Ratiometric Fluorescent pH Probe Research Articles

Latent pH-responsive ratiometric fluorescent cluster based on self-assembled photoactivated SNARF derivatives

We have developed a self-assembled fluorescent cluster comprising a seminaphthorhodafluor (SNARF) derivative protected by a photoremovable o-nitrobenzyl group. Prior to UV irradiation, a colorless and nonfluorescent cluster was spontaneously assembled in aqueous solution. After UV irradiation, the self-assembled cluster remained intact and showed a large enhancement in pH-responsive fluorescence. The unique pH responsive fluorescent cluster could be used as a dual-emissive ratiometric fluorescent pH probe not only in the test tube but also in HeLa cell cultures.

A colorimetric and ratiometric fluorescent pH probe based on ring opening/closing approach and its applications in monitoring cellular pH change

A cell permeable colorimetric and ratiometric fluorescent pH probe with a pKavalue of 6.0 has been developed.

Ratiometric fluorescent pH probes based on aggregation-induced emission-active salicylaldehyde azines

AIE-active salicylaldehyde azines (SAs) display strong blue, green, and red fluorescence with large Stokes shifts in MeCN–water and solid medium and can be used as ratiometric fluorescent pH probes in water and test paper.

Indole-based pH probe with ratiometric fluorescence behavior for intracellular imaging

3-[3-(4-Fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-(E)-2-propenal(FMIP) was used as a ratiometric fluorescent pH probe with favorable optical properties. The probe has excellent cell membrane permeability and is applied successfully to monitor pH in living cells.

A highly selective ratiometric fluorescent pH probe based on a PAMAM wavelength-shifting bichromophoric system

A novel PAMAM wavelength-shifting bichromophoric system has been successfully developed. Novel compound was configured as a light harvesting antenna where the system surface is labeled with yellow–green emitting 4-(N,N-dimethylamino)ethylamino-1,8-naphthalimide “donor” units capable of absorbing light and efficiently transferring the energy to a focal Rhodamine 6G “acceptor”. The periphery of the system was designed on the “fluorophore–spacer–receptor” format, capable of acting as a molecular fluorescence photoinduced electron transfer based probe. Due to the both effects, photoinduced electron transfer in the periphery of the system and pH dependent rhodamine core absorption, novel antenna is able to act as a selective ratiometric pH fluorescence probe in aqueous medium. Thus, the distinguishing features of the fluorescence resonance energy transfer systems were successfully combined with the properties of classical ring-opening charge transfer systems, which may be beneficially for monitoring pH variations in complex samples.

A two-photon ratiometric fluorescent probe enables spatial coordinates determination of intracellular pH

We reported a two-photon ratiometric fluorescent probe for detecting intracellular pH. When excited with 800nm laser, an optimal output of laser as the routine equipment of two-photon fluorescence microscopy, the two-photon excited fluorescence of this probe showed distinct emission peak shift as large as 109nm upon the change of pH values in vitro. Very importantly, the experiment results show that this probe has large two-photon absorption cross-section at pH 4.5 at 800nm of 354g, which ranks it as one of the best two-photon ratiometric fluorescent pH probes, and its working pH value is between 4.0 and 8.0 which could fit the intracellular pH range. Moreover, utilizing this probe, the two-photon ratiometric fluorescent images in living cells have been obtained, and the spatial coordinates of intracellular pH can be mapped. At the same time, the probe also exhibited selectivity, photostability and membrane permeability. And the photophysical properties of this probe in various solvents indicated that these photophysical properties variations are due to an intramolecular charge transfer process. At last, the imaging depth of the probe in liver biopsy slices was investigated. The experimental results demonstrated the maximum imaging depth can arrive 66µm in living rat liver tissues.

A novel strategy to design latent ratiometric fluorescent pH probes based on self-assembled SNARF derivatives

