Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.269
Filtrar
1.
Food Chem ; 340: 127835, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33002825

RESUMO

A simple, sensitive and rapid fluorometric system has been developed for the detection of aldicarb (ALD) based on inner filter effect (IFE) of gold nanoparticles (AuNPs) on fluorescence (FL) intensity of carbon quantum dots (CQDs). Addition of CQDs into AuNPs, gets them aggregated due to electrostatic interaction resulting in quenching the FL intensity of CQDs. With addition of ALD into AuNPs, an intercalated layer was formed between them through Au-N and Au-S bond which reduced IFE of AuNPs. Hence, CQDs FL intensity recovered along with ALD concentration varying between 3.8 and 76 µg L-1 with lower detection limit of 3.02 µg L-1. The spiked real samples study in fruits, vegetables and soft drinks revealed that this sensing platform was repeatable and effective for real samples. The validation of proposed method indicates that the ALD sensor is promising and adaptable for everyday on spot environment and food safety monitoring.


Assuntos
Aldicarb/análise , Análise de Alimentos/métodos , Substâncias Intercalantes/química , Nanopartículas Metálicas/química , Pontos Quânticos/química , Aldicarb/química , Carbono/química , Fluorescência , Análise de Alimentos/instrumentação , Contaminação de Alimentos/análise , Frutas/química , Ouro/química , Reprodutibilidade dos Testes , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Verduras/química
2.
Biosens Bioelectron ; 169: 112604, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32980805

RESUMO

Virus severely endangers human life and health, and the detection of viruses is essential for the prevention and treatment of associated diseases. Metal-organic framework (MOF), a novel hybrid porous material which is bridged by the metal clusters and organic linkers, has become a promising biosensor platform for virus detection due to its outstanding properties including high surface area, adjustable pore size, easy modification, etc. However, the MOF-based sensing platforms for virus detection are rarely summarized. This review systematically divided the detection platforms into nucleic acid and immunological (antigen and antibody) detection, and the underlying sensing mechanisms were interpreted. The nucleic acid sensing was discussed based on the properties of MOF (such as metal ion, functional group, geometry structure, size, porosity, stability, etc.), revealing the relationship between the sensing performance and properties of MOF. Moreover, antibodies sensing based on the fluorescence detection and antigens sensing based on molecular imprinting or electrochemical immunoassay were highlighted. Furthermore, the remaining challenges and future development of MOF for virus detection were further discussed and proposed. This review will provide valuable references for the construction of sophisticated sensing platform for the detection of viruses, especially the 2019 coronavirus.


Assuntos
Técnicas Biossensoriais/métodos , Estruturas Metalorgânicas/química , Viroses/virologia , Vírus/isolamento & purificação , Animais , Anticorpos Antivirais/análise , Antígenos Virais/análise , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Modelos Moleculares , Impressão Molecular/instrumentação , Impressão Molecular/métodos , Ácidos Nucleicos/análise , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Viroses/diagnóstico
3.
Food Chem ; 328: 127091, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32474237

RESUMO

Methanol is highly toxic for human, so methanol detection is valuable especially in water and ethanol medium without complicated and time consuming procedure. In this work, we present a new fluorescence probe for direct detection of methanol in aqueous and ethanol medium based on the ZnS:Mn2+ quantum dot (QDs) and soluble N-methylpolypyrrole (NMPPy) hybrid. Moreover, the number of spectroscopic techniques were used to study the chemical composition and optical properties of the resultant QDs as well as investigation on the sensing mechanism toward methanol. Also, methanol can be determined by using ZnS:Mn2+ QDs/NMPPy hybrid based switchable fluorescence sensing system, with high sensitivity, high selectivity and a very good detection limit of 1 mM with linearity in the concentration range of 25-230 mM (~0.1-0.9% v/v) in aqueous solution. Finally, the ZnS:Mn2+ QDs/NMPPy hybrid as optical sensor was successfully utilized to determine the amount of methanol in real alcoholic beverage samples.


