pH-sensitive Fluorophore Research Articles

Development of an O2‐Sensitive Fluorescence‐Quenching Assay for the Combinatorial Discovery of Electrocatalysts for Water Oxidation

High kinetic barriers associated with the oxidation of water to O2 [4,5] and the common use of high-cost electrocatalytic materials are among the challenges that limit the utility of photoelectrochemical energy storage. Improved electrocatalysts that operate at lower overpotential and avoid the use of expensive precious-metal or rare-earth elements are needed. While valuable progress is being made toward this goal, the rational design of optimal catalysts from first principles remains infeasible, and the number of possible catalyst compositions, even those with well-defined metal stoichiometry, far exceeds the number that can be tested in a traditional sequential fashion. Combinatorial methods can play an important role in the discovery of new electrocatalysts, and we describe herein a fluorescence-based assay for spatially resolved, direct detection of O2 across an array of metal-oxide electrocatalysts. Initial implementation of this technique has led to the identification of new electrocatalysts, which are composed entirely of earth-abundant elements (e.g., Ni/Al/ Fe) and warrant further investigation. Combinatorial methods for the discovery of electrocatalysts have been pursued previously. For example, a soluble fluorescent pH indicator has been used to screen electrocatalysts for reactions that consume or generate protons, including water oxidation mediated by platinum-group-metal electrocatalysts. Potential complications with this method include the use of poorly buffered electrolytes to ensure sensitivity to pH changes, and the instability of organic pHsensitive fluorophores under conditions required for water oxidation. Various combinatorial approaches have been used to probe photoelectrocatalytic performance of mixed-metaloxide materials. In most of these assays, the oxides are required to act simultaneously as a light-harvesting semiconductor and as the electrocatalyst for one or both watersplitting half-reactions. The most efficient PECs, however, will probably integrate separate photovoltaic (PV) and catalytic materials. Therefore, we targeted an assay that would enable rapid assessment of electrocatalysts for water oxidation independent of the other PEC functions. Catalysts discovered by such methods could then be used in indirect PECs (Figure 1A) or developed further for integration with PV semiconductors in direct PECs (Figure 1B). The essential feature of electrocatalytic water oxidation is O2 production, and an ideal catalyst-screening assay would directly monitor O2 evolution. In addition, a fluorescencebased assay seemed appealing because such methods are often compatible with parallel, rather than serial, analysis of activity, and they avoid the need for costly specialized analytical instrumentation. Fluorescent pressure-sensitive paints are well suited to meet these criteria. These paints are used in the automobile and aerospace industries to study aerodynamics in wind tunnels, and their utility arises from the sensitivity of their fluorescence intensity to the partial pressure of O2 (pO2). [23] Quantitative measurements are improved by incorporating two fluorophores into the paint, one that is insensitive to O2 as a background reference, and another that exhibits fluorescence quenching in proportion to the pO2. Our assay takes advantage of a commercially Figure 1. Schematic representations of indirect (A) and direct (B) PEC configurations for water splitting. The former employs a PV solar cell coupled to an electrolysis cell, whereas the latter features direct integration of the electrocatalysts with the charge-separating PV semiconductor.

Na+,HCO3−‐cotransport is crucial for intracellular pH control in human breast cancer

Single nucleotide polymorphisms in Na+,HCO3−‐cotransporter NBCn1 (SLC4A7) are associated with breast cancer. Here, we studied the expression of NBCn1 and the importance of Na+,HCO3− ‐cotransport in human and mouse breast carcinomas. NBCn1 expression was detected at mRNA‐ and protein level in freshly obtained human breast tumors. Immunohistochemical membrane staining for NBCn1 was increased in human primary breast carcinomas and metastases compared to matched healthy breast epithelium. Using pH‐sensitive fluorophores, we found that Na+,HCO3 −‐cotransport and Na+/H+‐exchange are the only important mechanisms of net acid extrusion from human and ErbB2‐overexpressing mouse primary breast carcinomas. In the near‐physiological pHi range (pHi>6.60), CO2/HCO3−‐dependent mechanisms were responsible for >90% of total net acid extrusion. Steady‐state pHi of human breast carcinomas was 0.35±0.06 units lower in the absence than in the presence of CO2/HCO3−. In conclusion, NBCn1 expression is increased in human breast carcinomas where Na+,HCO3−‐cotransport is a prominent mechanism of acid extrusion and crucial for steady‐state pHi control. Support: Danish Council for Independent Research and Danish Cancer Society.

