Fluorescent pH Probe Research Articles

High-Yield, Stable, and Ultrasensitive Carbon Dots for pH Sensing and Fe2+ Detection Are Synthesized at Room Temperature

Fluorescent carbon quantum dots (r-CDs) were synthesized safely and readily by stirring at room temperature with a yield of 76.5%. The synthesized r-CDs exhibit bright green emission with an absolute quantum yield of 12.0% under the excitation of ultraviolet light. Within the pH range of 3–12, the r-CDs exhibit excellent fluorescence stability. Interestingly, the fluorescence intensity of r-CDs faded promptly when the pH value diminished to 2 and then regained when the pH value returned to 3. This means that r-CDs can be used as “on–off–on” fluorescent probes for pH sensing. The mechanism of this sensitive pH response behavior was discussed in detail, and a possible mechanism of protonation–deprotonation–protonation processes was proposed. In addition, we have proposed a green preparation route for eco-friendly filter paper using anticounterfeiting. Interestingly, the presence of Fe2+ significantly quenched the fluorescence of r-CDs. Experimental results demonstrated the r-CDs can also be used to detect the content of Fe2+ in water, and the detection limit was 8.82 μM/L.

Near-Infrared Ratiometric Hemicyanine-Based pH Fluorescence Probe with Bone Targetability for Monitoring Bone Resorption

Accurate detection of bone resorption is extremely important in the orthodontic treatment process as it can provide a basis for clinical treatment strategies. Recently, pH-responsive fluorescence probes have received tremendous attention in bone resorption monitoring owing to their high sensitivity, good specificity, and in situ and real-time detection capabilities, but there are still some shortcomings like the increase in the risk of osteonecrosis of the jaw by use of bisphosphonate as the bone-targeting moiety and the insufficient monitoring accuracy due to susceptibility to interference. Herein, we designed and synthesized a near-infrared ratiometric hemicyanine-based pH fluorescence probe (Hcy-Asp6) with fluorescence-imaging and pH-determining capabilities, and bone targetability for more reliably and safely monitoring the bone resorption in orthodontic treatment. In vitro optical performance tests of Hcy-Asp6 revealed that the probe had high sensitivity, excellent photostability, reversibility, and strong resistance to interference, and the probe suggested excellent bone-binding ability and biocompatibility in the bone-targeting evaluation and the cytotoxicity test. Furthermore, in vitro and in vivo bone resorption monitoring assays demonstrated that this probe can detect bone resorption by fluorescence imaging and quantitative monitoring of pH associated with the bone resorption. Thus, the results indicated that this probe possessing bone targetability and accurate bone resorption-monitoring capability has an extraordinarily great clinical potential to be employed for real-time monitoring of bone resorption in orthodontic treatment and could also serve as a reference in bone resorption monitoring for other bone resorption-related diseases.

Converting pH probes into "turn-on" fluorescent receptors for anions.

Recognition of anions by synthetic receptors is an integral part of supramolecular chemistry continuing to expand and find new application areas in our daily life. Many applications require visualization of anion recognition events, and the generated analytical signal is used to quantify anions in solution. Transferring a binding event to a measured signal is a challenging task. The design of a synthetic receptor must involve not only the perfectly positioned binding sites with complementary noncovalent interactions for a guest but should also realize the sensing mechanism that generates a strong analytical response upon guest binding. This feature article outlines the design concept for the construction of "turn-on" fluorescent receptors for anions involving fluorescent pH probes. Applications of this concept for the construction of synthetic fluorescent receptors for inorganic anions and nucleotides are described. Features of the obtained receptors and possible competing binding and sensing processes in solution are analyzed to understand the scope and limitations of the approach.

FRET-based carbazole-fluorescein ionic nanoparticle for use as an effective bioimaging agent.

Fӧrster resonance energy transfer (FRET)-based systems are widely applicable in many areas of interest. In this study, a novel FRET-based ionic material (IM) was synthesized by pairing carbazole imidazolium cation (CI+) with fluorescein anion (Fl2-) through a simple ion-exchange method. The resulting IM ([CI]2[Fl]) was converted into an ionic nanoparticle (INP) in aqueous media for practical use for bioimaging application. The photophysical properties of the parent dyes, [CI]2[Fl], and INP were studied in detail. All FRET parameters were calculated in the synthesized material. [CI]2[Fl] exhibited a significant spectral overlap integral and an ideal theoretical FRET distance. The presence of the FRET mechanism was verified by the observed decrease in donor fluorescence lifetime and a moderate FRET efficiency in [CI]2[Fl]. The INP formed from [CI]2[Fl] was evaluated for use as a fluorescent pH probe and bioimaging agent. FRET efficiency of INP is calculated in a series of pH studies which indicates the highest efficiency at physiological pH. Whereas no FRET phenomenon is observed in highly acidic and basic conditions. The pH-dependent photophysical properties of [CI]2[Fl] are monitored and allow for the potential application as a fluorescent probe for the detection of acidic tissues in biological systems. The FRET-capable INP showed superior bioimaging capability in vitro as compared to the parent dye.

