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1.
Talanta ; 206: 120215, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514903

RESUMO

This article described the fabrication of novel magnetic carbon nanotube modified with polymeric deep eutectic solvent (M-CNT@PDES) and its application as extractant for the magnetic solid phase extraction (MSPE) of bovine serum albumin (BSA). The physicochemical properties and morphology of M-CNT@PDES were characterized by X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermo-gravimetric analysis (TGA), zeta potentials, fourier transform infrared spectrometry (FT-IR) and transmission electron microscope (TEM). Afterwards, several parameters such as pH value, initial concentration of BSA, extraction time, ionic strength and extraction temperature were optimized. The results indicated that the modification of PDES significantly improved the extraction performance for BSA, and the maximum extraction capacity was 225.15 mg/g under the optimized conditions. In addition, 0.20 mol/L NaCl-PBS solution was chosen as the appropriate eluent, and favourable elution rate (81.22%) was obtained. Circular dichroism spectroscopy (CD) indicated that the secondary structure of BSA has not changed during extraction and elution. The regenerative experiment and application in real calf serum confirmed the outstanding durability and practical application ability of M-CNT@PDES. All of above verified that the proposed M-CNT@PDES coupled with MSPE method has great application potential for the pre-concentration of biomolecules.


Assuntos
Nanotubos de Carbono/química , Soroalbumina Bovina/análise , Solventes/química , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Limite de Detecção , Fenômenos Magnéticos , Compostos de Amônio Quaternário/química , Extração em Fase Sólida/métodos , Xilitol/química
2.
Anal Bioanal Chem ; 411(27): 7055-7059, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31598742

RESUMO

A new perspective on the relevant problem-creating simple, rapid, and efficient protein sensors based on microstructured optical fibers using a simple homogeneous analysis format-was proposed. Commercially available long-period grating hollow core microstructured optical fibers (LPG HCMOF) were used to determine bovine serum albumin (BSA) and albumin from chicken eggs (OVA) in binary mixtures as well as immunoglobulin G (IgG) in the presence of BSA and OVA. LPG HCMOF transmission spectra allowed the detection of both BSA and OVA up to 10 mg/mL with LOD as low as 0.1 and 0.8 µg/mL, respectively. Partial least squares regression (PLS) was utilized for modeling of LPG HCMOF spectral data and quantitative analysis of BSA, OVA, total protein, and IgG in binary and ternary mixtures. Rather high coefficients of determination (R2) and low root mean square error for the calibration (RMSEC) (15%) and prediction (RMSEP) (20%) were obtained for all PLS models. The proposed approach was tested in the analysis of BSA in spiked horse blood hemolyzed (HBH). The results demonstrated the functionality of the proposed approach and offered the opportunity for the creation of a wide range of sensors for protein determination in complex mixtures. Graphical abstract.


Assuntos
Imunoglobulina G/análise , Ovalbumina/análise , Soroalbumina Bovina/análise , Animais , Análise Química do Sangue/métodos , Bovinos , Galinhas , Cavalos , Análise dos Mínimos Quadrados , Camundongos , Modelos Moleculares , Fibras Ópticas
3.
Int J Biol Macromol ; 138: 602-617, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319084

RESUMO

Albumin is a globular protein which plays a pivotal role in maintaining plasma pressure and the nutritional balance. Different compounds are transported by binding to albumin in the blood. Also, human health is closely related to the serum albumin concentration in blood plasma or other biological fluids. Due to the high structural similarity with human serum albumin (HSA), bovine serum albumin (BSA) has been widely investigated as a model protein in different fields. Importantly, albumin detection has recently gained huge interest, as this protein serves as an important indicator of cow health, and its milk and meat quality. Also, it is also known as an allergenic and a carrier protein. As a result, it is highly essential to determine bovine albumin in various industries, such as medicine, pharmaceutical, clinical and food. Therefore, the development of new, efficient, fast and straightforward methods for selective detection of BSA is critical. This review seeks to highlight different characteristics of BSA and its importance. Then, by focusing on recent developments made in the last two decades in BSA biosensing and determination methods, the use of different biomaterials/nanomaterials is discussed.