A fluorescence based assay with pyranine labeled hexa-histidine tagged organophosphorus hydrolase (OPH) for determination of organophosphates

Organophosphorus hydrolase has been employed extensively for catalysis based biosensor synthesis with optical, potentiometric and amperometric based detection mechanism. The present study reports the response of bioconjuagtes prepared by utilizing OPH and pH sensitive polyanionic fluorophore i.e. pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) in different molar ratios by the aid of electrostatic force. Two types of bioconjugates were prepared, one of them contained mature form of OPH obtained from Escherichia coli cells expressing organophosphate degrading (opd) gene from a tac promoter, whereas the variant OPH6His, has 6× histidine tail at C-terminus. The investigation was carried out utilizing the bioconjugates of different molar ratios prepared with both the enzymes and ratio showing maximum degree of labeling with pyranine was selected for the detection of organophosphates in standard samples. The lower limit of detection for paraoxon was ∼20ppb and for methyl parathion and coumaphos it was ∼50ppb under optimized conditions (55–60°C) with the reaction time of 3min. These features of the prepared conjugate make it a strong contender for the development of the field deployable biosensor for organophosphates estimation.

Imaging Techniques for Quantifying Passive Diffusion Across Lipid Bilayer Membranes

Passive diffusion of small-molecule species across the cell membranes is an essential step in drug delivery since molecules that can easily permeate the plasma membrane have high oral bioavailability. There are several techniques for characterizing the passive transmembrane transport of small molecules; however, there are many instances in which these techniques produce widely varying results. For instance, literature values for the permeabilities of low-molecular-weight carboxylic acids across phosphatidylcholine membranes span three orders of magnitude.We have developed a technique for precisely quantifying lipid membrane permeability to small molecules based on imaging of the concentration field surrounding giant unilamellar lipid vesicle (GUV) membranes. High-speed spinning disk confocal microscopy can be used to image dynamic changes in the concentration field and precisely measure permeabilities as fast as 0.2 cm/s. This technique can be used to measure the transport of both dye-containing species and species that result in a change of fluorescence intensity of a dye molecule. For instance, by using a pH-sensitive fluorophore, the transport of non-fluorescent carboxylic acid molecules can by observed.This technique can be combined with direct imaging of the membrane to simultaneously measure distinct transport rates across different phase-separated regions of a lipid vesicle. We have also imaged transport across GUVs with compositionally asymmetric membranes. These GUVs are formed in a multiphase microfluidic flow system in which the composition of each leaflet of the membrane can be controlled independently. We have measured proton and carboxylic acid transport across membranes with charge asymmetry. Early results show that the permeability of membranes with charge asymmetry depends on the direction in which small molecules and proton species are moving with respect to the membrane.

Error analysis of ratiometric imaging of extracellular pH in a window chamber model

Ratiometric fluorescence-imaging technique is commonly used to measure extracellular pH in tumors and surrounding tissue within a dorsal skin-fold window chamber. Using a pH-sensitive fluorophore such as carboxy SNARF-1 one can measure pH distributions with high precision. However, it is often observed that the measured pH is lower than expected, with a bias that varies from one image to another. A comprehensive analysis of possible error sources is presented. These error sources include photon noise, estimator bias, instrument errors, temperature, and calibration errors from biological factors.