In this study, we describe a rational design strategy to develop a latent ratiometric fluorescent pH probe from self-assembled seminaphthorhodafluor (SNARF) derivatives in aqueous conditions. Analysis of the characteristics of SNARF derivatives with protected phenolic groups allowed classification of the threshold between the assembled and monomeric states according to the Hansch–Fujita hydrophobic parameters for a substituent inserted as a SNARF-OH phenolic-protecting group. The esterase-activated latent ratiometric fluorescent pH probe SNARF-OBn(OAc) was characterized in the application of tuneable small-molecule aggregation and disaggregation. Before esterase treatment, SNARF-OBn(OAc) formed self-assembled clusters, emitting no fluorescence. The significant fluorescence was observed after esterase treatment because the self-assembled clusters of SNARF-OBn(OAc) diffuse as SNARF-OH monomers, demonstrating the intracellular application of SNARF-OBn(OAc) as a latent ratiometric fluorescent pH probe for floating cells. Furthermore, to directly visualize the state of self-assembled SNARF derivatives for investigating the mechanism of their cellular uptake, SNARF-Dan was rationally designed and applied for cellular imaging. Through the cellular application of SNARF-Dan, it was suggested that the self-assembled SNARF derivatives were introduced into the cell via macropinocytosis.

Development of a ratiometric fluorescent pH probe for cell imaging based on a coumarin–quinoline platform

It is highly desirable to develop ratiometric pH probes with a well-resolved emission spectrum for biological imaging applications. In this work the design, synthesis, photophysical properties and cellular imaging studies of a new linked coumarin–quinoline probe is described. The major features of the novel probe include two well-resolved (>100 nm) emission peaks, strong fluorescence under acidic conditions, a pKa of 4.69, low cytotoxicity and good cell membrane permeability; features which render the probe useful for monitoring pH variations from neutral to acidic conditions in living cells.

Two‐Photon Ratiometric Fluorescent Mapping of Intracellular Transport Pathways of pH‐Responsive Block Copolymer Micellar Nanocarriers

Ultrasensitive ratiometric fluorescent pH probes based on dual dye-labeled pH-responsive diblock copolymer micellar scaffold are constructed. The pH-sensitive emitting nature of BTPE dyes and emission turn-on of CMA moieties triggered by pH-actuated micelle-to-unimer of diblock scaffold synergistically contribute to the observed ≈250-fold changes of BTPE/CMA emission intensity ratios in the whole pH range. Two-photon ratiometric fluorescent pH mapping of intracellular gradients subjected by pH-responsive micellar nanoparticles in their endocytic pathway has been thus achieved.

Ratiometric spiropyran-based fluorescent pH probe

Spiropyran (SP) is an attractive molecular platform for the design of fluorescent OFF-ON probes for the detection of cations and anions operative on a ring-opening/closing mechanism. By incorporating a benzimidazole moiety at the ortho-position of the oxygen atom of the pyran unit, a ratiometric fluorescent probe in the NIR range for the measurement of pH change in aqueous ACN solution was designed and investigated. The ring-opening mechanism and cis–trans interconversion of the probe are also substantiated by 1H NMR titration. The probe showed a ratiometric change in the pH range from 7.0 to 5.5 and the pKa of the probe was calculated to be 5.9. The relative intensity ratio (I690 nm/I517 nm) changed from 0.08 to 2.11 with a more than 26-fold enhancement in response to the pH variation.

Development of FRET‐Based Dual‐Excitation Ratiometric Fluorescent pH Probes and Their Photocaged Derivatives

Dual-excitation ratiometric fluorescent probes allow the measurement of fluorescence intensities at two excitation wavelengths, which should provide a built-in correction for environmental effects. However, most of the small-molecule dual-excitation ratiometric probes that have been reported thus far have shown rather limited separation between the excitation wavelengths (20-70 nm) and/or a very small molar absorption coefficient at one of the excitation wavelengths. These shortcomings can lead to cross-excitation and thus to errors in the measurement of fluorescence intensities and ratios. Herein, we report a FRET-based molecular strategy for the construction of small-molecule dual-excitation ratiometric probes in which the donor and acceptor excitation bands exhibit large separations between the excitation wavelengths and comparable excitation intensities, which is highly desirable for determining the fluorescence intensities and signal ratios with high accuracy. Based on this strategy, we created a coumarin-rhodamine FRET platform that was then employed to develop the first class of FRET-based dual-excitation ratiometric pH probes that have two well-resolved excitation bands (excitation separations>160 nm) and comparable excitation intensities. In addition, these pH probes may be considered as in a kind of "secured ratioing mode". As a further application of these pH probes, the dual-excitation ratiometric pH probes were transformed into the first examples of photocaged dual-excitation ratiometric pH probes to improve the spatiotemporal resolution. It is expected that the modular nature of our FRET-based molecular strategy should render it applicable to other small-molecule dual-dye energy-transfer systems based on diverse fluorescent dyes for the development of a wide range of dual-excitation ratiometric probes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.

