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1.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443466

RESUMO

Bees and their products are useful bioindicators of anthropogenic activities and could overcome the deficiencies of air quality networks. Among the environmental contaminants, mercury (Hg) is a toxic metal that can accumulate in living organisms. The first aim of this study was to develop a simple analytical method to determine Hg in small mass samples of bees and beehive products by cold vapor atomic fluorescence spectrometry. The proposed method was optimized for about 0.02 g bee, pollen, propolis, and royal jelly, 0.05 g beeswax and honey, or 0.1 g honeydew with 0.5 mL HCl, 0.2 mL HNO3, and 0.1 mL H2O2 in a water bath (95 °C, 30 min); samples were made up to a final volume of 5 mL deionized water. The method limits sample manipulation and the reagent mixture volume used. Detection limits were lower than 3 µg kg-1 for a sample mass of 0.02 g, and recoveries and precision were within 20% of the expected value and less than 10%, respectively, for many matrices. The second aim of the present study was to evaluate the proposed method's performances on real samples collected in six areas of the Lazio region in Italy.


Assuntos
Abelhas/química , Monitoramento Biológico/métodos , Mercúrio/análise , Espectrometria de Fluorescência/métodos , Animais , Temperatura Baixa , Cucumis melo/química , Confiabilidade dos Dados , Poluição Ambiental/análise , Ácidos Graxos/análise , Mel/análise , Itália , Pólen/química , Própole/análise , Espectrofotometria Atômica/métodos , Ceras/análise
2.
Nat Commun ; 12(1): 4654, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341342

RESUMO

Ligand-oligonucleotide transduction provides the critical pathway to integrate non-nucleic acid molecules into nucleic acid circuits and nanomachines for a variety of strand-displacement related applications. Herein, a general platform is constructed to convert the signals of ligands into desired oligonucleotides through a precise kinetic control. In this design, the ligand-aptamer binding sequence with an engineered duplex stem is introduced between the toehold and displacement domains of the invading strand to regulate the strand-displacement reaction. Employing this platform, we achieve efficient transduction of both small molecules and proteins orthogonally, and more importantly, establish logical and cascading operations between different ligands for versatile transduction. Besides, this platform is capable of being directly coupled with the signal amplification systems to further enhance the transduction performance. This kinetically controlled platform presents unique features with designing simplicity and flexibility, expandable complexity and system compatibility, which may pave a broad road towards nucleic acid-based developments of sophisticated transduction networks.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Cinética , Ligantes , Modelos Genéticos , Modelos Moleculares , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Transdução de Sinais/genética , Espectrometria de Fluorescência/métodos
3.
J Photochem Photobiol B ; 221: 112251, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34229147

RESUMO

Blood is one of the most common body fluids discovered at crime scenes involving violent actions. It is one of the most important types of forensic evidence since it allows for the identification of the individual providing that there is a match with a known DNA profile. Determining the time since deposition (TSD) can assist investigators in establishing when the crime occurred or if a bloodstain present is actually related to the investigated event. To develop a forensically sound method for determining the TSD of a bloodstain, it is necessary to understand the underlying biochemical mechanisms occurring during aging. As biochemical processes occurring in blood are necessary for the continued survival of living organisms, they are important subjects of human biology and biomedicine and are well understood. However, the biochemistry of bloodstain aging ex vivo is primarily of interest to forensic scientists and has not yet been thoroughly researched. This preliminary study utilizes steady-state fluorescence spectroscopy to probe the changes in fluorescence properties of peripheral and menstrual blood up to 24-h post deposition. Peripheral and menstrual blood exhibited similar kinetic changes over time, assigned to the presence of the fluorophores: tryptophan, nicotinamide adenine dinucleotide (NADH), and flavins in both biological fluids. The biochemical mechanism of blood aging ex vivo is discussed.


Assuntos
Manchas de Sangue , Espectrometria de Fluorescência/métodos , Análise Química do Sangue , Feminino , Flavinas/química , Humanos , Masculino , NAD/química , Oxirredução , Fatores de Tempo , Triptofano/química
4.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298920

RESUMO

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.