One step synthesis of highly photoluminescent red light-emitting carbon dots from O-phenylenediamine and 2,4-diaminophenol as fluorescent probes for the detection of pH and Cr(VI).

In this study, new red light-emitting carbon dots (R-CDs) that can selectively recognize Cr(VI) were prepared using a strategy that utilizes 2,4-diaminophenol to enhance the fluorescence of O-phenylenediamine based carbon dots. The results showed that 2,4-diaminophenol increased the quantum yield (QY) of the carbon quantum dots (CDs), and that the QY of the CDs increased from their original value of 8.7% to 20.1% (R-CDs). The R-CDs show sensitivity to acidic conditions and maintain good linearity between pH = 1.00-4.00, making them useful as pH probes. Furthermore, the prepared R-CDs possess good solubility in water and are responsive to changes in Cr(VI) concentrations in aqueous environments. The quenching of the R-CDs fluorescence was linearly correlated with the Cr(VI) concentration within a range of 0-20 μM, with a lower detection limit of 66 nM. The detection mechanism is attributed to the formation of hydrogen bonds between Cr(VI) and the R-CDs, resulting in the fluorescence quenching of the R-CDs. The R-CDs can be considered effective multifunctional fluorescent probes for both pH and Cr(VI) in aqueous environments. This study will provide new R-CD design strategies for probes that selectively identify specific target substances.

A water-soluble fluorescent pH probe and its application for monitoring lysosomal pH changes in living cells.

Intracellular pH plays a crucial role in many cellular processes, and abnormal intracellular pH has been linked to common diseases such as cancer and Alzheimer's. To address this issue, a water-soluble fluorescent pH probe was designed based on the protonation/deprotonation of the 4-methylpiperazin-1-yl group, using dicyanoisophorone as the fluorophore. In the neutral form of the probe, fluorescence is quenched due to charge transfer from the 4-methylpiperazin-1-yl group to the fluorophore upon excitation. Under acidic conditions, protonation of the 4-methylpiperazin-1-yl group inhibits the photoinduced electron transfer process, leading to an increase in fluorescence intensity. Density-functional theory calculations also verified the fluorescence OFF-ON mechanism. The probe exhibits high selectivity, photostability, fast response to pH changes, and low cytotoxicity to cells. Additionally, the probe selectively accumulates in lysosomes, with a high Pearson coefficient (0.95) using LysoTracker Green DND-26 as a reference. Notably, the probe can monitor lysosomal pH changes in living cells and track pH changes stimulated by chloroquine. We anticipate that the probe has potential for diagnosing pH-related diseases.

Simple and commercially available imidazo[1,2-a]pyridine-8-carboxylic acid-based fluorescent pH probe for basic conditions

Searching for new probes for pH detection is useful for the diagnosis of related diseases. However, the fluorescent probes that can respond to pH value less than 4 or greater than 8 are scarce and multisteps are needed to synthesize such probes. Thus, exploration of simple pH fluorescent probes for strong acid or basic system is an urgent task. Herein, we report a commercially available compound 6-chloroimidazo [1,2-a]pyridine-2-carboxylic acid IP-YC as the pH probe (> 7) and its bioimaging in S. cerevisiae and HepG2 cells.

Synthesis of N,S co-doped carbon dots for fluorescence detection of Fe3+ ions and intracellular pH imaging

• N,S co-doped dual emission carbon dots (DECDs) were prepared. • The DECDs has high accuracy and selectivity for the determination of Fe 3+ in tap water. • The dual emission of DECDs is contributed by core and surface. • The DECDs have low cytotoxicity and can be used to monitor pH changes in cell. Using citric acid and thiourea as precursors, sulfur-nitrogen co-doped dual emission carbon dots (DECDs) were prepared by a simple solvothermal method. At the excitation wavelength of 360 nm, DECDs have two emission peaks located at 465 nm (blue) and 592 nm (yellow-green) which are attributed by core and surface of DECDs. It is found that Fe 3+ ions can coordinate with oxygen-containing groups on DECDs, and then the electrons transfer from DECDs to Fe 3+ ions, which quench the fluorescence of DECDs. The fluorescence intensity is linear with the concentration of Fe 3+ ions in the range of 0–100 μM and the detection limit is 0.77 μM. Moreover, the designed sensor can be applied to detect Fe 3+ ions in tap water with high selectivity, which provides a basis for the quantitative detection of Fe 3+ ions. At the same time, DECDs can be used as a pH fluorescence probe. In the pH 2.5–11.5 range, the fluorescence increases with pH whilst the solution color changes, providing a visualization platform for pH detection. Moreover, the pH fluorescence probe can be used for intracellular pH fluorescence imaging in living cells, and DECDs have an excellent biocompatibility (e.g., cell viability remains about 80% after treatment for 24 h at 200 μg·mL −1 of DECDs). Therefore, DECDs has a certain application prospect in food detection, biological detection and determination of intracellular pH change.