Assuntos
Etanol/química , Metanol/análise , Polímeros/química , Pirróis/química , Pontos Quânticos/química , Espectrometria de Fluorescência/instrumentação , Sulfetos/química , Água/química , Compostos de Zinco/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Magnésio/química , Metanol/química
4.
Food Chem ; 328: 127112, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470778

RESUMO

A ratiometric fluorescent probe (Probe 1) was developed for the sensitive detection of ß-galactosidase (ß-gal) activity. Probe 1 detected ß-gal activity in the range 0-1.0 U/mL, with a limit of detection of 0.025 U/mL. In addition, as different activities of ß-gal added, the luminescent intensity of Probe 1 gradually increased, as observed under a 365 nm ultraviolet lamp. Moreover, this method is low-volume, 20 µL, and time-efficient, 45 min per measurement. Probe 1 was successfully used to measure the ß-gal activity in real fruit samples in a qualitative manner, by the naked eye, fast semi-quantitative manner, by smartphone, or quantitative manner, by fluorescence spectrometer.


Assuntos
Corantes Fluorescentes/química , Análise de Alimentos/métodos , Frutas/enzimologia , Proteínas de Plantas/análise , beta-Galactosidase/análise , Limite de Detecção , Proteínas de Plantas/metabolismo , Smartphone , Espectrometria de Fluorescência/instrumentação , beta-Galactosidase/metabolismo
5.
Food Chem ; 326: 126935, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32447160

RESUMO

Carbon dots (CDs) have been a new class of fascinating carbon-based fluorescent nanomaterials. In the present work, new N-doped CDs with highly crystalline graphite structures are prepared from renewable precursors, chitosan and tartaric acid, and are well characterized. The prepared CDs are applied as a biocompatible fluorescent sensor for the sequential detection of Fe3+ and AA. Among various transition metal ions, Fe3+ can selectively quench the fluorescence of CDs. Upon the further addition of AA, the quenched fluorescence of CDs is then restored as Fe3+ is reduced to Fe2+ by AA, which can be utilized for the fluorescent determination of AA. A good linear relationship in the range of 0-150 µM of AA concentration is established with a low detection limit of 0.02 µM. Moreover, the practical applications of this fluorescent sensing method in measurement of AA in food samples are successfully realized with satisfactory results.


Assuntos
Ácido Ascórbico/análise , Carbono/química , Compostos Férricos/análise , Nanoestruturas/análise , Espectrometria de Fluorescência/métodos , Fluorescência , Limite de Detecção , Nitrogênio/química , Pontos Quânticos/química , Espectrometria de Fluorescência/instrumentação
6.
Food Chem ; 319: 126575, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32172051

RESUMO

A core-shell molecular imprinting fluorescence nanosensor was developed for the ratiometric fluorescence and visual detection of folic acid (FA). The nanosensor was prepared by anchoring imprinting shell on the silica nanoparticles, and embedding the CdTe quantum dots in imprinted shell to provide FA-dependent fluorescence signals. Under the optimum conditions, a favorable linearity relationship between the fluorescence intensities ratio (I449/I619) and the FA concentration over 0.23-113 µM was offered with a detection limit (LOD) of 48 nM. The visual detection for FA was realized by evaluating profuse fluorescence color change from red to pink to purple to final blue. The proposed sensor possessed excellent sensing performances of rapid response, high precision, super sensitivity and selective recognition. Furthermore, endogenous FA was detected in real samples ranging from 37.4 to 265.8 µg/100 g; satisfactory spiked recoveries were obtained within 94.8-104.2%, which conformed to the measurement results by HPLC-UV.


Assuntos
Ácido Fólico/análise , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Impressão Molecular/métodos , Compostos de Cádmio/química , Cor , Ácido Fólico/química , Limite de Detecção , Nanopartículas/química , Propilaminas/química , Pontos Quânticos/química , Sensibilidade e Especificidade , Silanos/química , Dióxido de Silício/química , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Telúrio/química
7.
J Fluoresc ; 30(1): 181-191, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31940104

RESUMO

Desi ghee, obtained by buffalo and cow milk, is highly expensive because it contains valuable vitamins and conjugated linoleic acid (CLA). Its high demand and cost result in to its adulteration with inferior banaspati ghee. In this study, Fluorescence spectroscopy along with multivariate analysis has been utilised for the detection and quantification of adulteration. Spectroscopic analysis showed that buffalo ghee contains more vitamins and CLA than cow, whereas cow ghee is enriched with beta-carotene. For multivariate analysis, principle component analysis (PCA) and partial least square regression (PLSR) have been applied on the spectral data for the determination of adulteration. PLSR model was authenticated by predicting 23 unknown samples including 3 commercial brands of desi ghee. The root mean square error in prediction (RMSEP) of unknown samples was found to be 1.7 which is a reasonable value for quantitative prediction. Due to non-destructive and requiring no sample pre-treatment, this method can effectively be employed as on line characterization tool for the food safety assurance.