Assuntos
Multimerização Proteica/fisiologia , Proteínas/metabolismo , Células Cultivadas , Citosol/metabolismo , Dictyostelium/metabolismo , Difusão , Dimerização , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Fótons , Espectrometria de Fluorescência/métodos
5.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299377

RESUMO

The results of time-resolved fluorescence measurements of flavin mononucleotide (FMN) in rigid polyvinyl alcohol films (PVA) demonstrate that fluorescence intensity decays are strongly accelerated in the presence of fluorescent dimers and nonradiative energy transfer processes. The fluorescence decay originating both from H and J dimer states of FMN was experimentally observed for the first time. The mean fluorescence lifetimes for FMN dimers were obtained: τfl = 2.66 ns (at λexc = 445 nm) and τfl = 2.02 (at λexc = 487 nm) at λobs = 600 nm and T = 253 K from H and J state of dimers, respectively. We show that inhomogeneous orientational broadening of energy levels (IOBEL) affects the shape of the fluorescence decay and leads to the dependence of the average monomer fluorescence lifetime on excitation wavelength. IOBEL affected the nonradiative energy transfer and indicated that different flavin positioning in the protein pocket could (1) change the spectroscopic properties of flavins due to the existence of "blue" and "red" fluorescence centers, and (2) diminish the effectiveness of energy transfer between FMN molecules.


Assuntos
Mononucleotídeo de Flavina/química , Álcool de Polivinil/química , Dimerização , Transferência de Energia , Polarização de Fluorescência/métodos , Polímeros/química , Espectrometria de Fluorescência/métodos
6.
J Am Chem Soc ; 143(30): 11544-11553, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34288684

RESUMO

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here, we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a nonfluorescent eosin Y derivative (EYH3-) under green light. The deactivated photocatalyst is stable and rapidly activated under low-intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH3- is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e-/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH3- degradation, we successfully improved EYH3--to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photoinduced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/análise , Amarelo de Eosina-(YS)/análogos & derivados , Espectrometria de Fluorescência/métodos , 3,3'-Diaminobenzidina/química , Biomarcadores/química , Catálise/efeitos da radiação , Amarelo de Eosina-(YS)/síntese química , Amarelo de Eosina-(YS)/efeitos da radiação , Fluorescência , Luz , Limite de Detecção , Oxirredução/efeitos da radiação , Fosfoproteínas/análise , Polietilenoglicóis/química , Polimerização , Estudo de Prova de Conceito , SARS-CoV-2/química
7.
ACS Appl Mater Interfaces ; 13(24): 27991-27998, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34110123

RESUMO

Porphyria is a group of genetic photodermatoses that cause too much porphyrin to accumulate in the blood, skin, and liver, resulting in skin photosensitivity and damage, liver disease, or potential liver failure. Conventional detection methods include high-performance liquid chromatography and fluorescence spectrometry. However, these methods usually require complicated pretreatment and time-consuming processes. Therefore, efficient and fast detection of porphyria is urgently needed. Herein, we develop a molecular afterglow reporter-based sensing scheme for the detection of porphyrins in whole blood. The afterglow reporter can respond to the production of singlet oxygen (1O2) of porphyrins after light excitation, and the detection signals can be amplified through adjusting the amount of singlet oxygen and afterglow reporter molecules. Moreover, without the use of a real-time excitation source, afterglow signals can avoid the scattering and autofluorescence interference in biological samples, thereby reducing background noise. More importantly, we prove the applicability of the afterglow reporter in the quantitative detection of porphyrins in whole blood and demonstrate its great clinical potential.