Assuntos
Técnicas Biossensoriais , Soroalbumina Bovina/análise , Animais , Bovinos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Nanopartículas Metálicas/química , Modelos Moleculares , Estrutura Molecular , Nanotecnologia , Conformação Proteica , Soroalbumina Bovina/química , Albumina Sérica Humana/análise , Albumina Sérica Humana/química , Espectrometria de Fluorescência
4.
Talanta ; 204: 613-625, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357343

RESUMO

A modified CUPRAC (cupric reducing antioxidant capacity) method was developed for the simultaneous estimation of protein oxidation and counteracting antioxidant defense, and the results were compared with those of a modified 2,4-dinitrophenylhydrazine (DNPH) carbonyl assay. The alkaline carbonyl method was cleared off interferences by solvent extraction using a cationic surfactant. Both solution and Nafion membrane sensor CUPRAC methods were used to measure the oxidative hazard in protein solutions. Bovine serum albumin, fetal bovine serum and egg white were used as protein probes, exposed to oxidation by Fe(II)-induced Fenton reaction in the absence and presence of selected antioxidants (ascorbic acid, cysteine, gallic acid, glutathione, and N-acetyl cysteine). Protein probes were initially unreactive toward the CUPRAC and DNPH reagents, but produced colored products upon Fenton oxidation which were bleached by antioxidants, enabling an indirect measurement of antioxidant activity (AOA) by difference. Spearman's rank test for antioxidants demonstrated that there was a strong correlation (+0.7 to +0.9) between the modified CUPRAC and carbonyl assays. There was also a strong correlation between the results of the solution phase and optical sensing CUPRAC methods (R2 > 0.95). As opposed to conventional antioxidant assays not using biologically relevant probes, this work utilizes protein probes for AOA assessment.


Assuntos
Proteínas Sanguíneas/análise , Proteínas do Ovo/análise , Carbonilação Proteica , Soroalbumina Bovina/análise , Animais , Antioxidantes/química , Proteínas Sanguíneas/química , Bovinos , Citrus sinensis , Colorimetria/métodos , Cobre/química , Proteínas do Ovo/química , Sucos de Frutas e Vegetais , Hidrazinas/química , Fenantrolinas/química , Aves Domésticas , Soroalbumina Bovina/química
5.
J Am Soc Mass Spectrom ; 30(9): 1643-1653, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31168746

RESUMO

Chemical cross-linking combined with mass spectrometry (CL-MS) is a powerful method for characterizing the architecture of protein assemblies and for mapping protein-protein interactions. Despite its proven utility, confident identification of cross-linked peptides remains a formidable challenge, especially when the peptides are derived from complex mixtures. MS cleavable cross-linkers are gaining importance for CL-MS as they permit reliable identification of cross-linked peptides by whole proteome database searching using MS/MS information. Here we introduce a novel class of MS cleavable cross-linkers called isotopomeric cross-linkers (ICLs), which allow for confident and efficient identification of cross-linked peptides by whole proteome database searching. ICLs are simple, symmetrical molecules that asymmetrically incorporate heavy and light stable isotopes into the two arms of the cross-linker. As a result of this property, ICLs automatically generate pairs of isotopomeric cross-linked peptides, which differ only by the positions of the heavy and light isotopes. Upon fragmentation during MS analysis, these isotopomeric cross-linked peptides generate unique isotopic doublet ions that correspond to the individual peptides in the cross-link. The doublet ion information is used to determine the masses of the two cross-linked peptides from the same MS2 spectrum that is also used for peptide spectrum matching (PSM) by sequence database searching. Here we present the rationale for and mechanism of cross-linked peptide identification by ICL-MS. We describe the synthesis of the ICL-1 reagent, the ICL-MS workflow, and the performance characteristics of ICL-MS for identifying cross-linked peptides derived from increasingly complex mixtures by whole proteome database searching.


