Ratiometric pH Sensor Research Articles

A ratiometric fluorescent pH sensor based on nanoscale metal-organic frameworks (MOFs) modified by europium(III) complexes.

We report a new ratiometric fluorescent pH sensor based on postsynthetically modified nanoscale MOF-253. Two types of Eu(3+) with different characteristic excitation wavelength are realized in MOF-253 by a postsynthetic method (PSM). The pH sensor requires no calibration because only one of the two types of Eu(3+) is affected by the pH variation.

Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments.

Fabrication of triple-labeled polyelectrolyte microcapsules for localized ratiometric pH sensing

Encapsulation of pH sensitive fluorophores as reporting molecules provides a powerful approach to visualize the transportation of multilayer capsules. In this study, two pH sensitive dyes (fluorescein and oregon green) and one pH insensitive dye (rhodamine B) were simultaneously labeled on the microcapsules to fabricate ratiometric pH sensors. The fluorescence of the triple-labeled microcapsule sensors was robust and nearly independent of other intracellular species. With a dynamic pH measurement range of 3.3–6.5, the microcapsules can report their localized pH at a real time. Cell culture experiments showed that the microcapsules could be internalized by RAW 246.7cells naturally and finally accumulated in acidic organelles with a pH value of 5.08±0.59 (mean±s.d.; n=162).

Development of ratiometric fluorescent pH sensors based on chromenoquinoline derivatives with tunable pKa values for bioimaging

In this work, we first studied the pH-dependent characteristic of chromenoquinoline. Based on this, we then designed and synthesized two novel chromenoquinoline derivatives that can act as fluorescent pH sensors. The pKa values of two novel chromenoquinoline derivatives can be modulated from 2.32 to 4.38 and 6.27 by introducing EDG on the backbone of chromenoquinoline. Furthermore, we demonstrate that the sensor 4 can be used as a ratiometric fluorescent pH sensor for fluorescence imaging in living cells.

Rational design of a colorimetric pH sensor from a soluble retinoic acid chaperone.

Reengineering of cellular retinoic acid binding protein II (CRABPII) to be capable of binding retinal as a protonated Schiff base is described. Through rational alterations of the binding pocket, electrostatic perturbations of the embedded retinylidene chromophore that favor delocalization of the iminium charge lead to exquisite control in the regulation of chromophoric absorption properties, spanning the visible spectrum (474-640 nm). The pKa of the retinylidene protonated Schiff base was modulated from 2.4 to 8.1, giving rise to a set of proteins of varying colors and pH sensitivities. These proteins were used to demonstrate a concentration-independent, ratiometric pH sensor.

In Vivo Intracellular pH Measurements in Tobacco andArabidopsisReveal an Unexpected pH Gradient in the Endomembrane System

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

A fluorescent colorimetric pH sensor and the influences of matrices on sensing performances

A fluorescent colorimetric pH sensor was developed by a polymerization of a monomeric fluorescein based green emitter (SM1) with a monomeric 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran derived red emitter (SM2) in poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrices. Polymerized SM1 (PSM1) in the polymer matrices showed bright emissions at basic conditions and weak emissions at acidic conditions. Polymerized SM2 (PSM2) in the polymer matrices exhibited a vastly different response when compared to PSM1. The emissions of PSM2 are stronger under acidic conditions than those under basic conditions. When SM1 and SM2 were polymerized in the same polymer matrix, a dual emission sensor acting as a ratiometric pH sensor (PSM1,2) was successfully developed. Because the PSM1 and PSM2 exhibited different pH responses and separated emission windows, the changes in the emission colors were clearly observed in their dual color sensor of PSM1,2, which changed emission colors dramatically from green at pH 7 to red at pH 4, which was detected visually and/or by using a color camera under an excitation of 488nm. In addition to the development of the dual color ratiometric pH sensor, we also studied the effects of different matrix compositions, crosslinkers, and charges on the reporting capabilities of the sensors (sensitivity and pKa).

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system

Within the nervous system, intracellular Cl− and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl− and pH are often co-regulated, and network activity results in the movement of both Cl− and H+. Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl− and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN—a new genetically-encoded ratiometric Cl− and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl− and H+ concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons.

