Monitoring Of Intracellular pH Research Articles

Biocompatible Re-Containing Block Copolymers for Intracellular pH Mapping in the PLIM Mode.

Monitoring of intracellular pH is of great importance since deviation of this parameter from the "normal" magnitudes can be considered as an indicator of various pathologies. Thus, the development of new efficient and biocompatible sensors suitable for application in biological systems and capable of quantitative pH estimation remains an urgent chemical task. Herein, we report the synthesis of a series of phosphorescent rhenium [Re(NN)(CO)2(PR3)2]+ complexes based on the NN diimine ligands containing pH-responsive carboxylic groups and styrene-containing phosphine ligands. The complexes, which display the highest pH sensitivity, were copolymerized with polyvinylpyrrolidone using the RAFT protocol to impart water solubility and to protect the chromophores from interaction with molecular oxygen. The resulting copolymers show an emission lifetime response onto pH variations in the physiological range. Cellular experiments with Chinese hamster ovary cells (CHO-K1) reveal easy internalization of the probes in cell culture and an approximately uniform distribution in cells, with some preference for location in acidic compartments (late endosomes and lysosomes). Using nigericin to homogenize intra- and extracellular pH, we built a calibration of lifetime versus pH in live CHO-K1 cells. Analysis of the phosphorescence lifetime imaging microscopy (PLIM) data confirms the applicability of the obtained sensors for monitoring the intracellular pH in cell cultures.

Multi-purpose nitrogen and phosphorous codoped carbon nanodots for fluorescence-based sensing and bioimaging

A single-step, straightforward hydrothermal method is put forward to synthesize multi-purpose nitrogen and phosphorous co-doped carbon nanodots (N,P-CNDs). Endowed with excellent water-solubility, chemical, and optical stability, the synthesized CΝDs showed high prospects for utilization in chemical and physicochemical probing and bioimaging. Specifically, the CNDs were successfully applied as a pH and benzophenone-1 probe with satisfactory precision and accuracy, as well as to assess the activity of urease. The fluorescence intensity of CNDs decreases linearly with the pH rising within the range of 3.0–12.0. Based on the benzophenone-induced fluorescence quenching of the CNDs, an acceptable linear regression for detecting benzophenone-1 was achieved over the concentration range of 1.7–76 μM. Furthermore, the synthesized CNDs demonstrated exceptional potential for cell imaging, especially by selectively labeling cell nuclei. Capitalizing on the bioimaging capabilities and pH-sensing properties of the CNDs, we developed an integrated system for intracellular pH monitoring, which can enable improved understanding of cellular processes. This versatile system opens up new opportunities for the application of CNDs in sensing various chemical and physicochemical parameters, thereby expanding the horizons of their utilization in diverse fields. The fact that the synthesized CNDs exhibit all the above capabilities enables the development of a multi-purpose probing system. The findings of this study contribute to the expanding body of knowledge of CNDs applications, whereas the CNDs can be used in diverse fields, such as environmental monitoring and analysis, as well as in biological and medical research. Environmental implicationTo date, there have been reports about the endocrine-disrupting properties of benzophenones and their bioaccumulation in human tissues, genotoxicity, etc. Despite the importance in their detection, the number of analytical methods developed for the analysis of benzophenones is limited. In this study, the development of a fluorescence probe for the sensitive and selective determination of benzophenone-1 is described. Moreover, the developed carbon dots could also serve as a pH probe. This property was utilized for monitoring cellular pH changes, which may alter due to environmental conditions.Benzophenones have raised concerns due to their reported endocrine-disrupting properties, bioaccumulation in human tissues, and genotoxicity. Addressing the gap of limited analytical methods, our study focuses on the development of a highly sensitive and selective fluorescence probe for the determination of benzophenone-1. By successfully synthesizing carbon dots with unique optical properties, we not only enable the detection of benzophenone-1 but also harness the pH-sensing capabilities of these carbon dots. This dual functionality allows the monitoring of cellular pH changes, which can be influenced by environmental conditions. Through our research, we provide a valuable tool for the detection of benzophenones and pH monitoring, thereby supporting efforts towards environmental monitoring and risk assessment.

Multimode Sensing Platform Based on Turn-On Fluorescent Silicon Nanoparticles for Monitoring of Intracellular pH and GSH.

