Fluorescent pH Probe Research Articles

A ratiometric fluorescent probe based on FRET mechanism for pH and its cell imaging

pH balance is a prerequisite for maintaining cell function optimally and performs an important role in diverse physiological activities. Even subtle changes in cellular pH can influence many cell behaviors. More specifically, for mitochondria, its unique functions under physiological conditions depend largely on its pH values. Therefore, an effective analytical method needs to be developed to detect the alteration of intracellular pH values, especial in mitochondria. In this work, a ratiometric pH fluorescent probe (DPM) based on fluorescence resonance energy transfer (FRET) mechanism was developed by constructing coumarin and hemicyanine fluorophore. DPM could respond to pH changes by the FRET-off and FRET-on process depending on the cyclization and ring-opening reaction. DPM exhibited high energy transfer efficiency (89.1 %) and responded to pH ranges of 6.0–8.0 rapidly and selectively (pKa value of 7.6 in solution and 7.0 in cells). Moreover, DPM exhibited nearly non-toxicity to living cells and excellent photostability. DPM showed great pH-responsive feature in cells and achieved detection of mitochondrial acidification. The present work not only provided a new structure for pH fluorescence probe and simple synthetic method for the FRET-based probe but also found that the probe could target mitochondria in living cells.

Monitoring cancer treatments in melanoma cells using a fluorescence lifetime nanoprobe based on a CdSe/ZnS quantum dot functionalized with a peptide containing D-penicillamine and histidine

Anticancer therapies with cisplatin and volasertib (BI-6727) were monitored by fluorescence lifetime imaging microscopy (FLIM) in live SK-Mel-2 melanoma cells. A CdSe/ZnS quantum dot functionalized with a peptide containing D-penicillamine and histidine (CdSe/ZnS-PH) was used as intracellular pH fluorescent probe. A faster cytosol acidification was observed for cells treated with cisplatin when compared to volasertib. The first changes in the intracellular pH were found after 2 hours of treatment with cisplatin and 8 hours with volasertib. Additionally, the relationship between cytosol acidification and apoptosis was investigated using an innovative methodology based on time-resolved fluorescence measurements. Similar low percentages of apoptotic cells were observed after short incubation periods (2 – 8 hours) with both drugs. In contrast, late apoptosis and death were found for a large fraction of cells during 24-hour incubation with cisplatin but not volasertib. Thus, the early acidification observed in cisplatin treatment could accelerate apoptosis and cell death. Despite volasertib treatment shows slower mechanism of action than cisplatin, similar inhibitory effects were found for both drugs at longer incubation periods (72 hours). This study proves the potential of CdSe/ZnS-PH nanoparticle as a fluorescence lifetime probe in the study of the mechanism of action of antitumor drugs.

Bioimaging research of a turn-on homodimer fluorescent probes for pH and viscosity: A new turn-on strategy

A turn-on homodimer fluorescent probe (BL) that responded to pH and viscosity was facilely synthesized based on homo-FRET. The probe exhibited strong fluorescence intensity and color changes in alkaline pH. In addition, the probe's fluorescence had altered with changes in viscosity. What's more, Mitochondrial localization and pH bioimaging in E. coli cell experiments confirmed that BL was a novel multifunctional probe that could be applied to mitochondrial-targeted fluorescence imaging under normal conditions and real-time fluorescence imaging of E. coli under alkaline conditions.

PH-Responsive Aminobenzocoumarins as Fluorescent Probes for Biological Acidity.

Regulation of pH plays an essential role in orchestrating the delicate cellular machinery responsible for life as we know it. Its abnormal values are indicative of aberrant cellular behavior and associated with pathologies including cancer progression or solid tumors. Here, we report a series of bent and linear aminobenzocoumarins decorated with different substituents. We investigate their photophysical properties and demonstrate that the probes display strong pH-responsive fluorescence "turn on" behavior in highly acidic environments, with enhancement up to 300-fold. In combination with their low cytotoxicity, this behavior enabled their application in bioimaging of acidic lysosomes in live human cells. We believe that these molecules serve as attractive lead structures for future rational design of novel biocompatible fluorescent pH probes.

