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1.
Ecotoxicol Environ Saf ; 202: 110895, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615496

RESUMO

Halogenated phenols, such as 2,4-dichlorophenol (2,4-DCP) and 4-bromophenol (4-BP) are pollutants generated by a various industrial sectors like chemical, dye, paper bleaching, pharmaceuticals or in an agriculture as pesticides. The use of Horseradish peroxidase (HRP) in the halogenated phenols treatment has already been mentioned, but it is not well understood how the different phenolic substrates can bind in the peroxidase active site nor how these specific interactions can influence in the bioremediation potential. In this work, different removal efficiencies were obtained for phenolic compounds investigated using HRP as catalyst (93.87 and 59.19% to 4BP and 2,4 DCP, respectively). Thus, to rationalize this result based on the interactions of phenols with active center of HRP, we combine computational and experimental methodologies. The theoretical approaches utilized include density functional theory (DFT) calculations, docking simulation and quantum mechanics/molecular mechanics (QM/MM) technique. Michaelis Menten constant (Km) obtained through experimental methodologies were 2.3 and 0.95 mM to 2,4-DCP and 4-BP, respectively, while the specificity constant (Kcat/Km) found was 1.44 mM-1 s-1 and 0.62 mM-1 s-1 for 4-BP and 2,4-DCP, respectively. The experimental parameters appointed to the highest affinity of HRP to 4-BP. According to the molecular docking calculations, both ligands have shown stabilizing intermolecular interaction energies within the HRP active site, however, the 4-BP showed more stabilizing interaction energy (-53.00 kcal mol-1) than 2,4-dichlorophenol (-49.23 kcal mol-1). Besides that, oxidative mechanism of 4-BP and 2,4-DCP was investigated by the hybrid QM/MM approach. This study showed that the lowest activation energy values for transition states investigated were obtained for 4-BP. Therefore, by theoretical approach, the compound 4-BP showed the more stabilizing interaction and activation energy values related to the interaction within the enzyme and the oxidative reaction mechanism, respectively, which corroborates with experimental parameters obtained. The combination between experimental and theoretical approaches was essential to understand how the degradation potential of the HRP enzyme depends on the interactions between substrate and the active center cavity of the enzyme.


Assuntos
Biodegradação Ambiental , Peroxidases/metabolismo , Fenóis/metabolismo , Catálise , Poluentes Ambientais , Peroxidase do Rábano Silvestre/química , Cinética , Simulação de Acoplamento Molecular , Oxirredução
2.
Food Chem ; 331: 127368, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32569962

RESUMO

A novel strategy for AFB1 detection in grains was proposed based on DNA tetrahedron-structured probe (DTP) and horseradish peroxidase (HRP) triggered polyaniline (PANI) deposition. Briefly, the DNA tetrahedron nanostructures were assembled on the gold electrode, with carboxylic group designed on top vertex of them. The carboxylic group was conjugated with the AFB1 monoclonal antibody (mAb) to form DTP. The test sample and a known fixed concentration of HRP-labeled AFB1 were mixed and they compete for binding to DTP. The HRP assembled on the gold electrode catalyzed the polymerization of aniline on DTP. AFB1 in grains could be determined by using PANI as electrochemical signal molecules. Interestingly, DNA tetrahedron-structure, which has mechanical rigidity and structural stability, can improve antigen-antibody specific recognition and binding efficiency through the use of mAb ordered assembly. Meanwhile, nucleic acid backbone with a large amount of negative charge is good template for aniline polymerization under mild conditions.