Assuntos
Reagentes para Ligações Cruzadas/química , Espectrometria de Massas/métodos , Peptídeos/análise , Peptídeos/química , Reagentes para Ligações Cruzadas/síntese química , Isótopos/química , Proteoma/análise , Proteoma/química , RNA Polimerase II/análise , RNA Polimerase II/química , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Espectrometria de Massas por Ionização por Electrospray/métodos
6.
PLoS One ; 14(4): e0215863, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31002721

RESUMO

In many biology- and chemistry-related research fields and experiments the quantification of the peptide and/or protein concentration in samples are essential. Every research environment has unique requirements, e.g. metal ions, incubation times, photostability, pH, protease inhibitors, chelators, detergents, etc. A new protein assay may be adequate in different experiments beyond or instead of the well-known standard protocols (e.g. Qubit, Bradford or bicinchoninic acid) in related conceptions. Based on our previous studies, we developed a novel protein assay applying the 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (BisANS) fluorescent dye. This molecule has several advantageous properties related to protein detection: good solubility in water, high photostability at adequate pH, quick interaction kinetics (within seconds) with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients. The protocol described in this work is highly sensitive in a large spectrum to detect protein (100-fold diluted samples) concentrations (from 0.28 up to more than 100 µg/mL). The BisANS protein assay is valid and applicable for quantification of the amount of protein in different biological and/or chemical samples.


Assuntos
Naftalenossulfonato de Anilina/química , Bioensaio/normas , Corantes Fluorescentes/química , Proteínas de Saccharomyces cerevisiae/análise , Soroalbumina Bovina/análise , Animais , Bovinos , Detergentes/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Saccharomyces cerevisiae/química , Solubilidade , Água/química
7.
Food Chem ; 289: 1-6, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955590

RESUMO

A new protein quantification method based on first order derivative spectrophotometry was established to eliminate various interferences, mainly chitosan, to the utmost using bovine serum albumin (BSA) as a model food protein. Absorbance spectra of BSA solutions were recorded and their first order derivative calculated. The values of derivative absorbance at 288 nm were used to generate linear calibration curve of BSA. The new method was applied in entrapping BSA into chitosan-tripolyphosphate beads. A general calibration curve was established with a CI (width of 95% confidence interval of three repeat measurements of unknown samples) less than 0.0262 g/L and a LOQ (limit of quantification) of 0.11 g/L, showing excellent tolerance to various interferences, which was further verified by the good mass balance of BSA during encapsulation. Overall, the method successfully eliminated the interferences from chitosan and other factors to facilitate the measurement of protein in complicated environments.


Assuntos
Quitosana/química , Soroalbumina Bovina/análise , Espectrofotometria , Adsorção , Animais , Calibragem , Bovinos , Quitosana/análogos & derivados , Limite de Detecção , Soroalbumina Bovina/química , Espectrofotometria/normas
8.
Chemosphere ; 227: 662-669, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31015087

RESUMO

This work investigated the synergistic effect of polyvinylpyrrolidone (PVP) and hydroxypropyl-beta-cyclodextrin (HP-ß-CD) as dual pore forming agents on the properties and performance of polysulfone (PSf) ultrafiltration membranes. A fixed concentration of PVP and varying concentrations of HP-ß-CD were used to prepare the membranes using the phase inversion technique. The results showed that the inclusion of these additives in the dope solution increased its thermodynamic instability and promoted instantaneous demixing. Overall, an increase was observed in the hydrophilicity, open porous structure and mechanical strength of the membranes. Cross-flow filtration tests demonstrated that the pure water permeability of the fabricated membrane was 891 LMH bar-1, about 4.37 times higher than the pristine membrane, while bovine serum albumin (BSA) rejection was relatively constant (about 93%) for all the fabricated membranes. This work proposed that the addition of HP-ß-CD and PVP as dual pore formers can produce a viable ultrafiltration membrane with improved water permeability without a middle ground on rejection potential.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Membranas Artificiais , Polímeros/química , Povidona/química , Sulfonas/química , Ultrafiltração/métodos , Purificação da Água/métodos , Interações Hidrofóbicas e Hidrofílicas , Permeabilidade , Porosidade , Soroalbumina Bovina/análise , Água/análise
9.
Artigo em Inglês | MEDLINE | ID: mdl-30925315

