Development of ratiometric fluorescent pH sensors based on chromenoquinoline derivatives with tunable pKa values for bioimaging

In this work, we first studied the pH-dependent characteristic of chromenoquinoline. Based on this, we then designed and synthesized two novel chromenoquinoline derivatives that can act as fluorescent pH sensors. The pK_a values of two novel chromenoquinoline derivatives can be modulated from 2.32 to 4.38 and 6.27 by introducing EDG on the backbone of chromenoquinoline. Furthermore, we demonstrate that the sensor 4 can be used as a ratiometric fluorescent pH sensor for fluorescence imaging in living cells.     

A series of simple curcumin-derived colorimetric and fluorescent probes for ratiometric-pH sensing and cell imaging

Colorimetric and fluorescent probes have emerged as a potent tool for pH sensing due to easy operation and high sensitivity. However, most of the existing bimodal probes require complicated synthesis,which greatly limits their wide applications. Herein, a simple fluorescent dye（called BFCUR） featuring a D-π-A-π-D conjugated system was developed from the natural polyphenol curcumin（CUR）. BFCUR exhibited significant red-shift in UV absorption and fluorescence emission as pH increased because of th...     

Determination of Phosphoamino Acids by 6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein and MEKC with LIF Detection

The fluorescent reagent 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein (SAMF), has been newly synthesized for use as a label for characterization of phosphoamino acids by micellar electrokinetic capillary electrophoresis (MEKC) with laser-induced fluorescence (LIF) detection. The conditions for derivatization and separation of the phosphoamino acids, including pH, concentration of electrolyte, and Brij-35 concentration were optimized in detail. Derivatization was performed at 35 °C for 10min in borate buffer (pH 8.0). The derivatives were separated to baseline by use of running buffer containing 50 mM borate and 20 mM Brij-35 at pH 9.3. Detection limits ranged from 5 × 10^?11 to 1 × 10^?9 mol L^?1 (signal to noise ratio = 3). The method was used for characterization of the phosphoamino acids in a sample from hydrolysis of a novel protein kinase from tobacco cells.     

Monitoring the pH fluctuation of lysosome under cell stress using a near-infrared ratiometric fluorescent probe

Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organelle-specific pH fluctuation. To understand the lysosomal pH variation under cell stress, a novel NIR ratiometric pH-responsive fluorescent probe (BLT) with lysosomes localization capability was developed. The quinoline ring of BLT combined with hydrogen ion which triggered the rearrangement of π electrons conjugated at low pH medium, meanwhile, the absorption and fluorescent spectra of BLT showed a red-shifts, which gived a ratiometric signal. Moreover, the probe BLT with a suitable pK_a value has the potential to discern changes in lysosomal pH, either induced by heat stress or oxidative stress or acetaminophen-induced (APAP) injury stress. Importantly, this ratiometric fluorescent probe innovatively tracks pH changes in lysosome in APAP-induced liver injury in live cells, mice, and zebrafish. The probe BLT as a novel fluorescent probe possesses important value for exploring lysosomal-associated physiological varieties of drug-induced hepatotoxicity.     

New fluorescent and colorimetric chemosensors based on the rhodamine and boronic acid groups for the detection of Hg

Novel rhodamine B (RB) derivatives bearing mono and bis-boronic acid groups were investigated as Hg²⁺ selective fluorescent and colorimetric sensors. These derivatives are first examples of reversible fluorescent chemosensors for Hg²⁺ which utilized boronic acid groups as binding sites. Two new RB-boronic acid derivatives displayed selective ‘Off-On’-type fluorescent enhancements and distinct color changes with Hg²⁺. Selective fluorescent enhancement of two rhodamine derivatives was attributed to ring opening from the spirolactam (nonfluorescent) to ring-opened amide (fluorescent).     

Influence of micellar media on the fluorescence of various benzo- and methyl-quinolizinium salts

The bebaviour of several different micelar systems (anionic, cationic and non-ionic) on the fluorescence of quinolizinium salts is studied. Important factors, such as pH and ionic strength that influence fluorescence parameters, are discussed. Fourteen quinolizinium salts (benzo and methyl derivatives) are examined as fluorescent probes in micellar media. All of them showed a marked increase of fluorescence intensity when sodium dodecyl sulfate solutions of critical micelle concentration (CMC) are added. The presence of non-ionic surfactants did not change the fluorescent emission of the probes. The emission intensity is much decreased when N-cetyl-N,N,N- trimethylammonium bromide concentrations are above the CMC. Changes in pH ido not significantly affect the fluorescence intensity of the benzo derivatives. Increasing the ionic strength decreases the fluorescence. For 9-cyanobenzo[a]phenanthro [9,10-g] quinolizinium chloride, the spectrum changes when the surfactant concentration is high than the CMC thereforre this compound is considered to be a good fluorescent probe in icell     

