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1.
Molecules ; 26(7)2021 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-33917350

RESUMEN

Ratiometric near-infrared fluorescent probes (AH+ and BH+) have been prepared for pH determination in mitochondria by attaching dithioacetal and formal residues onto a hemicyanine dye. The reactive formyl group on probe BH+ allows for retention inside mitochondria as it can react with a protein primary amine residue to form an imine under slightly basic pH 8.0. Probes AH+ and BH+ display ratiometric fluorescent responses to pH changes through the protonation and deprotonaton of a hydroxy group in hemicyanine dyes with experimentally determined pKa values of 6.85 and 6.49, respectively. Calculated pKa values from a variety of theoretical methods indicated that the SMDBONDI method of accounting for solvent and van der Waals radii plus including a water molecule located near the site of protonation produced the closest overall agreement with the experimental values at 7.33 and 6.14 for AH+ and BH+ respectively.


Asunto(s)
Carbocianinas/química , Colorantes Fluorescentes/química , Mitocondrias/metabolismo , Muerte Celular , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Fenómenos Ópticos , Espectrometría de Fluorescencia , Agua/química
2.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804002

RESUMEN

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.


Asunto(s)
Proteína con Homeodominio Antennapedia/genética , Complejos de Proteína Captadores de Luz/genética , Procesos Fototróficos/genética , Agregado de Proteínas/genética , Proteína con Homeodominio Antennapedia/química , Clorofila/química , Clorofila/genética , Clorofila/efectos de la radiación , Análisis por Conglomerados , Fluorescencia , Concentración de Iones de Hidrógeno , Luz/efectos adversos , Complejos de Proteína Captadores de Luz/química , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/efectos de la radiación , Espectrometría de Fluorescencia , Tilacoides/química , Tilacoides/genética , Tilacoides/efectos de la radiación , Zeaxantinas/genética
3.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800923

RESUMEN

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.


Asunto(s)
Nucleótidos de Desoxiuracil/metabolismo , Modelos Teóricos , Espectrometría de Fluorescencia/métodos , Electricidad Estática , Timidilato Sintasa/metabolismo , Sustitución de Aminoácidos , Animales , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/metabolismo , Nucleótidos de Desoxiuracil/química , Fluorodesoxiuridilato/metabolismo , Ratones , Modelos Moleculares , Análisis Multivariante , Conformación Proteica , Timidilato Sintasa/química
4.
Am J Vet Res ; 82(5): 417-424, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33904802

RESUMEN

OBJECTIVE: To compare progesterone (P4) concentrations measured with surface plasmon field-enhanced fluorescence spectroscopy (SPFS) and chemiluminescence immunoassay (CLIA) in serum and plasma samples of client-owned bitches of various ages and breeds and to determine reference ranges for P4 concentrations at various stages of the estrous cycle. SAMPLES: 102 serum samples and 104 plasma samples. PROCEDURES: In experiment 1, 1 aliquot each of serum and plasma was analyzed for P4 concentration by use of SPFS incorporated in a veterinary-specific point-of-care immunologic analyzer and CLIA. In experiment 2, serum collected from bitches in various stages of the estrous cycle was analyzed for P4 concentration by use of SPFS to establish reference ranges for each stage. RESULTS: In experiment 1, P4 concentrations measured by SPFS and CLIA were highly correlated (serum, r = 0.966; plasma, r = 0.968). In experiment 2, ranges of serum basal (proestrous) P4 concentrations (n = 114) and P4 concentrations at the estimated time of ovulation (76), during pregnancy or diestrus (107), and during the prepartum period (50) measured with SPFS were 0.42 to 1.46 ng/mL, 3.69 to 7.85 ng/mL, 11.73 to 28.24 ng/mL, and 1.54 to 3.22 ng/mL, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Because serum and plasma P4 concentrations measured with SPFS were highly correlated with those measured with CLIA and ranges of serum P4 concentrations measured with SPFS for each of phase of the estrous cycle were well-defined for the large sample size, veterinarians may be able to accurately use this veterinary-specific point-of-care immunologic analyzer with SPFS methodology to determine P4 concentrations of bitches in their daily practice.


