An ESIPT-Based Ratiometric Fluorescent Probe for Highly Sensitive and Rapid Detection of Sulfite in Living Cells

The novel ratiometric fluorescent probe HPQRB with an ESIPT effect based on Michael addition for highly sensitive and fast detection of sulfite in living HepG2 cells is reported. HPQRB can be easily synthesized by a two-step condensation reaction. HPQRB has a large emission shift (Δλ=116 nm), which is beneficial for fluorescence imaging research, and its sulfite-responsive site is based on a rhodamine-like structure with the emission peak at 566 nm, which decreases with increasing sulfite concentration. and its HPQ structure always has an ESIPT effect throughout the reaction process, keeping the emission peak at 450 nm as a self-reference. In particular, HPQRB has high selectivity for sulfite and responds quickly (within 30 s) with a low detection limit (44 nM). Furthermore, HPQRB has been successfully used for fluorescence imaging of sulfite in HepG2 cells, demonstrating the superior ability to detect sulfite under physiological conditions.     

Developing a novel ratiometric fluorescent probe based on ESIPT for the detection of pH changes in living cells

As an important parameter of intracellular metabolism, pH plays important roles in maintaining normal physiological processes. The abnormal pH could cause disorder of cell function which may cause neurological diseases. Herein, we present two novel ratiometric fluorescent probes to detect pH changes. The probes employed 2-(2′-hydroxyphenyl)benzothiazole as fluorescent platform, and displayed desirable fluorescence response to pH on the basis of excited state intramolecular proton transfer (ESIPT) process. The probe BtyC-1 showed green fluorescence at 546 nm under acidic conditions, while it displayed strong blue fluorescence at 473 nm and weak green fluorescence at 546 nm under alkaline conditions. Biological experiments demonstrated that the probe BtyC-1 could be successfully applied for the ratiometric imaging of cellular pH and the NH₄Cl-induced pH changes in living cells.     

Novel fluorescent 2‐(2‐aminofluorophenyl)benzoxazoles: Syntheses and photophysical properties

2‐(2‐Amino‐3,4,5,6‐tetrafluorophenyl)benzoxazole ( 2 ) absorbs in long wavelength band (λ_abs^max = 346 nm in methanol) and in the normal wavelength band (λ_abs^max = 285.5 nm), and emits blue fluorescence. The emission intensity is highly affected by the solvent polarity and is large in a polar solvent such as methanol. 2‐(2‐Pentafluorobenzamido‐3,4,5,6‐ tetrafluorophenyl)benzoxazole ( 5 ) emits green fluorescence along with the short wavelength emission around 380 nm and their relative intensity depends on the solvent polarity. Green fluorescence is enhanced in nonpolar solvents such as chloroform and toluene, resulting in the considerably large Stokes shift.     

A Highly Selective Two‐Photon Fluorescent Probe for Detection of Cadmium(II) Based on Intramolecular Electron Transfer and its Imaging in Living Cells

A new quinoline‐based probe was designed that shows one‐photon ratiometric and two‐photon off–on changes upon detecting Cd²⁺. It exhibits fluorescence emission at 407 nm originating from quinoline groups in Tris‐HCl (25 mM , pH 7.40), H₂O/EtOH (8:2, v/v). Coordination with Cd²⁺ causes quenching of the emission at 407 nm and simultaneously yields a remarkable redshift of the emission maximum to 500 nm with an isoemissive point at 439 nm owing to an intramolecular charge‐transfer mechanism. Thus, dual‐emission ratiometric measurement with a large redshift (Δλ=93 nm) and significant changes in the ratio (F₅₀₀/F₄₃₉) of the emission intensity (R/R₀ up to 27) is established. Moreover, the sensor H₂L displays excellent selectivity response, high sensitive fluorescence enhancement, and strong binding ability to Cd²⁺. Coordination properties of H₂L towards Cd²⁺ were fully investigated by absorption/fluorescence spectroscopy, which indicated the formation of a 2:1 H₂L/Cd²⁺ complex. All complexes were characterized by X‐ray crystallography, and TD‐DFT calculations were performed to understand the origin of optical selectivity shown by H₂L. Two‐photon fluorescence microscopy experiments have demonstrated that H₂L could be used in live cells for the detection of Cd²⁺.     

