Ratiometric Fluorescent pH Probe Research Articles

Preparation of near-infrared photoacoustic imaging and photothermal treatment agent for cancer using a modifiable acid-triggered molecular platform.

Ratiometric near-infrared fluorescent pH probes with various pKa values were innovatively designed and synthesized based on cyanine with a diamine moiety. The photochemical properties of these probes were thoroughly evaluated. Among the series, IR-PHA exhibited an optimal pKa value of approximately 6.40, closely matching the pH of cancerous tissues. This feature is particularly valuable for real-time pH monitoring in both living cells and living mice. Moreover, when administered intravenously to tumor-bearing mice, IR-PHA demonstrated rapid and significant enhancement of near-infrared fluorescence and photoacoustic signals within the tumor region. This outcome underscores the probe's exceptional capability for dual-modal cancer imaging utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) modalities. Concurrently, the application of a continuous-wave near-infrared laser efficiently ablated cancer cells in vivo, attributed to the photothermal effect induced by IR-PHA. The results strongly indicate that IR-PHA is well-suited for NIRF/PA dual-modality imaging and photothermal therapy of tumors. This makes it a promising candidate for theranostic applications involving small molecules.

A Ratiometric Fluorescent Probe for pH Measurement over a Wide Range Composed of Three Types of Fluorophores Assembled on a DNA Scaffold

The desirable properties of the sophisticated fluorescent pH probe are ratiometric detection properties and a wide detection range. In this study, three types of fluorophores with different fluorescence properties were assembled on a DNA origami nanostructure. DNA nanostructure has the advantage of being a scaffold that can assemble different types of fluorophores with control over their number and position. The defined number of three different fluorophores, i.e., pH-sensitive fluorescein (CF) and Oregon Green (OG), and pH-insensitive tetramethylrhodamine (CR), assembled on the DNA scaffold provided a ratiometric fluorescent pH probe with a wide pH detection range that could cover the variation of intracellular pH.

Micellar Ratiometric Fluorescent Blood pH Probe Based on Triplet-Sensitized Upconversion and Energy-Transfer Behaviors.

The measurement of pH is greatly significant in monitoring physiological and biochemical states. In this work, a novel micellar ratiometric fluorescent probe featuring sophisticated energy-transfer (ET) behaviors with p-nitrophenol (PNP) as the energy acceptor and a triplet-triplet annihilation upconversion (TTA-UC) system as the energy donor was designed. The pH-induced molecular configuration of PNP determined the process for the transfer of energy from TTA-UC to PNP. The introduction of the TTA-UC system enabled probe excitation under a long wavelength and afforded a ratiometric signal for pH detection with excellent reliability over diverse interfering factors. This TTA-UC/ET pH probe demonstrated a high sensitivity to hydronium below nanomolar concentrations and an excellent anti-interference ability in serum samples, which provided a novel significant strategy for rapid and accurate detection of blood pH in vitro.

An AIE-Active probe for detection and bioimaging of pH values based on lactone hydrolysis reaction.

Cellular pH homeostasis is essential for many physiological and pathological processes. pH monitoring is helpful for the diagnosis, treatment and prevention of disorders and diseases. Herein, we developed a ratiometric fluorescent pH probe (TCC) based on a coumarin derivative containing a highly active lactone ring. TCC exhibited a typical AIE effect and emitted blue fluorescence under weak acidic condition. When under weak basic condition, the active lactone moiety underwent a hydrolysis reaction to afford a water-soluble product, which gave red-shifted emission. The emission color change from blue through cyan and then to yellow within pH 6.5-9.0 which is approximate to the biological pH range. And the fluorescence color change along with pH value is reversible. Furthermore, TCC was successfully utilized in the detection of the intracellular pH change of live HeLa cells, which indicated that TCC had practical potential in biomedical research.

An FRET-ICT-based ratiometric fluorescent and colorimetric probe for pH monitoring in lysosomes and water

A novel FRET-ICT-based ratiometric fluorescent and colorimetric pH probe (BDMNP) was developed. BDMNP exhibited 96% energy transfer efficiency and good linear response in the pH ranges of 5.4–6.8 (pKa = 6.10) with high selectivity and sensitivity. BDMNP has been successfully applied to monitor lysosomal pH changes in living cells. Meanwhile, BDMNP can also be used to detect the subtle pH changes of a variety of environmental water samples.