Assuntos
Adamantano/análogos & derivados , Corantes Fluorescentes/química , Porfirias/diagnóstico , Protoporfirinas/análise , Quinolinas/química , Espectrometria de Fluorescência/métodos , Adamantano/síntese química , Corantes Fluorescentes/síntese química , Humanos , Limite de Detecção , Porfirias/sangue , Protoporfirinas/efeitos da radiação , Quinolinas/síntese química , Oxigênio Singlete/metabolismo
8.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067835

RESUMO

The potential of tannic acid (TA) as a dispersing agent for graphene (G) in aqueous solutions and its interaction with riboflavin have been studied under different experimental conditions. TA induces quenching of riboflavin fluorescence, and the effect is stronger with increasing TA concentration, due to π-π interactions through the aromatic rings, and hydrogen bonding interactions between the hydroxyl moieties of both compounds. The influence of TA concentration, the pH, and the G/TA weight ratio on the quenching magnitude, have been studied. At a pH of 4.1, G dispersed in TA hardly influences the riboflavin fluorescence, while at a pH of 7.1, the nanomaterial interacts with riboflavin, causing an additional quenching to that produced by TA. When TA concentration is kept constant, quenching of G on riboflavin fluorescence depends on both the G/TA weight ratio and the TA concentration. The fluorescence attenuation is stronger for dispersions with the lowest G/TA ratios, since TA is the main contributor to the quenching effect. Data obey the Stern-Volmer relationship up to TA 2.0 g L-1 and G 20 mg L-1. Results demonstrate that TA is an effective dispersant for graphene-based nanomaterials in liquid medium and a green alternative to conventional surfactants and synthetic polymers for the determination of biomolecules.


Assuntos
Grafite/química , Riboflavina/química , Taninos/química , Fluorescência , Grafite/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Radical Hidroxila , Riboflavina/metabolismo , Espectrometria de Fluorescência/métodos , Tensoativos , Taninos/metabolismo , Água
9.
Molecules ; 26(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067563

RESUMO

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.


Assuntos
Benzotiazóis/química , Técnicas Biossensoriais/métodos , DNA/química , Benzotiazóis/metabolismo , Fluorescência , Corantes Fluorescentes/química , Limite de Detecção , Sondas Moleculares , Hibridização de Ácido Nucleico/métodos , Proteínas/química , Proteínas/metabolismo , Espectrometria de Fluorescência/métodos
10.
Anal Bioanal Chem ; 413(15): 3925-3932, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33932155

RESUMO

Acid phosphatase has become a significant indicator of prognostic and medical diagnosis, and its dysfunction may lead to a series of diseases. A novel dual-signal fluorescence method for acid phosphatase detection based on europium polymer (europium-pyridine dicarboxylicacid-adenine) and pyridoxal phosphate (PLP) was proposed. PLP coordinated with europium polymer via Eu3+ and P-O bonds, and the fluorescence of europium polymer was quenched due to the photoinduced electron transfer (PET) effect between aldehyde and europium polymer. Upon addition of acid phosphatase, the PLP was transformed to phosphate (Pi) and pyridoxal (PL). The PL was released from the surface of europium polymer, and the blue emission was enhanced due to the formation of internal hemiacetal, while the fluorescence of europium polymer recovered. The blue (PL) and red emission (Eu3+) were positively correlated with acid phosphatase activity; thus the sensitive assay of acid phosphatase was effectively achieved. The two signals were applied to determine the acid phosphatase with limits of detection (LOD) of 0.04 mU/mL and 0.38 mU/mL, and the linear ranges were 0.13-5.00 mU/mL and 1.25-20.00 mU/mL, respectively. The probe can be used to trace the acid phosphatase in biological systems and holds promise for use in clinical diagnosis and early prevention.


Assuntos
Fosfatase Ácida/análise , Corantes Fluorescentes/química , Limite de Detecção , Espectrometria de Fluorescência/métodos
11.
Anal Bioanal Chem ; 413(15): 3847-3859, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33934191