γ-Fe2O3 Nanoparticles Covered with Glutathione-Modified Quantum Dots as a Fluorescent Nanotransporter

The present paper describes the synthesis, characterization, and utilization of multi-functional magnetic conjugates that integrate optical and magnetic properties in a single structure for use in many biomedical applications. Spontaneous interaction with eukaryotic cell membrane (HEK-239 cell culture) was determined using fluorescence microscopy, and fluorescence analyses. Both, differences in excitation, and emission wavelength were observed, caused by glutathione intake by cells, resulting in disintegration of core–shell structure of quantum dots, as well as adhesion of conjugate onto cell surface. When compared with quantum dots fluorescent properties, HEK-239 cells with incorporated nanoconjugate exhibited two excitation maxima (λ _ex = 430 and 390 nm). Simultaneously, application of ideal λ _ex for quantum dots (λ _ex = 430 nm), resulted in two emission maxima (λ = 740 and 750 nm). This nanoconjugate fulfills the requirements of term theranostics, because it can be further functionalized with biomolecules as DNA, proteins, peptides or antibodies, and thus serves as a tool for therapy in combination with simultaneous treatment.

     

FRET‐Based Mitochondria‐Targetable Dual‐Excitation Ratiometric Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells

Hydrogen sulfide (H₂S) is connected with various physiological and pathological functions. However, understanding the important functions of H₂S remains challenging, in part because of the lack of tools for detecting endogenous H₂S. Herein, compounds Ratio‐H₂S 1/2 are the first FRET‐based mitochondrial‐targetable dual‐excitation ratiometric fluorescent probes for H₂S on the basis of H₂S‐promoted thiolysis of dinitrophenyl ether. With the enhancement of H₂S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I₄₀₂/I₅₇₀) exhibit a drastic change from 0.048 in the absence of H₂S to 0.36 in the presence of 180 μM H₂S; this is a 7.5‐fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H₂S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria‐targetable dual‐excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

A colorimetric and ratiometric fluorescent turn-on fluoride chemodosimeter and application in live cell imaging: high selectivity via specific SiO cleavage in semi aqueous media and prompt recovery of ESIPT along with the X-ray structures

A ratiometric fluorescent turn-on probe for fluoride ion, based on modulation of the excited-state intramolecular proton transfer (ESIPT) process by chemodosimetric desilylation pathway is reported. The probe SNBT (silyl protected hydroxynaphthalene benzothiazole moiety) shows a significant increase of ratiometric absorption band at 440 nm and emission band at 477 nm by the deprotection of fluoride mediated silyl bond cleavage in CH₃CN–H₂O (8/2, v/v, 25 °C). The test strips based on SNBT and F⁻ are fabricated, which can act as a convenient and efficient F⁻ test kits. Furthermore, the biological application shows that it can be very useful as a selective fluoride probe in the fluorescence imaging of living cells.     

Water‐Soluble Luminescent Cyclometalated Gold(III) Complexes with cis‐Chelating Bis(N‐Heterocyclic Carbene) Ligands: Synthesis and Photophysical Properties

A new class of cyclometalated Au^III complexes containing various bidentate C‐deprotonated C^N and cis‐chelating bis(N‐heterocyclic carbene) (bis‐NHC) ligands has been synthesized and characterized. These are the first examples of Au^III complexes supported by cis‐chelating bis‐NHC ligands. [Au(C^N)(bis‐NHC)] complexes display emission in solutions under degassed condition at room temperature with emission maxima (λ_max) at 498–633 nm and emission quantum yields of up to 10.1 %. The emissions are assigned to triplet intraligand (IL) π→π* transitions of C^N ligands. The Au^III complex containing a C^N (C‐deprotonated naphthalene‐substituted quinoline) ligand with extended π‐conjugation exhibits prompt fluorescence and phosphorescence of comparable intensity with λ_max at 454 and 611 nm respectively. With sulfonate‐functionalized bis‐NHC ligand, four water‐soluble luminescent Au^III complexes, including those displaying both fluorescence and phosphorescence, were prepared. They show similar photophysical properties in water when compared with their counterparts in acetonitrile. The long phosphorescence lifetime of the water‐soluble AuIII complex with C‐deprotonated naphthalene‐substituted quinoline ligand renders it to function as ratiometric sensor for oxygen. Inhibitory activity of one of these water‐soluble Au^III complexes towards deubiquitinase (DUB) UCHL3 has been investigated; this complex also displayed a significant inhibitory activity with IC₅₀ value of 0.15 μM .     