Assuntos
Contaminação de Alimentos/análise , Ghee/análise , Animais , Bovinos , Análise dos Mínimos Quadrados , Leite/química , Análise Multivariada , Análise de Componente Principal , Espectrometria de Fluorescência/instrumentação
8.
Mikrochim Acta ; 187(2): 146, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31970525

RESUMO

An optical fiber nanoprobe is presented for fluorometric determination of copper(II). The method based on the use of water-dispersible AgInZnS quantum dots (QDs) deposited at the end of an optical fiber in a poly(vinyl alcohol) matrix. The fluorescnece of the QDs, best measured at excitation/emisssion wavelengths of 365/570 nm, is quenched by Cu(II) due to both static and electron transfer from the QDs to Cu(II). This is experimentally confirmed by photoluminescence and UV-vis absorption spectra, and measurement of luminescence lifetimes. The probe is highly selective and possesses a linear detection range that extends from 2.5 to 800 nM. Graphical abstractSchematic representation of an optical fiber nanoprobe based on hydrophilic AgInZnS quantum dots for fluorometric determination of copper(II). The fluorescence is quenched by Cu(II) due to static quenching and dynamic quenching. It has a detection range of 2.5-800 nM.


Assuntos
Cobre/análise , Corantes Fluorescentes/química , Fibras Ópticas , Pontos Quânticos/química , Índio/química , Lagos/análise , Prata/química , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Sulfetos/química , Poluentes Químicos da Água/análise , Zinco/química
9.
Analyst ; 145(4): 1227-1235, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-31898707

RESUMO

Rifampicin is a common antibiotic used in human and veterinary medicine to treat tuberculosis and other diseases caused by numerous pathogenic bacteria. However, the excessive or improper use of rifampicin usually leads to a series of problems, including bacterial resistance, excessive drug-resistance and water pollution. Thus, it is of great importance to develop selective and sensitive assays for monitoring rifampicin in biological systems. In this study, we designed a fluorescence "turn-off" strategy for the trace detection of rifampicin based on a glutathione-stabilized copper nanoclusters (GSH-Cu NC) sensor. In an aqueous solution, the fluorescence of the GSH-Cu NCs at 632 nm can be quenched effectively and selectively by rifampicin due to the inner-filter effect (IFE) of fluorescence mechanism. Distinctively, this GSH-Cu NC sensor exhibited excellent fluorescence sensing capability for the trace detection of rifampicin with a very low limit of detection (LOD) of 16 pM in a wide linear range from 50 to 10 000 pM. It is not only more sensitive than the other methods previously reported for the detection of rifampicin, but also has an outstanding selectivity and strong anti-interference in complex samples. Furthermore, the as-developed GSH-Cu NCs were also successfully applied to determine rifampicin in different real samples with quantitative spike recoveries ranging from 97% to 105%.


Assuntos
Cobre/química , Glutationa/química , Limite de Detecção , Nanoestruturas/química , Rifampina/análise , Espectrometria de Fluorescência/instrumentação , Humanos , Soluções Oftálmicas/química , Rifampina/sangue , Rifampina/química
10.
Biosens Bioelectron ; 148: 111791, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31677526

RESUMO

A smartphone-integrated ratiometric fluorescent sensing system (DPA-Ce-GMP-Eu) for visual and point-of-care testing (POCT) of tetracycline with high sensitivity and accuracy was developed. The blue fluorescence of DPA-Ce-GMP was changed into red by doping with Eu3+ duo to the energy transfer from Ce3+ to Eu3+. Upon exposure to tetracycline, coordination between Eu3+ and tetracycline blocks energy transfer from Ce3+ to Eu3+, converting the fluorescent color from red to blue. The tetracycline detection can be realized within a wide concentration range from 0.01 µM to 45 µM. The limit of detection (LOD) reaches as low as 6.6 nM. To realize quantitative point-of-care detection in real samples, a portable device with smartphone as signal reader and analyzer is further designed to integrate with the DPA-Ce-GMP-Eu sensing platform. The Color Picker APP installed in the smartphone can convert the Red, Green and Blue (RGB) channels of the fluorescence images into digital values. With milk as real sample, tetracycline can be on-site detected with LOD of 10.8 nM. This developed platform presents a great promise for POCT in practical application with merits of low cost, easy carry, simple operation, and excellent selectivity and repeatability.