Assuntos
Aflatoxina B1/análise , Compostos de Anilina/química , Técnicas Eletroquímicas/métodos , Contaminação de Alimentos/análise , Nanoestruturas/química , Aflatoxina B1/imunologia , Anticorpos Monoclonais/química , DNA/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Peroxidase do Rábano Silvestre/química , Polimerização , Sensibilidade e Especificidade
3.
Chemosphere ; 258: 127306, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32540533

RESUMO

The threat of antibiotics in the environment causing antibiotics resistance is a global health concern. Enzymes catalyze pollutant transformations, and how commercially available enzymes like horseradish peroxidase (HRP), with or without a redox mediator, may be used to degrade antibiotics in water treatment is of great interest. This work demonstrates tetracycline transformation by HRP, and how it is significantly enhanced by free radicals created from the mediator 2,2-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Water temperature and pH strongly influence the tetracycline removal rate due to their correlation with the enzyme activity, abundance and stability of ABTS•+. Four transformation products were identified in the pure HRP system using a liquid chromatography tandem mass spectrometry hybrid quadrupole-orbitrap mass spectrometer system. Addition of 25 µmol L-1 ABTS not only accelerated the degradation of tetracycline, but also expanded the range of degradation pathways. Potential tetracycline transformation pathways are proposed based on these observations, which include a range of mechanisms such as hydroxylation, demethylation, dehydration, decarbonylation and secondary alcohol oxidation. Despite of decreased efficiency, the HRP/ABTS system was able to degrade tetracycline in a domestic wastewater treatment plant effluent matrix, which demonstrates the potential of the system to be utilized in wastewater treatment.


Assuntos
Peroxidase do Rábano Silvestre/química , Tetraciclina/química , Poluentes Químicos da Água/química , Antibacterianos , Benzotiazóis , Catálise , Cromatografia Líquida , Radicais Livres/química , Oxirredução , Ácidos Sulfônicos , Água , Purificação da Água/métodos
4.
Mikrochim Acta ; 187(3): 195, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32124063

RESUMO

A dual functionalized magnetic nanomaterial was fabricated by combining the magnetic core, titania shell, and hydrophilic molecules (denoted as Fe3O4@TiO2-IDA). Based on the mechanism of metal oxide affinity chromatography and hydrophilic interaction liquid chromatography, the sorbent shows excellent one-step separation capacity for both glycopeptides and phosphopeptides. For phosphopeptide enrichment, Fe3O4@TiO2-IDA exhibits high sensitivity and selectivity. The concentration of ß-casein in the digests can be as low as 0.0125 ng µL-1; the mass ratio of ß-casein and BSA digest can reach 1:800. For glycopeptide enrichment, Fe3O4@TiO2-IDA also exhibits good performance. The concentration of horseradish peroxidase (HRP) in the digested sample can be as low as 0.04 ng µL-1; the mass ratio of HRP and BSA digest can reach 1:100. Following the one-step enrichment, elution, and nano LC-MS/MS analysis, 550 unique phosphopeptides and 330 glycopeptides were identified from 100 µg mouse brain sample through a single run of LC-MS/MS. Graphical abstract Multipurpose Fe3O4@TiO2-IDA nanomaterials are employed in simultaneous enrichment and separation of glycopeptides and phosphopeptides.


Assuntos
Ácidos Dicarboxílicos/química , Glicopeptídeos/análise , Iminoácidos/química , Nanopartículas de Magnetita/química , Fosfopeptídeos/análise , Titânio/química , Animais , Encéfalo/metabolismo , Peroxidase do Rábano Silvestre/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
5.
Chemistry ; 26(40): 8714-8719, 2020 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-32134164

RESUMO

In the development of colorimetric biosensors, the use of electrochromic mediators has been accepted and widely used during decades. The main drawback of these types of enzymatic substrates is the difficult recovery of the initial redox state of the molecule, which can be done electrochemically or by antioxidants addition, complicating the initially simple structure of the biosensor. those strategies are rarely followed Actually, being the disposable biosensor configuration the most extended for this detection mechanisms. Alternatively, we propose the first reported use of a diacid dithienylethene 1,2-bis(5-carboxy-2-methylthien-3-yl)cyclopentene (DTE) photoelectrochromic compound as a substrate of the horseradish peroxidase (HRP). The photoisomerization between the open (DTEo) and closed (DTEc) forms of the molecule and the respective shift in the redox potential allowed the light-induced enzymatic detection of glucose in the glucose oxidase [(GOx)]-HRP cascade system. This fast and easy control over the enzymatic substrate availability by light pulses permits a gradually consumption and the light-regeneration of the biosensor for a number of cycles. We consider the presented results transcendent in the development of reusable and light-controlled photonic biosensing systems.