Highly Durable Double Sol–Gel Layer Ratiometric Fluorescent pH Optode Based on the Combination of Two Types of Quantum Dots and Absorbing pH Indicators

Long-term stable ratiometric fluorescent optical pH sensors (optodes) based on double sol-gel silica layers are prepared with the first layer embedding two types of quantum dots (QDs) and the second layer embedding light-absorbing pH indicators. The sensors are fabricated by a simple general sol-gel spin-coating method. The resulting double-layer pH optodes are designed as having long Stokes shift as well as ratiometric fluorescence emission response to pH in aqueous solutions of varying pH values. This optode has high durability against continuous light exposure, even under severe acidic condition (1 M HCl), and the storage stability is over a period of more than 6 months. These results indicate that the double-layer ratiometric fluorescence-based pH optode allows for long-term pH sensing. When two pH indicators of different pK(a) values with an optimized mixing ratio are embedded into the second layer, a double-layer pH optode with reproducible linear response in a wide pH range of over 6 pH units (from pH 4 to 10) can be designed and fabricated.

A sprayable luminescent pH sensor and its use for wound imaging in vivo

Non-invasive luminescence imaging is of great interest for studying biological parameters in wound healing, tumors and other biomedical fields. Recently, we developed the first method for 2D luminescence imaging of pH in vivo on humans, and a novel method for one-stop-shop visualization of oxygen and pH using the RGB read-out of digital cameras. Both methods make use of semitransparent sensor foils. Here, we describe a sprayable ratiometric luminescent pH sensor, which combines properties of both these methods. Additionally, a major advantage is that the sensor spray is applicable to very uneven tissue surfaces due to its consistency. A digital RGB image of the spray on tissue is taken. The signal of the pH indicator (fluorescein isothiocyanate) is stored in the green channel (G), while that of the reference dye [ruthenium(II)-tris-(4,7-diphenyl-1,10-phenanthroline)] is stored in the red channel (R). Images are processed by rationing luminescence intensities (G/R) to result in pseudocolor pH maps of tissues, e.g. wounds.

Iron(II) Complexes Containing Octadentate Tetraazamacrocycles as ParaCEST Magnetic Resonance Imaging Contrast Agents

Iron(II) complexes of the macrocyclic ligands 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC) and (1S,4S,7S,10S)-1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (STHP) contain a highly stabilized Fe(II) center in the high-spin state, which is encapsulated by an octadentate macrocycle. The complexes are resistant to acid, metal cations, phosphate, carbonate, and oxygen in aqueous solution. [Fe(TCMC)](2+) contains exchangeable amide protons, and [Fe(STHP)](2+) contains exchangeable protons attributed to alcohol OH donors, which give chemical exchange saturation transfer (CEST) peaks at physiological pH and 37 °C at 50 and 54 ppm from bulk water, respectively. The distinct pH dependence of the CEST peak of the two complexes over the range of pH 6-8 shows that these two groups may be useful in the development of ratiometric pH sensors based on iron(II).

A Tunable Ratiometric pH Sensor Based on Carbon Nanodots for the Quantitative Measurement of the Intracellular pH of Whole Cells

A Tunable Ratiometric pH Sensor Based on Carbon Nanodots for the Quantitative Measurement of the Intracellular pH of Whole Cells

A Tunable Ratiometric pH Sensor Based on Carbon Nanodots for the Quantitative Measurement of the Intracellular pH of Whole Cells

The whole picture: Carbon nanodots labeled with two fluorescent dyes have been developed as a tunable ratiometric pH sensor to measure intracellular pH. The nanosensor shows good biocompatibility and cellular dispersibility. Quantitative determinations on intact HeLa cells and pH fluctuations associated with oxidative stress were performed.

Quantum Dot–Fluorescent Protein FRET Probes for Sensing Intracellular pH

Intracellular pH (pH(i)) plays a critical role in the physiological and pathophysiological processes of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to assess the pH(i) of intact cells and subcellular compartments. Here we describe a nanoparticle-based ratiometric pH sensor, comprising a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging. We found that Förster resonance energy transfer between the QD and multiple FPs modulates the FP/QD emission ratio, exhibiting a >12-fold change between pH 6 and 8. The modularity of the probe enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the probe through mutagenesis of the fluorescent protein. The QD-FP probes facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. These probes have the potential to enjoy a wide range of intracellular pH imaging applications that may not be feasible with fluorescent proteins or organic fluorophores alone.

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.

A Nanocrystal-based Ratiometric pH Sensor for Natural pH Ranges.

A ratiometric fluorescent pH sensor based on CdSe/CdZnS nanocrystal quantum dots (NCs) has been designed for biological pH ranges. The construct is formed from the conjugation of a pH dye (SNARF) to NCs coated with a poly(amido amine) (PAMAM) dendrimer. The sensor exhibits a well-resolved ratio response at pH values between 6 and 8 under linear or two-photon excitation, and in the presence of a 4% bovine serum albumin (BSA) solution.