RESUMO

An efficient and novel 2,5-bis(benzo[d]thiazol-2-yl)phenol scaffold-based ratiometric fluorescent probe BTP-Cys for the sensing of cysteine has been developed. The probe BTP-Cys with acrylates moiety, as recognition site, has been successfully constructed on account of the excited state intramolecular proton transfer (ESIPT) mechanism. Upon the treatment with Cys (0-250 µM), this probe BTP-Cys exhibits a dramatic fluorescent intensity ratios enhancement (from 0.03 to 18.3) and a large emission shift (113 nm). The detection limit of this probe is as low as 3.8 × 10-7 M. Importantly, the concentration and time dependent of Cys in bovine serum albumin (BSA) has also been measured, indicating that BTP-Cys could be a biocompatible and rapid probe for Cys in vitro.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Fenóis/química , Soroalbumina Bovina/análise , Animais , Bovinos , Cisteína/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Técnicas de Sonda Molecular , Soroalbumina Bovina/química , Espectrometria de Fluorescência
10.
Biosens Bioelectron ; 132: 162-170, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30875628

RESUMO

We demonstrate a swarm biosensing platform that detects analyte based on the change in plasmonic signal from thousands of single nanoparticles sensors, leading to increased quantitative accuracy. Following dark field microscopy, we perform computational image registration and analyses to compile the hue change from thousands of single gold nanoparticles acting as individual quantitative biosensors. This platform demonstrated a limit of detection of 10 pM with a dynamic range of at least 4 orders of magnitude in buffer solution, and the successful detection of c-reactive protein (CRP) in serum compatible with 3-tier clinical cutoffs within a 10-fold difference without the need for a blocking step. By analyzing the before-and-after status of each plasmonic sensor, our sensing scheme provides informative sensing capabilities with the flexibility to select a subset of nanoparticles with optimal performance based on their initial states. Hue comparisons within and among devices also render the platform tolerant to particle and device variation. In addition, the simplicity of the readout instrumentation based on optical imaging and the implementation of microfluidics make it promising for future adaptation into point-of-care systems.


Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/instrumentação , Proteína C-Reativa/análise , Colorimetria/instrumentação , Ouro/química , Nanopartículas Metálicas/química , Animais , Técnicas Biossensoriais/métodos , Bovinos , Colorimetria/métodos , Desenho de Equipamento , Humanos , Processamento de Imagem Assistida por Computador , Nanopartículas Metálicas/ultraestrutura , Microscopia/instrumentação , Microscopia/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Sistemas Automatizados de Assistência Junto ao Leito , Soroalbumina Bovina/análise
11.
Talanta ; 198: 55-62, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876598