RESUMO

Graphdiyne (GDY) adsorbed DNA probes have been used as a fluorescent sensing platform, but topics including DNA adsorption affinities, DNA probe displacement, and fluorescence quenching ability were rarely researched. Herein, the adsorption affinity of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) on a tremella-like GDY was tuned by modulating the surface chemistry of GDY. The fluorescence quenching ability of GDY with different oxidation degrees was compared. The nonspecific displacement of DNA probes on GDY was studied. Under the same concentrations, GDY with low oxidation degree exhibited stronger adsorption affinity and higher adsorption capacity to both ssDNA and dsDNA than highly oxidized GDY. DNA adsorbed on low-oxidized GDY was more resistant to displacement by other DNAs. Protein showed strong interaction with different GDY and could displace DNA probes on GDY. Based on these findings, an ideal GDY with proper oxidation degree, exhibiting high surface affinity for ssDNA and low affinity for dsDNA, was used as scavenger of redundant ssDNA fluorescent probe in an enzyme-assisted amplification system for sensitive ochratoxin (OTA) detection. This study has enhanced our fundamental understanding of DNA adsorption by GDY. It also provided a rational way to apply GDY for fluorescence sensing in a complicated system.


Assuntos
DNA/química , Grafite/química , Espectrometria de Fluorescência/métodos , Adsorção , Corantes Fluorescentes/química , Limite de Detecção , Microscopia Eletrônica de Varredura , Ocratoxinas/análise , Análise Espectral/métodos , Propriedades de Superfície
12.
Anal Bioanal Chem ; 413(15): 3945-3953, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33954830

RESUMO

Novel spherical polymer nanoparticles were synthesized by hyperbranched polyethylenimine (hPEI) and 6-hydroxy-2-naphthaldehyde (HNA) via Schiff base reaction (one-pot reaction), which had great advantages in water solubility and green synthesis. Meanwhile, probe PEI-HNA could quickly detect Cu2+ in the range of 0-60 µM in 30 s with the detection limit of 243 nM. The fluorescence of PEI-HNA-Cu2+ could be recovered by the addition of S2- in 50 s with the detection limit of 227 nM. Based on the excellent optical properties, PEI-HNA has been used in the bioimaging of living cells with excellent cell penetrability and low toxicity. More importantly, PEI-HNA has been doped into filter paper, hydrogel, and nanofibrous film to prepare solid-phase sensors, displaying rapid response and excellent sensitivity. Moreover, the low-cost and simple preparation of these sensors offers great potential and possibilities for industrialization, which could help accelerate the development of sensors in environmental and biological fields.


Assuntos
Cobre/análise , Corantes Fluorescentes/química , Nanopartículas/química , Polímeros/química , Enxofre/análise , Células HeLa , Humanos , Espectrometria de Fluorescência/métodos
13.
Anal Bioanal Chem ; 413(16): 4255-4265, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33988741

RESUMO

Anderson-type polyoxometalate containing Fe3+ and Mo6+, (NH4)3[H6Fe(III)Mo6O24] (FeMo6), was found to work as an oxidase-mimicking nanoenzyme for the first time, exhibiting the ability of catalytic oxidation of o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTs), and 3,3',5,5'-tetramethylbenzidine (TMB), which features easy synthesis, low cost, simple operation, and low consumption. Attributed to the nature of FeMo6 and Fenton-like effect, a novel sensor based on two consecutive "turn on" fluorescence was developed for detecting dopamine (DA) by employing the FeMo6-OPD system, and the linear range was from 1 to 100 µM with the detection limit 0.0227 µM (3σ/s). Moreover, to increase oxidase-mimic activity of FeMo6, reduced graphene oxide (rGO) loading FeMo6 composites (FeMo6@rGO (n), n = 5%, 10%, 15%) was fabricated, and results show that oxidase-like activities of FeMo6@rGO (n) are dependent on the mass ratio of FeMo6/rGO, and FeMo6@rGO (10%) exhibits the highest oxidase-mimic activity and the fastest respond time (4 min) among all reported oxidase mimic of DA to date. Graphical abstract Anderson-type Mo-POMs FeMo6 was found to work as an oxidase-mimicking nanoenzyme for the first time and was used to detect DA for two consecutive "turn on" fluorescence sensor modes.