Stability of new optical pH sensing material based on cross-linked poly(vinyl alcohol) copolymer

Cross-linked poly(vinyl alcohol) (PVA)-silica gel copolymer has been employed as a optical pH sensor substrate for immobilisation of fluorescein. Cross-linking was carried out by the sol-gel process incorporating PVA in initial sol-gel solution of tetra-methoxysilane (TMOS) under acidic conditions. Three dimensional network formation could be achieved using compositions of PVA/TMOS=80-90/20-10 vol.% to result in crack-free films. The fluorescent sensor layers were prepared by dip-coating of gel solution onto glass slides. The dynamic fluorescence response towards different pH values was investigated in terms of the influence of sample ionic strength, membrane composition as well as age of sol-gel layers. Depending on the composition of the matrix pK_a values of 6.50, 6.68 and 7.06 were found 18 days after continues storage in buffer.     

Fluorescent response of sol-gel derived ormosils for optical ammonia sensing film

Optical sensing films for ammonia have been investigated based on a fluorescent indicator aminofluorescein (AF). AF was immobilized in diverse organically modified silicates (ORMOSILSs) obtained by copolymerizing various proportions of methyltrimethoxysilane (Me-TriMOS), phenyltrimethoxysilane (Ph-TriMOS), dimethyldimethoxysilane (DiMe-DiMOS), and diphenyldimethoxysilane (DiPh-DiMOS) with tetramethoxysilane (TMOS). The effective polarities of ormosils were probed by using the solvatochromic dye E_T (30) [2,6-diphenyl-4-(2,4,6-triphenyl-N-pyridino)]phenolate. Compared with the fluorescent responses of fluorescein and fluorescein isothiocyanate, the fluorescent intensity enhancement of AF for ammonia was caused by the reaction between ammonia and the NH₂ group on AF. The reaction may cause the reduction of the intermolecular self-quenching of AF to make a fluorescence enhancement of sensing film in NH₃ solution. The ammonia sensing range and rate of response were found to highly depend on the type and content of organosilicon precursors employed. Films prepared with a 1:3, 1:1.5, 1:1.2, or 1:1 mole ratio (TMOS:Me-TriMOS, TMOS:Ph-TriMOS, TMOS:DiMe-DiMOS, or TMOS:DiPh-DiMOS) was found to be the best in terms of stability and response. Their detection limits for ammonia in water was 0.01, 0.5, 0.2, and 0.5 μg ml⁻¹, respectively.     

Three-dimensional imaging of proton gradients at microelectrode surfaces using confocal laser scanning microscopy

The first use of fluorescence confocal laser scanning microscopy (CLSM) to image three-dimensional pH gradients at electrode surfaces is described, using the reduction of benzoquinone (BQ) to hydroquinone in aqueous solution as an example. The associated local pH changes accompanying the process have been imaged using a trace amount of fluorescein, which has a pH-dependent fluorescent signal. Images recorded in x–y–z space, allow pH profiles to be obtained as a function of applied electrode potential. Experimentally determined profiles measured when BQ is reduced at a diffusion-limited rate are shown to be in good agreement with predictions from numerical simulation. Future applications of CLSM for pH imaging at electrode surfaces and its use in scanning electrochemical microscopy (SECM) are highlighted briefly.     

Photophysical behavior of lipophilic xanthene dyes without the involvement of photoinduced electron transfer mechanism

The correlation of dibutyl-ether-ester of xanthene dye structures with their photophysical properties is discussed with respect to their capability as fluorescent probes based on ultraviolet–visible absorption, fluorescence spectra and fluorescence lifetimes measured in different solvents. It was found that the dibutyl-ether-ester of fluorescein is very weakly emissive in aprotic solvents, but fairly strong fluorescent in alcohols. The dependence of fluorescence quantum yield (Φ_f) and lifetime (τ_f) on solvent polarity suggests non-involvement of the intra-molecular photoinduced electron transfer (PeT) mechanism, suggested previously to account for the emission efficiency of fluorescein derivatives. The xanthene dyes intend to self-assemble in aprotic solvents, less polar solvents facilitate the aggregation while hydrogen bonding disfavor it. The formation of non-emissive H-aggregates is proposed to be responsible for their fluorescent behavior. The esterification showed stronger influences on the photophysics than the etherification, i.e. the former caused larger reduction of Φ_f owing to the internal conversion. The halogenation decreases the fluorescence quantum yield and lifetime of the xanthene dyes, owing to the enhancement of inter-system crossing process.     