Evaluating visually a new apoptosis-induced reagent by a ratiometric two-photon fluorescent pH probe

The level of apoptosis is directly related to the occurrence of cancer and other diseases. Real-time continuous monitoring of apoptotic process of living cells has great potential for early diagnosis of related diseases, as well as evaluation of therapeutic effect and screening of apoptotic drugs. This work proposed a ratiometric two-photon fluorescent probe Lyso-PCE equipped with a dual-function pyridine, as a pH-sensitive and specific lysosome-targeted group. A clear linearity relationship between two-channel fluorescence intensity ratios of Lyso-PCE and pH values was established in both solutions and living cells. Thanks to its ability of quantitative detection of pH, probe Lyso-PCE can detect the dynamic pH changes of lysosomes treated by chloroquine, and also can be used to in situ monitor dexamethasone-induced apoptosis. More importantly, reagent potassium bisperoxo (1,10-phenanthroline) oxovanadate (bpV(phen)) was evaluated and testified as a new apoptotic drug by Lyso-PCE, and the real-time visualization of bpV(phen)-induced apoptosis was realized under two-photon excitation. Our results provide a new and promising strategy for evaluating and screening apoptosis-related drugs.

Single-wavelength Excited Ratiometric Fluorescence pH Probe to Image Intracellular Trafficking of Tobacco Mosaic Virus

As a typical plant virus which has biocompatibility and high transfection efficiency, tobacco mosaic virus (TMV) has shown broad application potential in drug or gene delivery field. Elucidating its intracellular trafficking is of great importance in investigation of its cytotoxicity, targeting site, and delivery efficiency, and is advantageous to designing new TMV-based drug delivery systems with different targets. By taking advantage of the regulated pH value of different organelles in a mammalian cell, we exploit a pH detection strategy to investigate the intracellular trafficking pathway of TMV. Here, we report a single-wavelength excited ratiometric fluorescent pH probe. This probe is constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate (FITC) and pH-insensitive rhodamine B isothiocyanate (RBIRC) onto the inner surface of TMV. The fluorescence intensity ratio of FITC to RBITC excited at 488 nm responds specifically towards pH value over other interferential agents. By taking use of this single-wavelength excited ratiometric pH probe and confocal laser scanning microscopy, it is shown that the endocytosed TMV is located in a pH decreasing microenvironment and eventually enters lysosomes. This work may provide important guidance on construction of TMV-based nano carriers.

An intermolecular pyrene excimer-based ratiometric fluorescent probes for extremely acidic pH and its applications

Fluorescent ratiometric probes based on monomer/excimer of pyrene group are often used to detect cations. However, only a few pyrene-containing ratiometric pH probes have been reported so far, and almost all of them produce intramolecular pyrene excimer. In this work, three new ratiometric fluorescent pH probes based on intermolecular pyrene excimer, pyrene-1-carboxamide (1), N-(2-hydroxyethyl)pyrene -1-carboxamide (2) and N,N-bis(2-hydroxyethyl) pyrene-1-carboxamide (3), were synthesized. These probes, 1, 2, and 3, have pKa value of 1.93, 1.85 and 2.07, respectively, and can fluorescently determine the pH value of the extremely acidic aqueous solution with a detection range of about 1.0–2.5. When the pH value was decreased from 2.5 to 1.0, these probes showed a ratiometric fluorescent response signal due to the conversion of monomer/excimer of pyrene group. Therefore, intermolecular excimer of the pyrene group from these probes can be produced in extremely acidic environment. Further tests show that these probes have potential as excellent pH fluorescent probes, with high sensitivity, fast response time, good photostability, strong anti-ion interference ability and high repeatability, etc. The applicability of these probes was demonstrated with environment water samples. In addition, fluorescent pH test paper was successfully developed, which can monitor pH values in the solution through pH-related change of fluorescent color of the probes.

Liposome-Enveloped Molecular Nanogels.

Novel hydrogel@liposome particles were prepared by pH-triggered molecular gel formation inside of liposomes loaded with a low-molecular weight gelator derived from l-valine (1). Liposome formation was carried out using l-α-phosphatidylcholine (PC) and cholesterol as components of the lipid bilayer. Molecular hydrogelator 1 and pyranine, a ratiometric fluorescent pH probe, were entrapped in the liposomes at pH 9 and posterior acidification with d-glucono-1,5-lactone to pH 5-6 provoked intraliposomal gel formation. Removal of the lipid bilayer with sodium dodecyl sulfate yielded naked nanogel particles. The systems were characterized by transmission electron microscopy and dynamic light scattering. The hydrogel@liposomes were loaded with doxorubicin, showing a similar release than that observed for liposomes. The hybrid particles described here are the first case of nonpolymeric hydrogel@liposome systems reported. This type of nanocarriers merges the benefits of liposomal vehicles with the inherent stimuli responsiveness and enhanced biocompatibility of hydrogels formed by low-molecular weight molecules, foretelling a potential use in environmentally sensitive drug release.

