Fluorescent pH Research Articles

A Patient-Ready Wearable Transcutaneous CO2 Sensor

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.

Excitation Wavelength‐Dependent pKa Extends the pH Sensing Range of Fluorescence Lifetime Sensors

Biological activity is strongly dependent on pH, which may fluctuate within a variety of neutral, alkaline, and acidic local environments. The heterogeneity of tissue and subcellular pH has driven the development of sensors with different pKa values, and a huge assortment of fluorescent sensors have been developed to measure and visualize pH in living cells and tissues. In particular, sensors that report based on fluorescence lifetime are advantageous for quantitation. Here, we apply a theoretical framework to determine how the apparent pKa of lifetime‐base pH sensors depends on fluorescence excitation wavelength. We then demonstrate that theory accurate predicts the behavior of two different fluorescent protein‐base pH sensors. Furthermore, we show that this behavior has great practical value in matching the appropriate detection parameters with the physiological pH range. More broadly, our results show that the versatility of a single lifetime‐based sensor has been significantly underappreciated and our approach provides a means to use a single sensor across a range of pH environments.

Pulmonary Arterial Smooth Muscle Cell Monocarboxylate Transporter 1/4 Contributes to Extracellular Acidosis following Chronic Hypoxia‐induced Pulmonary Hypertension

Pulmonary hypertension (pHTN) is defined by mean pulmonary arterial pressure at rest exceeding 20 mmHg with mortality often resulting from right ventricular failure that is secondary to increased afterload. During pHTN, increased reliance on aerobic glycolysis and inhibition of mitochondrial oxidative respiration is observed in pulmonary arterial smooth muscle cells (PASMCs). This metabolic shift is associated with hyperproliferation and resistance to apoptosis contributing to the pathogenesis of pHTN. Along with these phenotypic changes, cells undergoing aerobic respiration exhibit increased glucose uptake and subsequent lactate and H+ efflux, contributing to extracellular acidosis. Acid‐sensing ion channel 1 (ASIC1) is a voltage‐independent, proton‐gated cation channel that contributes to the development of chronic hypoxia (CH)‐induced pHTN. While the function of ASIC1 is well characterized in pHTN, it has not been determined whether the metabolic shift observed during pHTN contributes to the activation of ASIC1. Therefore, we hypothesized that enhanced glucose uptake and subsequent acidification of the extracellular microenvironment in PASMCs lead to the activation of ASIC1 in CH‐induced pHTN. As an initial step in testing this hypothesis, rats were housed in a hypobaric chamber (barometric pressure: ~380 mmHg) to induce pHTN and compared to age‐matched normoxic controls. After 4 weeks, PASMCs were collected and transiently cultured under normoxia for 3‐4 days. As expected, PASMCs from CH rats showed a reduced oxygen consumption rate (p<0.0001) and greater extracellular acidification (p<0.0001) compared to those from control animals, indicating increased glycolytic activity. Utilizing the fluorescent pH indicators SNARF‐5/AM, we found that PASMCs from CH rats exhibit intracellular alkalization (p<0.0001 vs. control) and extracellular acidosis (p<0.001 vs. control). We further examined the H+ transporter(s) involved in mediating extracellular acidosis by inhibiting carbonic anhydrase IX (CA‐IX), the Na+/H+ exchanger (NHE1), the vacuolar‐type H+‐type ATPase (V‐ATPase), or monocarboxylate transporters 1 and ‐4 (MCT1 and MCT4). The dual MCT1/4 inhibitor, syrosingopine, prevented intracellular alkalization and extracellular acidosis following CH (Figure 1A), which corresponded with an increase in MCT4, but not MCT1, expression (Figure 1B). These data suggest MCT plays an important role in regulating PASMC pH homeostasis. Future studies will assess the role of MCT to activate ASIC1 and contribute to pHTN.