Asunto(s)
Ovulación , Progesterona , Animales , Ciclo Estral , Femenino , Embarazo , Valores de Referencia , Espectrometría de Fluorescencia/veterinaria
5.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806656

RESUMEN

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein-ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein-ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor-kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor-kinase interactions.


Asunto(s)
Proteínas/química , Espectrometría de Fluorescencia/métodos , Polarización de Fluorescencia/métodos , Colorantes Fluorescentes/química , Ligandos , Bibliotecas de Moléculas Pequeñas/química , Termodinámica
6.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809594

RESUMEN

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.


Asunto(s)
Cuerpos de Inclusión/metabolismo , Metaloproteinasa 9 de la Matriz/química , Proteínas Recombinantes/química , Animales , Bovinos , Cromatografía de Afinidad , Dicroismo Circular , Dispersión Dinámica de Luz , Escherichia coli/metabolismo , Lactobacillus/metabolismo , Metaloproteinasa 9 de la Matriz/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Solubilidad , Espectrometría de Fluorescencia , Temperatura , Triptófano/química
7.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804193

RESUMEN

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a' was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a' in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.


Asunto(s)
Colorantes Fluorescentes/química , Compuestos de Piridinio/química , Coloración y Etiquetado/métodos , Animales , Colorantes Fluorescentes/síntesis química , Ratones , Microscopía Fluorescente , Fenotiazinas/química , Fotones , Compuestos de Piridinio/síntesis química , Solventes/química , Espectrometría de Fluorescencia/métodos
8.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-33797236

RESUMEN

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Asunto(s)
Fluorescencia , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Imagen Molecular , ARN Mensajero/análisis , ARN Mensajero/metabolismo , /tratamiento farmacológico , Línea Celular Tumoral , Citosina/análogos & derivados , Citosina/análisis , Citosina/síntesis química , Citosina/química , Colorantes Fluorescentes/síntesis química , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Humanos , Estructura Molecular , ARN Mensajero/química , ARN Mensajero/uso terapéutico , Espectrometría de Fluorescencia
9.
Adv Exp Med Biol ; 1310: 1-30, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834430

RESUMEN

Confocal laser scanning microscopy (CLSM) and related microscopic techniques allow a unique and versatile approach to image and analyze living cells due to their specificity and high sensitivity. Among confocal related techniques, fluorescence correlation methods, such as fluorescence correlation spectroscopy (FCS) and dual-color fluorescence cross-correlation spectroscopy (FCCS), are highly sensitive biophysical methods for analyzing the complex dynamic events of molecular diffusion and interaction change in live cells as well as in solution by exploiting the characteristics of fluorescence signals. Analytical and quantitative information from FCS and FCCS coupled with fluorescence images obtained from CLSM can now be applied in convergence science such as drug delivery and nanomedicine, as well as in basic cell biology. In this chapter, a brief introduction into the physical parameters that can be obtained from FCS and FCCS is first provided. Secondly, experimental examples of the methods for evaluating the parameters is presented. Finally, two potential FCS and FCCS applications for convergence science are introduced in more detail.


Asunto(s)
Microscopía Confocal , Color , Difusión , Espectrometría de Fluorescencia , Coloración y Etiquetado
10.
Adv Exp Med Biol ; 1310: 31-58, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834431

RESUMEN

Number and brightness (N&B) analysis helps to visualize protein oligomer and its localization in a living cell. N&B analysis provides apparent brightness, which reflects the oligomeric state of a fluorescently labeled protein, by analyzing the temporal intensity fluctuation at each pixel. N&B analysis is useful in understanding the dynamic oligomerization in signal transduction and neurodegenerative diseases. Furthermore, it also helps in gaining useful insights regarding the controlling mechanisms in protein function. In this chapter, we describe the basic theory and notations of N&B analysis implemented with confocal laser scanning microscopy for quantitative analyses.


