RESUMEN
8-Hydroxypyrene-1,3,6-trisulfonate (HPTS) is a small, hydrophilic fluorescent molecule. Since the pKa of the hydroxyl group is close to neutrality and quickly responds to pH changes, it is widely used as a pH-reporter in cell biology for measurements of intracellular pH. HPTS fluorescence (both excitation and emission spectra) at variable pH was measured in pure water in the presence of NaCl solution or in the presence of different buffers (PBS or hepes in the presence or not of NaCl) and in a solution containing BSA. pKa values have been obtained from the sigmoidal curves. Herein, we investigated the effect of mono-, di-, and trivalent cations (Na+, Ca2+, La3+, Gd3+) on fluorescence changes and proposed its use for the quantification of trivalent cations (e.g., gadolinium ions) present in solution as acqua-ions. Starting from the linear regression, the LoD value of 6.32 µM for the Gd3+ detection was calculated. The effects on the emission were also analyzed in the presence of a combination of Gd3+ at two different concentrations and the previously indicated mono and di-valent ions. The study demonstrated the feasibility of a qualitative method to investigate the intracellular Gd3+ release upon the administration of Gd-based contrast agents in murine macrophages.
Asunto(s)
Medios de Contraste , Gadolinio , Animales , Cationes , Medios de Contraste/farmacología , Fluorescencia , Gadolinio/química , Imagen por Resonancia Magnética , Ratones , Cloruro de SodioRESUMEN
Pyranine (HPTS) is a remarkably interesting pH-sensitive dye that has been used for plenty of applications. Its high quantum yield and extremely sensitive ratiometric fluorescence against pH change makes it a very favorable for pH-sensing applications and the development of pH nano-/microsensors. However, its strong negative charge and lack of easily modifiable functional groups makes it difficult to use with charged substrates such as silica. This study reports a methodology for noncovalent HPTS immobilization on silica microparticles that considers the retention of pH sensitivity as well as the long-term stability of the pH microsensors. The study emphasizes the importance of surface charge for governing the sensitivity of the immobilized HPTS dye molecules on silica microparticles. The importance of the immobilization methodology, which preserves the sensitivity and stability of the microsensors, is also assessed.
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Colorantes Fluorescentes , Dióxido de Silicio , Arilsulfonatos , Concentración de Iones de Hidrógeno , Espectrometría de FluorescenciaRESUMEN
A practical demonstration of pH measurement in real biological samples with an in-house developed fiber-optic pH sensor system is presented. The sensor uses 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) fluorescent dye as the opto-chemical transducer. The dye is immobilized in a hybrid sol-gel matrix at the tip of a tapered optical fiber. We used 405 nm and 450 nm laser diodes for the dye excitation and a photomultiplier tube as a detector. The sensor was used for the measurement of pH in human aqueous humor samples during cataract surgery. Two groups of patients were tested, one underwent conventional phacoemulsification removal of the lens while the other was subjected to femtosecond laser assisted cataract surgery (FLACS). The precision of the measurement was ±0.04 pH units. The average pH of the aqueous humor of patients subjected to FLACS and those subjected to phacoemulsification were 7.24 ± 0.17 and 7.31 ± 0.20 respectively.
Asunto(s)
Terapia por Láser , Facoemulsificación , Humor Acuoso , Humanos , Concentración de Iones de Hidrógeno , Estudios ProspectivosRESUMEN
We disclose the results of our investigations on the influence that the insertion method of aryl-extended calix[4]pyrrole into liposomal membranes exerts on their properties as anion carriers. We use the standard HPTS assay to assess the transport properties of the carriers. We show that the post-insertion of the carrier, as DMSO solution, assigns better transport activities to the "two-wall" α,α-aryl-extended calix[4]pyrrole 1 compared to the "four-wall" α,α,α,α-counterpart 2. Notably, opposite results were obtained when the carriers were pre-inserted into the liposomal membranes. We assign this difference to an improved incorporation of carrier 2 into the membrane when delivered by the pre-insertion method. On the other hand, carrier 1 shows comparable levels of transport independently of the method used for its incorporation. Thus, an accurate comparison of the chloride transport activities featured by these two carriers demands their pre-incorporation in the liposomal membranes. In contrast, using the lucigenin assay with the pre-insertion method both carriers displayed similar transport efficiencies.
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Electrochemical reactions are the unrivaled backbone of next-generation energy storage, energy conversion, and healthcare devices. However, the real-time visualization of electrochemical reactions remains the bottleneck for fully exploiting their intrinsic potential. Herein, for the first time, a universal approach to direct spatiotemporal-dynamic in situ optical visualization of pH-based as well as specific byproduct-based electrochemical reactions is performed. As a highly relevant and impactful example, in-operando optical visualization of on-catalyst water splitting processes is performed in neutral water/seawater. HPTS (8-hydroxypyrene-1,3,6-trisulfonicacid), known for its exceptional optical capability of detecting even the tiniest pH changes allows the unprecedented "spatiotemporal" real-time visualization at the electrodes. As a result, it is unprecedentedly revealed that at a critical cathode-to-anode distance, the bulk-electrolyte "self-neutralization" phenomenon can be achieved during the water splitting process, leading to the practical realization of enhanced additive-free neutral water splitting. Furthermore, it is experimentally unveiled that at increasing electrolyte flow rates, a swift and severe inhibition of the concomitantly forming acidic and basic 'fronts', developed at anode and cathode compartments are observed, thus acting as a "buffering" mechanism. To demonstrate the universal applicability of this elegant strategy which is not limited to pH changes, the technique is extended to visualization of hypochlorite/ chlorine at the anode during electrolysis of sea water using N-(4-butanoic acid) dansylsulfonamide (BADS). Thus, a unique experimental tool that allows real-time spatiotemporal visualization and simultaneous mechanistic investigation of complex electrochemical processes is developed that can be universally extended to various fields of research.