Time resolved fluorescence in the picosecond region

Time resolved fluorescence studies of four organic molecules in solution show that, contrary to an earlier report, the relaxed fluorescent S₁ states are formed within 10 ps of excitation even when excitation is to higher states. Fluorescence lifetimes for fluorescein and its halogen substituted derivatives vary over a factor of 40 owing to enhanced intersystem crossing. Measured radiative lifetimes agree with those calculated from integrated absorption intensities and are relatively constant.     

Monitoring intracellular pH fluctuation with an excited-state intramolecular proton transfer-based ratiometric fluorescent sensor

Intracellular pH is a key parameter related to various biological and pathological processes. In this study, a ratiometric pH fluorescent sensor ABTT was developed harnessing the amino-type excited-state intramolecular proton transfer (ESIPT) process. Relying on whether the ESIPT proceeds normally or not, ABTT exhibited the yellow fluorescence in acidic media, or cyan fluorescence in basic condition. According to the variation, ABTT behaved as a promising sensor which possessed fast and reversible response to pH change without interference from the biological substances, and exported a steady ratiometric signal (I₄₇₈/I₅₄₆). Moreover, due to the ESIPT effect, large Stokes shift and high quantum yield were also exhibited in ABTT. Furthermore, ABTT was applied for monitoring the pH changes in living cells and visualizing the pH fluctuations under oxidative stress successfully. These results elucidated great potential of ABTT in understanding pH-dependent physiological and pathological processes.     

Two chemodosimeters for fluorescence recognition of biothiols in aqueous solution and their bioimaging application

Two fluorescein derivatives containing 2,4-dinitrobenzenesulfonyl group have been developed as fluorescent probes to detect the biothiols (Cys, Hcy and GSH) in aqueous solution. Probes 1 and 2 can distinguish these biothiols in the presence of other amino acids. While probe 1 can recognize the biothiols in PBS/DMSO (v:v = 95:5, pH = 7.40) solution, notably probe 2 could be used in PBS buffer solution (pH = 7.40). The detection limit of Cys for probe 2 reached at 0.021 μM in aqueous solution, which was lower than the intracellular concentration of Cys. In the recognition process, a reaction between the probes and the biothiols occurred, in which the S–O bond was cleaved to remove 2, 4-dinitrobenzenesulfonyl group. The data of ¹H NMR, MS and DFT/TD-DFT calculation further confirmed the detection mechanism. Moreover, two probes were successfully applied to the HeLa cell imaging.     

A new Vilsmeier-type reaction for one-pot synthesis of pH sensitive fluorescent cyanine dyes

A new Vilsmeier-type reaction is suggested for the synthesis of novel indocarbocyanine pH sensors, which are fluorescent when protonated but nonfluorescent upon proton abstraction. These sensors show significant ratiometric UV-visible as well as fluorescence spectral changes upon subtle variation of pHs with pK_a values near neutral.     

Ratiometric Near-Infrared Fluorescent Probes Based on Hemicyanine Dyes Bearing Dithioacetal and Formal Residues for pH Detection in Mitochondria

Ratiometric near-infrared fluorescent probes (AH⁺ and BH⁺) have been prepared for pH determination in mitochondria by attaching dithioacetal and formal residues onto a hemicyanine dye. The reactive formyl group on probe BH⁺ allows for retention inside mitochondria as it can react with a protein primary amine residue to form an imine under slightly basic pH 8.0. Probes AH⁺ and BH⁺ display ratiometric fluorescent responses to pH changes through the protonation and deprotonaton of a hydroxy group in hemicyanine dyes with experimentally determined pKa values of 6.85 and 6.49, respectively. Calculated pK_a values from a variety of theoretical methods indicated that the SMD_BONDI method of accounting for solvent and van der Waals radii plus including a water molecule located near the site of protonation produced the closest overall agreement with the experimental values at 7.33 and 6.14 for AH⁺ and BH⁺ respectively.     