Asunto(s)
Transducción de Señal , Proteínas Fluorescentes Verdes , Microscopía Confocal , Multimerización de Proteína , Espectrometría de Fluorescencia
11.
Adv Exp Med Biol ; 1310: 449-473, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834445

RESUMEN

Semiconductor nanocrystals (SNCs) are a nano-sized inorganic material. Due to the quantum confinement effect, these crystals exhibit unique optical and electrical properties. This chapter focuses on biological applications of SNCs, ranging from in vitro single-molecule tracking to in vivo fluorescence imaging. The following fundamental properties and technical procedures related to SNCs are also described: structures of SNCs, synthetic procedures and shape control of SNCs, preparation methods of water-soluble SNCs, and conjugation methods of biomolecules.


Asunto(s)
Puntos Cuánticos , Espectrometría de Fluorescencia
12.
Adv Exp Med Biol ; 1310: 475-493, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834446

RESUMEN

Fluorescence imaging of nucleic acids is a key technology needed to understand gene expression and the resulting changes in cellular function. This chapter focuses on sequence-specific fluorescence imaging of nucleic acids in cells using fluorescent nucleic acids. The design and preparation of fluorescent nucleic acids and their application to fluorescence imaging of intracellular nucleic acids are introduced.


Asunto(s)
Ácidos Nucleicos , ARN , ADN/genética , Fluorescencia , Colorantes Fluorescentes , Ácidos Nucleicos/genética , Imagen Óptica , ARN/genética , Espectrometría de Fluorescencia
13.
Int J Nanomedicine ; 16: 2751-2759, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33859476

RESUMEN

Purpose: Sulfamethazine (SMZ) exposed in the environment can enter the human body through the food chain and pose a serious threat to human health. Therefore, it is important to develop a rapid and sensitive method for detecting SMZ in environmental samples. In order to fastly and quantitatively detect SMZ in environmental samples, we developed a label-free fluorescent aptasensor based on specific aptamer (SMZ1S) and fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and rhodamine B (RhoB). Methods: In the absence of SMZ, SMZ1S was adsorbed on the surface of AuNPs, which led to dispersion of the AuNPs in high concentration saline solution, thus effectively quenching the fluorescence of RhoB. With the increase of the SMZ concentration, the specific binding of SMZ1S and SMZ led to the aggregation of AuNPs in the presence of NaCl, which reduced the quenching of RhoB fluorescence and increased the fluorescence intensity. The sensitivity and linearity curve of the label-free fluorescent aptasensor were determined with different concentrations of sulfamethazine standard solutions. The specificity of this fluorescent aptasensor was determined by replacing sulfamethazine with different antibiotics. In addition, the actual water and soil samples were spiked and recovered. Results: Under optimized conditions, the proposed fluorescent aptasensor demonstrated a good linear detection of SMZ in binding buffer from 1.25 ng mL-1 to 40 ng mL-1 and the limit of detection was 0.82 ng mL-1. The spiked recoveries for SMZ were 94.4% to 108.8% with a relative standard deviation of 1.8-10.3% in water and soil samples, respectively. Conclusion: The label-free fluorescent aptasensor investigated in the current study is a promising tool to detect and quantify SMZ in water and soil samples.


Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Sulfametazina/análisis , Dispersión Dinámica de Luz , Oro/química , Humanos , Límite de Detección , Nanopartículas del Metal/química , Suelo/química , Espectrometría de Fluorescencia , Coloración y Etiquetado , Agua/química
14.
Food Chem ; 355: 129633, 2021 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-33819808

RESUMEN

In the presented study, a horseradish peroxidase (HRP)-mediated ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) for zearalenone (ZEN) was reported based on fluorescence quenching of gold-silver bimetallic nanoclusters (Au-Ag NCs). HRP-antibody was used as a bridge in this immunoassay, linking the ratiometric fluorescence signal to the ZEN concentration. HRP catalyzed the oxidization of o-phenylenediamine in the presence of H2O2, leading to the formation of 2,3-diaminophenazine, which not only delivered a new peak at 580 nm but also quenched Au-Ag NCs fluorescence at 690 nm. Under optimal conditions, the detection limit for the proposed ELISA was 0.017 ng/mL, which was approximately 6.6-fold lower than conventional ELISA. Moreover, analytical performances were evaluated fully including specificity, accuracy, precision, and practicability, and showed that this method provides a potential platform for sensitive and reliable detection of ZEN.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Oro/química , Peroxidasa de Rábano Silvestre/metabolismo , Nanopartículas del Metal/química , Plata/química , Zearalenona/análisis , Fluorescencia , Peróxido de Hidrógeno/química , Nanopartículas del Metal/ultraestructura , Espectrometría de Fluorescencia/métodos
15.
Molecules ; 26(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802020