Assuntos
Antibacterianos/análise , Técnicas Biossensoriais/instrumentação , Contaminação de Alimentos/análise , Smartphone/instrumentação , Tetraciclina/análise , Animais , Cério/química , Desenho de Equipamento , Európio/química , Fluorescência , Corantes Fluorescentes/química , Análise de Alimentos/instrumentação , Limite de Detecção , Leite/química , Carne de Porco/análise , Espectrometria de Fluorescência/instrumentação
11.
Ecotoxicol Environ Saf ; 187: 109821, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31677572

RESUMO

In this study, a porous framework MOF-74(Zn) (Zn2 (DHBDC)(DMF)(H2O)2, H4dondc = 1, 5-dioxido-2, 6-naphthalenedicarboxylic acid) with open metal sites was successful synthesized. MOF-74(Zn) as a template was grafted on the open metal sites with ethylenediamine (en) named MOF-74(Zn)-en to develop a highly selective and sensitive fluorescence detector for rapid determination of tetrabromobisphenol A (TBBPA). The obtained MOF-74(Zn)-en was well characterized by Fourier Transform Infrared (FT-IR), Scanning Electron Microscopy (SEM) and showed ideal properties of photoluminescence. The fluorescence enhancement showed a good linear relationship with the concentrations of TBBPA in the range of 50-400 µg/L, and its limit of detection could reach to 0.75 µg/L. Furthermore, the possible sensing mechanism of the fluorescence enhancement could be attributed to Förster resonance energy transfer (FRET). The results will provide a convenient and quick method for detection of TBBPA. To the best of my knowledge, this is the first case to detect TBBPA by fluorescence enhancement with MOF derivatives.


Assuntos
Fluorescência , Estruturas Metalorgânicas/química , Bifenil Polibromatos/análise , Poluentes Químicos da Água/análise , Zinco/química , Etilenodiaminas/química , Limite de Detecção , Bifenil Polibromatos/química , Espectrometria de Fluorescência/instrumentação , Poluentes Químicos da Água/química
12.
Luminescence ; 35(2): 284-291, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31762136

RESUMO

The direct determination of alogliptin benzoate (ALO) using fluorescence has not yet been accomplished because ALO cannot fluoresce naturally. Accordingly, it should be derivatized first with a fluorogenic reagent to enhance the sensitivity required for its bioanalysis. This method is the first spectrofluorimetric assay for ALO quantification exploiting the nucleophilic nature of its amino group to react with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer at pH 8.5 to produce a strong fluorescent compound that is excited at and emits at wavelengths 470 and 527 nm, respectively. Experimental variables concerning the conditions of reaction and fluorogenic intensity were carefully investigated and optimized. Linearity was from 1-250 ng ml-1 with a lower detection limit of 0.29 ng ml-1 and a lower quantification limit of 0.88 ng ml-1 . Validation of the current study was accomplished with mean per cent recovery of 100.62 ± 1.59 in tablets and 99.86 ± 0.82 in human plasma. Furthermore, the current method has been utilized in the bioanalysis of ALO in real rat plasma after oral administration with a simple specimen preparation. The developed method has proven to be a promising alternative method for ALO analysis in bioequivalence studies.


Assuntos
4-Cloro-7-nitrobenzofurazano/química , Benzoatos/sangue , Corantes Fluorescentes/química , Piperidinas/sangue , Espectrometria de Fluorescência , Uracila/análogos & derivados , Animais , Benzoatos/química , Benzoatos/farmacocinética , Humanos , Masculino , Estrutura Molecular , Piperidinas/química , Piperidinas/farmacocinética , Teoria Quântica , Ratos , Ratos Wistar , Espectrometria de Fluorescência/instrumentação , Uracila/sangue , Uracila/química , Uracila/farmacocinética
13.
Luminescence ; 35(2): 260-265, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31733037

RESUMO

A highly accurate, simple and sensitive spectrofluorimetric analytical method for dapagliflozin (DGF) quantitation was developed. The proposed method was successively applied to DGF analysis in both its pure and pharmaceutical dosage forms. This method was developed to investigate DGF stability in its degradation products, as laid out in International Council for Harmonisation (ICH) rules. Kinetics of alkaline degradation of DGF was also calculated. The half-life time (t1/2 ) of the reaction was 75.32 min. An alkaline degradation pathway was described. The present study involved measurement of the second-derivative synchronous fluorescence intensity of DGF at Δλ = 30 nm. Peak amplitude was measured at 322 nm. Linear range of the calibration curve was 0.1-1.0 µg ml-1 . Lower detection and quantitation limits were 0.023 and 0.071 µg ml-1 , respectively, and indicated good sensitivity of the proposed method. Mean per cent recovery was 99.78 ± 1.78%. The proposed analytical approach was successfully applied to DGF in the quality control laboratory and would be suitable as a stability-indicating assay.