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Few post-graduate training programs offer a comprehensive curriculum that includes structured clinical experiences to teach interprofessional care. To address this need, the United States Department of Veterans Affairs, Office of Academic Affiliations funded the Centers of Excellence in Primary Care Education (CoEPCE) from 2011-2019 to provide interprofessional curricula for health profession trainees (HPTs), including physician residents, nurse practitioner residents, pharmacy residents, and psychology residents. We examined changes over time in curricular domains, system impacts, and program practices based on HPT survey data and the qualitative evaluation of narrative feedback. An annual survey was administered to participants. Indirect standardized ratios were calculated for interprofessional professional education (IPE) program domains, system impacts, and program practices. Qualitative responses were coded based on curricular domains and key program components. The study cohort included 369 HPTs. Site and profession standardized indirect ratios across all professions indicated improvements in curricular domains, system impacts, and program practices, with significant differences observed for associated health HPTs as compared to other HPTs for performance improvement. Qualitative data indicated that profession was associated with differences in perceptions of the curriculum. Although improvements occurred over time, our findings support the need for the thoughtful consideration of profession-specific identity characteristics when designing interprofessional curricula.
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A simple, accurate, precise spectrofluorimetric method has been proposed for the determination of three cephalosporins, namely, cefixime (cefi), cephalexine (ceph), and cefotaxime sodium (cefo) in pharmaceutical formulations. This method is based on a reaction between cephalosporins with 8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt (HPTS) in alkaline medium, at pH 12.0 for cefi and 13.0 for ceph and cefo to give highly fluorescent derivatives extracted with chloroform and subsequent measurements of the formed fluorescent products at 520, 500 and 510 nm after excitation at 480, 470 and 480 nm for cefi, ceph and cefo respectively. The optimum experimental conditions have been studied. Beer's law is obeyed over concentrations of 10-60 ng/mL, 5-35 ng/mL and 10-60 ng/mL for cefi, ceph and cefo, respectively. The detection limits were 4.20 ng/mL, 2.54 ng/mL and 4.09 ng/mL for cefi, ceph and cefo, respectively, with a linear regression correlation coefficient of 0.99783, 0.99705 and 0.9978 and recoveries in ranges 96.96-105.77, 96.13-102.55 and 95.45-105.39% for cefi, ceph and cefo, respectively. This method is simple and can be applied for the determination of cefi, ceph and cefo in pharmaceutical formulations in quality control laboratories.
Asunto(s)
Cefalosporinas/análisis , Preparaciones Farmacéuticas/química , Espectrometría de Fluorescencia/métodos , Estructura MolecularRESUMEN
Continuously monitoring transcutaneous CO2 partial pressure is of crucial importance in the diagnosis and treatment of respiratory and cardiac diseases. Despite significant progress in the development of CO2 sensors, their implementation as portable or wearable devices for real-time monitoring remains under-explored. Here, we report on the creation of a wearable prototype device for transcutaneous CO2 monitoring based on quantifying the fluorescence of a highly breathable CO2-sensing film. The developed materials are based on a fluorescent pH indicator (8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt or HPTS) embedded into hydrophobic polymer matrices. The film's fluorescence is highly sensitive to changes in CO2 partial pressure in the physiological range, as well as photostable and insensitive to humidity. The device and medical-grade films are based on our prior work on transcutaneous oxygen-sensing technology, which has been extensively validated clinically.
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Dióxido de Carbono , Dispositivos Electrónicos Vestibles , Humanos , Humedad , Oxígeno , Polímeros/químicaRESUMEN
Coordination polymers (CPs), new functional hybrid materials, have received important attention due to their structural features and many different applications such as gas storage, catalysis, energy storage, small molecule adsorption, luminescence, and chemical sensors. In this study, two newly synthesized metal-organic frameworks (MOFs); [Mn2(µ4-dmg)-(µ4-dmg)(µ-bipy)]n (CP1) and [Mn2(µ4-dmg)(µ4-dmg)(µ-dpeten)]n (CP2); were used for sensing of gaseous carbon dioxide with a spectrofluorometric method with a dye, 8-hydroxypyrene-1, 3, 6-trisulfonic acid (HPTS) as matrix additive material for the first time. The ion-pair form of the HPTS was used in the polymethyl methacrylate matrix as a thin film form. When the HPTS based sensing slides along with the CPs, resulted in many advances such as high relative signal change and larger linear response range, improved sensor dynamics, and higher sensitivity with respect to the additive-free form. More specifically, in the presence of silver nanoparticles (AgNPs), the variation in relative signal intensities of CP1 and CP2 doped HPTS sensing agents were measured as 60 and 40% for the concentration range of 0-10% pCO2, respectively. The aim of this study is to enhance the CO2 response of HPTS with doped CPs due to their useful attributes and potential applications containing selective gas absorption.