Synthesis, optical properties, and application of novel chalcone skeleton as pH fluorescent probe: Based AIE + ESIPT strategy

A series of “turn off” pH fluorescence probes with chalcone skeleton for basic system have been developed. The molecules emitted bright yellow fluorescence under acidic condition, resulting AIE coupled ESIPT characteristic and ICT process. What’s more, the compounds exhibited excellent sensitivity and selectivity for detecting pH as a facile “On-Off” fluorescence probe, and the fluorescence of them were quenched with the ESIPT process interrupted under alkaline condition. Theoretical calculation for the related compounds also performed to verify the electron effect on photophysical properties and confirm the rational speculation on the mechanism.

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.

ICT-based fluorescent probes for intracellular pH and biological species detection.

Fluorescent probes, typically based on the intramolecular charge transfer (ICT) mechanism, have received considerable research attention in cell detection due to their non-invasiveness, fast response, easy regulation, high sensitivity, and low damage tolerance for in vivo bio-samples. Generally, intracellular pH and biological species such as various gases, metal ions, and anions constitute the foundation of cells and participate in the basic physiological processes, whose abnormal level can lead to poisoning, cardiovascular disease, and cancer in living organisms. Therefore, monitoring of their quantity plays an essential role in understanding the status of organisms and preventing, diagnosing, and treating diseases. In the last decades, remarkable progress has been made in developing ICT probes for the detection of biological elements. In this review, we highlight the recent ICT probes focusing primarily on the detection of intracellular pH, various gases (H2S, CO, H2O2, and NO), metal ions (Cu2+, Hg2+, Pb2+, Zn2+, and Al3+), and anions (ClO-, CN-, SO3 2-, and F-). In addition, we discuss the issues and limitations of ICT-based fluorescent probes for in vivo detection and explore the clinical translational potential and challenges of these materials, providing valuable guidance and insights for the design of fluorescent materials.

Nitrogen, sulfur-doped carbon quantum dots with large Stokes shift for real-time monitoring of pH in living cells

Construction of carbon quantum dots-based (CQDs) fluorescent probes for real-time monitoring pH in cells is still unsatisfied. Here, we propose the synthesis of nitrogen, sulfur-doped CQDs (N,S-CQDs) using one-pot hydrothermal treatment, and serve it as fluorescent probes to realize the real-time sensing of intracellular pH. These pH-responsive N,S-CQDs were proved exhibited a diversity of admirable properties, including great photostability, nontoxicity, favorable biocompatibility, and high selectivity. Particularly, due to the doping of nitrogen and sulfur, N,S-CQDs possessed long-wavelength emission and large Stokes Shift (190 nm), which could avoid self-absorption of tissue to realize high contrast and resolution bioimaging. The response of the probes to pH showed a good linear in range of 0.93–7.00 with coefficient of determination of 0.9956. Moreover, with advantages of high signal-to-noise ratio and stability against photobleaching, the as-prepared N,S-CQDs were successfully applied to monitor pH in living cells via bioimaging. All findings suggest that N,S-CQDs have significant potential for practical application for sensing and visualizing pH fluctuation in living systems.

Sensing Mechanism and Excited-State Dynamics of a Widely Used Intracellular Fluorescent pH Probe: pHrodo.

The pHrodo with an "off-on" response to the changes of pH has been widely used as a fluorescent pH probe for bioimaging. The fluorescence off-on mechanism is fundamentally important for its application and further development. Herein, the sensing mechanism, especially the relevant excited-state dynamics, of pHrodo is investigated by steady-state and time-resolved spectroscopy as well as quantum chemical calculations, showing that pHrodo is best understood using the bichromophore model. Its first excited state (S1) is a charge transfer state between two chromophores. From S1, pHrodo relaxes to its ground state (S0) via an ultrafast nonradiative process (∼0.5 ps), which causes its fluorescence to be "off". After protonation, S1 becomes a localized excited state, which accounts for the fluorescence being turned "on". Our work provides photophysical insight into the sensing mechanism of pHrodo and indicates the bichromophore model might be relevant to a wide range of fluorescent probes.

Rhodamine-Based Cyclic Hydroxamate as Fluorescent pH Probe for Imaging of Lysosomes.

Monitoring the microenvironment within specific cellular regions is crucial for a comprehensive understanding of life events. Fluorescent probes working in different ranges of pH regions have been developed for the local imaging of different pH environments. Especially, rhodamine-based fluorescent pH probes have been of great interest due to their ON/OFF fluorescence depending on the spirolactam ring's opening/closure. By introducing the N-alkyl-hydroxamic acid instead of the alkyl amines in the spirolactam of rhodamine, we were able to tune the pH range where the ring opening and closing of the spirolactam occurs. This six-membered cyclic hydroxamate spirolactam ring of rhodamine B proved to be highly fluorescent in acidic pH environments. In addition, we could monitor pH changes of lysosomes in live cells and zebrafish.