Assuntos
Ânions/química , Dopamina/sangue , Polieletrólitos/química , Materiais Biomiméticos/química , Dopamina/análise , Grafite/química , Humanos , Ferro/química , Molibdênio/química , Oxirredução , Oxirredutases/química , Espectrometria de Fluorescência/métodos
14.
Anal Bioanal Chem ; 413(15): 4049-4061, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34057557

RESUMO

In the clinical diagnosis of tumors, a single-marker immunoassay may lead to false results. Thus there is a need for an effective and valid method for the simultaneous measurement of multiple tumor markers. In this work, an efficient fluorescence immunosensor for the simultaneous measurement of CA125 and CA15-3 tumor markers was fabricated by utilizing the high selectivity of magnetic molecularly imprinted polymers (MMIPs) and the high sensitivity of a fluorescence (FL) method. Ni nanoclusters (Ni NCs) and noble Cd nanoclusters (Cd NCs) were introduced as efficient and economic emitters, and magnetic graphene oxide (GO-Fe3O4) was applied as a support material for surface molecularly imprinted polymers. Under the most favorable experimental conditions, the fluorescence intensity of the Cd NCs and Ni NCs gradually increased with increasing concentration of CA125 and CA15-3 antigens at a range of 0.0005-40 U mL-1, respectively, with a limit of detection (LOD) of 50 µU mL-1. The developed method had excellent properties including a broad linear range, good reproducibility, and simple operation for the clinical diagnosis of CA 125 and CA 15-3 tumor markers. This molecularly imprinted fluorescence sensor has the potential to be an effective clinical tool for the timely screening of breast cancer in human serum samples and OVCAR-3 and MCF-7 cell lines, and can be applied in clinical diagnostics.


Assuntos
Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Cádmio/química , Corantes Fluorescentes/química , Mucina-1/sangue , Níquel/química , Espectrometria de Fluorescência/métodos , Linhagem Celular Tumoral , Humanos , Limite de Detecção , Impressão Molecular , Reprodutibilidade dos Testes
15.
ACS Appl Mater Interfaces ; 13(19): 22361-22367, 2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-33969689

RESUMO

Heavy metals, such as lead ions, are regarded as the main environmental contaminants and have a negative impact on human bodies, making detection technologies of lead ions critical. However, most existing detection methods suffer from time consumption, complicated sample pretreatment, and expensive equipment, which hinder their broad use in real-time detection. Herein, we show a new fluorescence sensor for detecting lead ions derived from liquid crystals doped with an aggregation-induced emission luminogen. The mechanism is based on the variation of fluorescence intensity caused by the disturbance of an ordered liquid crystal configuration in the presence of Pb2+, induced by DNAzyme and its catalytic cleavage. The proposed fluorescence sensor exhibits a low detection limit of 0.65 nM, which is 2 orders of magnitude lower than that previously reported in an optical sensor based on liquid crystals. The detection range of the Pb2+ fluorescence sensor is broad, from 20 nM to 100 µM, and it also selects lead ions from numerous metal ions exactly, resulting in a highly sensitive, highly selective, simple, and low-cost detection strategy of Pb2+ with potential applications in chemical and biological fields. This approach to designing a liquid crystal fluorescence sensor offers an inspiring stage for detecting biomacromolecules or other heavy metal ions by varying decorated molecules.


Assuntos
Corantes Fluorescentes/química , Chumbo/análise , Cristais Líquidos/química , Técnicas Biossensoriais/métodos , Fluorescência , Limite de Detecção , Espectrometria de Fluorescência/métodos
16.
Toxicol Lett ; 348: 18-27, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34023437

RESUMO

In the lungs, asbestos develops an Fe-rich coating (Asbestos Body, AB) that becomes the actual interface between the foreign fibers and the host organism. Conventional approaches to study ABs require an invasive sample preparation that can alter them. In this work, a novel combination of x-ray tomography and spectroscopy allowed studying unaltered lung tissue samples with chrysotile and crocidolite asbestos. The thickness and mass density maps of the ABs obtained by x-ray tomography were used to derive a truly quantitative elemental analysis from scanning x-ray fluorescence spectroscopy data. The average mass density of the ABs is compatible with that of highly loaded ferritin, or hemosiderin. The composition of all ABs analyzed was similar, with only minor differences in the relative elemental fractions. Silicon concentration decreased in the core-to-rim direction, indicating a possible partial dissolution of the inner fiber. The Fe content in the ABs was higher than that possibly contained in chrysotile and crocidolite. This finding opens two opposite scenarios, the first with Fe coming from the fiber bulk and concentrating on the surface as long as the fiber dissolves, the second where the Fe that takes part to the formation of the AB originates from the host organism Fe-pool.