Ratiometric Fluorescent Nanosensors for Copper(II) Based on Bis(rhodamine)‐Derived PMOs with J‐Type Aggregates

By using pentyl‐linked bis(rhodamine)‐derived tetra‐siloxane (PRh‐Si₄) as the organosilica precursor, highly ordered PRh‐bridged periodic mesoporous organosilicas (PRhPMOs) were prepared. When excited at λ=500 nm, the PRhPMO suspension that contained metal ions showed two separate emission peaks at λ=550 and 623 nm. The first peak, located at λ=550 nm, was due to ring‐opening of the spiro structure in the rhodamine moiety and the second, located at λ=623 nm, originated from fluorescent aggregates of the PRh units embedded in the silica framework of the PRhPMO. By using the different intensity ratios of the two fluorescence signals (FI_550/623), PRhPMOs could be used as turn‐ON type fluorescent ratiometric chemosensors for Cu²⁺. Furthermore, based on the single‐exciton theory, it was deduced that the fluorescent aggregates formed were of the J‐type and had a coplanar configuration. Consequently, PRhPMOs display a longer fluorescence lifetime and greater fluorescent quantum yield than the respective monomers dissolved in solution, which is consistent with the experimental results.     

Imaging of Colorectal Cancers Using Activatable Nanoprobes with Second Near‐Infrared Window Emission

Fluorescent probes in the second near‐infrared window (NIR‐II) allow high‐resolution bioimaging with deep‐tissue penetration. However, existing NIR‐II materials often have poor signal‐to‐background ratios because of the lack of target specificity. Herein, an activatable NIR‐II nanoprobe for visualizing colorectal cancers was devised. This designed probe displays H₂S‐activated ratiometric fluorescence and light‐up NIR‐II emission at 900–1300 nm. By using this activatable and target specific probe for deep‐tissue imaging of H₂S‐rich colon cancer cells, accurate identification of colorectal tumors in animal models were performed. It is anticipated that the development of activatable NIR‐II probes will find widespread applications in biological and clinical systems.     

A ratiometric fluorescent probe for iron(III) and its application for detection of iron(III) in human blood serum

A simple and versatile ratiometric fluorescent Fe³⁺ detecting system, probe 1, was rationally developed based on the Fe³⁺-mediated deprotection of acetal reaction. Notably, this reaction was firstly employed to design fluorescent Fe³⁺ probe. Upon treatment with Fe³⁺, probe 1 showed ratiometric response, with the fluorescence spectra displaying significant red shift (up to 132 nm) and the emission ratio value (I₅₂₂/I₃₉₀) exhibiting approximately 2362-fold enhancement. In addition, the probe is highly sensitive (with the detection limit of 0.12 μM) and highly selective to Fe³⁺ over other biologically relevant metal ions. The sensing reaction product of the probe with Fe³⁺ was confirmed by NMR spectra and mass spectrometry. TD-DFT calculation has demonstrated that the ratiometric response of probe 1 to Fe³⁺ is due to the regulation of intramolecular charge transfer (ICT) efficiency. Moreover, the practical utility in fluorescence detection of Fe³⁺ in human blood serum was also conducted, and probe 1 represents the first ratiometric fluorescent probe that can be used to monitor Fe³⁺ level in human blood serum. Finally, probe 1 was further employed in living cell imaging with pancreatic cancer cells, in which it displayed low cytotoxicity, satisfactory cell permeability, and selective ratiometric response to Fe³⁺.     

Development of an AND Logic‐Gate‐Type Fluorescent Probe for Ratiometric Imaging of Autolysosome in Cell Autophagy

The concomitant detection of two biological events facilitates the highly selective and sensitive analysis of specific biological functions. In this article, we report an AND logic‐gate‐type fluorescent probe that can concurrently sense two biological events in living cells: H₂O₂ accumulation and acidification. The probe exhibits a unique fluorescence sensing mechanism, in which a xanthene fluorophore is oxidatively transformed to a xanthone derivative by H₂O₂, thereby resulting in a clear dual‐emission change. This transformation is significantly accelerated under weak acidic conditions, which enables the selective and sensitive detection of H₂O₂ production in an acidic cellular compartment. This unique sensing property was successfully applied to the ratiometric fluorescence imaging of autolysosome formation in selective mitochondrial autophagy (mitophagy), which highlights the utility of this novel probe in autophagy research.     