Assuntos
Glucose Oxidase/metabolismo , Glucose/química , Peroxidase do Rábano Silvestre/metabolismo , Técnicas Biossensoriais/métodos , Colorimetria/métodos , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Oxirredução
6.
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
7.
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
8.
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
9.
Talanta ; 209: 120505, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31891997

RESUMO

Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H2O2. Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10-15 M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research.


Assuntos
Eletroforese em Microchip/métodos , MicroRNAs/análise , Armoracia/enzimologia , Linhagem Celular Tumoral , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Exodesoxirribonucleases/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Luminescência , Medições Luminescentes , Luminol/química , MicroRNAs/genética , Neoplasias/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico
10.
Talanta ; 209: 120465, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892037

RESUMO

A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3',5,5'-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22 pmol L-1), below the maximum levels allowed by the legislation, with a wide response range (50-10000 pmol L-1). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use.


Assuntos
Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Nanopartículas Metálicas/química , Neonicotinoides/análise , Nitrocompostos/análise , Praguicidas/análise , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Armoracia/enzimologia , Benzidinas/química , Citrullus/química , Água Potável/análise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Contaminação de Alimentos/análise , Ouro/química , Peroxidase do Rábano Silvestre/química , Limite de Detecção , Lycopersicon esculentum/química , Neonicotinoides/imunologia , Nitrocompostos/imunologia , Praguicidas/imunologia , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/imunologia
11.
Talanta ; 208: 120445, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816708

RESUMO

Multi-channel capillaries (MC) formed from thousands individual microcapillaries with diameters ranging 10-100 µm are of a great interest for their use as platforms for molecular imprinting due to their relatively large surface area, high mechanical stability and possibility of facile integration in sensor systems. The manuscript proposes a new format of immunoassay based on imprinted protein immobilized on a MC inner surface modified with poly-l-lysine. The combination of the environmentally friendly, easy-to-produce and cheap recognition element with the carrier allowing to increase the assay sensitivity makes the described technique a perspective alternative for the existing screening tests. Two bioimprinting approaches were described. The imprinted protein (ovalbumin, OVA) primarily prepared separately and later immobilized on a MC structure was compared to the imprinted OVA directly prepared on the MC surface. Detection of a food contaminant zearalenone was chosen as a proof-of-concept. In a case of the immobilization of the primarily prepared imprinted OVA the reached limit of detection (LOD) was 0.8 ng/mL, and for the in-situ imprinted OVA the LOD was 0.12 ng/mL. The sensitivity of the developed bioimprinted assay was comparable to the commercially available ELISA kits for ZEN detection. The OVA in-situ imprinted on the MC surface was tested for the detection of ZEN in artificially spiked wheat samples. The high recovery values (88-112%) and good repeatability (RSD of 8.5-9.6%) were demonstrated allowing to conclude that the IPs-based MC-ELISA is a promising tool for analysis of the mycotoxin in complex matrices.


Assuntos
Contaminação de Alimentos/análise , Imunoensaio/métodos , Impressão Molecular , Ovalbumina/química , Triticum/química , Zearalenona/análise , Vidro , Peroxidase do Rábano Silvestre/química , Proteínas Imobilizadas/química , Polilisina/química , Soroalbumina Bovina/química , Zearalenona/química
12.
Mater Sci Eng C Mater Biol Appl ; 107: 110273, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761219

RESUMO

A novel electrospinning approach is proposed for the fabrication of copper (Cu)-nanoflower decorated gold nanoparticles (AuNPs)-graphene oxide (GO) nanofiber (NF) as an electrochemical biosensor for the glucose detection. In this study, GO was mixed with poly(vinyl alcohol) (PVA) and used as a fiber precursor, which greatly improves the electrochemical properties. The above solution was uniformly coated onto the surfaces of gold chip to form GO NFs via electrospinning. AuNPs were coated onto the surface of GO NFs and then incorporated organic-inorganic hybrid nanoflower [Cu nanoflower-glucose oxidase (GOx) and horseradish peroxidase (HRP)]. The electrochemical experiments revealed that Cu-nanoflower@AuNPs-GO NFs exhibited outstanding electrochemical catalytic nature, and selectivity for the conversion of glucose to gluconic acid in the presence of GOx-HRP-Cu nanoflower. The Cu-nanoflower@AuNPs-GO NFs coated Au chip exhibited good linear range 0.001-0.1 mM, with a detection limit of 0.018 µM. The Cu-nanoflower@AuNPs-GO NFs modified Au chip exhibited higher catalytic properties, which are attributed to the coating of unique organic-inorganic nanostructured materials on the surfaces of Au chip. These results indicate that the nano-bio hybrid materials can be applied as a promising electrochemical biosensor to monitor glucose levels in biofluids.