Mitochondria-Immobilized Near-Infrared Ratiometric Fluorescent pH Probe To Evaluate Cellular Mitophagy.

Mitochondria, powerhouses of cells, possess a weakly alkaline environment. Various stress stimulations may lead to mitophagy, which further gives a rise to mitochondrial acidification and disfunction. Therefore, monitoring mitochondrial pH alterations is of great importance to better elucidate their role in the cellular metabolism. Toward this end, a number of mitochondrial fluorescent pH probes have been proposed, but most of them are based on electrostatic attraction and readily leak out from the mitochondria during mitophagy with decreased membrane potential, thus failing to accurately measure the pH changes. In this work, we report a mitochondria-immobilized ratiometric fluorescent pH probe, which allows the quantitative measurements of mitochondrial pH. The probe was designed and prepared by introducing a reactive benzyl chloride into a positively charged near-infrared hydroxyl-hemicyanine. The cationic property facilitates the probe to be quickly enriched into mitochondria, the hydroxyl group is responsible for producing a reversible ratiometric fluorescence signal, and benzyl chloride is used to react with nucleophiles for immobilizing the probe in mitochondria. Taking these advantages of the probe, the mitochondrial pH variations during mitophagy caused by rapamycin and hypoxia have been determined quantitatively for the first time. The observed severe acidification of mitochondria under these stimulations, together with the rationally designed probe, may be useful for studying the detailed function of mitochondria in some bioprocesses.

A ratiometric 4-amido-1,8-naphthalimide fluorescent probe based on excimer-monomer emission for determination of pH and water content in organic solvents

The pH and water content in organic solvents are some of the most important parameters that play a key role in chemical laboratories, clinical analysis, food processing, biomedical and environmental monitoring, and life sciences. Herein we are reporting on the design, synthesis and sensor activity of a novel 4-amido-1,8-naphthalimide ratiometric fluorescent pH probe based on excimer-monomer switching mechanism. The fluorescence chemosensing behavior of the synthesized compound was studied in solvents of different polarity and the results obtained clearly demonstrates the great opportunity of the novel probe selectively to determine pH in aqueous solutions and water content in organic environment.

A through-bond energy transfer-based ratiometric fluorescent pH probe: For extreme acidity and extreme alkaline detection with large emission shifts

A ratiometric fluorescence pH probe 1 based on through-bond energy transfer (TBET) with a 2-(2-hydroxyphenyl)benzoxazole (HBO) as donor and a Rhodamine derivative as acceptor is developed through simple condensation reaction. The probe exhibits a ratiometric fluorescence emission (I593/I422) characteristics and linear response to extreme acidity range of 5.00–2.88, and a ratiometric fluorescence emission (I555/I422) characteristics and linear response to extreme alkaline range of 10.00–13.78, respectively. Moreover, 1 possesses highly selective response to pH over metal ions, good reversibility and excellent photostability. Probe 1 is cellpermeable and can distinguish near pH 5.55 fluctuations in Hela cells. Furthermore, 1 can be immobilized on a test paper, which shows a rapid and reversible colorimetric response to HCl/NH3 vapor by the naked-eye.

A FRET-based two-photon probe for in vivo tracking of pH during a traumatic brain injury process

Traumatic brain injury (TBI) is a cause of neurodegenerative diseases accompanied by intracellular pH decrease. Herein, a FRET-based ratiometric two-photon fluorescent pH probe is designed to monitor pH change and understand TBI process.

Lysosome-targeting ratiometric fluorescent pH probes based on long-wavelength BODIPY.

BODIPY-based probes were developed for the ratiometric fluorescence detection of pH values. They showed high sensitivity under acidic conditions via a protonation-modulated ICT mechanism and could selectively stain lysosomes, and thus could be applied for imaging and detecting abnormal pH decreases in live cells.

Novel benzimidazole-based ratiometric fluorescent probes for acidic pH

2,6-Bis(1-alkyl-1H-benzo[d]imidazol-2-yl)naphthalene (2a-2c) as three new benzimidazole molecules have been designed, synthesized and applied as ratiometric fluorescent pH probes in the acidic pH region with high-efficiency and fast response. For probe 2a, upon decreasing the pH from 7.5 to 2.5, the emission spectra exhibit a large red shift from 402 to 472 nm, and the emission ratio (I472/I402) changes dramatically from 0.10 to 3.42 with a pKa value of 2.92. No significant interference on emission behavior is observed in the presence of various metal ions. Similar results are obtained for probes 2b and 2c. 1H NMR and DFT calculations studies demonstrate that the ratiometric response of the probes to acidic pH is due to H+ binding with one N atom in C=N of benzimidazole ring and the subsequent enhancement of the intramolecular charger transfer (ICT) process. In addition, these probes can be used in real-time and reversible pH sensing. Finally, the probes have been successfully applied to the visual detection of pH not only in solution, but also in test paper.

