pH-sensitive Fluorescent Probe Research Articles

PH-Sensitive Fluorescent Probe in Nanogel Particles as Theragnostic Agent for Imaging and Elimination of Latent Bacterial Cells Residing Inside Macrophages.

Rhodamine 6G (R6G) and 4-nitro-2,1,3-benzoxadiazole (NBD) linked through a spacer molecule spermidine (spd), R6G-spd-NBD, produces a fluorescent probe with pH-sensitive FRET (Förster (fluorescence) resonance energy transfer) effect that can be useful in a variety of diagnostic applications. Specifically, cancer cells can be spotted due to a local decrease in pH (Warburg effect). In this research, we applied this approach to intracellular infectious diseases-namely, leishmaniasis, brucellosis, and tuberculosis, difficult to treat because of their localization inside macrophages. R6G-spd-NBD offers an opportunity to detect such bacteria and potentially deliver therapeutic targets to treat them. The nanogel formulation of the R6G-spd-NBD probe (nanoparticles based on chitosan or heparin grafted with lipoic acid residues, Chit-LA and Hep-LA) was obtained to improve the pH sensitivity in the desired pH range (5.5-7.5), providing selective visualization and targeting of bacterial cells, thereby enhancing the capabilities of CLSM (confocal laser scanning microscopy) imaging. According to AFM (atomic force microscopy) data, nanogel particles containing R6G-spd-NBD of compact structure and spherical shape are formed, with a diameter of 70-100 nm. The nanogel formulation of the R6G-spd-NBD further improves absorption and penetration into bacteria, including those located inside macrophages. Due to the negative charge of the bacteria surface, the absorption of positively charged R6G-spd-NBD, and even more so in the chitosan derivatives' nanogel particles, is pronounced. Additionally, with a pH-sensitive R6G-spd-NBD fluorescent probe, the macrophages' lysosomes can be easily distinguished due to their acidic pH environment. CLSM was used to visualize samples of macrophage cells containing absorbed bacteria. The created nanoparticles showed a significant selectivity to model E. coli vs. Lactobacillus bacterial cells, and the R6G-spd-NBD agent, being a mild bactericide, cleared over 50% E.coli in conditions where Lactobacillus remained almost unaffected. Taken together, our data indicate that R6G-spd-NBD, as well as similar compounds, can have value not only for diagnostic, but also for theranostic applications.

Biochar as ammonia exchange biofilm carrier for enhanced aerobic nitrification in activated sludge

The effects of biochar on aerobic nitrification in activated sludge were investigated in sequencing batch reactors. Biochar amended reactors exhibited 87–94 % lower ammonia in effluent and 16–71 % greater removal of total Kjeldahl nitrogen compared to control reactors. Quantitative qPCR analyses revealed that the relative abundance of ammonia oxidizing bacteria (AOB, amoA/16S rRNA genes) was greater in biochar than in control reactors. AOB were enriched on biochar surfaces, with biochar particles having up to 12.1 times greater relative abundance of AOB compared to suspended biomass. Biochar’s maximum ammonia sorption capacity of 4.4 mg N/g at pH 7 decreased with decreasing pH, however a pH-sensitive fluorescent probe was used to show that biofilms growing on biochar surfaces maintain a median pH of > 6.7 despite reactor acidification by nitrification. Microbial colonization of biochar in activated sludge creates a pH-sheltered environment that sustains biochar’s ammonia sorption capacity, resulting in enrichment of AOB on biochar particles and improved nitrification.

Acidic preconditioning induced intracellular acid adaptation to protect renal injury via dynamic phosphorylation of focal adhesion kinase-dependent activation of sodium hydrogen exchanger 1