Donor manipulation for constructing a pH sensing thermally activated delayed fluorescent probe to detect alkaliphiles

pH homeostasis is essential for alkaliphiles, given their widespread use in biotechnological applications. However, quantitative monitoring of alkaline pH in alkaliphiles remains challenging. Here, we synthesized for the first time, a thermally activated delayed fluorescent (TADF) pH probe: NI-D-OH. Our probe exhibits a good linear relationship between fluorescence intensity and pH in the neutral to alkaline range (pH 7.0–8.6), as well as long-lived TADF emission. We further show that NI-D-OH can be used to monitor intracellular pH in living organisms, and evaluate the effect of Na+ on the pH homeostasis, demonstrating the potential for alkaline pH monitoring and time-resolved fluorescence imaging.

Imaging and Measuring Vesicular Acidification with a Plasma Membrane-Targeted Ratiometric pH Probe.

Tracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging.

Fluorescent sensing (Cu2+ and pH) and visualization of latent fingerprints using an AIE-active naphthaldehyde-pyridoxal conjugated Schiff base

Tunable light-emissive materials have achieved an extensive research attention due to their potential applications in the environmental and biomedical fields. This work introduced an aggregation-induced emission (AIE) active naphthaldehyde-pyridoxal (NPPY) conjugated Schiff base for the fluorescence sensing of pH and Cu2+, and visualization of latent fingerprints (LFPs). The weakly emissive NPPY in freely soluble DMSO converted to a bright yellow fluorescent upon increasing the poor solvent water fraction above 50% in DMSO. The DLS and SEM analysis supported the formation of self-aggregates of NPPY, and conversion of NPPY into a bright yellow fluorescent Schiff base due to the restriction of intramolecular rotation. The yellow fluorescent NPPY between pH 2.7 to 10.3 became green fluorescent above pH 10.3 with an estimated pKa of 10.8. The metal ions recognition ability of the AIE active NPPY was explored in HEPES buffer (5% DMSO, 10 mM HEPES, pH = 7.4). The fluorescent intensity at 570 nm of NPPY was quenched upon complexation with Cu2+. The chelation enhanced fluorescence quenching was occurred due to the electron transfer from NPPY to the paramagnetic Cu2+. The detection limit reached the nanomolar level of 32.9 nM. Sensor NPPY was applied for the quantification of Cu2+ in real environmental and biological samples. Also, NPPY coated paper test strips and cotton buds were developed for the qualitative visual detection of Cu2+. Additionally, the aggregates of NPPY was applied for the visualization of LFPs and also fluorescent gelatine gel was developed for future applications in fabricating light-emitting devices.

Algae based green biocomposites for uranium removal from wastewater: Kinetic, equilibrium and thermodynamic studies

Industrial wastewater contained a large amount of uranium U(VI) generally produced by uranium mining, nuclear electrical generation causes severe damage to the environment. Plant biomass and microbial available in abundant amounts in nature could be used for U(VI) adsorbent. This research represents, the initiation of biocomposites sorbent composed of two algae, Spirogyra (geen algae) and Vaucheria (yellow green algae) utilized in a batch system for U(VI) adsorption. Separation of the U(VI) from the industrial wastewater was analysed with initial pH, biocomposites' dose, contact time, initial temperature, isothermal, and kinetic behaviour. The prepared biocomposites characterization was performed by Fourier transform infrared spectroscopy (FTIR), particle size distribution (PSA), energy dispersive X-ray fluorescence spectroscopy (EDXRF) and pH point zero charges (pHpzc). The separation of U(VI) particles was favourable at pH 2 with 100 mg of adsorbent dose to 50 mg/L U(VI) initial concentration, within only 40 min at 25 °C with adsorption capability of 24.70 mg/g. The removal efficiency of the synthesized biocomposites was measured in terms of dosage 90.82% and pH 92.44%. The obtained values of correlation regression coefficients for used isotherm models show that the sorption process placed well to Langmuir's and Freundlich's models. According to calculated kinetic studies, experimental results fitted very well to the intraparticle diffusion models and pseudo-second-order (phase I). The enthalpy (ΔH°), entropy (ΔS°) and Gibbs free energy (ΔG°) were ascertained as thermodynamic parameters. Thermodynamic data showed that the sorption process onto algae-based biocomposites is spontaneous and exothermic. Moreover, in biosorption process mixture of algae with silica gel enhances the interaction properties with uranium ions. The developed algae-based biocomposites are bi-functionalised cost-effective and feasible adsorbent to eliminate U(VI) from water.