Assuntos
Compostos Benzidrílicos/análise , Glucosídeos/análise , Espectrometria de Fluorescência , Calibragem , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Conformação Molecular , Espectrometria de Fluorescência/instrumentação
14.
Biosens Bioelectron ; 148: 111811, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31678822

RESUMO

Alkaline phosphatase (ALP) is a membrane-bound enzyme widely present in biological tissues and is extensively used as a biomarker in clinical diagnosis because abnormal ALP levels in organisms are often closely related to many diseases. In order to meet the urgent needs of human health and the development of precision medicine, the classic method of detecting ALP seems to be unable to meet a "tailor-made" treatment plan for patients. In order to get the best therapeutic effect and the lowest side effects, the researchers have made considerable efforts and established numerous strategies for detecting ALP. In this review, we focus on the current development and basic principles of fluorescence strategy to detect ALP, and compare the advantages and disadvantages of detecting ALP based on different fluorescent detection modes. In addition, to more intuitively understand the performance of different materials for detecting ALP, a table is presented. Finally, the future trends and prospective in this field will be discussed, it could be speculated that invent and design the detection mechanism based on the relationship between the new material and ALP is the key to effective detection of ALP, and process data with a computer analog digital platform will be a welcome and obsessive technology. It is hoped that some insights and inspiration for future research work will be provided.


Assuntos
Fosfatase Alcalina/análise , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodos , Animais , Técnicas Biossensoriais/instrumentação , Humanos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Espectrometria de Fluorescência/instrumentação
15.
ACS Sens ; 4(12): 3283-3290, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31736294

RESUMO

A digital fluorescence detector (DFD), a handheld fluorescence detection device, can convert the fluorescence signal of samples into the corresponding fluorescer concentration. Herein, by adopting a DFD as the readout, a novel intelligent platform was developed based on a ratiometric paper-based device (RPD) for multiple aminoglycoside detection. There are five layers and four parallel channels contained in the designed RPD, functioning as reagent storage, fluidic path control and signal processing, respectively. The rationale of this design lies in the fact that aptamer/graphitic carbon nitride nanosheet (Apt/g-C3N4 NS) modified layers can catalyze o-phenylenediamine to fluorescent 2,3-diaminophenazine (DAP) in the presence of H2O2. When Apt was removed from nanosheets via the Apt-target reaction, the peroxidase-like activity would be decreased, thus decreasing the production of DAP. All the changes of the fluorescence DAP signal can be read out using a portable DFD. Based on the DFD signal change related to the concentration of the target, a quantitative reaction platform was established. Furthermore, the sample flow and Apt-target reaction time can be reasonably regulated using the H2O2-cleavable hydrophobic compound modified layer placed between the target recognition region and detection region. Then, the practicality of this platform was verified through realizing sensitive analysis of streptomycin, tobramycin, and kanamycin simultaneously. Overall, with merits including portability and ease of operation, the platform shows great potential in on-site simultaneous detection of multiple targets, especially in resource-limited settings.


Assuntos
Antibacterianos/análise , Canamicina/análise , Papel , Espectrometria de Fluorescência/métodos , Estreptomicina/análise , Tobramicina/análise , Animais , Antibacterianos/química , Aptâmeros de Nucleotídeos/química , Fluorescência , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Grafite/química , Peróxido de Hidrogênio/química , Canamicina/química , Limite de Detecção , Leite/química , Nanoestruturas/química , Compostos de Nitrogênio/química , Fenazinas/química , Fenilenodiaminas/química , Reprodutibilidade dos Testes , Rios/química , Espectrometria de Fluorescência/instrumentação , Estreptomicina/química , Tobramicina/química , Poluentes Químicos da Água/análise
16.
Sci Rep ; 9(1): 17131, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748592