RESUMO

Immunoassay as a primary analytical tool is widely applied in the field of analysis. However, biological antibodies used in the routine immunoassay exhibit high cost and poor stability. Herein, in this study, a biomimetic ELISA method using molecularly imprinted polymers as a alternative to antibodies was developed. The molecularly imprinted polymers were prepared through dopamine polymerization using Fe3O4 nanoparticles as magnetic nuclei, bovine serum albumin as a template, dopamine as a functional monomer and crosslinking agent. The molecularly imprinted polymers were characterized by scanning electron micrographs, transmission electron microscope, X-ray photoelectron spectroscopy, thermogravimetric analysis, vibrating sample magnetometer and X-ray diffractometer, respectively. The detection range of the established biomimetic ELISA method was 5-1000 µg mL-1. The method exhibited high selectivity for bovine serum albumin compared with other proteins and good recovery ranging from 89.0% to 102.3% was obtained from spiked bovine serum samples. The results showed that the method by using molecularly imprinted polymers as biomimetic antibodies could be used to detect bovine serum albumin rapidly in bovine serum with a high sensitivity and accuracy. This study demonstrates that the preparation of molecularly imprinted polymers by dopamine polymerization can produce material with high affinity and potential to replace antibodies in biomimetic ELISA for protein detection.


Assuntos
Avidina/química , Materiais Biomiméticos/química , Biotina/química , Ensaio de Imunoadsorção Enzimática , Impressão Molecular , Polímeros/química , Soroalbumina Bovina/análise , Animais , Bovinos
12.
Anal Biochem ; 569: 31-38, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30707897

RESUMO

The effects of incubation temperature and acetonitrile (ACN) amount on microwave-assisted tryptic digestion of horse skeletal muscle myoglobin (MYG) and bovine serum albumin (BSA) were investigated. Microwave-assisted tryptic digestion was performed on BSA or MYG solutions containing different amounts (0, 10, and 20%) of ACN for different times (10, 20, 30, 40, and 50 min) at different temperatures (25, 37, and 55 °C). Conventional overnight tryptic digestion was also conducted with gentle mixing at 37 °C for 16 h. Digested samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Similar sequence coverage (SC) values were obtained under most conditions except when the protein sample solutions were digested with 20% ACN at 55 °C, which provided the lowest SC for both MYG and BSA for all investigated digestion times. Considering the missed cleavage (MC) ratios for 50-min microwave-assisted digestion, the highest MC ratio, (i.e., lowest trypsin activity) was observed for the digestion condition of 20% ACN at 55 °C for both proteins, while the lowest MC ratio was observed with 0% ACN at 25 °C for MYG and with 0% ACN at 55 °C for BSA. Conventional overnight tryptic digestion at 37 °C provided more completely cleaved peptides than 50-min microwave-assisted tryptic digestion at the same temperature.


Assuntos
Acetonitrilos/química , Micro-Ondas , Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tripsina/metabolismo , Animais , Bovinos , Cavalos , Mioglobina/análise , Mioglobina/metabolismo , Peptídeos/metabolismo , Soroalbumina Bovina/análise , Soroalbumina Bovina/metabolismo , Temperatura Ambiente
13.
Talanta ; 196: 402-407, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683384

RESUMO

Through years of extensive research and development, molecularly imprinted polymers (MIPs) are still inferior to their biological rivals such as antibodies, enzymes etc. In this study we report a protein-imprinted cryogel, showing antibody-like affinity and selectivity against the protein template (bovine serum albumin, BSA). The MIP was synthesized from the co-polymerization of acrylamide, N,N-methylenebisacrylamide, acrylic acid and diallylamine. Due to the participation of the ionizable monomers (acrylic acid and diallylamine), imprinted cavities with inner surface-clung charged groups were created to recognize BSA. Therefore each cavity appears like a molecular capacitor charged by carboxyl and amino groups. As the cavities are all of a molecule-size volume, a membrane made of the MIP contains a huge array of the molecular capacitors. This will produce a synergistic effect and greatly amplify the impedance signal deviations when template sorption/desorption takes place on the sensor. When the MIP was used as an artificial antibody to make an electrochemical sensor, high sensitivity and selectivity were achieved at the same time. Results indicate that BSA could be determined in a linear range of 1.5 × 10-16-10-12 mol-L-1. Meanwhile a low limit of detection was achieved at 7.2 × 10-18 mol L-1. Conclusively protein-imprinted amphoteric polyacrylamide cryogels are materials of a great potential to sense and determine charged objects like molecules, cells, microorganisms or other particles.