A tri-response colorimetric-fluorescent probe for pH and lysosomal imaging

Intracellular pH has been associated with multiple biological processes. However, fluorescent probes for pH are limited. Herein, a new pH fluorescent probe was developed based on reversible structural changes. This probe provided three reversible pH response modes within 2.00–12.00. Under the acidic conditions, there is no-fluorescence, under the neutral conditions, the emission is at 650 nm, under the alkaline conditions, the emissions at 650 nm decreases and at 460 nm appears and increases simultaneously. The probe solution presented distinct colors response to the pH values from 2.00 to 12.00. The fluorescent response from pH 2.00–12.00 makes the probe suitable for the monitoring pH in living cells. Moreover, the imaging results confirm that probe S can be applied in fluorescence imaging in living cells.

Spiropyran isomerization triggering ESIPT for visualization of pH fluctuations during oxidative stress in living cells

Intracellular pH homeostasis is foundation of maintaining normal physiological functions. More and more evidences show that intracellular pH fluctuations were usually associated with many diseases (such as cancer, epilepsy and neurodegenerative diseases). It is very important to develop in situ real-time determination of pH. In recent years, it has been verified that pH can regulate the isomerization process of spiropyran. Thus, we report a pH fluorescent probe BSL, which is a closed loop spiropyran structure by coupling benzothiazole derivatives with indole salts. We utilizes the process of spiropyran isomerization as the trigger of excited state intramolecular proton transfer (ESIPT) effect, and adjust the process of spiropyran isomerization through pH, and then the molecular transformation from enol to ketone (enol: 525 nm, ketone: 677 nm) through the ESIPT effect. This process achieved accurate measurement of pH. The probe BSL showed sensitive and reversible fluorescence response to pH in vitro. Ultimately, BSL was successfully applied to detect pH fluctuations in cell oxidative stress model.

A tunable pH probe scaffold based on sulfonamide rhodamine and its application in mitochondrial pH research

The mitochondrial pH is a vital microenvironment factor of mitochondria. Whether the mitochondria can play their unique function in a cell largely depends on what the mitochondrial pH is. Most of the influence factors of mitochondrial pH remain unclear because of the lack of pH sensitive probes for quantitative measurement of mitochondrial pH changes in vivo. Here, we report a pH fluorescence probe scaffold by which we can modify and accurately predict the pKa of the probe just according to the Hammett substituent constants. To verify the adjustability of the scaffold, 4-fluorophenylsulfonyl rhodamine was inspected as a test sample. The research showed that the predicted pKa value of the sample (5.586) was consistent with the measured value (5.584). Based on the scaffold, a two-photon ratio fluorescent pH probe 8 was designed, which can well target mitochondria and precisely measure the changes of mitochondrial pH. Probe 8 was successfully applied to study the effect of glucose content and oxidative stress on mitochondrial pH. For the first time, we measured the huge difference in mitochondrial pH between normal cells and cancer cells under a high glucose concentration with oxidative stress. The difference implied that cancer cells have lower adaptability to oxidative stress than normal cells under high glucose concentration.

Rational Design of Oxazolidine-Based Red Fluorescent pH Probe for Simultaneous Imaging Two Subcellular Organelles.

A reversible pH-responsive fluorescent probe, BP, was rationally designed and synthesized, based on protonation and deprotonation gave rise to oxazolidine ring open and close. The fluorescence response of BP against pH ranges from 3.78 to 7.54, which is suitable for labeling intracellular pH-dependent organelles. BP displayed strong red emission at a relatively high pH in living HeLa cells and U87 cells. More importantly, this probe exhibited good colocalization with both mitochondria and lysosomes in these two cell lines, attributing to pH-induced structure tautomerism resulting in an oxazolidine ring open and close that triggered effective targeting of these two organelles. As organelle interactions are critical for cellular processes, this strategy of targeting dual organelles through the structure tautomerism is conducive to further developing more effective and advanced probes for real-time imaging of the interaction between mitochondria and lysosomes.