BackgroundDisruptions in intracellular pH (pHi) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pHi to normalcy remains unclear. Early research identified H+ as a critical mediator of ischemic preconditioning (IPC), leading to the concept of acidic preconditioning (AP). This concept proposes that short-term, repetitive acidic stimulation can enhance a cell’s capacity to withstand subsequent adverse stress. While AP has demonstrated protective effects in various ischemia-reperfusion (I/R) injury models, its application in kidney injury remains largely unexplored.MethodsAn AP model was established in human kidney (HK2) cells by treating them with an acidic medium for 12 h, followed by a recovery period with a normal medium for 6 h. To induce hypoxia/reoxygenation (H/R) injury, HK2 cells were subjected to hypoxia for 24 h and reoxygenation for 1 h. In vivo, a mouse model of IPC was established by clamping the bilateral renal pedicles for 15 min, followed by reperfusion for 4 days. Conversely, the I/R model involved clamping the bilateral renal pedicles for 35 min and reperfusion for 24 h. Western blotting was employed to evaluate the expression levels of cleaved caspase 3, cleaved caspase 9, NHE1, KIM1, FAK, and NOX4. A pH-sensitive fluorescent probe was used to measure pHi, while a Hemin/CNF microelectrode monitored kidney tissue pH. Immunofluorescence staining was performed to visualize the localization of NHE1, NOX4, and FAK, along with the actin cytoskeleton structure in HK2 cells. Cell adhesion and scratch assays were conducted to assess cell motility.ResultsOur findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pHi homeostasis by AP involved the upregulation of Na+/H+ exchanger 1 (NHE1) expression and activity. The activity of NHE1 was regulated by dynamic changes in pHi-dependent phosphorylation of Focal Adhesion Kinase (FAK) at Y397. This process was associated with NOX4-mediated reactive oxygen species (ROS) production. Furthermore, AP induced the co-localization of FAK, NOX4, and NHE1 in focal adhesions, promoting cytoskeletal remodeling and enhancing cell adhesion and migration capabilities.ConclusionsThis study provides compelling evidence that AP maintains pHi homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells.

Exploring biorelevant conditions and release profiles of ritonavir from HPMCAS-based amorphous solid dispersions

Development of a release test for amorphous solid dispersions (ASDs) that is in vivo predictive is essential to identify optimally performing formulations early in development. For ASDs containing an enteric polymer, consideration of buffer properties is essential. Herein, release rates of hydroxypropyl methyl cellulose acetate succinate (HPMCAS) and ritonavir from ASDs with a 20% drug loading were compared in phosphate and bicarbonate buffers with different molarities, at pH 6.5. The bioaccessibility of ritonavir from the ASD in the tiny-TIM apparatus was also evaluated and compared to that of the crystalline drug. The surface pH at the dissolving solid: solution interface was evaluated using a pH-sensitive fluorescence probe for HPMCAS and ASD compacts in phosphate and bicarbonate buffers. Drug and polymer were found to release congruently in all buffer systems, indicating that the polymer controlled the drug release. Release was slowest in 10 mM bicarbonate buffer, and much faster in phosphate buffers with molarities typically used in release testing (20-50 mM). Release from the 10 mM bicarbonate buffer was matched in a 5 mM phosphate buffer. The surface pH of HPMCAS and HPMCAS:ritonavir ASDs was found to be lower than the bulk solution pH, where surface pH differences largely explained release rate differences seen in the different buffer systems. Ritonavir was highly bioaccessible from the ASD, as assessed by the tiny-TIM system, and much less bioaccessible when crystalline drug was used. The observations highlight the need for continued development of biorelevant assays tailored for ASD formulation assessment.

Design, synthesis and application of polarity- and pH-sensitive dual-responsive small molecule fluorescent probe for early cancer diagnosis

Cancer is one of the deadliest and most complex diseases globally. It is well known that cancer cells exhibit a unique intracellular microenvironment characterized by low pH and low polarity compared to normal cells. Thus, dual-responsive fluorescent probe are more suitable for such complex and variable physiological environments than single-response probe. In this study, we designed and synthesized a dual-responsive fluorescent probe, CCT, which responds to both pH and polarity based on intramolecular charge transfer (ICT) and photoelectron transfer (PET) mechanisms.We synthesized probe CCT by combining naphthylimide, a coumarin unit, and morpholine through a six-step process. The PET mechanism between morpholine and naphthylimide renders probe CCT pH-sensitive. Additionally, the direct linkage of the polarity-sensitive units, coumarin and naphthylimide, creates a larger polarity-sensitive system. When both low pH and low polarity conditions are present, the fluorescence intensity of probe CCT is significantly higher than under separate conditions. This increased fluorescence intensity matches the low pH and low polarity microenvironment of cancer cells, facilitating bioimaging.Fluorescence imaging results demonstrate that probe CCT effectively distinguishes between cancer and normal cells, suggesting its potential as a powerful tool for early cancer diagnosis.