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.

PKa-Tunable fluorescent probes for visualizing minor pH fluctuations in different subcellular organelles and in vivo

Visualizing minor pH fluctuations (0.1–0.2 unit) in subcellular organelles is in great demand for dissecting physiological events in living organism. Here, we judiciously constructed a family of coumarin-indolenium based fluorescent pH probes to visualize minor pH fluctuations (0.1–0.2 unit) in different subcellular organelles. The probe response to pH was based on a nucleophilic addition/elimination reaction induced ring closing/opening process. Notably, pKa values of the probes could be tuned by synergistic effect of multiple approaches, including change of ring size, alteration of nucleophilic ability of nucleophilic group in their ring-opened form, and variation of electrophilic ability of electrophilic atom in their ring-opened form. The probes have been utilized for tracking minor pH fluctuation in mitochondria, lysosome and endoplasmic reticulum (ER). Moreover, acidification of ER under 1,4-Dithiothreitol (DTT) triggered ER stress, which was visualized. In addition, as a proof of concept study, the probes have been applied for imaging pH variation in zebrafish under high level of exogenous CO2.

Advances in Small-Molecule Fluorescent pH Probes for Monitoring Mitophagy.

Mitochondria play a crucial role in regulating cellular energy homeostasis and cell death, making them essential organelles. Maintaining proper cellular functions relies on the removal of damaged mitochondria through a process called mitophagy. Mitophagy is associated with changes in the pH value and has implications for numerous diseases. To effectively monitor mitophagy, fluorescent probes that exhibit high selectivity and sensitivity based on pH detection have emerged as powerful tools. In this review, we present recent advancements in the monitoring of mitophagy using small-molecule fluorescence pH probes. We focus on various sensing mechanisms employed by these probes, including intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), through bond energy transfer (TBET), and photoelectron transfer (PET). Additionally, we discuss disease models used for studying mitophagy and summarize the design requirements for small-molecule fluorescent pH probes suitable for monitoring the mitophagy process. Lastly, we highlight the remaining challenges in this field and propose potential directions for the future development of mitophagy probes.

WITHDRAWN: One-pot hydrothermal synthesis of a carbon dot-based pH fluorescent probe

WITHDRAWN: One-pot hydrothermal synthesis of a carbon dot-based pH fluorescent probe

A novel turn-on fluorescent probe for detection of pH in extremely acidic environment and its application

A water-soluble turn-on fluorescent probe PNAP for pH has been designed and synthesized. PNAP was consist of pyrene as fluorophore and morpholine as receptor. Owing to the photoinduced electron transfer (PET) effect, the fluorescence of PNAP was quenched, while PNAP exhibited a remarkable “turn-on” fluorescence with the increase of acidity. Notably for its pKa of 2.15, PNAP was one of the pH fluorescent probes used in extremely acidic environments. Furthermore, PNAP also displayed good repeatability, strong anti-ion interference ability, high sensitivity and selectivity toward pH. In addition, PNAP has been successfully applied to the test strips and monitor the pH of environment water samples and realistic samples, showing its good promising prospect.

Synthesis and Optical Properties of 1,3-Bis(Arylimino) isoindoline-4,7-diols

A novel, simple and efficient CuBr2-catalyzed reaction of 3,6-dihydroxyphthalonitrile with arylamines to produce 1,3-bis(arylimino)isoindolines has been developed. Under the optimal conditions, thirteen 1,3-bis-(arylimino)isoindoline derivatives and one 2,3-bis(1H-benzo [d]- imidazole-2-yl)benzene-1,4-diol were obtained in yields among 51–93%. A reasonable mech-anism involving promotion of CuBr2 to 3,6-dihydroxyphthalonitrile is proposed based on experimental results. The optical properties of some products were determined, which resulted in a potential proton transferring dye, 3e (1,3-bis(2-methoxyphenylimino)-isoindoline-4,7-diol), and a pH fluorescent probe in pH range of 7.00–9.30, 3f (1,3-bis(4-hydroxyphenylimino)-isoindoline-4,7-diol).