A new rhodamine B-based lysosomal pH fluorescent indicator

We designed and synthesized a new pH fluorescent probe, RCE, based on structural changes of rhodamine dye at different pH values. The probe exhibits high selectivity, high sensitivity and quick response to acidic pH, as well as low cytotoxicity, excellent photostability, reversibility and cell membrane permeability. Fluorescence intensity at 584 nm was increased more than 150-fold within pH range 7.51–3.53. This probe has pK_a value 4.71, which is valuable for studying acidic organelles. Because of its long absorption and emission wavelengths, RCE can avoid associated cell damage. The probe can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

Exhaustive syntheses of naphthofluoresceins and their functions

Naphthofluorescein and/or seminaphthofluorescein derivatives possessing the additional benzene units to one or both sides of fluorescein were exhaustively constructed through Friedel-Crafts type reactions between corresponding aroylbenzoic acids and dihydroxynaphthalenes. Compound 4 works as a one-dye pH indicator, which shows red in strong acid condition and blue in basic solution. Compound 23 (diacetate of compound 4) shows good transitivity to the HEK 293 cells and acts as a fluorescent pigment for the living cell imaging. Compounds 5, 6, and 9 show fluorescent emission in the NIR region (>700 nm) and imply the potentialities of NIR fluorescent probes.     

Functional Hybrid Molecules for the Visualization of Cancer: PESIN-Homodimers Combined with Multimodal Molecular Imaging Probes for Positron Emission Tomography and Optical Imaging: Suited for Tracking of GRPR-Positive Malignant Tissue**

We describe multimodal imaging probes for gastrin-releasing peptide receptor (GRPR)-specific targeting suited for positron emission tomography and optical imaging (PET/OI), consisting of PESIN (PEG₃-BBN_7-14) dimers connected to multimodal imaging subunits. These multimodal agents comprise a fluorescent dye for OI and the chelator ((1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) (NODA-GA) for PET radiometal isotope labelling. Special focus was put on the influence of the used dyes on the properties of the whole bioconjugates. For this, several compounds with different fluorescent dyes and non-dye carrying subunits were synthesized and investigated. As fluorescent dyes, dansyl, NBD, derivatives of fluorescein, coumarin and rhodamine as well as three pyrilium-based dyes were employed. Considerable influence of the charge of the colored unit on hydrophilicity as well as in vitro target receptor binding was observed and classified. High radiochemical yields and purities were found during radiolabeling of the multimodal imaging subunits as well as their GRPR-specific bioconjugates with ⁶⁸Ga. Examinations of the photophysical properties of both molecule species displayed no loss or alteration of fluorescence characteristics.     

Ferric hydrogensulphate as a recyclable catalyst for the synthesis of fluorescein derivatives

Polycondensation reactions of phenols with phthalic anhydride were carried out in the presence of ferric hydrogensulphate under melt conditions. The reactions proceeded in short reaction times by using a catalytic amount of Fe(HSO₄)₃ and the corresponding fluorescein derivatives were obtained in high yields. The simplicity, scale-up, along with the use of an inexpensive, non-toxic, recyclable catalyst of an environmentally benign nature, are other remarkable features of the procedure. The absorption and emission properties of these fluorescein derivatives were studied.     

Reagentless pH-based biosensing using a fluorescently-labelled dextran co-entrapped with a hydrolytic enzyme in sol-gel derived nanocomposite films

We report on the development of reagentless fluorescence-based sensing films utilizing hydrolytic enzymes co-entrapped with polymers that are labelled with pH sensitive fluorophores. Aqueous solutions of a hydrophilic enzyme (urease) or a lipophilic enzyme (lipase) containing fluorescein or carboxy-seminaphtharhodafluor-1 (SNARF-1), either free or conjugated to a dextran polymer backbone, were mixed with hydrolyzed alkoxysilane solutions and cast onto planar surfaces to form thin, biologically active sol-gel derived films (ca. 500 nm thick). The films also contained various additives, such as methyltrimethoxysilane, dimethyldimethoxysilane, polyethylene glycol or polyvinyl alcohol, to optimize the activity of the entrapped enzymes. The photostability, leaching, pK_a and pH response of the entrapped probes were characterized, as was the performance of the entrapped enzymes, and an optimal set of processing conditions was obtained for each different sensing film. In general, the results indicated that SNARF-labelled dextran was the most useful pH sensitive dye owing to insensitivity to leaching and photobleaching. Furthermore, it was observed that the pK_a and pH response of this probe was insensitive to preparation conditions. The performance of the co-entrapped enzymes was highly dependent on the type and level of additive, but in all cases, it was possible to obtain active enzymes with good performance characteristics. Reagentless sensing films for urea and glyceryl tributyrate (GTB) are demonstrated based on the detection of enzyme-mediated pH changes from films coated onto planar substrates.