Distal Renal Tubular Acidosis Mutants of the Kidney Anion Exchanger Disrupt Cytosolic pH and Cause Abnormal Autophagy in Renal Epithelial Cells

Background: Distal renal tubular acidosis (dRTA) is a disease caused by impaired renal acid secretion leading to metabolic acidosis and subsequent biochemical derangements which facilitate formation of kidney stones. This can result from a mutation in the kidney anion exchanger 1 (kAE1) protein, a chloride bicarbonate exchanger in the alpha intercalated cells (A-ICs) of the kidney collecting duct. Studies in human dRTA patients and mouse dRTA models have shown an unexplained depletion in A-ICs. dRTA mouse kidney sections also showed an accumulation of autophagy markers in the remaining A-ICs. Objective and Hypothesis: To better understand the link between dRTA kAE1 mutant expression and A-IC depletion, we hypothesised that dRTA mutant expression activates IC loss through abnormal autophagy and apoptosis. Methods: We used mouse inner medullary collecting duct (mIMCD3) cells expressing two dRTA kAE1 mutants (R589H or S525F) for in vitro studies and two dRTA kAE1 knockin mice L919X and R607H, the murine equivalents of human R901X and R589H mutations, respectively for in vivo studies. We assessed intracellular pH using the ratiometric pH-sensitive fluorescent probe BCECF-AM. Using live cell confocal microscopy, we measured autophagy flux with the eGFP-RFP-LC3 construct. Real time measurements of extracellular acidification and oxygen consumption rates were used to determine cellular glycolytic and oxidative ATP production rates. Results: In vitro, kAE1 R589H and S525F dRTA mutants had significantly more alkaline intracellular pH compared to wild type (WT). Both mutants had higher autophagy induction and accumulation of autolysosomes at steady state. Chemically acidifying cytosolic pH of mutants reversed abnormal autophagy in mutant cells. The kAE1 R589H mutant had significantly lower oxidative ATP production rate whereas S525F mutant had significantly lower glycolytic ATP production rate compared to WT. In vivo, dRTA R607H homozygous mutant mice showed reduced abundance of the vacuolar proton ATPase and of mature Cathepsin D compared to WT. Conclusion: These findings confirm that kAE1 mutant expression alters cytosolic pH and induces abnormal autophagy as a result of reduced overall ATP production and defective lysosomal function. Future work will analyse the processing/traffcking of lysosomal cathepsins and mitochondrial structure and function in mutant cells, and assessing collecting duct lysosome numbers in kidney sections by immunohistochemistry. Our work provides a lead explaining the loss of A-ICs observed in dRTA patients. Canadian Institutes of Health Research (CIHR), Graduate Student Engagement Scholarship — University of Alberta. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Visualizing dynamic interactions between mitochondria and lysosomes in living cells using novel pH-sensitive fluorescent probes in dual-channel

The neutral fluorescent probes 1a-1c based on D-π-A-π-D structure, with benzopyranoquinoline as the fluorophore and the electron-donating groups at both ends, were developed, which emitted dual-channel fluorescence in different pH environments. Among them, probe 1a with both N,N-diethyl group and morpholine ring as electron-donating groups exhibited the best fluorescence performance, showing a red shift of 85 nm in dual emission at 505 nm and 590 nm, and a Stokes shift of 95 nm in both channels. The neutral 1a displayed green fluorescence specifically targeting mitochondria, while the protonated form 1a+H+ resulting in red fluorescence localized in lysosomes. The ability to distinguish healthy and pathological cells such as cancer or inflammation could be achieved by observing the differences in red fluorescence within lysosomes. Probe 1a could migrate and retarget between mitochondria and lysosomes, and it was discovered by the physiological effects of chloral hydrate experiments. In addition, the dynamic changes in morphology and movement of mitochondria and lysosomes during cellular autophagy through the variation of fluorescence intensity in green and red channels induced by rapamycin and starvation conditions had been investigated deeply.

Design, synthesis, and in vitro gene transfer efficacy of novel ionizable cholesterol derivatives

ABSTACT The medicinal properties of genetic drugs are highly dependent on the design of delivery systems. Ionizable cationic lipids are considered core materials in delivery systems. However, there has not yet been a widespread consensus on the relationship between the wide diversity of lipid structure design and gene delivery efficiency. The aims of the research work were to synthesize ionizable cholesterol derivatives (iChol-lipids) and to evaluate their potential applications as gene delivery vector. A series of iChol-lipids with different head groups were synthesized with carbamate bond spacer. The chemical structures were characterized by 1H NMR, MS, melting range, and pK a. The interactions between iChol-lipids and MALAT1-siRNA were studied by molecular dynamics simulations and compared with market available DC-Chol, which revealed that hydrogen bonds, salt-bridge, and electrostatic interaction were probably involved. The self-assemble behaviors of these lipids were intensively investigated and evaluated by dynamic laser scattering in the presence of different helper lipids and PEGylated lipids. Their plasmid binding ability, transfection efficiency, hemolytic toxicity, and cytotoxicity were fully studied. IZ-Chol-LNPs was proved to be highly potential to effectively complex with DNA, and endosome escape mechanisms mediated by proton sponge effect was verified by pH-sensitive fluorescence probe BCFL.