A fluorescent pH probe for evaluating the freshness of chicken breast meat

A fluorescent probe, Nap-MOR, based on the naphthalimide fluorophore, was designed and developed for pH measurement in aqueous solutions. Nap-MOR had a close linear relationship between fluorescence intensity and pH, in the range 4.5–8, which covers the full range of pH found in normal fresh, defective and spoiled meat. pH measurement with Nap-MOR was free from interference by a wide range of ions and biochemicals found in meat and the results were not significantly different in comparison with a pH meter. Therefore, Nap-MOR is a robust and convenient way to evaluate the freshness of chicken breast meat by measuring its pH.

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.

Anagliptin promotes apoptosis in mouse colon carcinoma cells via MCT-4/lactate-mediated intracellular acidosis.

Cancer cells frequently exhibit an acidic extracellular microenvironment, where inversion of the transmembrane pH gradient is associated with tumor proliferation and metastasis. To elucidate a new therapeutic target against cancer, the current study aimed to determine the mechanism by which the dipeptidyl peptidase-4 inhibitor anagliptin regulates the cellular pH gradient and concomitant extracellular acidosis during cancer progression. A total of 5x105 CT-26 cells (resuspended in phosphate buffer saline) were injected subcutaneously in the right flank of male BALB/c mice (weighing 25-28 g). The tumor samples were harvested, and lactate was detected using a lactate assay kit. Immunohistochemistry was used to detect the Ki67 and PCNA. MTT assay and flow cytometric were used to detect cell viability. Intracellular pH was detected by fluorescence pH indicator. The results revealed that anagliptin effectively reduced tumor growth, but did not affect the body weight of treated mice. Anagliptin reduced the accumulation of lactate in tumor sample. Treatment with anagliptin stimulated the apoptosis of CT-26 cells. And lactate excretion inhibition is accompanied by an increase in extracellular pH (pHe) after treatment with anagliptin. Furthermore, anagliptin induced intracellular acidification and reversed the low pHe gradient via monocarboxylate transporter-4 (MCT-4)-mediated lactate excretion. Additionally, anagliptin reversed the aberrant transmembrane extracellular/intracellular pH gradient by suppressing MCT-4-mediated lactate excretion, while also reducing mitochondrial membrane potential and inducing apoptosis. These data revealed a novel function of anagliptin in regulating lactate excretion from cancer cells, suggesting that anagliptin may be used as a potential treatment for cancer.

Solid Fluorescence pH Sensors Based on 1,8-Naphthalimide Copolymers Synthesized by UV Curing

Novel water-swollen photo-crosslinked membranes were obtained by copolymerization of the N-vinylpyrrolidone, butyl acrylate and ethyl methacrylate monomers functionalized with naphthalimide groups, as pH sensitive fluorescence probes. For that purpose, two monomers with pending naphthalimide groups anchored to ethyl methacrylate through alkyl chains with different length, were previously synthesized. The membranes were characterized using different techniques. The pH dependence of absorbance and the corresponding quenching of fluorescence were investigated and related to the structure of naphthalimide substituents linked to the membrane. The new solid sensors exhibited sensitive fluorescence changes at pH < 3, and lower time response was determined for membranes where the sensing group was linked through longer alkyl chain to the polymer matrix. The membranes were solid, thermally stable and easily handled to be applied as sensor materials, and showed a reversible behavior, which is an important feature for further fabrication of an economical on-site tool for the acidity detection in aqueous environments.

The Sensitive Optical pH Sensor Based on the Complex of Nanosheet and Carbon Dots

AbstractThe requirement to non‐invasively quantify H+ is gradually increasing. In this regards, optical pH sensors based on fluorescent materials have advantages. However, the stability and sensitivity of currently used chromophores still need to be further improved to meet the continuous increase in actual demand for pH measurement. Here, we proposed to employ Montmorillonite (MMT) nanosheets as matrix and carbon dots (CD) as chromophores to fabricate a new complex. The as‐prepared MMT‐CD have the following advantages: 1) the aggregation of CD is effectively reduced, leading to the effectively improved fluorescence performance; 2) MMT‐CD has good light stability and storage stability; 3) as‐prepared the fluorescence pH sensor shows good response to pH, high sensitivity, and wide linear range. Therefore, MMT‐CD materials have great potential in the rapid and sensitive sensing of pH value.