RESUMO

In this study, we developed a portable smartphone-based diffusometry for analyzing the C-reactive protein (CRP) concentration. An optimized fluorescence microscopic add-on system for a smartphone was used to image the 300 nm fluorescent beads. Sequential nanobead images were recorded for a period and the image data were used for fluorescence correlation spectrometric (FCS) analysis. Through the analysis, the nanobeads' diffusion coefficient was obtained. Further, the diffusion coefficients of the anti-CRP-coated nanobeads, which were suspended in the samples with various CRP concentrations, were estimated using smartphone-based diffusometry. After 10 min of reaction, the anti-CRP-coated nanobeads in a higher CRP concentration solution led to a lower diffusion coefficient. Based on the experiments, a linear sensing range of 1~8 µg/mL was found.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Proteína C-Reativa/química , Imunoensaio/instrumentação , Imunoensaio/métodos , Fluorescência , Testes Imunológicos/instrumentação , Testes Imunológicos/métodos , Nanopartículas/química , Smartphone , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos
17.
ACS Sens ; 4(11): 2908-2914, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31599572

RESUMO

In all eukaryotic cells, modifications of proteins by polymers of ubiquitin (polyUb) are signals used in diverse biological processes. To better understand how polyUb signals are read and promote their different functions, quantitative measurements of their interactions with receptor proteins are needed. However, affinities and selectivities of different forms of polyUb with various receptors have been difficult to determine because the availability of well-defined polyUb chains can be limiting and there is a lack of general, sensitive methods to assay their interactions. We have addressed this challenge by developing a series of fluorescent protein sensors for polyUb; by competition of the sensors against receptor proteins in vitro for limiting amounts of polyUb, receptor·polyUb affinities can be quantified. Due to the high affinities of the polyUb sensors (Kd ∼ 10-9 M), binding assays using this competition format require much less polyUb (<0.1%) than would be needed in direct titrations of the polyUb ligands. Furthermore, the high sensitivity and large dynamic range of the sensor fluorescence readout allow for precise measurements even for very tight interactions (i.e., nanomolar Kd). Importantly, as demonstrated here with Ub2 and Ub3 ligands, the assay does not require labeling of either the receptor protein or the polyUb, and it can be used with polyUb ligands composed of virtually any Ub-Ub linkage type.


Assuntos
Técnicas Biossensoriais , Proteínas Luminescentes/análise , Poliubiquitina/química , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Ligantes , Espectrometria de Fluorescência/instrumentação
18.
Anal Bioanal Chem ; 411(27): 7251-7260, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31612256

RESUMO

Chloride widely exists in the environment and will cause serious interference for arsenic speciation analysis. The determination of four arsenic species including arsenite (As(III)), arsenate (As(V)), monomethylarsenate (MMA), and dimethylarsonate (DMA) in samples containing high concentrations of Cl- was carried out in this work by coupling of liquid chromatography (LC) with hydride generation atomic fluorescence spectrometry (HG-AFS). The interference of Cl- was successfully eliminated by coupling two anion-exchange chromatographic columns in series and eluting with 35.0 mmol L-1 (NH4)2HPO4 (pH = 6.00). A novel pre-treatment system was subsequently developed to realize on-line column switch and pre-reduction of As(V). The analysis time was shortened by an isocratic elution but programmed flow rate method, and the sensitivity of As(V) was also enhanced by the introduction of pre-reduction using the developed system. The proposed method can resist at least 10 g L-1 Cl- without any pre-treatment operations. Since LC-HG-AFS is low-cost and can be afforded or self-assembled by most labs, the developed method can be adopted as a routine analysis method for arsenic species in chloride-bearing samples, such as urine and seawater. Graphical abstract.


Assuntos
Arseniatos/análise , Arsenicais/análise , Arsenitos/análise , Poluentes Químicos da Água/análise , Arseniatos/urina , Arsênico/análise , Arsênico/urina , Arsenicais/urina , Arsenitos/urina , Cloretos/análise , Cloretos/urina , Cromatografia Líquida de Alta Pressão/instrumentação , Desenho de Equipamento , Humanos , Limite de Detecção , Metilação , Água do Mar/análise , Espectrometria de Fluorescência/instrumentação , Espectrofotometria Atômica/instrumentação , Poluentes Químicos da Água/urina
20.
Nano Lett ; 19(10): 7434-7442, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31526002

RESUMO

Single molecule detection provides detailed information about molecular structures and functions but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labeling. However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguide nanoapertures enable the observation of the UV fluorescence of single label-free ß-galactosidase proteins with increased brightness, microsecond transit times, and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient, and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. Although the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.


Assuntos
Escherichia coli/enzimologia , Espectrometria de Fluorescência/instrumentação , Triptofano/química , beta-Galactosidase/química , Alumínio/química , Escherichia coli/química , Fluorescência , Nanoestruturas/química , Raios Ultravioleta
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA