Fluorescent pH Sensor Research Articles

Development of novel optical pH sensors based on coumarin 6 and nile blue A encapsulated in resin particles and specific support materials.

Inspired by traditional pH papers, two types of ratiometric fluorescent pH sensors for neutral and alkaline pH ranges were developed in this study by using two fluorescent dyes, coumarin 6 (C6) and nile blue A (NB). These dyes were encapsulated into melamine-formaldehyde (MF) resin particles, which were then incorporated with polymer Nafion (Nf) or polyurethane hydrogel (PU) to prepare pH-sensing membranes. MF-C6 particles immobilized into polymer Nafion (i.e., MF-C6-Nf membrane) showed a dynamic ratiometric pH detection range of 4.5-7.5 through shift in fluorescence emission spectra at acidic and neutral pH solutions, as well as distinct color transition under normal visual sense from pink to yellow color. By contrast, MF-NB particles immobilized into polyurethane hydrogel (i.e., MF-NB-PU membrane) displayed a dynamic ratiometric fluorescence detection range of pH 9 - pH 12 via change in ratiometric fluorescence intensity at two emission band edges. The membrane also showed a clear change from blue to purple color under sunlight at high pH values. These pH-sensing membranes also exhibited high sensitivity, good reversibility, and stability. They were then applied to measure pH values in real urine samples and fermentation media.

Evidence That Ion-Based Signaling Initiating at the Cell Surface Can Potentially Influence Chromatin Dynamics and Chromatin-Bound Proteins in the Nucleus.

We have developed tools and performed pilot experiments to test the hypothesis that an intracellular ion-based signaling pathway, provoked by an extracellular stimulus acting at the cell surface, can influence interphase chromosome dynamics and chromatin-bound proteins in the nucleus. The experimental system employs chromosome-specific fluorescent tags and the genome-encoded fluorescent pH sensor SEpHluorinA227D, which has been targeted to various intracellular membranes and soluble compartments in root cells of Arabidopsis thaliana. We are using this system and three-dimensional live cell imaging to visualize whether fluorescent-tagged interphase chromosome sites undergo changes in constrained motion concurrently with reductions in membrane-associated pH elicited by extracellular ATP, which is known to trigger a cascade of events in plant cells including changes in calcium ion concentrations, pH, and membrane potential. To examine possible effects of the proposed ion-based signaling pathway directly at the chromatin level, we generated a pH-sensitive fluorescent DNA-binding protein that allows pH changes to be monitored at specific genomic sites. Results obtained using these tools support the existence of a rapid, ion-based signaling pathway that initiates at the cell surface and reaches the nucleus to induce alterations in interphase chromatin mobility and the surrounding pH of chromatin-bound proteins. Such a pathway could conceivably act under natural circumstances to allow external stimuli to swiftly influence gene expression by affecting interphase chromosome movement and the structures and/or activities of chromatin-associated proteins.

Self-assembly, protonation-dependent morphology, and photophysical properties of perylene bisimide with tertiary amine groups

It is crucial to tune the morphology of aggregates to obtain functional photoelectric properties in perylene bisimide (PBI). The associated properties can be controlled by appropriate molecular design and by tuning the self-assembly conditions. However, many studies only focused on the modification with easily-protonated groups at the bay positions of perylene bisimide, which greatly limited their potential applications. In this work, four PBI derivates bearing tertiary amine groups at the imide positions were synthesized and their self-assembly with different concentrations of hydrochloric acid were performed. The final morphology and the properties of the aggregates were tuned by modifying the charge interaction in addition to π-π stacking and hydrophobic interactions. The evolution of the specific morphology with the acidity of the self-assembly conditions was discussed and the molecular structure was optimized. The photophysical properties of the aggregates were characterized by UV–visible and fluorescence spectroscopy. The shapes of the spectra varied with the molecular structures and the acid concentration. Moreover, the emission spectra of the aggregates experienced a large Stokes shift. The color of the aggregates formed from the three derivates (MDI-PBI, EDI-PBI, and AEP-PBI) changed from red to blue upon protonation. These derivatives may have potential applications in organic photoelectric functional devices where a strict morphology or size is needed. The protonation-dependent morphology, the color change, and the large Stokes shift makes the aggregates potential candidate for fluorescent probes or pH sensors.

Pyranine-Modified Amphiphilic Polymer Conetworks as Fluorescent Ratiometric pH Sensors.

The fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonate (pyranine) combines high photostability with ratiometric pH detection in the physiological range, making it a prime candidate for optical sensors in biomedical applications, such as pH-based chronic wound monitoring. However, pyranine's high water solubility and the difficulty of covalent attachment pose severe limitations in terms of leaching from sensor matrices. Herein, pyranine-modified nanophase-separated amphiphilic polymer conetworks (APCNs) are reported as fluorescent ratiometric pH sensors. The thin, freestanding APCN membranes composed of one hydrophilic and one hydrophobic polymer provide an optically transparent, flexible, and stable ideal matrix that enables contact between dye and aqueous environment. An active ester-based conjugation approach results in a highly homogeneous and stable pyranine modification of the APCN's hydrophilic phase. This concept effectively solves the leaching challenge for pyranine without compromising its functionality, which is demonstrated by ratiometric pH detection in the range of pH 5-9.

Metabolic activity and intracellular pH in induced pluripotent stem cells differentiating in dermal and epidermal directions

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

Preparation, characterization, pH titration, and electrochemical properties of an anthracene-bridged binuclear ruthenium complex containing imidazole

By using an anthryl-phenyl-imidazo-phen as a pH responsive ligand, a binuclear ruthenium complex has been synthesized as a pH luminescence probe under visible light excitation at 460 nm. The ground- and excited-state acid–base properties of the complex were studied by UV–visible and emission spectrophotometric pH titrations. The excited-state ionization constants of pKa2* = 6.92 is in a physiologically relevant regime and the complex is an “on-off” type fluorescent pH sensor. In addition, electrochemical studies of the binuclear ruthenium complex show one Ru(II)-centered oxidation at 1.29 V and three ligand-centered reductions.

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor.

Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 ns lifetime change from pH 5.5 to 9.0. Critically, we find that the sensor maintains a p Ka of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are robustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time-resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent sensor, we also demonstrate that mCherryTYG is spectrally compatible with the ATeam sensor and EGFP for simultaneous dual-color measurements of intracellular pH, ATP, and extracellular pH. In a proof-of-concept, we quantify acute respiration-dependent pH homeostasis that exhibits a stoichiometric relationship with the ATP-generating capacity of the carbon fuel choice in E. coli. Broadly speaking, our work presents a previously unemployed methodology that will greatly facilitate continuous pH quantification.

Bimetal-organic framework nanocomposite based point-of-care visual ratiometric fluorescence pH microsensor for strong acidity

pH is of great importance in understanding physiological and pathologic processes. Abnormal gastric juice pH is an indicator of many gastric diseases such as peptic ulcer and gastric cancer. In this work, a nanocomposite 1-hydroxypyrene@ Co/Tb- dipicolinic acid metal-organic framework (1-OHP@Co/Tb-DPA MOF) was constructed as a ratiometric fluorescent pH sensor for strong acidity. The nanocomposite-based pH sensor was response-rapid (30 s), linearly responsive in the broad pH range of 0.3–7.8 (r > 0.95) and ultra-sensitive in the pH range of 0.3–5.0. Importantly, the nanocomposite-loaded fiber paper was prepared as a visual microsensor for point-of-care pH detection via ratiometric chromaticity, coupled with a homemade portable determination device. The paper-based visual microsensor is response-rapid and sensitive, long-term stable (at least 30 days), and suitable for pH assay of microvolume (as little as 5 μL) samples at pH 0.3–5.0. Both the nanocomposite-based ratiometric fluorescent sensor and the paper-based visual microsensor can accurately determine the pH of artificial gastric juices (relative error <5.4%). To our knowledge, this is the first time to report a MOF-based ratiometric fluorescent pH sensor for strong acidity (pH<1) and a paper-based pH microsensor for point-of-care visual assay via ratiometric chromaticity.

Core-shell polymeric nanoparticles comprising BODIPY and fluorescein as ultra-bright ratiometric fluorescent pH sensors.

A new ratiometric fluorescent pH nanosensor is presented. It is based on ultrabright nanoparticles containing two spatially separated fluorophores: BODIPY covalently linked to the polystyrene core and fluorescein grafted to the nanoparticle shell. The nanoparticles comprise a large number (≥2500) of both fluorescent moieties. Their spectroscopic characteristics were studied at different pH and ionic strength. They could successfully be used to determine the solution pH between 5.5 and 7.5 by measuring the fluorescence intensity ratio of the sensor molecule (fluorescein) relative to the reference dye (BODIPY).

Polymer Microarrays for the Discovery and Optimization of Robust Optical-Fiber-Based pH Sensors.

Polymer microarrays were utilized for the high-throughput screening and discovery of optimal polymeric substrates capable of trapping functional ratiometric fluorescence-based pH sensors. This led to the identification of poly(methyl methacrylate- co-2-(dimethylamino) ethyl acrylate) (PA101), which allowed, via dip coating, the attachment of fluorescent pH sensors onto the tips of optical fibers, resulting in robust, rapid, and reproducible sensing of physiological pHs.

Development of fluorescent pH sensors based on a sol-gel matrix for acidic and neutral pH ranges in a microtiter plate

In this study, two typical pH-sensitive dyes, fluorescein amine (FA) and 8‑hydroxypyrene‑1,3,6‑trisulfonic acid trisodium salt (HPTS), were doped with two types of silica particles. The dye-doped silica particles were entrapped in a mixture of 3‑glycidoxypropyltrimethoxysilane (GPTMS) and 3-aminopropyltrimethoxysilane (APTMS) sol-gel matrix (GA) and a polyurethane hydrogel (PU) to prepare stable pH-sensitive membranes in a microtiter plate. Four pH-sensing membranes prepared at the bottom of wells in 24-well microtiter plates showed high sensitivity, fast recoverability, and good photostability. Supporting materials with fast ion transport properties were used to produce a pH-sensing membrane with rapid response time. The membranes entrapped with FA-doped silica particles had a dynamic detection range from pH 2 to pH 6, while the membranes entrapped with HPTS-doped silica particles had a dynamic detection range from pH 5 to pH 8. Dye-leaching from the pH-sensing membranes did not occur due to tight binding of functional groups in the dyes (FA and HPTS) to the sol-gel GA matrix, the doping of dyes in the silica particles, and the addition of an anti-fading agent (DABCO). Four pH-sensing membranes were used to measure the pH of wastewater. The measured results were consistent with standard pH values.

Two pH-responsive fluorescence probes based on indole derivatives

Two new pH fluorescent probes 2,8-bis((E)-2-(1,1-dimethyl-1H-benzo[e]indol-2-yl)vinyl)-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine (1) and 2,8-bis((E)-2-(3,3-dimethyl-3H-indol-2-yl)vinyl)-6H,12H-5,11-methanodibenzo[b,f] [1,5]diazocine (2) were designed and synthesized via ethylene bridging of the Tröger's Base (TB) framework and indole derivatives. As the protonation of nitrogen atoms of indole derivatives, both probes displayed the significant pH-dependent spectral properties. The probing behavior toward pH variations indicated that probes exhibited excellent pH dependent behavior in the pH ranges of 2.07–7.01 (1) and 1.96–7.01 (2), especially possessing good linear response in the pH ranges of 2.50–4.00. The emissions of 1 can be reversibly switched between brilliant yellow and dark states by protonation and deprotonation. Also, the yellow-green fluorescence emission of 2 could be quenched upon protonation. Such behaviors enable them to work as turn-off fluorescent pH sensors in the solution state. Furthermore, protonation and deprotonation on indole moiety nitrogen of the probe 1 and 2 provided a sensing mechanism for pH changes. In addition, both probes performed good photostability, high selectivity and excellent reversibility.

Water Electrolysis Carried Out on Microelectrodes to Obtain New Insights into the Regulation of Cytosolic pH

AbstractIn this study, the use of electrochemistry in cell biology is extended by showing that electrochemically generated microscale pH gradients can be used to gain new insights into the regulation of cytosolic pH of normal and cancer cells. The developed procedure involves positioning a carbon fiber microelectrode into the extracellular space of adherently growing cells and setting its potential to values suitable for electrooxidation or electroreduction of water. While the electrooxidation of water decreases the pH of the solution surrounding the microelectrode (because it produces hydronium ions), the electroreduction of water increases the pH of the same solution (because it produces hydroxide ions). Fluorescence microscopy is then used to observe the impact of the electrochemically generated microscale pH gradient on the cytosolic pH of cells loaded with a fluorescent pH sensor. The obtained results indicate that electrochemically induced acid stress affects the cytosolic pH of normal cells significantly faster than that of cancer cells while electrochemically induced alkaline stress appears to have very limited impact on the cytosolic pH of both cell types. In comparison to classic experiments concerning the regulation of cytosolic pH using perfusion chambers, the developed, electrochemistry‐based, approach has the advantages of a better spatial and temporal resolution and elimination of the flow‐induced shear stress.