A novel pH-sensitive hemi-cyanine containing tetrahydropyridine ring near-infrared fluorescence probe with lysosome-targeting ability

In recent years, hemi-cyanine dyes have been widely used as biological probes due to their red-light emission characteristics and high fluorescence quantum yield. In this study, we synthesized a novel hemi-cyanine dye containing a tetrahydropyridine ring. A lysosomal target was introduced into its structure to create a new pH-sensitive near-infrared fluorescent probe that successfully targeted lysosomes. The results showed that when the probe solution was excited at the absorption wavelength of 650 nm, its fluorescence emission wavelength was about 700 nm, and the peak intensity changed with different pH values in a wide range. Therefore, this probe enabled non-invasive detection of changes in the acidic environment of lysosomes in living organisms and showed good imaging capabilities. Moreover, the probe displays high sensitivity and good stability. The theoretical calculation of a probe structure has also been completed to discuss the relationship between structure and property.

A hemicyanine-based fluorescent probe for detection of pH and its applications in live cells imaging and food monitoring

pH as an important parameter is closely related to many pathological processes and food freshness. In this paper, a new pH-sensitive fluorescent probe 1 based on hemicyanine dye was synthesized and used for the detection of pH in live cells and food spoilage. Under neutral and alkaline conditions, the phenolic hydroxyl group of the probe deprotonates, forming a closed spirocyclic structure and resulting in fluorescence quenching. Under acidic conditions, the probe maintains an open-loop structure and has intense fluorescence. The probe possessing good selectivity and stability can target lysosomes and visualize the exogenous pH in live cells. Furthermore, by using this probe, we successfully achieved the monitoring of pH in metamorphic process of shrimp and pork. This probe is expected to provide a good imaging tool for real-time detection of pH in live cells and food monitoring.

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.

Ratiometric fiber optic fluorescent pH sensor for hydroxide diffusion measurements in concrete

This article delineates the development of an advanced ratiometric pH optode designed for protracted monitoring of pH dynamics in cementitious materials—a crucial aspect of structural management. The optode features a dual-layer Hydromed™ D4 polymer pellet incorporating a pH-sensitive Naphth-AlkyneOMe fluorescent probe (pKa ≈ 12) and CdSe quantum dots as an internal reference. The polymer matrix, optimized with diamond powder and carbon black, enhances the signal while minimizing light artifacts. Demonstrating a robust response to pH fluctuations, the optode, utilizing ratiometric measurements, enables steadfast monitoring for up to 75 days.The experimental setup includes a Delrin® sample compartment housing a glass slide with the optode connected to an optical fiber via an SMA connector. HydroMed™ D4 films, forming the pH-sensitive layer, are deposited on the glass slide and secured. The optode configuration is fine-tuned to optimize signal intensity by reducing its length. For optode-detector coupling, a 1000 µm core diameter optical fiber links the optode, and a 405 nm LED illuminates the sensing film. The emission signal is collected through the optical fiber, and signals are processed using computer-controlled lock-in amplifiers.In the investigation of hydroxide ion diffusion through a concrete sample, the optode's efficacy was validated, aligning with pH electrode measurements. Addressing long-term stability, a degradation model uncovered leisurely kinetics with commendable stability. Slow degradation kinetics, with a reaction rate coefficient of 1.6 × 10⁻⁷ s⁻¹ and a half-life of 1180 h, exceeded expectations.This pH optode, showcasing an accurate and resilient response to pH variations within the 10 to 12.5 range, stands as a promising tool for monitoring pH dynamics in low-pH cementitious materials. The incorporation of a degradation model underscores fidelity over prolonged durations and offers a blueprint for enduring surveillance of concrete structures.