Quinolizinium-based tunable pH fluorescent probes for imaging in live cells

A new class of quinolizinium-based tunable pH fluorescent probes for cell imaging was developed. The probes showed turn “On” fluorescence response upon acidification brought by a modulation of photoinduced electron transfer (PET) from the N,N-dialkyl aniline substituent to the quinolizinium core skeleton. These novel PET-based pH fluorescent probes displayed tunable emission properties in between 478 and 509 nm. Also, the probes exhibited tunable pH responsiveness with pKa ranging from <2.1 to 6.1 contributed by the steric effect of N,N-dialkyl anilines. They were successfully applied to in vivo pH sensing in lysosome in live cells.

A mitochondrial suitable ratiometric fluorescent probe for tracking pH change during mitophagy

Intracellular pH is one of the important parameters of cellular metabolism. The function of mitochondria is highly dependent on pH. Developing ratiometric fluorescent probes with suitable alkaline pKa for the detection of mitochondrial pH is urgently demanded. Herein, a mitochondria-targeted ratiometric pH fluorescent probe SNRF was designed based on 1,6-dihydroxynaphthalene and 4-diethylaminoketo acid. SNRF contains a hydroxyl electron donor and a nitrogen positron acceptor with excellent molecular charge transfer (ICT) performance. The hydroxyl group is a pH-sensitive group, which is prone to be protonated under acidic conditions and deprotonated under alkaline conditions. The ratiometric performance of SNRF can avoid the interference from instrumental factors, concentration of probe and microenvironment around the probe. SNRF exhibits a suitable pKa (8.30) with a linear variation between pH 7.4–9.0, making it ideal for monitoring mitochondrial pH changes. And, SNRF can be used to detect pH changes in HeLa cells and zebrafish without interference from other biologically active molecules. Furthermore, SNRF showed excellent mitochondrial targeting and was successfully used to track pH changes during rapamycin-induced mitophagy.

A water-soluble 1,8-naphthalimide-based fluorescent pH probe for distinguishing tumorous tissues and inflammation in mice.

A water-soluble fluorescent probe BPN, by introducing a piperazine as the pH-sensitive fluorescence signaling motif to the hydrophilic propionic acid-substituted 1,8-naphthalimide fluorophore, is highly sensitive to pH changes within cytoplasm matrix in living cells, as well as pH-related diseases models. Owing to the protonation-induced inhibition of the photoinduced electron transfer (PET) from piperazine to naphthalimide fluorophore, BPN displayed a significant fluorescence enhancement (more than 131-fold) upon the pH decreasing from 11.0 to 3.0. The linear range was between pH 6.4 to 8.0 with a pKa value of 6.69 near the physiological pH, which was suitable for cytosolic pH research. Furthermore, BPN exhibited a large Stokes shift (142 nm), good water solubility, excellent photostability, high selectivity and low cytotoxicity. All these advantages were particularly beneficial for intracellular pH imaging. Using BPN, we demonstrated the real-time monitoring of cytosolic pH changes in living cells. Most importantly, BPN has not only been successfully applied for distinguishing inflammation in mice, but also the surgical specimens of cancer tissue, making it of great potential application in cancer diagnosis.

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.

Opto-Chemical pH Detection of Myocardial Ischaemia Using Fluorescent Hydrogels

In this research fluorescent optochemical pH probes for the detection of ischaemia have been investigated. Myocardial ischaemia is the most prominent risk during heart surgery. During open heart surgery the heart is temporarily arrested and, since there no blood flowing, oxygen supply and removal of waste products is stopped and heart cells can be damaged. In this paper we propose a novel method to monitor the condition of the heart by placing optochemical pH sensors on several strategic places around the heart during surgery. Low cost opto-chemical pH sensors, using a HPTS (8-hydroxy-1,3,6-pyrene trisulfonic acid trisodium salt) fluorescent dye encapsulated in a thin bio-compatible hydrogel layer, were investigated for this application. Our research started with an extensive optical characterization of several types of hydrogel layers at different pH levels. Secondly a reflection probe prototype using several of these layers was designed, built and tested. Dual wavelength excitation and ratiometric detection of the fluorescent signals was used to detect the pH level. Typical output signals of 35% to 53% per pH in the range from 6.5-8.0 pH have been measured and a response time of typically 400 seconds was obtained for the prototypes. Finally based on our measurements on the HPTS layers and the reflection probe we propose an improved type of pH probe for the detection of ischaemia during open heart surgery.

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.

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.