Assuntos
Asbestos/química , Asbestose/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Espectrometria de Fluorescência/métodos , Tomografia por Raios X/métodos , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino
17.
Food Chem ; 359: 129960, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33945987

RESUMO

The interaction between lipase and quercetin 3-rhamnoside was studied by fluorescence spectroscopy, enzyme kinetics, and molecular dynamics simulation. The results showed that quercetin 3-rhamnoside had a strong quenching effect on the intrinsic fluorescence of lipase. The binding constant decreased with increasing temperature, and the number of binding sites approached 1. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces are the dominant forces when the interaction occurs. Circular dichroism spectroscopy and infrared spectroscopy proved that the ligand perturbed the structure of lipase. Enzyme kinetics results showed that quercetin 3-rhamnoside inhibited lipase, and the inhibitory effect was dose-dependent. Molecular dynamics simulation further explained the interaction mechanism and inhibitory effect. This study confirmed the inhibitory effect of quercetin 3-rhamnoside on lipase explained their binding mechanism, which will contribute to guiding the development of fat-reducing functional foods.


Assuntos
Lipase/metabolismo , Quercetina/metabolismo , Sítios de Ligação , Fluorescência , Ligação de Hidrogênio , Lipase/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Quercetina/química , Espectrometria de Fluorescência/métodos , Temperatura , Termodinâmica
18.
Anal Bioanal Chem ; 413(17): 4441-4450, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34002275

RESUMO

Hypochlorite (ClO-) is an important reactive oxygen species (ROS) in organisms. In this work, a fluorescent probe DBTM based on triphenylamine was synthesized successfully and characterized by spectral methods. The designed probe can rapidly respond to ClO- in just 1 min, followed by the apparent color change from red to yellow. The colorimetric and ratiometric absorbance change of DBTM was attributed to the strong oxidation of ClO-, which broke the connected double bonds and destroyed the conjugate system. The probe DBTM showed an excellent selectivity towards ClO- in comparison with other ROS probes. Besides, the DBTM probe exhibited a highly sensitive response to ClO-, with the detection limits calculated to be 3.3 nM. The probe can be applied in the form of cotton swabs and test strips that could detect ClO- easily, suggesting its potential use as imaging agents for realistic ClO- detection. In particular, DBTM exhibited very low background fluorescence in living cells and was able to detect the minor variation of endogenous hypochlorite in L929 cells. Based on these advantages, the probe DBTM could be a good candidate for detecting ClO- in biological systems.


Assuntos
Compostos de Anilina/química , Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Animais , Ânions/análise , Linhagem Celular , Camundongos , Microscopia de Fluorescência/métodos , Modelos Moleculares , Imagem Óptica/métodos , Espectrometria de Fluorescência/métodos
19.
Methods Mol Biol ; 2274: 3-14, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050457

RESUMO

The nuclear envelope (NE), a double membrane that separates nuclear components from the cytoplasm, undergoes a breakdown and reformation during cell division. To trace NE dynamics, the NE needs to be labeled with a fluorescent marker, and for this purpose, markers based on inner nuclear membrane (INM) proteins are normally used. However, NE labeling with INM proteins has some limitations. Here, we introduce a protocol for fluorescent labeling and imaging of NE that does not rely on INM proteins, along with protocols for simultaneously imaging two nuclear components and for time-lapse imaging of labeled cells.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Membrana Nuclear/metabolismo , Espectrometria de Fluorescência/métodos , Células HeLa , Humanos
20.
J Vis Exp ; (170)2021 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-33970128

RESUMO

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.


Assuntos
Doenças Neurodegenerativas/fisiopatologia , Agregados Proteicos/fisiologia , Espectrometria de Fluorescência/métodos , Humanos
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