Various physiological activities and metabolic reactions of cells need to be carried out under the corresponding pH environment. Intracellular GSH as an acid tripeptide and an important reducing substance also plays an important role in maintaining cellular acid-base balance and redox balance. Therefore, developing a method to monitor pH and GSH and their changes in cells is necessary. Herein, we developed a novel turn-on fluorescent silicon nanoparticles (SiNPs) using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane as the silicon source and dithiothreitol as the reducing agent via a one-pot hydrothermal method. It was worth mentioning that the fluorescence intensity of the SiNPs increased along with the acidity increase, making the SiNPs have excellent pH and GSH sensing capability. Furthermore, the pH and GSH sensing performance of the SiNPs in the cell was verified by confocal imaging and flow cytometry experiment. Based on the above, the prepared SiNPs had the potential to be used as an intracellular pH and GSH multimode fluorescent sensing platform and exhibited the ability to distinguish between normal cells and cancer cells.

Ratiometric Sensing of Intracellular pH Based on Dual Emissive Carbon Dots.

Accurate monitoring of intracellular pH in living cells is critical for developing a better understanding of cellular activities. In the current study, label-free carbon dots (p-CDs), which were fabricated using a straightforward one-pot solvothermal treatment of p-phenylenediamine and urea, were employed to create a new ratiometric pH nanosensor. Under single-wavelength excitation (λex = 500nm), the p-CDs gave dual emission bands at 525 and 623nm. The fluorescent intensity ratio (I525/I623) was linearly related to pH over the range 4.0 to 8.8 in buffer solutions, indicating that the ratiometric fluorescence nanoprobe may be useful for pH sensing. In pH measurements, the p-CDs also demonstrated outstanding selectivity, reversibility, and photostability. Owing to the advantages outlined above, the nanoprobe was used to monitor the pH of HeLa cells effectively. The label-free CD-based ratiometric nanoprobe features comparatively easy manufacturing and longer excitation and emission wavelengths than the majority of previously reported CD-based ratiometric pH sensors, which is ultimately beneficial for applications in biological imaging.

Two-photon ratiometric carbon dot-based probe for real-time intracellular pH monitoring in 3D environment

• Design and development of novel pH-sensitive two-photon ratiometric carbon dot-based probe was presented. • Probe possessed remarkable thermal stability, photostability, two-photon NIR excitation capability, and biocompatibility. • Probe demonstrated superb reversibility and ratiometric response to pH changes (4–10), upon single wavelength excitation. • Precise real-time monitoring and quantification of the intracellular pH values in 2D and 3D environments was performed. Designing a two-photon ratiometric pH-sensitive nanoprobe for real time monitoring of intracellular pH in biological environment is of great importance for better understanding the pathogenesis of diseases and the design of intracellular drug delivery-based system. However, the development of such probe remains a challenge. Here, we report for the first time the design and development of the two-photon ratiometric carbon dot-based nanoprobe and demonstrate its capability to monitor and quantify the intracellular cytoplasmic pH value, in real time. The nanoprobe comprised of a fluorescent carbon dot functionalized with a pH-responsive fluorescein dye (FACD). With increasing pH, FACD exhibits a clear ratiometric change in the emission intensity ratio, with sensitivity across a wide pH range in both extracellular and intracellular compartments. FACD is non-toxic to adipose stem cells in cell imaging experimental conditions and exhibits remarkable thermal stability, photostability, and two-photon near-infrared excitation capability. Using real-time dual-channel two-photon confocal microscopy we demonstrate the great potential of FACD as an efficient nanoprobe with high accuracy for the intracellular sensing of pH in living adipose stem cells seeded on either cell-culture dishes or on a 3D printed bioactive scaffold.

Fluorescein isothiocyanate-doped conjugated polymer nanoparticles for two-photon ratiometric fluorescent imaging of intracellular pH fluctuations

Herein, we report a two-photon ratiometric fluorescent pH nanosensor based on conjugated polymer poly(9,9-di-n-octylfluorenyl-2,7-diyl) (PFO) nanoparticles loaded with pH-sensitive fluorescein isothiocyanate (FITC) for intracellular pH monitoring. The obtained nanosensor (FITC-PFO NPs) possesses high sensitivity, excellent stability, good reversibility, favorable two-photon excitability and low cytotoxicity. The ratiometric fluorescence of FITC and PFO (F517/F417) in FITC-PFO NPs solution shows an efficient pH-sensitive response over the pH range from 3 to 10 (pKa = 6.43) under two-photon excitation. Additionally, the FITC-PFO NPs is successfully applied for ratiometric imaging of intracellular pH and its fluctuation in both one-photon and two-photon excitation modes. Overall, the two-photon pH nanosensor based on FITC-PFO NPs exhibits great potential in crucial physiological and biological processes related to intracellular pH fluctuations.