Intracellular chloride channel protein CLIC1 regulates macrophage functions via modulation of phagosomal acidification

Intracellular chloride channel protein 1 (CLIC1) is a 241 amino acid protein of the glutathione S transferase fold family with redox- and pH-dependent membrane association and chloride ion channel activity. Whilst CLIC proteins are evolutionarily conserved in Metazoa, indicating an important role, little is known about their biology. CLIC1 was first cloned on the basis of increased expression in activated macrophages. We therefore examined its subcellular localisation in murine peritoneal macrophages by immunofluorescence confocal microscopy. In resting cells, CLIC1 is observed in punctate cytoplasmic structures that do not colocalise with markers for endosomes or secretory vesicles. However, when these macrophages phagocytose serum-opsonised zymosan, CLIC1 translocates onto the phagosomal membrane. Macrophages from CLIC1(-/-) mice display a defect in phagosome acidification as determined by imaging live cells phagocytosing zymosan tagged with the pH-sensitive fluorophore Oregon Green. This altered phagosomal acidification was not accompanied by a detectable impairment in phagosomal-lysosomal fusion. However, consistent with a defect in acidification, CLIC1(-/-) macrophages also displayed impaired phagosomal proteolytic capacity and reduced reactive oxygen species production. Further, CLIC1(-/-) mice were protected from development of serum transfer induced K/BxN arthritis. These data all point to an important role for CLIC1 in regulating macrophage function through its ion channel activity and suggest it is a suitable target for the development of anti-inflammatory drugs.

Polymer‐Coated Nanoparticles: A Universal Tool for Biolabelling Experiments

Water solubilization of nanoparticles is a fundamental prerequisite for many biological applications. To date, no single method has emerged as ideal, and several different approaches have been successfully utilized. These 'phase-transfer' strategies are reviewed, indicating key advantages and disadvantages, and a discussion of conjugation strategies is presented. Coating of hydrophobic nanoparticles with amphiphilic polymers provides a generic pathway for the phase transfer of semiconductor, magnetic, metallic, and upconverting nanoparticles from nonpolar to polar environments. Amphiphilic polymers that include maleimide groups can be readily functionalized with chemical groups for specific applications. In the second, experimental part, some of the new chemical features of such polymer-capped nanoparticles are demonstrated. In particular, nanoparticles to which a pH sensitive fluorophore has been attached are described, and their use for intracellular pH-sensing demonstrated. It is shown that the properties of analyte-sensitive fluorophores can be tuned by using interactions with the underlying nanoparticles.

Real time intracellular pH dynamics in Listeria innocua under CO 2 and N 2O pressure

This article investigates the inactivation mechanism of high-pressure food treatment, considered as alternative to conventional biocidal processes. We aimed to determine intracellular pH decrease under CO 2 and N 2O pressure, so far postulated as one of the main causes of inactivation. Working with a lab-scale bioreactor in mild conditions – 25 °C and pressures up to 8 MPa – we monitored – for the first time during pressurization – cytoplasmic pH variations of Listeria innocua labeled with pH-sensitive fluorophores based on fluorescein. We show that carbonic acid, due to solubilization of CO 2 into the aqueous phase, causes a rapid pH drop in the cytosol, reaching pH 4.8 at 1 MPa and falls below the detection limit of the indicator fluorophore of pH 4.0. This correlates with a reduced viability (below 90%) in all the pressure ranges investigated. Contrarily, treatment under N 2O pressure reduces cell viability without significant pH-drop neither of intra- nor extra-cellular liquid at any pressure investigated. The pH value remains between 7 and 6 while an inactivation of more than 80% is achieved at 8 MPa. Our data clearly demonstrate that, as a critical pressure is achieved, microbial inactivation is mainly due to pressure-induced membrane permeation – stimulated by non-acidifying fluids as well, rather then cytoplasmic acidification, as widely argued so far. A definitive understanding of the microbial inactivation mechanism due to CO 2/N 2O under pressure has been advanced significantly.

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK(a) to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome-lysosome pathway.

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Polyelectrolyte multilayers generated in a microfluidic device with pH gradients direct adhesion and movement of cells

In this study, multilayers from polyethylene imine, heparin and chitosan are prepared at three different pH values of 5, 7 and 9. Water contact angle and quartz microbalance measurements show that resulting multilayers differ in terms of wetting behaviour, layer mass and mechanical properties. The multilayer is then formed within a gradient generation microfluidic (μFL) device. Polyethylene imine or heparin solutions of pH 5 are introduced into one inlet and the same solutions but at pH 9 into another inlet of the μFL device. The pH gradient established during the multilayer formation can be visualized inside the microchamber by pH sensitive fluorophores and confocal laser scanning microscopy. From this setup it is expected that properties of multilayers displayed at distinct pH values can be realised in a gradient manner inside the μFL device. Behaviour of the osteoblast cell line MG-63 seeded and cultured on top of multilayers created inside the μFL device support this hypothesis. It is observed that more cells adhere and spread on multilayers build-up at the basic side of the μFL channel, while those cells on top of multilayers built at pH 5 are fewer and smaller. These results are consistent with the behaviour of MG-63 cells seeded on multilayers formed at discrete pH values. It is particularly interesting to see that cells start to migrate from multilayers built at pH 5 to those built at pH 9 during 6 h of culture. Overall, the presented multilayer formation setup applying pH gradients leads to surfaces that promote migration of cells.

Dual-fluorophore ratiometric pH nanosensor with tuneable pKa and extended dynamic range

Ratiometric pH nanosensors with tuneable pK(a) were prepared by entrapping combinations of two pH-sensitive fluorophores (fluorescein isothiocyanate dextran (FITC-D) and Oregon Green(®) dextran (OG-D)) and a reference fluorophore (5-(and-6)-carboxytetramethylrhodamine dextran (TAMRA-D)), in a biocompatible polymer matrix. Dual-fluorophore pH nanosensors permit the measurement of an extended dynamic range, from pH 4.0 to 7.5.

Multiwell plates loaded with fluorescent hydrogel sensors for measuring pH and glucose concentration

Fluorescent hydrogels were polymerized directly in multi-well plates at ambient temperature and in the presence of air, producing sensors for measuring pH and glucose concentration. The plates were rapidly analyzed using a fluorescence plate reader. Multiwell pH sensors with good reproducibility among different wells and a dynamic range from pH 6 to 9 were prepared by incorporating a polymerizable pH sensitive fluorophore in the hydrogel. Non-enzymatic glucose sensors comprising a boronic acid-appended fluorescence quencher together with an aminopyrene fluorophore were prepared in a matter of hours in multiwell plates. The sensors showed good reproducibility in response to solutions of glucose at physiological pH. Dried glucose sensors rehydrated with analyte solution performed similarly to freshly prepared hydrogels. The loaded plates are designed for use in high throughput screening applications. Plates were prepared using the redox initiator system metabisulfite/persulfate/iron(II) to generate hydrogels of N,N-dimethylacrylamide crosslinked with N,N′-methylenebisacrylamidein situ.

Conformation transition and electric potential of single weak polyelectrolyte: molecular weight dependence

The nature of the conformation transition of a hydrophobic weak polyelectrolyte (PE) chain is critically determined by local electrostatic environment in vicinity of the PE backbone. In this work, we examine the coil-to-globule conformation transition (CGT) and electric potential of a model weak PE, poly(2-vinyl pyridine) (P2VP) of varied molecular weight (Mn), in aqueous solutions at a single molecule level by fluorescence correlation spectroscopy (FCS) and photon counts histogram (PCH). By using FCS, the CGT of P2VP in aqueous solutions is observed upon increasing solution pH, yet showing a strong Mn dependence. The observed CGT is gradual and continuous at low Mn, in sharp contrast to an abrupt, discontinuous one at high Mn. Measured critical transition pH, pHCGT, to induce CGT is found to decrease as increasing Mn. To further elucidate the effect of Mn on CGT and local electrostatic interaction, PCH is employed with pH-sensitive fluorophore probes attached to a P2VP chain to determine the local pH, i.e. the local proton concentration immediately adjacent to an expanded P2VP coil. The electric potential, φ for a single P2VP chain is thereby estimated from the difference between local and bulk pHs, exhibiting a scaling with P2VP polymerization degree, N as φ ∼ N0.1. The increase in φ with increasing Mn suggests an enhanced electrostatic repulsion between local protons and a longer P2VP backbone, leading to the lowering of pHCGT.