Assuntos
Técnicas Biossensoriais/métodos , Cobre/química , Glucose/análise , Ouro/química , Grafite/química , Nanoestruturas/química , Biocatálise , Técnicas Eletroquímicas , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Limite de Detecção , Nanopartículas Metálicas/química , Nanofibras/química , Álcool de Polivinil/química
13.
Anal Chim Acta ; 1096: 44-52, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31883590

RESUMO

In this paper, horseradish peroxidase (HRP) was successfully immobilized on heated Au disk electrode (HAuDE) by biotin-streptavidin specific interaction through HS-ssDNA-biotin self-assembled on HAuDE for investigation the electrocatalytic activity of HRP. With elevated electrode temperature, the significant temperature effect of the electrocatalytic activity of HRP for H2O2 reduction was demonstrated by using this bio-sensing platform. With an electrode temperature of 40 °C, a detection limit of 1.5 × 10-6 mol L-1 for H2O2 reduction could be obtained, which was more than one magnitude lower than that with an electrode temperature of 0 °C. Because HRP can be widely used as an enzyme label for amplification detection, this sensing platform can be broadly applied to analytical chemistry such as nucleic acid detection, and aptamer-based biosensors.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Ouro/química , Peróxido de Hidrogênio/análise , Tampões (Química) , Eletrodos , Enzimas Imobilizadas/química , Desenho de Equipamento , Peroxidase do Rábano Silvestre/química , Temperatura Alta , Temperatura , Água/análise
14.
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
15.
Anal Biochem ; 589: 113493, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31682794

RESUMO

Reduced nicotinamide adenine dinucleotide (NADH) plays a pivotal role in the electron-transfer chain of biological system. Analysis of many biological markers is based on the detection of the enzymatically generated NADH. In this paper, a sensitive hydrogen peroxide (H2O2) biosensor, fabricated by carbon nanotubes (CNTs)/tetrathiafulvalene (TTF)/horseradish peroxidase (HRP), was applied for detecting the NADH in a buffer containing methylene blue (MB) at low operating potential of - 0.3 V (vs. Ag/AgCl). Since the NADH could be oxidized by MB to release H2O2, the electrochemical biosensor enables to detect the NADH in the MB buffer. And the low working potential made the biosensor avoid the interference from other electroactive substances. Linear response ranges from 10 µM to 790 µM, with a sensitivity of 4.76 µA mM-1 and a detection limit of 1.53 µM were obtained under the optimum conditions. The proposed sensor provided a promising approach for sensitively detecting the NADH.


Assuntos
Técnicas Biossensoriais/métodos , NAD/análise , Eletroquímica , Eletrodos , Enzimas Imobilizadas/química , Compostos Heterocíclicos/química , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Azul de Metileno/química , Nanotubos de Carbono/química
16.
Mikrochim Acta ; 186(12): 840, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31768650