Rational Design of Ratiometric Near-Infrared Fluorescent pH Probes with Various pKa Values, Based on Aminocyanine

Novel ratiometric, near-infrared fluorescent pH probes with various pK(a) values have been designed and synthesized on the basis of aminocyanine bearing a diamine moiety, and their photochemical properties were evaluated. Under acidic conditions, these pH probes showed a 46- to 83-nm red shift of the absorption maximum. This change is sufficiently large to permit their use as ratiometric pH probes, and is reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic conditions. Furthermore, the pK(a) values of these probes can be predicted from the calculated pK(a) values of the diamine moieties, obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH probes for various applications.

Ultrasensitive ratiometric fluorescent pH and temperature probes constructed from dye-labeled thermoresponsive double hydrophilic block copolymers

We report on the fabrication of highly sensitive ratiometric fluorescent pH and temperature probes based on thermoresponsive double hydrophilic block copolymers (DHBCs) with the two blocks labeled with two types of dyes possessing different pH-switchable emission characteristics. P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs were synthesized via consecutive reversible addition–fragmentation chain transfer (RAFT) polymerizations in combination with post-modifications, where NIPAM, OEGMA, FITC, and RhBAM are N-isopropylacrylamide, oligo(ethylene glycol) monomethyl ether methacrylate, fluorescein isothiocyanate, and rhodamine B-based derivatives, respectively. Due to that FITC and RhBAM moieties exhibit prominent decrease and increase in emission intensities with decreasing solution pH, respectively, intensity ratios of characteristic RhBAM and FITC emission bands, I582/I522, of P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) unimers at 25 °C exhibit ∼39-fold changes in the range of pH 2–10. At elevated temperatures, thermo-induced formation of PNIPAM-core micelles enables effective fluorescence resonance energy transfer (FRET) between FITC and RhBAM moieties respectively located within micellar cores and coronas, and I582/I522 exhibits ∼52.5-fold changes in the same pH range. The reported dually modulated multicolor-emitting P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs are capable of ultrasensitive fluorometric detection of solution pH and temperature in a ratiometric manner, which augurs well for their practical applications in sensing, imaging, and the fabrication of new generation of theranostic systems.

A ratiometric fluorescent pH probe based on aggregation-induced emission enhancement and its application in live-cell imaging

A ratiometric fluorescent pH probe, 4-carboxylaniline-5-chlorosalicylaldehyde Schiff base (1) was synthesized via a facile reaction and used for pH sensing in live cells. While exhibiting weak fluorescence when dispersed in solution, 1 displayed aggregation-induced emission enhancement (AIEE) characteristics in its aggregate/solid state, as a result of the restriction of free intramolecular rotation of a C–N bond and the non-planar configuration in the aggregate/solid state. The integration of hydroxyl and carboxyl groups provided 1 with a ratiometric fluorescent response to pH based on AIEE and the pH-dependent spectral characteristics of compound 1, with proton dissociation constants pKa1 and pKa2 of 4.8 ± 0.1 and 7.4 ± 0.1, respectively. The probe exhibited a significant fluorescence color change from orange to green with an intensity ratio (I516 nm/ I559 nm) enhanced when the pH increased from 5.0 to 7.0 in aqueous solution. Confocal fluorescence imaging of intracellular pH through ratiometric response was successfully achieved in live HepG2 cells. The results demonstrate that probe 1 is a good candidate for monitoring pH fluctuations in live cells with good selectivity, stability, and excellent membrane permeability.

Dye-concentrated organically modified silica nanoparticles as a ratiometric fluorescent pH probe by one- and two-photon excitation

Basic dye-concentrated nanoparticles (approximately 33 nm in diameter) show fluorescence-based ratiometric pH response, by one- and two-photon excitations, with improved proton sensing ability (pKa approximately 6.4) through nanoscopic intraparticle energy transfer.

Simple and Efficient Ratiometric Fluorescent Probes for Near-neutral pH in Aqueous Solutions

Abstract Two ratiometric fluorescent pH probes of 2,6-diaminopyridine (DAPD) and 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND), though simple-structured, show good sensitivity to near-neutral pH range (6.0–8.0) in aqueous solutions. Further studies indicate that the 2-amino groups on pyridine or naphthyridine ring play an important role in the pH-dependent fluorescence spectral properties of these dyes.

Intracellular pH.

Intracellular pH.