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Nanopartículas del Metal , Polímeros , Dióxido de Carbono , Plata , Espectrometría de FluorescenciaRESUMEN
Polyion complexes have been known about for decades, with their applications mainly restricted to drug and gene delivery. In this study, we show that by the introduction of fluorescent charged molecules into a polyion complex, it can be used as a specific detection system for surfactants. The fluorescence of 8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt (HPTS) is quenched in the ionic complex, while it can be recovered with the addition of the surfactant sodium dodecylbenzenesulfonate (SDBS), due to the stronger interaction between SDBS and the polyelectrolyte. This leads to a drastic color change of the solution, and a recovery of the strong emission of HPTS. Specifically, the fluorescence is linearly proportional to the concentration of SDBS, thus it can be used for the qualitative detection of SDBS. Furthermore, the detection limit for SDBS can be up to the order of 10-10 M. We believe that competitive dissociation of the ionic complex can be used as a general approach for the construction of new functional materials.
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In plants, the extracellular space (apoplast) is one of the main places where exchange of molecules occurs between cells. Not only is this compartment involved in the storage of multiple metabolites and ions, including calcium and protons, but it also plays a role in the transmission of signaling molecules for cell-to-cell communication. It has recently been shown multiple times that these two aspects are linked and can influence each other. In particular, apoplast pH was shown as a primary regulator of auxin (IAA) transport in Arabidopsis thaliana. To prove the role of apoplastic pH, we have developed a protocol for apoplastic fluid extraction from Arabidopsis leaves, followed by pH determination using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescent dye. This technique successfully allows one to monitor apoplastic pH variations among different plant lines and to link changes in apoplastic pH to cellular responses in the plant.
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Arabidopsis/química , Arilsulfonatos , Espacio Extracelular/química , Colorantes Fluorescentes , Ácidos Indolacéticos/metabolismo , Hojas de la Planta/química , Transporte Biológico , Calcio/metabolismo , Fluorescencia , Concentración de Iones de Hidrógeno , Protones , Transducción de SeñalRESUMEN
Minimally invasive in vivo measurement of pH in microscopic biological samples of µm or µl size, e.g. plant cells, tissues and saps, may help to explain complex biological processes. Consequently, techniques to achieve such measurements are a focus of interest for botanists. This paper describes a technique for the in vivo measurement of pH in the range pH5.0 to pH7.8 in microscopic plant tissue samples of Arabidopsis thaliana based on a ratiometric fluorescence method using low-loss robust tapered fiber probes. For this purpose tapered fiber probes were prepared and coated with a detection layer containing ion-paired fluorescent pH-transducer 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (c-HPTS). A fluorescence ratiometric approach was employed based on excitation at 415 nm and 450 nm and on the comparison of the fluorescence response at 515 nm. The suitability of tapered fiber probes for local detection of pH between 5.0 and 7.8 was demonstrated. A pH sensitivity of 0.15 pH units was achieved within the pH ranges 5.0-5.9 and 7.1-7.8, and this was improved to 0.04 pH units within the pH range 5.9-7.1. Spatial resolution of the probes was better than 20 µm and a time response within 15-20s was achieved. Despite the minute dimensions of the tapered fiber probes the setup developed was relatively robust and compact in construction and performed reliably. It has been successfully employed for the in vivo local determination of pH of mechanically resistant plant tissues of A. thaliana of microscopic scale. The detection of momentary pH gradients across the intact plant seems to be a good tool for the determination of changes in pH in response to experimental treatments affecting for example enzyme activities, availability of mineral nutrients, hormonal control of plant development and plant responses to environmental cues.
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Arabidopsis/metabolismo , Arilsulfonatos/química , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Espectrometría de FluorescenciaRESUMEN
Poly(methacrylic acid)-b-poly(ethylene oxide) are double hydrophilic block copolymers, which are able to form micelles by complexation with a counter-polycation, such as poly-l-lysine. A study was carried out on the ability of the copolymers to interact with model membranes as a function of their molecular weights and as a function of pH. Different behaviors were observed: high molecular weight copolymers respect the membrane integrity, whereas low molecular weight copolymers with a well-chosen asymmetry degree can induce a membrane alteration. Hence by choosing the appropriate molecular weight, micelles with distinct membrane interaction behaviors can be obtained leading to different intracellular traffics with or without endosomal escape, making them interesting tools for cell engineering. Especially micelles constituted of low molecular weight copolymers could exhibit the endosomal escape property, which opens vast therapeutic applications. Moreover micelles possess a homogeneous nanometric size and show variable properties of disassembly at acidic pH, of stability in physiological conditions, and finally of cyto-tolerance.