The development and characterisation of novel hybrid sol–gel-derived films for optical pH sensing

This paper focuses on the development and detailed characterisation of a novel hybrid sol–gel material derived from 3-glycidoxypropyltrimethoxysilane (GPTMS) and ethyltriethoxysilane (ETEOS). The material has been doped with a fluorescent pH-sensitive dye, modified 8-hydroxy-1,3,6-pyrene trisulfonic acid (HPTS) and its application as a high-performance optical ratiometric pH sensor has been demonstrated. The optimum sol–gel-based material composition has been characterised using atomic force microscopy (AFM), scanning electron microscopy in combination with energy dispersive X-ray spectroscopy (SEM-EDX), thermal gravimetric-infrared (TGA-IR) analysis, Fourier transform infrared (FT-IR) and Raman spectroscopy, solid state 29Si and 13C nuclear magnetic resonance (NMR). AFM and SEM images demonstrated a high degree of homogeneity of the pH films. FT-IR, Raman and NMR results have shown that hydrolysis and condensation reactions were almost completed and extensive crosslinking occurred in the final material. They also revealed that an opening of the epoxy ring in GPTMS took place which was key to optimum pH response. The pH sensor produced using the hybrid material compares very well with the current state-of-the-art and exhibits very good reversibility, reproducibility, stability, short response time and no leaching. The dynamic range extends from pH 5.0 to 8.0 which is compatible with bioprocessing, environmental and clinical monitoring applications.

Fluorescent Carbon Dots (CDs) as a pH Sensor

The pH-dependent properties of carbon dots (CDs), decorated with surface carboxylic acid functional groups, are described. Absorption, fluorescence emission measurements were used to study the reversible pH-dependent spectral response. The pH-dependent emissions of CDs demonstrate a strong dependence on the excitation wavelengths and make the as-prepared CDs a good ratiometric pH-sensor candidate. This green material CDs-based probe shows promising potential for probing pH in real sample and in vivo intracellular imaging.

Influence of pH on luminescence from water-soluble colloidal Mn-doped ZnSe quantum dots capped with different mercaptoacids

Water-soluble ZnSe/ZnS core–shell quantum dots with ZnSe core doped by manganese ions show different luminescence response to pH changes in aqueous solutions depending on the type of solubilizing agents (thioglycolic acid, mercaptoundecanoic acid, sodium mercaptopropylsulfonate). In the case of long-chain mercaptoundecanoic acid only excitonic emission is affected by pH changes. Short-chain thioglycolic acid brings about equal excitonic/Mn emission variations with pH, while mercaptopropylsulfonate-stabilized quantum dots are insensitive to pH. The mechanism discussed here is based on the competition between different relaxation channels for excited excitons in ZnSe: excitonic radiative recombination, energy transfer to Mn ion and the photogenerated electron trapping due to the presence of protonated carboxyl group. ZnSe:Mn/ZnS quantum dots stabilized with long-chain mercaptoacids may be used as a new type of fluorescence ratiometric pH-sensor or indicator.

Development and In Vivo Application of a Novel Family of Dendrimer-Based Fluorescent Biosensensors

Fluorescent sensors are powerful tools to unveil dynamical changes of ions or biomolecules concentration in vivo. Both Green Fluorescent Proteins (GFPs) and fluorescent dyes were employed to measure biological relevant species. Synthetic fluorophores were largely used to measure a wide range of biological relevant species such as pH, Ca2+, K+, Cl-. However these molecules suffer of several limitations: i)difficulties in intracellular targeting; ii)no ratiometric imaging allowed; iii)poor solubility; iv)time dependent cell leakage. To overcome some limitations of fluorescent biosensors we developed a nano-sized platform able to carry multiple copies of different dyes inside living cells. Synthesis and targeted intracellular delivery of ratiometric fluorescent pH sensors is reported. We choose dendrimers as scaffold for this new family of reporters. Dendrimers are multifunctional hyper branched polymers recently applied in sensing and drug delivery thanks to their interesting features such as multifunctionality, tunable chemical properties and low toxicity. Ratiometric pH sensors were realized by conjugating dendrimers to a pH sensitive dye and a pH insensitive dye in order to allow signal normalization for sensor concentration. Different sensors with tuned affinity for H+ were developed demonstrating the general applicability of this modular approach. Dendrimer-based sensors were calibrated in vitro and then applied to living cells. We demonstrate the ability of the dendritic carrier to selectively deliver the sensing moieties to different intracellular compartments such as cytoplasm, nucleus, nucleoli, lysosomes and plasma membrane. Confocal microscopy was employed to acquire targeted pH maps with high spatial resolution in many cell lines. In particular the sensor was successfully used to detect dynamic changes of pH in different organelles. In conclusion with our work we proposed a new family of fluorescent reporters with improved properties for the investigation of a number of biological processes.