A pH-sensitive supermolecular fluorescent probe for albumin-mediated cell imaging

A supermolecular fluorescent probe with favorable pH-sensitive serum albumin responsiveness was successfully fabricated based on the assembly of a binary host-guest complex of water-soluble pillar [5]arene (WP5) and ICT probe DPI. DPI possessed a favorable fluorescence "turn-on" property towards bovine serum albumin (BSA). This property was retained after the binary amphipathic complex formation between WP5 and DPI. Moreover, due to the excellent pH responsiveness of WP5, WP5⊃DPI complex exhibited favorable pH-sensitive BSA responsiveness. The WP5⊃DPI complex also possessed strong BSA binding ability. Common commercial drugs had a negligible adverse effect on the BSA responsiveness of WP5⊃DPI complex. Furthermore, the amphiphilic WP5⊃DPI complex assembled into stable spherical nanoparticles in solution. Such a pH-sensitive supramolecular fluorescent probe had negligible cytotoxicity but exhibited excellent cell permeability and favorable cell imaging properties, demonstrating potential for albumin-mediated precise tumor imaging.

Live-Cell Imaging Quantifies Changes in Function and Metabolic NADH Autofluorescence During Macrophage-Mediated Phagocytosis of Tumor Cells

ABSTRACT Background The immune system has evolved to detect foreign antigens and deliver coordinated responses, while minimizing “friendly fire.” Until recently, studies investigating the behavior of immune cells were limited to static in vitro measurements. Although static measurements allow for real-time imaging, results are often difficult to translate to an in vivo setting. Multiphoton microscopy is an emerging method to capture spatial information on subcellular events and characterize the local microenvironment. Previous studies have shown that multiphoton microscopy can monitor changes in single-cell macrophage heterogeneity during differentiation. Therefore, there is a need to use multiphoton microscopy to monitor molecular interactions during immunological activities like phagocytosis. Here we investigate the correlation between phagocytic function and changes in endogenous optical reporters during phagocytosis. Methods In vitro autofluorescence imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) was used to detect metabolic changes in macrophages during phagocytosis. More specifically, optical redox ratio, mean NADH fluorescence lifetime and ratio of free to protein-bound NADH were used to quantify changes in metabolism. Results Results show that IFN-γ (M1) macrophages showed decreased optical redox ratios and mean NADH lifetime while phagocytosing immunogenic cancer cells compared to metastatic cells. To validate phagocytic function, a fluorescence microscopy-based protocol using a pH-sensitive fluorescent probe was used. Results indicate that M0 and M1 macrophages show similar trends in phagocytic potential. Conclusion Overall, this work demonstrates that in vitro multiphoton imaging can be used to longitudinally track changes in phagocytosis and endogenous metabolic cofactors.

Revealing Mitochondrion-Lysosome Dynamic Interactions and pH Variations in Live Cells with a pH-Sensitive Fluorescent Probe.

Mitochondrion-lysosome interactions have garnered significant attention in recent research. Numerous studies have shown that mitochondrion-lysosome interactions, including mitochondrion-lysosome contact (MLC) and mitophagy, are involved in various biological processes and pathological conditions. Single fluorescent probes are termed a pivotal chemical tool in unraveling the intricate spatiotemporal interorganelle interplay in live cells. However, current chemical tools are insufficient to deeply understand mitochondrion-lysosome dynamic interactions and related diseases, Moreover, the rational design of mitochondrion-lysosome dual-targeting fluorescent probes is intractable. Herein, we designed and synthesized a pH-sensitive fluorescent probe called INSA, which could simultaneously light up mitochondria (red emission) and lysosomes (green emission) for their internal pH differences. Employing INSA, we successfully recorded long-term dynamic interactions between lysosomes and mitochondria. More importantly, the increasing mitochondrion-lysosome interactions in ferroptotic cells were also revealed by INSA. Further, we observed pH variations in mitochondria and lysosomes during ferroptosis for the first time. In brief, this work not only introduced a pH-sensitive fluorescent probe INSA for the disclosure of the mitochondrion-lysosome dynamic interplays but also pioneered the visualization of the organellar pH alternation in a specific disease model.

A pH-sensitive fluorescent probe based on hemicyanine dyes for responding microenvironment changes of cardiomyocytes induced by Zn2+

The physiological state of myocardium is closely related to human health. Adverse physiological processes will lead to significant fluctuations in cardiomyocytes microenvironment, and even myocardial damage. So, it is particularly necessary to achieve the goal that monitoring cardiomyocytes microenvironment changes. Herein, we developed a new pH-sensitive fluorescent probe (3-ethyl-2-(4-hydroxy-3,5-dimethoxystyryl) benzo[d]thiazol-3-ium hemicyanine, named ETHC). The probe could respond to the pH in solution via both single fluorescence channel and ratio fluorescence as the output signal with great stability and biocompatibility. Then, the effect of Zn2+ on cardiomyocyte microenvironment was successfully monitored by ETHC through the significant changes of red channel fluorescence, which proved that Zn2+, just as resveratrol (Res), had excellent synergistic effect on cardiomyocyte protection. The subsequent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and ionomycin/Ca2+ treatment confirmed that the decrease of red channel fluorescence was indeed attributed to the acidification of mitochondria in cardiomyocytes. ETHC achieves the monitoring and biological imaging of cardiomyocyte microenvironment changes, which provides a valuable tool for the study of related life event models.