Flow Injection Small-volume Fiber-optic pH Sensor Based on Evanescent Wave Excitation and Fluorescence Determination

A small-volume fiber-optic pH sensor (FOEWS) based on evanescent wave excitation is developed and evaluated. The sensor is simply fabricated by inserting a decladded optical fiber into a transparent capillary tube. A microchannel between the optical fiber and the capillary inner wall was formed and acted as flow cell for solution flowing through. The pH-sensitive fluorophore of fluorescein can be excited by the evanescent wave field produced on the fiber core surface to produce emission fluorescence. pH value was then sensed by its enhancing effect on the emission fluorescence intensity. The response range of the sensor is from pH 2.09 to pH 8.85 and the linear range is from pH 3.25 to 8.85. The proposed sensor has a small detection volume of 2.5 μL and a short response time of 8 s. It has been applied to measure pH values of real water samples and was in good agreement with the results obtained by commercial pH meter.

Core-shell fluorescent silica nanoparticles for sensing near-neutral pH values

pH-responsive fluorescent core-shell silica nanoparticles (SiNPs) were prepared by encapsulating the pH-sensitive fluorophore 8-hydroxypyrene-1,3, 6-trisulfonate into their silica shell via a facile reverse microemulsion method. The resulting SiNPs were characterized by SEM, TEM, fluorescence lifetime spectroscopy, photobleaching experiments, and photoluminescence. The core-shell structure endows the SiNPs with reduced photobleaching, excellent photostability, minimized solvatachromic shift, and increased fluorescence efficiency compared to the free fluorophore in aqueous solution. The dynamic range for sensing pH ranges from 5.5 to 9.0. The nanosensors show excellent stability, are highly reproducible, and enable rapid detection of pH. The results obtained with the SiNPs are in good agreement with data obtained with a glass electrode.

Development of Fluorescent Array Based on Sol-Gel/Chitosan Encapsulated Acetylcholinesterase and pH Sensitive Oxazol-5-one Derivative

A highly sensitive fluorescent enzyme array for quantitative acetylcholine detection is developed. The enzyme array has been constructed by spotting of pH sensitive fluorophore 2-phenyl-4-[4-(1,4,7,10-tetraoxa-13-azacycloopentadecyl)benzylidene]oxazol-5-one and acetylcholinesterase doped in tetraethoxysilane/chitosan matrix via a microarrayer. The constructed tetraethoxysilane/chitosan network provided a microenvironment in which the enzyme molecule was active biologically. The optimal operational conditions for the array developed were investigated. The response of the developed biosensor array to acetylcholine was highly reproducible (RSD = 3.27%, n = 6). A good linearity was observed for acetylcholine in the concentrations up to 1 × 10(-8) M, with a detection limit of 0.27 × 10(-8) M.

Employing a pH Sensitive Fluorophore to Measure Intracellular pH in the In Vitro Brainstem Preparation of Rana catesbeiana~!2009-05-06~!2009-10-29~!2009-12-24~!

Employing a pH Sensitive Fluorophore to Measure Intracellular pH in the In Vitro Brainstem Preparation of Rana catesbeiana~!2009-05-06~!2009-10-29~!2009-12-24~!

Surface-coupled proton exchange of a membrane-bound proton acceptor

Proton-transfer reactions across and at the surface of biological membranes are central for maintaining the transmembrane proton electrochemical gradients involved in cellular energy conversion. In this study, fluorescence correlation spectroscopy was used to measure the local protonation and deprotonation rates of single pH-sensitive fluorophores conjugated to liposome membranes, and the dependence of these rates on lipid composition and ion concentration. Measurements of proton exchange rates over a wide proton concentration range, using two different pH-sensitive fluorophores with different pK(a)s, revealed two distinct proton exchange regimes. At high pH (> 8), proton association increases rapidly with increasing proton concentrations, presumably because the whole membrane acts as a proton-collecting antenna for the fluorophore. In contrast, at low pH (< 7), the increase in the proton association rate is slower and comparable to that of direct protonation of the fluorophore from the bulk solution. In the latter case, the proton exchange rates of the two fluorophores are indistinguishable, indicating that their protonation rates are determined by the local membrane environment. Measurements on membranes of different surface charge and at different ion concentrations made it possible to determine surface potentials, as well as the distance between the surface and the fluorophore. The results from this study define the conditions under which biological membranes can act as proton-collecting antennae and provide fundamental information on the relation between the membrane surface charge density and the local proton exchange kinetics.