The Efficient Ionization Reaction of DTBA Achieved by Surface Plasmon Catalysis Effect

4,4’-Dithiobisbenzoic acid (DTBA) is equivalent to two 4-mercaptobenzoic acid (pMBA) molecules connected together after losing H+, and this bimolecular mechanism of DTBA efficiently promotes the ionization reaction. Under the irradiation of laser light, DTBA molecules are broken to form bimolecules similar to pMBA, and this kind of bimolecular coupling greatly increases the probability of binding with Ag NPs. Also, this molecule has the carboxylic acid group, which leads to a certain sensitivity to pH. In this article, through the comparison of DTBA and pMBA parallel experiments, it is clear that DTBA has better Raman activity, higher reaction efficiency, and more stable reaction than pMBA. The occurrence of this highly efficient ionization reaction under the monitoring of surface-enhanced Raman spectroscopy (SERS) provides a certain value for the progress of further related reactions, and it also has a wide range of applications in pH sensors and intracellular pH monitoring.

Real-time monitoring of intracellular pH in live cells with fluorescent ionic liquid

Real-time monitoring of intracellular pH is of great significance due to its essential role in physiological and pathological processes. In present work, the ionic liquid (IL) N-methyl-6-hydroxyquinolinium bis(trifluoromethylsulfonyl) imide ([6MQc][NTf2]) is proposed as a fluorescence probe for the quantitative imaging of intracellular pH in response to external stimuli. The fluorescence of the IL [6MQc][NTf2] exhibits a sensitive response to pH variations, as the deprotonation of [6MQc][NTf2] generates the highly fluorescent zwitterionic product [6MQz]. pH fluctuations in the range of 6.0–7.5 can be accurately sensed by monitoring the fluorescence change at 555 nm. Moreover, this IL probe exhibits favorable biocompatibility, excellent anti-photobleaching properties, and high tolerance to ionic strength. Using the IL probe, real-time sensing of hypoxia- and drug-induced intracellular pH changes in MCF-7 cells is achieved.

Carbon Dots for Intracellular pH Sensing with Fluorescence Lifetime Imaging Microscopy.

The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6–8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In particular, a phasor FLIM analysis was used to improve the pH imaging. Moreover, the effects of the coenzymes, amino acids, and proteins on the fluorescence lifetime of CDs were examined in order to mimic the complex microenvironment inside the cells.

Strong acid-assisted preparation of green-emissive carbon dots for fluorometric imaging of pH variation in living cells.

New green-emissive carbon dots (G-CDs) are described here and shown to be viable fluorescent nanoprobes for the detection of changes in cellular pH values. By using m-phenylenediamine as the carbon source, G-CDs with an absolute quantum yield of 36% were solvothermally synthesized in the presence of strong H2SO4. The G-CDs have an average size of 2.3nm and display strong fluorescence with excitation/emission peaks at 450/510nm. The fluorescence intensity depends on the pH value in the range from 6.0 to 10.0, affording the capability for sensitive detection of intracellular pH variation. The nanosensor with excellent photostability exhibited good fluorescence reversibility in different pH solutions, and showed excellent stability against the influence of other biological species. The nanoprobe was successfully used in confocal fluorescence microscopy to determine pH values in SMMC-7721 cells. Graphical abstract Schematic presentation of green-emissive carbon dots (G-CDs)synthesized using m-phenylenediamine and sufuric acid through a solvothermal method for real-timefluorometric monitoring ofintracellular pH values. Mechanism can be ascribed to PET process from the electron lone pair in amino group to the CDs.