Turn a Shrimp into a Firefly: Monitoring Tissue pH in Small Crustaceans Using an Injectable Hydrogel Sensor with Infrared Excitation and Visible Luminescence

Various implantable optical sensors are an emerging tool in animal physiology and medicine that may provide real-time information about body fluids without tissue extraction. Such sensors are often fluorescence-based and require strong visible external illumination during signal acquisition, which causes anxiety or even stress for small animals and thus may influence the physiological parameters being measured. In order to overcome this obstacle, here, we combined a fluorescent molecular pH probe with upconverting particles within a hydrogel fiber suitable for injection into small crustaceans. The green luminescence of the particles under non-visible infrared illumination excited fluorescence of the molecular probe and allowed for pH measurements after correction of the probe readout for luminescence intensity. The developed optical setup based on a common microscope ensured effective visualization of the sensor and spectral pH measurements through the translucent exoskeleton of the amphipod (Amphipoda, Crustacea) Eulimnogammarus verrucosus, endemic to ancient Lake Baikal. Testing the sensors in these cold-loving crustaceans under environmentally relevant temperature increases showed alkalization of amphipod internal media by 0.2 soon after the start of the experiment, while further increases led to acidification by 0.5. The applied approach for simple sensor preparation can be useful in building other implantable optical sensors for light-sensitive organisms.

Detecting pH of Sub-Micrometer Aerosol Particles Using Fluorescent Probes.

Acidity is one of the most fundamental properties of atmospheric aerosol particles, influencing both chemical processes and environmental impacts. A couple of methods for measuring the pH of aerosol particles have been developed, yet no approach is currently available for quantifying the pH of suspending submicron particles. We developed an aerosol fluorescence cell (AFC) for quantifying the pH of aerosol particles in a continuous flow. Particles containing fluorescent pH probes (fluorescein and Oregon green 488) were injected into the AFC. Ratiometric analysis of pH for these particles was conducted by alternatively exciting them with two wavelengths of diode lasers (450 and 488 nm). The employment of the two types of fluorescent probes allowed the measurement of pH in the range of 2-7. The pH measurement by the AFC for submicron particles was compared with that measured by using pH indicator papers, providing a reasonably good agreement. Measurement of size-selected particles suggested that the AFC approach is applicable to particles of 80 nm in diameter. In combination with recent developments for analyzing renebulized water-soluble matter samples, we suggest that the AFC method can be applied not only for laboratory standards but also for atmospherically relevant samples in the future.

PH-sensing supramolecular fluorescent probes discovered by library screening

A new design concept for pH-sensing supramolecular fluorescent probes is reported. Supramolecular fluorescent pH probes based on pro-guest are designed and prepared. Pro-guests are designed to degrade under acidic condition and convert to competitive guests to displace encapsulated dyes, which leads to a significant enhancement in fluorescence intensity. A library of potential fluorescent pH probes is generated and screened to discover workable probes. These probes are capable of detecting the acidic pH in solution phase. We confirm that these supramolecular probes could detect the acidic environment in endosomal compartments in live cells.

Quinolinium-Based Fluorescent Probes for Dynamic pH Monitoring in Aqueous Media at High pH Using Fluorescence Lifetime Imaging.

Spatiotemporal pH imaging using fluorescence lifetime imaging microscopy (FLIM) is an excellent technique for investigating dynamic (electro)chemical processes. However, probes that are responsive at high pH values are not available. Here, we describe the development and application of dedicated pH probes based on the 1-methyl-7-amino-quinolinium fluorophore. The high fluorescence lifetime and quantum yield, the high (photo)stability, and the inherent water solubility make the quinolinium fluorophore well suited for the development of FLIM probes. Due to the flexible fluorophore-spacer-receptor architecture, probe lifetimes are tunable in the pH range between 5.5 and 11. An additional fluorescence lifetime response, at tunable pH values between 11 and 13, is achieved by deprotonation of the aromatic amine at the quinolinium core. Probe lifetimes are hardly affected by temperature and the presence of most inorganic ions, thus making FLIM imaging highly reliable and convenient. At 0.1 mM probe concentrations, imaging at rates of 3 images per second, at a resolution of 4 μm, while measuring pH values up to 12 is achieved. This enables the pH imaging of dynamic electrochemical processes involving chemical reactions and mass transport.