RESUMO

A colorimetric assay is described for simultaneous detection of multiple analytes related to food safety. It is based on the use of a sandwich aptasensor and terminal deoxynucleotidyl transferase (TdT) which produces a primer for subsequent rolling circle amplification (RCA). Two split aptamer fragments (Apt1 and Apt2) are firstly immobilized, Apt1 on gold nanoparticles (AuNPs), and Apt2 on magnetic beads (MBs). They are then used in a sandwich aptasensor. In the presence of analyte, two probes could specifically recognize target and form a ternary assembly, and the magnetic beads also act to separate rapidly and enrich the target. Then, the extension of template-free DNA is triggered by TdT at the exposed 3'-hydroxy terminals of Apt1. This produces polyA sequences that serve as primers for subsequent RCA. The product of RCA is hybridized with a complementary horse radish peroxidase (HRP) DNA probe. HRP catalyzes the H2O2-mediated oxidation of tetramethylbenzidine (TMB) and forms a blue chromogenic product. After magnetic separation, the absorption values of the blue product in the supernatant are measured at a wavelength of 600 nm. Based on this dual amplification mechanism, the assay was applied to multiplexed determination of enrofloxacin (ENR), lead(II), Escherichia coli O157:H7 and tropomyosin. Exemplarily, ENR is detectable at concentrations down to 2.5 pg mL-1 with a linear range that extends from 1 pg mL-1 to 1 µg·mL-1. The assay was validated by analysis of spiked fish samples. Recoveries range between 87.5 and 92.1%. Graphical abstractSchematic representation of a TdT-RCA based aptasensor for multiple analytes related to food safety. It makes use of sandwich aptasensors and TdT-produced universal primer-triggered RCA reaction. dATP: deoxyadenosine triphosphate, TdT: Terminal Deoxynucleotidyl Transferase, RCA: rolling circle amplification, TMB: 3,3',5,5'-Tetramethylbenzidine.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Colorimetria/métodos , DNA Nucleotidilexotransferase/química , Contaminação de Alimentos/análise , Animais , Aptâmeros de Nucleotídeos/genética , Armoracia/enzimologia , Benzidinas/química , Corantes/química , DNA/química , DNA/genética , Enrofloxacina/análise , Escherichia coli O157/isolamento & purificação , Ouro/química , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Chumbo/análise , Limite de Detecção , Nanopartículas Metálicas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Salmão , Alimentos Marinhos/análise , Tropomiosina/análise
17.
Molecules ; 24(21)2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31684005

RESUMO

Copper is the most common metal catalyst used in atom transfer radical polymerization (ATRP), but iron is an excellent alternative due to its natural abundance and low toxicity compared to copper. In this work, two new iron-porphyrin-based catalysts inspired by naturally occurring proteins, such as horseradish peroxidase, hemoglobin, and cytochrome P450, were synthesized and tested for ATRP. Natural protein structures were mimicked by attaching imidazole or thioether groups to the porphyrin, leading to increased rates of polymerization, as well as providing polymers with low dispersity, even in the presence of ppm amounts of catalysts.


Assuntos
Biomimética , Ferro/química , Polimerização , Porfirinas/química , Catálise , Cobre/química , Sistema Enzimático do Citocromo P-450/química , Hemoglobinas/química , Peroxidase do Rábano Silvestre/química , Imidazóis/química , Estrutura Molecular , Oxirredução , Polímeros/química , Sulfetos/química
18.
Analyst ; 144(23): 6914-6921, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31657376

RESUMO

Monitoring soluble immune checkpoints in circulating fluids has the potential for minimally-invasive diagnostics and personalised therapy in precision medicine. Yet, the sensitive detection of multiple immune checkpoints from small volumes of liquid biopsy samples is challenging. In this study, we develop a multiplexed immune checkpoint biosensor (MICB) for parallel detection of soluble immune checkpoints PD-1, PD-L1, and LAG-3. MICB integrates a microfluidic sandwich immunoassay using engineered single chain variable fragments and alternating current electrohydrodynamic in situ nanofluidic mixing for promoting biosensor-target interaction and reducing non-specific non-target binding. MICB provides advantages of simultaneous analysis of up to 28 samples in <2 h, requires as little as a single sample drop (i.e., 20 µL) per target immune checkpoint, and applies high-affinity yeast cell-derived single chain variable fragments as a cost-effective alternative to monoclonal antibodies. We investigate the assay performance of MICB and demonstrate its capability for accurate immune checkpoint detection in simulated patient serum samples at clinically-relevant levels. MICB provides a dynamic range of 5 to 200 pg mL-1 for PD-1 and PD-L1, and 50 to 1000 pg mL-1 for LAG-3 with a coefficient of variation <13.8%. Sensitive immune checkpoint detection was achieved with limits of detection values of 5 pg mL-1 for PD-1, 5 pg mL-1 for PD-L1, and 50 pg mL-1 for LAG-3. The multiplexing capability, sensitivity, and relative assay simplicity of MICB make it capable of serving as a bioanalytical tool for immune checkpoint therapy monitoring.