Thiophene aromatic amine derivatives with two-photon activities as probes for the detection of picric acid and pH

Sensors for explosive picric acid (PA) detection and intracellular pH monitoring have attracted significant attention. At present work, a series of fluorescent probes bearing two-photon activities (C1–C4) based on thiophene and aromatic amine were designed and synthesized. It was found that C3 can identify PA accompanied with the enhanced two-photon activities. Moreover, significant changes of fluorescent intensity were observed in a pH range of 6.0–8.5 for C3. Encouraged by the above properties, the potential applications were carried out for monitoring intracellular pH fluctuation in living cells benefiting from the small molecular weight and relatively low toxicity of C3.

Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring

Real-time monitoring of intracellular pH fluctuation is of great significance for diagnosis of diseases and study of related physiological and pathological processes. A novel near-infrared (NIR) pH-activated parent probe 4 was developed, and further functionalized to afford probes 6 and 7. With the decrease of pH from 11 to 2, they all exhibited a new absorption peak and strong fluorescence emission in NIR region, accompanied by rapid solution color change from pink to pale green. Moreover, the probes’ fluorescence responses toward pH fluctuations were reversible and stable, and the pseudo-linear relationship between the fluorescence intensity and pH value can be used to detect pH value in 2.76–6.45 range quantitatively. Finally, probe 6 and 7 were successfully applied to monitor the fluctuation of intracellular pH by a fluorescence turn-on mode, and the imaging results confirmed their low cytotoxicity and satisfactory cell-membrane permeability, as well as their application prospect in disease diagnosis (such as tumor specific imaging) or assist in study of pH-related biological processes in wide acidic environment.

Multifunctional fluorescent leucomalachite green derivatives for chemodosimetric detection of Fe3+, specific imaging of lipid droplets and intracellular pH monitoring

Fluorescent leucomalachite green derivatives detect micromolar concentrations of Fe3+, specifically stain lipid droplets in live cells and monitor intracellular pH variations in microorganisms.

Probing pH variation in living cells and assaying hemoglobin in blood with nitrogen enriched carbon dots

Nitrogen enriched carbon dots (CDs) are prepared by hydrothermal treatment with oxalic acid and ethylenediamine serving as raw materials. The obtained CDs display strong blue fluorescence emission at λex/λem = 320/410 nm, along with a quantum yield of 14%. It is interesting to obtain a linear increase of fluorescence with the variation of pH value within pH 5.01–8.94. Meanwhile, a favorable stability of the fluorescence is achieved in a high saline matrix, i.e., up to 1.0 mol L−1 NaCl, making the CDs particularly suitable for probing pH variation in living cells or real biological systems. In addition, hemoglobin (Hb) significantly quenched the fluorescence of CDs due to the inner filter effect and static quenching, providing a linear relationship between fluorescence and Hb concentration within 20–500 mg L−1. The CDs exhibit favorable selectivity for both pH sensing and hemoglobin detection, which are further demonstrated by selective and sensitive intracellular pH monitoring in living HeLa cells and Hb assay in human blood.

A dual site controlled probe for fluorescent monitoring of intracellular pH and colorimetric monitoring of Cu2+

A novel dual site controlled probe has been developed on the basis of rhodamine and biphenylcarbonitrile unit. The probe showed not only rapid fluorescence response to pH, but also good colorimetric recognition performance for copper ions in the HEPES buffer solution (pH 7.0). The optimal geometric configuration of the probe RhB-H with different structures and the [RhB-H-Cu2+] complex were calculated at B3LYP level by DFT method. On this basis, the response mechanism of pH was analysed combining with the 1H NMR titration experiment. Moreover, the cytotoxicity studies indicated that the probe had good biocompatibility and low toxicity. Then the probe was also successfully applied on real-time monitoring of the intracellular pH changes by exhibiting different fluorescence and emission intensity. These results demonstrated that the probe could be usefully involved in biological and biomedical research.

Microwave-assisted solid-phase synthesis of highly fluorescent carbon nanoparticles and its application in intracellular pH sensing

Fluorescent carbon nanoparticles (FCNPs) have been deeply researched and widely applied in recent years due to their good optics performance, chemical stability and biocompatibility. Herein, a green and rapid microwave-assisted solid-phase synthesis (solvent-free) approach was proposed for the fabrication of highly FCNPs in a very short period of time, 4 min. The as-prepared FCNPs can emit a blue emission with quantum yield of up to 63.2% in water solution and show yellow fluorescence in the solid state. The FCNPs also exhibit special solvent effect that the fluorescence emission can be adjusted by controlling the solvent ratio of ethanol and water. Most importantly, the FCNPs possess a narrow-range pH response. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.47–5.10 and the correlation coefficient is above 0.99. The proposed FCNPs also exhibit high photostability and reusability. As expected, the cell imaging and intracellular pH monitoring was achieved successfully in living SMMC 7721 hepatoma cells by this probe. The FCNPs is promising as a convenient and general fluorescent pH sensor for bioimaging applications.