RESUMEN

Glycyrrhizae Radix et Rhizoma (GRR) is one of the commonly used traditional Chinese medicines in clinical practice, which has been applied to treat digestive system diseases for hundreds of years. GRR is preferred for anti-gastric ulcer, however, the main active compounds are still unknown. In this study, GRR was used as precursor to synthesize carbon dots (CDs) by a environment-friendly one-step pyrolysis process. GRR-CDs were characterized by using transmission electron microscopy, high-resolution TEM, fourier transform infrared, ultraviolet-visible and fluorescence spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction and high-performance liquid chromatography. In addition, cellular toxicity of GRR-CDs was studied by using CCK-8 in RAW264.7 cells, and the anti-gastric ulcer activity was evaluated and confirmed using mice model of acute alcoholic gastric ulcer. The experiment confirmed that GRR-CDs were the spherical structure with a large number of active groups on the surface and their particle size ranged from 2 to 10 nm. GRR-CDs had no toxicity to RAW264.7 cells at concentration of 19.5 to 5000 µg/mL and could reduce the oxidative damage of gastric mucosa and tissues caused by alcohol, as demonstrated by restoring expression of malondialdehyde, superoxide dismutase and nitric oxide in serum and tissue of mice. The results indicated the explicit anti-ulcer activity of GRR-CDs, which provided a new insights for the research on effective material basis of GRR.


Asunto(s)
Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Puntos Cuánticos/química , Animales , Carbono/química , China , Cromatografía Líquida de Alta Presión/métodos , Glycyrrhiza/química , Masculino , Medicina China Tradicional/métodos , Ratones , Espectroscopía de Fotoelectrones/métodos , Pirólisis , Células RAW 264.7 , Espectrometría de Fluorescencia/métodos
16.
Nat Commun ; 12(1): 1898, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772017

RESUMEN

Triplet-triplet annihilation upconversion nanoparticles have attracted considerable interest due to their promises in organic chemistry, solar energy harvesting and several biological applications. However, triplet-triplet annihilation upconversion in aqueous solutions is challenging due to sensitivity to oxygen, hindering its biological applications under ambient atmosphere. Herein, we report a simple enzymatic strategy to overcome oxygen-induced triplet-triplet annihilation upconversion quenching. This strategy stems from a glucose oxidase catalyzed glucose oxidation reaction, which enables rapid oxygen depletion to turn on upconversion in the aqueous solution. Furthermore, self-standing upconversion biological sensors of such nanoparticles are developed to detect glucose and measure the activity of enzymes related to glucose metabolism in a highly specific, sensitive and background-free manner. This study not only overcomes the key roadblock for applications of triplet-triplet annihilation upconversion nanoparticles in aqueous solutions, it also establishes the proof-of-concept to develop triplet-triplet annihilation upconversion nanoparticles as background free self-standing biological sensors.


Asunto(s)
Glucosa Oxidasa/metabolismo , Glucosa/metabolismo , Nanopartículas/química , Oxígeno/química , Algoritmos , Catálisis/efectos de la radiación , Humanos , Luz , Modelos Químicos , Estructura Molecular , Nanopartículas/efectos de la radiación , Oxidación-Reducción/efectos de la radiación , Oxígeno/metabolismo , Espectrometría de Fluorescencia , Agua/química
17.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33672042