Assuntos
Antígenos CD/sangue , Antígeno B7-H1/sangue , Técnicas Biossensoriais/métodos , Receptor de Morte Celular Programada 1/sangue , Antígenos CD/imunologia , Armoracia/enzimologia , Antígeno B7-H1/imunologia , Benzidinas/química , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Colorimetria/métodos , Técnicas Eletroquímicas/métodos , Peroxidase do Rábano Silvestre/química , Humanos , Hidrodinâmica , Peróxido de Hidrogênio/química , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Receptor de Morte Celular Programada 1/imunologia , Anticorpos de Cadeia Única/imunologia
19.
Biosens Bioelectron ; 146: 111744, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31605986

RESUMO

MicroRNAs, essential for gene expression and physiological regulation, are considered to be reliable biomarkers for the early diagnosis and treatment of cancers. Herein, a sensitive biosensor that uses a synergistically catalytic nanoprobe and improved toehold strand displacement reaction (TSDR) has been fabricated, and successfully applied to microRNA-155 (miR-155) detection. A nanoscale copper-based metal organic framework assembled by Pt nanoparticles and horseradish peroxidase (Cu-NMOF@PtNPs/HRP) served as a co-catalytic nanoprobe and was coupled with improved TSDR to achieve multiple amplifications. In the absence of miR-155, the tetrahedral DNA nanostructures (TDNs) immobilized on the gold electrode were independent of the TSDR system because of the binding of the shielding region of the locked probe (LP) with the template probe (TP). Instead, the target would initiate the TSDR system, leading to the conformational change of TDNs and hybridization of the nanoprobe. Cu-NMOF@PtNPs/HRP exhibited extraordinary catalytic property towards the hydroquinone-hydrogen peroxide system, demonstrating that the nanoprobe exerted a concerted effect on the electrochemical performance of the biosensor. Under optimal conditions, the cathodic current exhibited a logarithmic relation over 0.50-1.0 × 105fM miR-155, with a detection limit of 0.13 fM, indicating that the constructed biosensor has considerable potential in the field of clinical disease diagnostics for miR-155.


Assuntos
Técnicas Biossensoriais/métodos , Estruturas Metalorgânicas/química , MicroRNAs/análise , Nanoestruturas/química , Platina/química , Linhagem Celular Tumoral , Cobre/química , Peroxidase do Rábano Silvestre/química , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Nanopartículas Metálicas/química , MicroRNAs/sangue
20.
Nanoscale ; 11(42): 19797-19805, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31621738

RESUMO

Enzymes are widely employed to reduce the environmental impact of chemical industries as biocatalysts improve productivity and offer high selectively under mild reaction conditions in a diverse range of chemical transformations. The poor stability of biomacromolecules under reaction conditions is often a critical bottleneck to their application. Protein engineering or immobilization onto solid substrates may remedy this limitation but, unfortunately, this is often at the expense of catalytic potency or substrate specificity. In this work, we show that the combinatorial approach of chemical modification and supramolecular nanoencapsulation can endow mechanistically diverse enzymes with apparent extremophilic behavior. A protein-polymer surfactant core-shell architecture facilitates construction of increasingly complex biofluids from individual biosynthetic components, each of which retain biological activity at hydration levels almost two orders of magnitude below solvation. The herein constructed multifunctional biofluids operate in tandem up to 150 °C and in the total absence of solvent under apparent diffusional mass-transport limitation. The biosynthetic promotion of extremophilic traits for enzymes with diverse catalytic motions and chemical functions highlights the extraordinary capacity for a viscous surfactant milieu to replace both hydration and bulk waters.


Assuntos
Enzimas Imobilizadas/química , Engenharia de Proteínas , Solventes , Tensoativos/química , Catálise , Peroxidase do Rábano Silvestre/química
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