A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH

The intracellular pH is an important modulator of various bio(techno)logical processes such as enzymatic conversion of metabolites or transport across the cell membrane. Changes of intracellular pH due to altered proton distribution can thus cause dysfunction of cellular processes. Consequently, accurate monitoring of intracellular pH allows elucidating the pH-dependency of (patho)physiological and biotechnological processes. In this context, genetically encoded biosensors represent a powerful tool to determine intracellular pH values non-invasively and with high spatiotemporal resolution.We have constructed a toolbox of novel genetically encoded FRET-based pH biosensors (named Fluorescence Biosensors for pH or FluBpH) that utilizes the FMN-binding fluorescent protein EcFbFP as donor domain. In contrast to many fluorescent proteins of the GFP family, EcFbFP exhibits a remarkable tolerance towards acidic pH (pKa∼3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pKa values of 5.7, 6.1 and 7.5 were used as pH-sensing FRET acceptor domains. The resulting biosensors FluBpH 5.7, FluBpH 6.1 and FluBpH 7.5 were calibrated in vitro and in vivo to accurately evaluate their pH indicator properties. To demonstrate the in vivo applicability of FluBpH, changes of intracellular pH were ratiometrically measured in E. coli cells during acid stress.

Self-Assembled Fluorescent Bovine Serum Albumin Nanoprobes for Ratiometric pH Measurement inside Living Cells.

In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6-8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol.

Fiber-Optic-Based Micro-Probe Using Hexagonal 1-in-6 Fiber Configuration for Intracellular Single-Cell pH Measurement.

Single-cell research is essential for understanding cell heterogeneity, cell differentiation, and carcinogenesis, among other important cellular processes. New techniques for intracellular pH monitoring are urgently needed to gain new insights into single-cell responses to external stimuli. In this study, fiber-optic reflection-based pH micro (μ)-probes (tip diameter: 500-3000 nm) were designed and fabricated using a novel hexagonal 1-in-6 fiber configuration. An organic-modified silicate (OrMoSils) sol-gel doped with a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), were coated onto the probe sensing tip for pH detection. These probes enabled neutral pH monitoring and quasi-real-time data acquisition (response time: 20 ± 5 s). The fluorescence signals of the newly developed probes were found to correlate linearly with pH (R(2) = 0.9869 when coupling laser power was at 8.2 mW) within a biologically relevant pH range (6.18-7.80). The pH resolution was 0.038 pH unit. The miniaturized probes were validated in single human lung cancer A549 cells to demonstrate applicability in single-cell experiments. In summary, novel pH μ-probes with excellent resolution and response times within a biologically relevant pH range were developed, and they can be used for measuring pH changes in single cells.

Intracellular pH-sensing using core/shell silica nanoparticles.

An in-depth understanding of biochemical processes occurring within biological systems is key for early diagnosis of disease and identification of appropriate treatments. Nanobiophotonics offers huge potential benefits for intracellular diagnostics and therapeutics. Intracellular sensing using fluorescent nanoparticles is a potentially useful tool for real-time, in vivo monitoring of important cellular analytes. This work is focused on synthesis of optical chemical nanosensors for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly silica matrix with co-encapsulated Texas Red, acting as a reference dye, and pH-sensitive fluorescein isothiocyanate enabling ratiometric quantitative environmental detection. In order to obtain silica-based nanoparticles -70 nm in size, a modified sol-gel-based Stöber method was employed. The potential of these nanosensors for intracellular pH monitoring is demonstrated inside a live human embryonic kidney cell line whereby a significant change in fluorescence is observed when the cell pH is switched from acidic to basic. High loading efficiencies of nanoparticles into the cells is seen, with little effect on cell morphology even following extended nanoparticle exposure (up to 72 h). Nanoparticle incubation time and the fast response of the nanosensor (-2 s) make it a very powerful tool in monitoring the processes occurring within the cytosol.