Multifunctional Probes with High Utilization Rates: Self-Assembled Merocyanine Nanoparticles in Water as Acid-Base Indicators and Mitochondrion-Targeting Chemotherapeutic Agents.

Multifunctional probes with high utilization rates have great value in practical applications in various fields such as cancer diagnosis and therapy. Here we have synthesized two organic molecules based on merocyanine. They can self-assemble in water to form ∼1.5 nm nanoparticles. Both of them have good application potential in fluorescent anticounterfeit printing ink and pH detection. More importantly, they have excellent mitochondrial targeting ability, intracellular red light and near-infrared dual-channel imaging ability, strong antiphotobleaching ability, and in vivo and in vitro near-infrared imaging capabilities, showing superior chemotherapy capabilities and biocompatibility in the 4T1 tumor-bearing mouse model.

Study of the Ph Effect on the Optical and Morphological Properties of S,N Self-Doped Carbon Dots Applied as Fluorescent Anti-Counterfeiting Ink and Ph Sensor

Study of the Ph Effect on the Optical and Morphological Properties of S,N Self-Doped Carbon Dots Applied as Fluorescent Anti-Counterfeiting Ink and Ph Sensor

Tunable fluorescent amino-functionalized Ti3C2Tx MXene quantum dots for ultrasensitive Fe3+ ion sensing.

The development of sensors with high sensitivity, good selectivity and reproducibility are of great importance for the detection of Fe3+ in contaminated water for environmental monitoring. In this work, a reflux approach has been adopted to synthesize Ti3C2Tx quantum dots (QDs) based on the cutting effect of tetramethylammonium hydroxide (TMAOH) on Ti3C2Tx at high temperature. The surface-functionalized Ti3C2Tx QDs contained abundant amino groups and exhibited tunable pH-dependent emission, which was attributed to the protonation and deprotonation of the surface terminations. The linearity of the radiometric fluorescence intensity versus pH indicates its great potential as a dual-emission ratiometric pH sensor. Additionally, the Ti3C2Tx QDs exhibited tunable excitation-dependent emission behavior, which was related to the degree of passivation by the amino groups on the surface. Furthermore, the fluorescence intensity of the Ti3C2Tx QDs shows a linear response toward Fe3+ in the nanomolar to micromolar range with a low detection limit of 2 nM, originating from the oxidation and reduction between Fe3+ and Ti3C2Tx. This ultra-sensitive and selective detection capability demonstrated the environmental application potential for Ti3C2Tx QDs as a nanoprobe to monitor Fe3+.

Revealing the sensing mechanism of a fluorescent pH probe based on a bichromophore approach.

Fluorescence sensing plays an increasingly important role in biology and biomedicine. For many practical applications of fluorescent probes, an "off-on" response is preferred. The question of how fluorescence quenching/enhancement occurs is fundamental and of high importance for application and design of new fluorescent probes. The sensing mechanism of an aminorhodamine (TMARh) pH probe is investigated using femtosecond transient absorption spectroscopy and quantum chemical calculations, showing that this probe is best understood using the bichromophore model rather than the more common models such as photoinduced electron transfer or intramolecular charge transfer. Under excitation in the main absorption band at 530 nm, a fast internal conversion to the first excited state (S1) is observed for TMARh; meanwhile, no new transient components are obtained when TMARh is excited directly to S1 in the weakly absorbing red tail at 630 nm. It is confirmed that the S1 of TMARh is a dark "off" state. Theoretical calculations show that the S1 "off" state is an intramolecular charge transfer state from an aminophenyl group to a rhodamine chromophore. After protonation of the aminophenyl group, to yield HTMARh, the transient S2/S1 internal conversion process that occurs in TMARh under 530 nm excitation is absent, suggesting that the charge transfer state becomes highly unfavorable. All calculations and spectral data confirm that HTMARh has localized transition in the rhodamine chromophore, in agreement with this being the bright "on" state of the pH probe. The current work not only provides a photophysical insight into the sensing mechanism of this specific probe, but also shows that the bichromophore model is useful and may be relevant for analyzing other probes or in the designing of new ones.