Assuntos
Técnicas Biossensoriais , Soroalbumina Bovina/análise , Técnicas Eletroquímicas , Impressão Molecular , Polímeros/química , Soroalbumina Bovina/química
14.
PLoS One ; 14(1): e0210286, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30650158

RESUMO

Insurmountable detection challenges will impede the development of many of the next-generation of lab-on-a-chip devices (e.g., point-of-care and real-time health monitors). Here we present the first membrane-based, microfluidic sample preconcentration method that is continuous, quantifiable, simple, and capable of working with any analyte. Forward osmosis rapidly concentrates analytes by removing water from a stream of sample fluid. 10-100X preconcentration is possible in mere minutes. This requires careful selection of the semi-permeable membrane and draw molecule; therefore, the osmosis performance of several classes of membranes and draw molecules were systematically optimized. Proof-of-concept preconcentration devices were characterized based on their concentration ability and fouling resistance. In-silico theoretical modeling predicts the experimental findings and provides an engineering toolkit for future designs. With this toolkit, inexpensive ready-for-manufacturing prototypes were also developed. These devices provide broad-spectrum detection improvements across many analytes and sensing modalities, enabling next-generation lab-on-a-chip devices.


Assuntos
Dispositivos Lab-On-A-Chip , Animais , Bovinos , Simulação por Computador , Desenho de Equipamento , Glucose/análise , Humanos , Membranas Artificiais , Osmose , Porosidade , Soroalbumina Bovina/análise
15.
Talanta ; 194: 643-648, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30609585

RESUMO

Serum albumin has a wide range of applications in biochemical experiments and pharmaceutical field. We found that a cyanine dye, dimethylindole red (Dir), could selectively interact with bovine serum albumin (BSA). Dir exhibited very weak red fluorescence, while the fluorescence intensity at 630 nm was enhanced up to 130-fold upon noncovalently interacting with 30 µM BSA. Besides, Dir showed a highly selective response to BSA over human serum albumin (HSA). For the detection of BSA, a limit of detection as low as 23 nM was obtained. Then biocompatible Dir-BSA nanoparticles were prepared by the desolvation technique. The Dir-BSA nanoparticles possess excellent fluorescence properties with a quantum yield of 32%. Furthermore, folic acid as a targeting group was conjugated to Dir-BSA nanoparticles and these nanoparticles were characterized by TEM and laser particle analyzer, etc. Folic acid-modified Dir-BSA nanoparticles were successfully used for tumor cell-targeted imaging.


Assuntos
Corantes Fluorescentes/química , Ácido Fólico/química , Nanopartículas/química , Imagem Óptica/métodos , Soroalbumina Bovina/análise , Albumina Sérica Humana/análise , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Células KB , Limite de Detecção , Camundongos , Células NIH 3T3 , Soroalbumina Bovina/química , Albumina Sérica Humana/química
16.
Mikrochim Acta ; 186(2): 68, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30627783

RESUMO

A novel magnetic nanomaterial for use in metal ion based affinity chromatography is described. It is based on the chelation between the phosphate groups of phytic acid (PA) and Ti(IV) ions. Due to the large number (6) of phosphate groups of PA, it has a large capacity for Ti(IV) ions. PA was first immobilized on magnetite nanoparticles (PA-MNPs) and then loaded with Ti(IV) ions to obtain the sorbent (Ti-PA-MNPs). The fraction of Ti(IV) ions on the surface of PA-MNPs that is exposed to the solution binds the phosphate groups of phosphopeptides. The bound phosphopeptides can then be magnetically separated. The method was applied to the enrichment of the phosphopeptides in a ß-casein tryptic digest. A tryptic digest of bovine serum albumin (BSA) was added at a molar ratio (ß-casein to BSA) of 1:2000 to study selectivity. The phosphopeptides were quantified by mass spectrometry. The limit of detection can be as low as 8 × 10-10 mol L-1. This sorbent has a high absorption capacity (53.5 µg mg-1) and shows good recoveries (90%). As many as 2145 phosphopeptides were isolated from 500 µg tryptic digest of a rat liver lysate after enrichment by Ti-PA-MNPs. This is superior to that (1568 phosphopeptides) of commercial TiO2 kit. Graphical abstract Schematic presentation of fabrication for a novel modified magnetic nanomaterial (Ti-PA-MNPs) based on the chelation of phytic acid (PA) with Ti(IV) ions. Ti-PA-MNPs were successfully applied to enriching low abundance phosphopeptides from biosamples in mass spectrometric analysis.