RESUMEN

The interactions of epoxiconazole and prothioconazole with human serum albumin and bovine serum albumin were investigated using spectroscopic methods complemented with molecular modeling. Spectroscopic techniques showed the formation of pesticide/serum albumin complexes with the static type as the dominant mechanism. The association constants ranged from 3.80 × 104-6.45 × 105 L/mol depending on the pesticide molecule (epoxiconazole, prothioconazole) and albumin type (human or bovine serum albumin). The calculated thermodynamic parameters revealed that the binding of pesticides into serum albumin macromolecules mainly depended on hydrogen bonds and van der Waals interactions. Synchronous fluorescence spectroscopy and the competitive experiments method showed that pesticides bind to subdomain IIA, near tryptophan; in the case of bovine serum albumin also on the macromolecule surface. Concerning prothioconazole, we observed the existence of an additional binding site at the junction of domains I and III of serum albumin macromolecules. These observations were corroborated well by molecular modeling predictions. The conformation changes in secondary structure were characterized by circular dichroism, three-dimensional fluorescence, and UV/VIS absorption methods.


Asunto(s)
Compuestos Epoxi/química , Simulación del Acoplamiento Molecular/métodos , Plaguicidas/química , Albúmina Sérica Bovina/química , Albúmina Sérica Humana/química , Triazoles/química , Animales , Sitios de Unión , Bovinos , Dicroismo Circular/métodos , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia/métodos , Electricidad Estática , Temperatura
18.
Nat Commun ; 12(1): 1748, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741958

RESUMEN

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.


Asunto(s)
Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Actinas/metabolismo , Animales , Células CHO , Membrana Celular , Cricetulus , Difusión , Receptores ErbB/metabolismo , Fluorescencia , Humanos , Espectrometría de Fluorescencia/métodos
19.
Molecules ; 26(5)2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33670879

RESUMEN

Four flavanone Schiff bases (E)-1-(2-phenylchroman-4-ylidene)thiosemicarbazide (FTSC) (1), N',2-bis((E)-2-phenylchroman-4-ylidene)hydrazine-1-carbothiohydrazide (FTCH) (2), (E)-N'-(2-phenylchroman-4-ylidene)benzohydrazide (FHSB) (3) and (E)-N'-(2-phenylchroman-4-ylidene)isonicotinohydrazide (FIN) (4) were synthesized and evaluated for their electronic and physicochemical properties using experimental and theoretical methods. One of them, (2), consists of two flavanone moieties and one substituent, the rest of the compounds (1, 3, 4) comprises of a flavanone-substituent system in relation to 1:1. To uncover the structural and electronic properties of flavanone Schiff bases, computational simulations and absorption spectroscopy were applied. Additionally, binding efficiencies of the studied compounds to serum albumins were evaluated using fluorescence spectroscopy. Spectral profiles of flavanone Schiff bases showed differences related to the presence of substituent groups in system B of the Schiff base molecules. Based on the theoretically predicted chemical descriptors, FTSC is the most chemically reactive among the studied compounds. Binding regions within human and bovine serum albumins of the ligands studied are in the vicinity of the Trp residue and a static mechanism dominates in fluorescence quenching.


Asunto(s)
Flavanonas/química , Bases de Schiff/química , Albúmina Sérica/química , Secuencia de Aminoácidos , Animales , Bovinos , Teoría Funcional de la Densidad , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Espectrometría de Fluorescencia , Espectrofotometría , Relación Estructura-Actividad
20.
Molecules ; 26(5)2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33670937

RESUMEN

The new symmetric acyclic N,N'-bis(1-pyrenyl) squaramide (H2L) functionalized with the pyrene moiety as a fluorogenic fragment has been designed and its ability to selectively detect specific anions and metals investigated. H2L selectively binds Cl- both in solution (DMSO 0.5% H2O and MeCN) and in the solid state, and allows to selectively detect Cu2+ in MeCN with the formation of a 2:1 metal-receptor complex, with a green intense emission appreciable by naked eye under the UV lamp. The H2L copper complex preserves its emission properties in the presence of Cl-. The addition of basic anions (OH-, CN-, and F-) up to 10 equivalents caused the deprotonation of the squaramide NHs and a dramatic change of the emission properties of the H2L copper complex.


Asunto(s)
Complejos de Coordinación/química , Cobre/química , Pirenos/química , Quinina/análogos & derivados , Acetonitrilos/química , Aniones/química , Teoría Funcional de la Densidad , Modelos Moleculares , Conformación Molecular , Quinina/química , Espectrometría de Fluorescencia
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