Color-Tunable Indolizine-Based Fluorophores and Fluorescent pH Sensor.

A new fluorescent indolizine-based scaffold was developed using a straightforward synthetic scheme starting from a pyrrole ring. In this fluorescent system, an N,N-dimethylamino group in the aryl ring at the C-3 position of indolizine acted as an electron donor and played a crucial role in inducing a red shift in the emission wavelength based on the ICT process. Moreover, various electron-withdrawing groups, such as acetyl and aldehyde, were introduced at the C-7 position of indolizine, to tune and promote the red shift of the emission wavelength, resulting in a color range from blue to orange (462–580 nm). Furthermore, the ICT effect in indolizine fluorophores allowed the design and development of new fluorescent pH sensors of great potential in the field of fluorescence bioimaging and sensors.

Whole-Plant Measure of Temperature-Induced Changes in the Cytosolic pH of Potato Plants Using Genetically Encoded Fluorescent Sensor Pt-GFP

Cytosolic pH (pHcyt) regulates a wide range of cellular processes in plants. Changes in pHcyt occurring under the effect of different stressors can participate in signal transmission. The dynamics of pHcyt under the action of external factors, including significant factors for open ground crops such as temperature, remains poorly understood, which is largely due to the difficulty of intracellular pH registration using standard methods. In this work, model plants of potato (one of the essential crops) expressing a fluorescent ratiometric pH sensor Pt-GFP were created. The calibration obtained in vivo allowed for the determination of the pHcyt values of the cells of the leaves, which is 7.03 ± 0.03 pH. Cooling of the whole leaf caused depolarization and rapid acidification of the cytosol, the amplitude of which depended on the cooling strength, amounting to about 0.2 pH units when cooled by 15 °C. When the temperature rises to 35–40 °C, the cytosol was alkalized by 0.2 pH units. Heating above the threshold temperature caused the acidification of cytosol and generation of variation potential. The observed rapid changes in pHcyt can be associated with changes in the activity of H+-ATPases, which was confirmed by inhibitory analysis.

Computational modeling predicts ephemeral acidic microdomains in the glutamatergic synaptic cleft

At chemical synapses, synaptic vesicles release their acidic contents into the cleft, leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction as glutamate, ATP, and protons (H+) were released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase activity and predicts substantial cleft acidification but only for fractions of a millisecond after neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 ms because the plasma membrane calcium-ATPase removes H+ from the cleft in exchange for calcium ions from adjacent pre- and postsynaptic compartments, thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis have little influence on the magnitude of acidification. Phosphate but not bicarbonate buffering is effective at suppressing the magnitude and time course of the acid spike, whereas both buffering systems are effective at suppressing cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH transients accompanying neurotransmission.

Multiple-color aggregation-induced emission (AIE) molecules: pH-responsive mechanism and fluorescence properties

A series of naphthalene-based Schiff base derivatives (L1-3) were successfully prepared. The Schiff base derivatives exhibit typical aggregation-induced emission (AIE) properties with various fluorescence emissions and pH responses in an alkaline environment. Through density functional theory (DFT) calculations, it is found that the electronegativity of the L1-3 changes with the change of the substituents on the 2-aminopyridine, which results in the L1-3 exhibiting multiple-color emission from green to orange. In addition, all naphthalene-based Schiff base derivatives exhibit green fluorescence emission under alkaline conditions. This is due to the intramolecular charge transfer (ICT) mechanism of the L1-3 which makes the L1-3 have a pH-response under alkaline conditions (pH at 7.0–12.0). The derivatives L1-3 exhibit unique optical properties, which indicated their potential applications in the field of optics and pH sensing materials.