Design of a pH-sensitive supramolecular fluorescent probe for selective cancer cell imaging

In this study, a pH-sensitive fluorescent probe was created through the host-guest assembly of an amphiphilic hydroxyphenyl benzotriazole derivative (HBT-11) and cucurbit[7]uril (CB7). The fluorescence of HBT-11 is highly dependent on the molar ratio (r) of CB7 to HBT-11. If r < 1, the peak positions remain unchanged while the intensity increases with r. If r > 1, the emission peaks shift to longer wavelengths. The complexation also results in a shift in the pKa of HBT-11. The 1:1 mixture of CB7 and HBT-11 has a pKa close to neutral pH. The supramolecular probe allows for discrimination of malignant A549 and benign HEK 293T cells owing to a pronounced 8-fold light-up effect in malignant A549 cells attributed to the pH-responsive fluorescence. Given that the selective light-up mechanism is based on the pH-sensitive fluorescence of the supramolecular probe, this approach is expected to be applicable to other cancer cells.

Constant Conversion Rate of Endolysosomes Revealed by a pH-Sensitive Fluorescent Probe.

Endolysosome dynamics plays an important role in autophagosome biogenesis. Hence, imaging the subcellular dynamics of endolysosomes using high-resolution fluorescent imaging techniques would deepen our understanding of autophagy and benefit the development of pharmaceuticals against endosome-related diseases. Taking advantage of the intramolecular charge-transfer mechanism, herein we report a cationic quinolinium-based fluorescent probe (PyQPMe) that exhibits excellent pH-sensitive fluorescence in endolysosomes at different stages of interest. A systematic photophysical and computational study on PyQPMe was carried out to rationalize its highly pH-dependent absorption and emission spectra. The large Stokes shift and strong fluorescence intensity of PyQPMe can effectively reduce the background noise caused by excitation light and microenvironments and provide a high signal-to-noise ratio for high-resolution imaging of endolysosomes. By applying PyQPMe as a small molecular probe in live cells, we were able to reveal a constant conversion rate from early endosomes to late endosomes/lysosomes during autophagy at the submicron level.

Evaluating the tumor stratification with a lysosomal pH sensitive-probe by fluorescence lifetime imaging

Owing to the anaerobic metabolism in the tumor, abundant acidic metabolites are produced and accumulated in the cells. Therefore, the cells in different tumor layers are directly linked to the pH micro-environment. Nevertheless, due to the lack of robust tools, the high-efficient evaluation of the acidic micro-environment of tumor stratification faces the challenge of accurate diagnosis. We designed a new pH sensitive fluorescent lifetime probe target to lysosomes. As we expected, the fluorescence lifetime of PLN possesses a good linear fit to the pH value, which could detect the pH change at a single lysosome level in real time, and then evaluate the different acidity of tumor stratification. The probe PLN is successfully used to evaluate the tumor stratification by fluorescence lifetime imaging microscopy (FLIM) for the first time, which is of great significance in the preoperative diagnosis of clinical tumor treatment or evaluation of drug delivery effect.

Design and application of lysosomal targeting pH-sensitive β-galactosidase fluorescent probe

β-galactosidase is a lysosomal enzyme that plays an essential biological function as a cancer marker in many physiological and pathological processes. Therefore, it is of great significance to study the detection and visualization methods of its activity in organelles. Here, we propose a feasible design strategy to switch fluorescence using an intramolecular photoinduced electron transfer (PET) process to obtain a pH-sensitive β-gal activable fluorescent probe (MLC). The probe was able to react selectively with β-gal and produce a strong fluorescence signal at low pH (4.5–6). Because of its special pH response mode, the probe has a similar function of double lock, with demonstrates high selectivity toward β-gal, lower cytotoxicity, lysosomal localization, and fast response time (＜10 min). In addition, with the help of MLC, we successfully visualized β-gal in ovarian cancer cells at different pH conditions. Notably, no β-gal fluorescent probe design using this strategy has yet been developed. We hope that these applications will make MLC a potentially powerful tool in the prevention and treatment of cancer.