Assuntos
Nanopartículas de Magnetita/química , Espectrometria de Massas/métodos , Fosfopeptídeos/análise , Adsorção , Animais , Caseínas/metabolismo , Bovinos , Limite de Detecção , Fígado/química , Ácido Fítico/química , Ratos , Soroalbumina Bovina/análise , Soroalbumina Bovina/metabolismo , Titânio/química
17.
J Dairy Sci ; 102(1): 672-677, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30343904

RESUMO

Recent studies report considerable variation in ruminal pH for lactating dairy cows even when fed the same diet. We hypothesized that blood metabolites would be indicators of low ruminal pH, and hence could be used as predictors to help manage this variability. The objective of the study was to determine whether blood metabolite concentrations, body reserves, and feed efficiency were associated with ruminal pH in high-producing dairy cows fed a high-concentrate diet. Seventy-eight individually fed lactating dairy cows (days in milk = 103 ± 27; body weight = 638 ± 77 kg at the start; mean ± SD) were fed a diet consisting of 35% forage and 65% concentrate (dry matter basis). Cows were adapted for 14 d and then were sampled for 10 d. Ruminal pH was measured by rumenocentesis for all cows at the end of the study 4 h after feeding, and reticular pH was measured on a subsample of 14 cows via indwelling sensors for 5 consecutive days. Cows were classified according to rumenocentesis pH as high (pH ≥ 6.0; n = 26), medium (5.8 ≤ pH < 6; n = 21), and low (pH < 5.8; n = 31). Cows were also classified according to reticular pH as high if pH <5.8 persisted <330 min/d (an average of 78 min/d; n = 5) or low if duration of pH <5.8 was ≥330 min/d (an average of 920 min/d; n = 9). The classification based on rumenocentesis pH revealed that serum activity of aspartate aminotransferase (AST) was greater in cows with low ruminal pH (70.7 U/L) than cows with high (56.6 U/L) and medium (59.9 U/L) ruminal pH. Also, the blood urea nitrogen concentration was greater in cows with low ruminal pH (13.6 mg/dL) than cows with medium (12.2 mg/dL) and high (12.5 mg/dL) ruminal pH. Blood albumin concentration was greater for cows with low ruminal pH than for cows with medium and high ruminal pH. The classification based on reticular pH also resulted in a trend of greater AST activity and greater blood urea nitrogen concentration in the blood of cows with low pH. Regression analysis showed high serum concentration of AST was associated with high valerate concentration in ruminal fluid (R2 = 0.14), low rumenocentesis pH (R2 = 0.10), and low milk fat percentage (R2 = 0.06). Glucose, triglyceride, cholesterol, globulin, alkaline phosphates, and serum amyloid A did not differ among the different ruminal pH classes. Low pH cows (reticular and ruminal) had less backfat thickness measured via ultrasound, and cows with low ruminal pH tended to have greater milk:feed ratio. Results indicated that cows that differ in ruminal pH also had different concentrations of blood metabolites and backfat thickness, and AST activity in blood may be a plausible indicator of ruminal pH in dairy cows. Further studies on the applicability of AST in blood as a biomarker for detecting low ruminal pH in dairy cows are warranted.