Reversible pH-induced fluorescence colour change of gold nanoclusters based on pH-regulated surface interactions.

To prepare water-dispersible, biocompatible, ratiometric pH nanosensors is challenging. We report here for the first time that the emission colour of NAD+-capped AuNCs responds to the mono-/bidentate anchoring of the phosphoric groups of the ligand. The AuNCs exhibit a high luminescence (21% quantum yield) and an outstanding performance as fluorescent ratiometric pH sensors over a broad pH range.

Short-chain lipid-conjugated pH sensors for imaging of transporter activities in reconstituted systems and living cells.

The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.

Sensitivity enhancement of a fluorescent pH sensor by double silanization of the sensing surface

The goal of this work concerns a new method to substantially increase the sensitivity of a fluorescence pH sensor. The method is based on a double silanization method. Two methods are compared: one using APTMS (3-Aminopropyl)trimethoxysilane) only and another one using both APTMS and APDMS (3-Aminopropyl)dimethoxymethylsilane). Using the second method, sensor’s sensitivity is improved by more than 500 %.

Microfluidic Free-Flow Isoelectric Focusing with Real-Time pI Determination.

Free-flow electrophoresis (FFE) may be used for continuous and preparative separation of a wide variety of biomolecules. Isoelectric focusing (IEF) provides for the separation of compounds according to their isoelectric point (pI). Here we describe a microfluidic chip-based protocol for the fabrication, application, and optical monitoring of free-flow isoelectric focusing (FFIEF) of proteins and peptides on the microscale with optical surveillance of the microscopic pH gradient provided by an integrated pH sensing layer. This protocol may be used with modifications also for the FFIEF of other biomolecules and may serve as template for the fabrication of microfluidic chips with integrated fluorescent or luminescent pH sensor layers for FFE and other applications.

Solvent-dependent and visible light-activated NIR photochromic dithienylethene modified by difluoroboron β-diketonates as fluorescent turn-on pH sensor

The development of near-infrared (NIR) photochromic materials, particularly activated by visible light, are of increasing interest for their potential application in photoresponsive optoelectronics and functional bio-materials. Here, a novel D-DTE-A type dithienylethene NDB, which was modified by difluoroboron β-diketonate (BF2bdk) and dimethylphenylamine (DMPA) moieties, has been developed. It exhibited solvent-dependent photophysical properties before irradiation, and the visible light-triggered NIR photochromic behavior with solvent-dependent features was only observed in Toluene. Moreover, it displayed intense fluorescent turn-on acid-sensing properties in less polar solvent [Toluene, Dichloromethane (DCM) and Ethyl acetate (EA)] through the blockage of photoinduced electron transfer (PET) effect. Specially, NDB with photochromic inactivity in DCM could perform a unique gated photochromism in the presence of TFA/TEA.

Rationally Designed Fluorescent pH Sensors for Measurements in Extremely Alkaline Media.

We investigated the pKa increasing effects on meso-phenol BODIPY to achieve fluorescent pH sensors working in extreme alkalinity. We demonstrated the effects of substituents on meso, α, γ and position of the hydroxyl group on pKa. By considering these parameters, we developed pH sensors working above 11.0 in aqueous medium (ethanol/water 1:1). One of these sensors had a pKa of 12.0 and enabled the measurement of pH up to 13.6. This highly photostable and bright sensor compound could be synthesized easily from cheap starting materials.

A sensitive pyridine-containing turn-off fluorescent probe for pH detection

A new pH fluorescent probe 2,8-(6H,12H-5,11-methanodibenzo[b,f]diazocineylene)-di(o-ethenyl-pyridine) (TBOP), which attaches electron-accepting pyridine groups into Tröger’s Base skeleton, was designed and synthesized. Compound TBOP exhibits excellent pH-dependent performance that its blue fluorescence emission can be quenched upon protonation and the fluorescence can be lighted by deprotonation. Accordingly, the color of the solution switched between colorless and yellow. Moreover, TBOP has high sensitivity, good selectivity and large Stokes’ shift (121 nm). Such property enables it to work as a fluorescent acidic pH sensor in solution state.