A ratiometric fluorescent pH probe based on keto-enol tautomerization for imaging of living cells in extreme acidity.

6-(Diethylamino)-2,3-dihydro-1H-xanthene-4-carbaldehyde (DDXC), a reported synthetic intermediate for near-infrared fluorescent dyes, was developed into a fluorescent pH probe for extreme acidity. The unique sensing mechanism of DDXC for pH is based on the reversible protonation of the carbonyl oxygen followed by keto-enol tautomerization. The probe displays a linear ratiometric fluorescence response (I512/I580) to H+ over the extremely acidic range of pH 2.0-4.0 with a pKa of 3.11, and features high fluorescence quantum yield (Φ = 0.60) and excellent selectivity. More importantly, the probe can be applied to ratiometric fluorescence imaging of pH changes in living cells, making it a potential molecular tool for pH-related cell biology study.

Fluorescent pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its Application in Human Cancer Cell Bioimaging.

A water-soluble, ratiometric fluorescent pH probe, L-SRhB, was synthesized via grafting spirolactam Rhodamine B (SRhB) to lignosulfonate (LS). As the ring-opening product of L-SRhB, FL-SRhB was also prepared. The pH-response experiment indicated that L-SRhB showed a rapid response to pH changes from 4.60 to 6.20 with a pKa of 5.35, which indicated that L-SRhB has the potential for pH detection of acidic organelle. In addition, the two probes were internalized successfully by living cells through the endocytosis pathway and could distinguish normal cells from cancer cells by different cell staining rates. In addition, L-SRhB showed obvious cytotoxicity to cancer cells, whereas it was nontoxic to normal cells in the same condition. L-SRhB might have potential in cancer therapy. L-SRhB might be a promising ratiometric fluorescent pH sensor and bioimaging dye for the recognition of cancer cells. The results also provided a new perspective to the high-value utilization of lignin.

3-AminoBODIPY dyes: unexpected synthesis from 2-borate derivatives and application as fluorescent probe for alkaline pH range

3-AminoBODIPY derivatives (5a–e) were unexpectedly synthesized by the coupling of amines with 2-BODIPY-borates in the presence of copper(II) acetate Cu(OAc)2 and tetramethylethylenediamine (TMEDA) in the air at room temperature. 5a (pKa=9.18) was found to be a novel colorimetric and ratiometric fluorescent pH probe at alkaline pH ranges.

Ratiometric Fluorescent pH Probes Based on Glycopolymers.

Effectively detecting pH changes plays a critical role in exploring cellular functions and determining physiological and pathological processes. A novel ratiometric pH probe based on a glycopolymer, armored with properties of serum-stability, tumor-targeting, and pH monitoring, is designed. Random copolymers of 2-(methacrylamido) glucopyranose and fluorescein O-methacrylate are first synthesized by reversible addition fragmentation chain transfer polymerization. Acryloxyethyl thiocarbamoyl rhodamine B is then attached to the polymer chain to prepare ratiometric fluorescent pH probes via a thiol-ene reaction. The synthesized polymeric probes are characterized by NMR, gel permeation chromatography, UV-vis spectroscopy, and transmission electron microscopy, and the fluorescence responses are examined in phosphate buffer at different pHs. The cytotoxicity and confocal imaging experiments of the probes are detected using HeLa cells, demonstrating a low toxicity and superior biocompatibility for detecting pH changes in bioapplications.

Thiazole-based ratiometric fluorescence pH probe with large Stokes shift for intracellular imaging

A ratiometric fluorescent pH probe 4-((1E, 3E)-4-(benzo[d]thiazol-2-yl)buta-1, 3-dienyl)-N, N-dimethylbenzenamine (BTDB) was synthesized via ethylene bridging of thiazol and cinnamaldehyde for extremely acidic sensing. BTDB exhibits ratiometric fluorescence emission (I425nm/I595nm) characteristics with pKa of 2.34 and a large Stokes shift of 177nm under acidic conditions. The linear response to pH is in the range of 2.3–4.0. Quantum chemical calculations with the B3LYP exchange functional theory demonstrated that the ratiometric response of the probe to acidic pH was due to H+ binding with the N of cinnamaldehyde and the induced decreases of the intramolecular charger transfer (ICT) process. The as-prepared probe displayed excellent cell membrane permeability was further applied successfully to monitor pH fluctuations in live cells with excellent lysosomal targeting ability, and extreme acidity in Escherichia coli cells.