Assuntos
Bovinos/fisiologia , Dieta/veterinária , Lactação/fisiologia , Rúmen/química , Ração Animal/análise , Animais , Aspartato Aminotransferases/sangue , Nitrogênio da Ureia Sanguínea , Líquidos Corporais/química , Doenças dos Bovinos/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Leite/química , Ácidos Pentanoicos/análise , Rúmen/metabolismo , Soroalbumina Bovina/análise
18.
Methods Mol Biol ; 1855: 491-494, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426443

RESUMO

We describe here an ultrafast method for electrophoresing proteins on SDS-PAGE. Previously we reported a method to complete SDS-PAGE and immunoblotting in an hour, including electrophoresing proteins at 70°C in 10 min. Here we show that we can electrophorese molecular weight standards and bovine serum albumin on a 4-20% gradient gel in well under 10 min using heated (44 °C) Laemmli running buffer and high voltage.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteínas/análise , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida/economia , Eletroforese em Gel de Poliacrilamida/instrumentação , Calefação , Peso Molecular , Proteínas/isolamento & purificação , Soroalbumina Bovina/análise , Soroalbumina Bovina/isolamento & purificação , Dodecilsulfato de Sódio/química , Fatores de Tempo , Trometamina/química
19.
Talanta ; 192: 14-23, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30348369

RESUMO

Bovine serum albumin (BSA) imprinted polyampholyte hydrogels (PAHs) were prepared by free radical polymerization using acrylamide (Am) as structural monomer, N-isopropylacrylamide (NIPAm), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (DMC) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) as functional monomers and N,N'-methylenebisacrylamide (MBA) as crosslinker in aqueous solution. The morphology of imprinted hydrogels and non-imprinted hydrogels were characterized by scanning electron microscope (SEM). The adsorption and recognition properties were evaluated as functions of Am monomer concentration, NIPAm/Am molar ratio, crosslinking structure and charge density ratio etc. The adsorption capacity and association constant of specific interaction between hydrogel and template protein were analyzed by Langmuir isotherm model and Freundlich model. The fitting experimental data suggested that this adsorption was better described as a monolayer adsorption. The specific adsorption on hydrogel with different crosslinking structure was investigated by selective binding BSA from single solution and binary mixture solution. The charge density ratio in molecular imprinting hydrogel had obvious influence for protein adsorption and recognition. The resultant of regeneration tests showed that elution had large impact on deterioration of the imprinting structure.


Assuntos
Hidrogéis/química , Impressão Molecular , Soroalbumina Bovina/análise , Adsorção , Animais , Bovinos , Estrutura Molecular , Tamanho da Partícula , Polimerização , Propriedades de Superfície , Termodinâmica
20.
Methods Mol Biol ; 1906: 133-142, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488391

RESUMO

This chapter describes the development of paper-based microchip electrophoresis (pME) devices for the separation of clinically relevant compounds. pME were fabricated by laser cut and thermal lamination process using polyester pouches. In addition, hand-drawn pencil electrodes were integrated to the device to perform capacitively coupled contactless conductivity detection (C4D). Finished device costs less than US$ 0.10 and did not require either sophisticated instrumentation or clean room facilities. Furthermore, pME is lightweight, easy to handle, flexible, and robust. pME-C4D device revealed an excellent capacity to separate BSA and creatinine in less than 150 s with baseline resolution. The device proposed in this chapter has proven to be a good alternative as a platform for the diagnosis of diseases from renal disorders such as diabetes mellitus and heart disease.


Assuntos
Creatinina/análise , Eletroforese em Microchip/instrumentação , Desenho de Equipamento/métodos , Soroalbumina Bovina/análise , Animais , Bovinos , Diabetes Mellitus/diagnóstico , Condutividade Elétrica , Eletroforese em Microchip/métodos , Cardiopatias/diagnóstico , Humanos , Nefropatias/diagnóstico , Lasers , Papel
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