γ-Fe2O3 Nanoparticles Covered with Glutathione-Modified Quantum Dots as a Fluorescent Nanotransporter

The present paper describes the synthesis, characterization, and utilization of multi-functional magnetic conjugates that integrate optical and magnetic properties in a single structure for use in many biomedical applications. Spontaneous interaction with eukaryotic cell membrane (HEK-239 cell culture) was determined using fluorescence microscopy, and fluorescence analyses. Both, differences in excitation, and emission wavelength were observed, caused by glutathione intake by cells, resulting in disintegration of core–shell structure of quantum dots, as well as adhesion of conjugate onto cell surface. When compared with quantum dots fluorescent properties, HEK-239 cells with incorporated nanoconjugate exhibited two excitation maxima (λ _ex = 430 and 390 nm). Simultaneously, application of ideal λ _ex for quantum dots (λ _ex = 430 nm), resulted in two emission maxima (λ = 740 and 750 nm). This nanoconjugate fulfills the requirements of term theranostics, because it can be further functionalized with biomolecules as DNA, proteins, peptides or antibodies, and thus serves as a tool for therapy in combination with simultaneous treatment.     

Two D-π-A type fluorescent probes based on isolongifolanone for sensing acidic pH with large Stokes shifts

Two novel isolongifolanone derivatives (2–3) with D-π-A configuration, which had a N,N-dimethylaniline unit attached to pyrazole and pyrimidine cores, were synthesized and characterized by IR, NMR and HRMS. As the protonation of the nitrogen atoms, the probes 2–3 displayed the significant pH-dependent spectral properties. The probe 2 exhibited a remarkable ratiometric fluorescence emission (I₄₄₅/I₃₇₃) characteristic with pK_a 2.59 and the linear response over the extremely acidic range of 1.5–4.0. The probe 3 showed an obvious emission quenching at 434?nm (λ_ex?=?300?nm) with a pK_a of 3.69 and responded linearly to monitor the pH fluctuations with the weakly acidic range of 3.5–7.0, while exhibited a linear emission enhancement at 519?nm (λ_ex?=?425?nm) over the extremely acidic range of 1.0–3.5. These pH probes also displayed favorable features including large Stokes shift under acidic conditions, high selectivity, rapid response, excellent photostability and good reversibility for sensing acidic pH which were further applied to response to acidic solid and gas phase using their solid-state samples, causing dramatic fluorescence color changes. In addition, the logic gates for probe 2 were constructed to develop its potential for practical applications.     

Colorimetric and ratiometric fluorescent chemosensor based on diketopyrrolopyrrole for selective detection of thiols: an experimental and theoretical study

A colorimetric and ratiometric fluorescent thiol probe was devised with diketopyrrolopyrrole (DPP) fluorophore. The probe gives absorption and emission at 523 and 666 nm, respectively. In the presence of thiols, such as cysteine, the absorption and emission band shifted to 479 and 540 nm, respectively. Correspondingly, the color of the probe solution changed from purple to yellow, and the fluorescence changed from red to yellow. The emission intensity at 540 nm was enhanced by 140-fold. The Stokes shift of probe 1 (107 nm) is much larger than the unsubstituted DPP fluorophore (56 nm). Mass spectral analysis demonstrated that besides the expected Michael addition of thiols to the C═C bonds, the CN groups of the malonitrile moieties also react with thiols to form 4,5-dihydrothiazole structure. Probe 1 was used for fluorescence imaging of intracellular thiols. In the presence of thiols, both the green and red channel of the microscopy are active. With removal of the intracellular thiols, signal can only be detected through the red channel; thus, ratiometric bioimaging of intracellular thiols was achieved. The ratiometric response of probe 1 was rationalized by DFT calculations. Our complementary experimental and theoretical studies will be useful for design of ratiometric/colorimetric molecular probes.     

A ratiometric fluorescent probe for the detection of hypochlorous acid in living cells and zebra fish with along wavelength emission

A ratiometric fluorescence probe, NClO, for the rapid and selective detection of HClO had been designed and synthesized based on a 1,8-naphthalimide derivative. Probe NClO displayed a red emission(λ_(max)= 615 nm). In the presence of HClO, the solution of probe NClO gave off a strong green fluorescence(λ_(em), _(max)= 520 nm) with a rapid response(within seconds). This probe had been applied to image HClO in living cells and zebra fish.     

Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity

Intracellular viscosity strongly influences transportation of mass and signal, interactions between the biomacromolecules, and diffusion of reactive metabolites in live cells. Fluorescent molecular rotors are recently developed reagents used to determine the viscosity in solutions or biological fluid. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY3) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. RY3 is a pentamethine cyanine dye substituted at the central (meso-) position with an aldehyde group (CHO). In nonviscous media, rotation of the CHO group gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous or low-temperature media results in strong fluorescence (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The specially designed molecular sensor has two absorption maxima (λ(abs) 400 and 613 nm in ethanol) and two emission maxima (in blue, λ(em) 456 nm and red, 650 nm in ethanol). However it is only the red emission which is markedly sensitive to viscosity or temperature changes, providing a ratiometric response (12-fold) as well as a large pseudo-Stokes shift (250 nm). A mechanism is proposed, based on quantum chemical calculations and (1)H NMR spectra at low-temperature. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and fluorescence lifetime imaging (FLIM). Although living cells are complex the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.     

Na+ Selective Fluorescent Tools Based on Fluorescence Intensity Enhancements,Lifetime Changes,and on a Ratiometric Response

Over the years, we developed highly selective fluorescent probes for K⁺ in water, which show K⁺-induced fluorescence intensity enhancements, lifetime changes, or a ratiometric behavior at two emission wavelengths (cf. Scheme 1, K1 – K4 ). In this paper, we introduce selective fluorescent probes for Na⁺ in water, which also show Na⁺ induced signal changes, which are analyzed by diverse fluorescence techniques. Initially, we synthesized the fluorescent probes 2 , 4 , 5 , 6 and 10 for a fluorescence analysis by intensity enhancements at one wavelength by varying the Na⁺ responsive ionophore unit and the fluorophore moiety to adjust different K_d values for an intra- or extracellular Na⁺ analysis. Thus, we found that 2 , 4 and 5 are Na⁺ selective fluorescent tools, which are able to measure physiologically important Na⁺ levels at wavelengths higher than 500 nm. Secondly, we developed the fluorescent probes 7 and 8 to analyze precise Na⁺ levels by fluorescence lifetime changes. Herein, only 8 (K_d=106 mm ) is a capable fluorescent tool to measure Na⁺ levels in blood samples by lifetime changes. Finally, the fluorescent probe 9 was designed to show a Na⁺ induced ratiometric fluorescence behavior at two emission wavelengths. As desired, 9 (K_d=78 mm ) showed a ratiometric fluorescence response towards Na⁺ ions and is a suitable tool to measure physiologically relevant Na⁺ levels by the intensity change of two emission wavelengths at 404 nm and 492 nm.     

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

The development of stimulated emission depletion (STED) microscopy represented a major breakthrough in cellular and molecular biology. However, the intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes, which significantly limits the performance and practical utility of STED microscopy. We herein developed a photoresistant fluorescent dye C‐Naphox as a practical tool for STED imaging. With excitation using either a λ=405 or 488 nm laser in protic solvents, C‐Naphox exhibited an intense red/orange fluorescence (quantum yield Φ_F>0.7) with a large Stokes shift (circa 5900 cm^?1). Even after irradiation with a Xe lamp (300 W, λ_ex=460 nm, full width at half maximum (FWHM)=11 nm) for 12 hours, 99.5 % of C‐Naphox remained intact. The high photoresistance of C‐Naphox allowed repeated STED imaging of HeLa cells. Even after recording 50 STED images, 83 % of the initial fluorescence intensity persisted.     

Thiazole Orange Dimers in DNA: Fluorescent Base Substitutions with Hybridization Readout

By using (S)‐2‐amino‐1,3‐propanediol as a linker, thiazole orange (TO) was incorporated in a dimeric form into DNA. The green fluorescence (λ=530 nm) of the intrastrand TO dimer is quenched, whereas the interstrand TO dimer shows a characteristic redshifted orange emission (λ=585 nm). Steady‐state optical spectroscopic methods reveal that the TO dimer fluorescence is independent of the sequential base contexts. Time‐resolved pump–probe measurements and excitation spectra reveal the coexistence of conformations, including mainly stacked TO dimers and partially unstacked ones, which yield exciton and excimer contributions to the fluorescence, respectively. The helicity of the DNA framework distorts the excitonic coupling. In particular, the interstrand TO dimer could be regarded as an excitonically interacting base pair with fluorescence readout for DNA hybridization. Finally, the use of this fluorescent readout was representatively demonstrated in molecular beacons.