based on the implementation of a sandwich immunoassay format on the surface of magnetic beads and their coupling onto disposable screen-printed electrodes to finally register the amperometric response at -200 mV vs. Ag pseudo-reference electrode, using H2O2 as enzymatic substrate and hydroquinone as redox mediator. The use of carboxyl-functionalized magnetic microbeads (MBs) and in-house made magnetic nanoparticles (MNPs) as solid supports have been evaluated and compared. Our experimental results confirm that the use of MBs, in addition to simplifying the test protocol, improves the resulting sensitivity, so they were selected for the implementation of the immunosensor. In the optimized experimental conditions, the developed immunosensor offered a LOD of 47 pg mL-1 for amperometric determination of shrimp TPM standards and great selectivity against TPM from other sources, thus allowing differentiation between crustaceans (shrimp) and mollusks (squid). Applicability studies demonstrated successful determination both in crude and cooked samples using very simple protocols. Additionally, processed foods based on fish and mollusks that could potentially include crustaceans in their composition have been analyzed using the sensor and compared to the declared ingredients. The sensitivity and specificity showed by the sensor in the analysis of heterogeneous food samples without a previous purification or enrichment stage, also outperforms existing solutions in terms of time and cost effectiveness and permits its direct and smooth implementation in the food industry for routine allergen analyses.


Assuntos
Alérgenos/análise , Contaminação de Alimentos/análise , Tropomiosina/análise , Alérgenos/imunologia , Animais , Anticorpos/imunologia , Armoracia/enzimologia , Técnicas Eletroquímicas/métodos , Análise de Alimentos/métodos , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Hidroquinonas/química , Imunoensaio/métodos , Limite de Detecção , Penaeidae/química , Tropomiosina/imunologia
18.
Analyst ; 144(19): 5794-5801, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31464300

RESUMO

We report here a highly sensitive sandwich type electrochemical aptasensor for lysozyme (lys) detection by the integration of an antifouling interface with HRP-based signal amplification. The biosensing interface with antifouling ability is designed, consisting of a lys-binding aptamer (LBA), dithiothreitol (DTT) and mercaptohexanol (MCH). When lys is captured by the immobilized LBA due to the specific recognition of the aptamer, gold nanoparticles (AuNPs) functionalized with HRP and LBA (HRP-AuNP-LBA) are further conjugated to the surface-bound lys, forming a sandwich assay format. HRP catalyzes the chemical oxidation of hydroquinone (HQ) by hydrogen peroxide (H2O2) to produce benzoquinone (BQ) which results in a large electrochemical reduction signal of BQ. Therefore, this reduction signal measured by differential pulse voltammetry (DPV) is used to detect lys. The catalytic behavior of HRP toward the reaction between HQ and H2O2, together with the high loading of HRP on AuNPs, remarkably amplifies the signal. A linear relationship between the DPV response and the logarithm of lys concentration from 0.01 pg mL-1 to 105 pg mL-1 with a detection limit of 0.003 pg mL-1 (S/N = 3) is obtained. The proposed biosensing platform combines antifouling ability and signal amplification, resulting in high sensitivity, providing an effective way for ultrasensitive assay of protein biomarkers in complex media.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Peroxidase do Rábano Silvestre/química , Muramidase/sangue , Armoracia/enzimologia , Sequência de Bases , Ouro/química , Humanos , Peróxido de Hidrogênio/química , Hidroquinonas/química , Limite de Detecção , Nanopartículas Metálicas/química , Muramidase/química , Reprodutibilidade dos Testes
19.
Analyst ; 144(19): 5748-5754, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31432061

RESUMO

A sensitive electrochemical immunoassay (e-ELISA) has been developed for the detection of the gastrointestinal parasitic nematode Ostertagia ostertagi (brown stomach worm) in infected and control serum samples. An antigen-indirect immunoassay format was employed to detect the presence of O. ostertagi antibodies, coupled with an anti-species monoclonal horseradish peroxidase (HRP) conjugate. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and TMB (3,3',5,5'-tetramethylbenzidine/hydrogen peroxide) were investigated as both chromogenic visualising reagents for optical ELISA and electroactive substrates for electrochemical ELISA in the HRP catalysed oxidation reaction. Coulometry was applied for the detection of O. ostertagi antibodies (via TMB electrochemistry) and compared with the commercial optical ELISA (ABTS based SVANOVIR® O. ostertagi-Ab ELISA kit). Cost-effective in-house sensors were designed and fabricated using polyester and chemical adhesive materials with the aid of stencil printing and laser machining techniques. The performance of the electrochemical ELISA and sensor was evaluated by investigating redox mediators (ABTS vs. TMB), stop solutions (sodium dodecyl sulfate vs. sulfuric acid) and incubation times (150 min vs. 70 min vs. 25 min). For a total assay incubation time of 70 minutes, the TMB/H2SO4 based e-ELISA was able to differentiate between positive (P) and negative (N) control serum samples, with a P/N70 control ratio 1.6 times higher than that of optical ELISA (TMB/H2SO4 combination) and 2.9 times higher than that of the commercial ELISA kit (ABTS/SDS combination). Furthermore, the e-ELISA approach is quicker and required only 25 min (total incubation time) with even better response (P/N25 = 14.7), which is approximately 4-fold higher than the optical immunoassay (P/N25 = 3.8). The proposed e-ELISA is specific (selective Ab-Ag interactions) and highly sensitive - capable of detecting up to 16-fold dilutions of a positive control serum sample. The electrochemical ELISA approach has the potential for rapid sample screening in a portable, disposable format, contributing to the quest for effective prevention and control of parasitic Ostertagia ostertagi infections in cattle.


Assuntos
Anticorpos Anti-Helmínticos/sangue , Doenças dos Bovinos/diagnóstico , Técnicas Eletroquímicas/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Ostertagíase/veterinária , Animais , Benzidinas/química , Benzotiazóis/química , Bovinos , Doenças dos Bovinos/parasitologia , Técnicas Eletroquímicas/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Imunoglobulina G/química , Ostertagia , Ostertagíase/diagnóstico , Ostertagíase/parasitologia , Ácidos Sulfônicos/química
20.
ACS Appl Mater Interfaces ; 11(32): 29158-29166, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31313570

RESUMO

Highly active, stable, and cost-effective enzyme-mimicking nanomaterials (nanozymes) hold the potential to be an alternative to replace natural enzymes for the catalysis of enzyme-like reactions in various applications. Here, novel 3D ruthenium-based metal-organic gels (Ru-MOGs) with fibrillar network structures have been successfully synthesized using a facile one-step strategy at room temperature. Surprisingly, the developed 3D fibrillar networked Ru-MOGs simultaneously possess intrinsic horseradish peroxidase and NADH peroxidase mimetic activities. Meanwhile, the horseradish peroxidase mimetic catalytic activity displays well in both acidic environment and alkaline condition. Kinetic analysis reveals that Ru-MOGs make an effective peroxidase mimic with exceptionally high catalytic velocity (Vm), substrate binding affinity (Km), and catalytic efficiency (Kcat/Km). Furthermore, as a proof-of-concept, the mimetic enzyme property of this material was further used to establish a chemiluminescent biosensing platform for glucose detection. These easily synthesized Ru-MOGs as highly active and novel nanozymes not only suggests a bright future for the nanomaterials as enzyme mimics but also provides new insights into the properties of MOGs, greatly broadening and advancing their applications in biocatalysis and bioassays.


Assuntos
Materiais Biomiméticos/química , Nanoestruturas/química , Peroxidases/química , Rutênio/química , Géis , Peroxidase do Rábano Silvestre/química
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