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.     

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.     

Ratiometric fluorescent Zn chemosensor constructed by appending a pair of carboxamidoquinoline on 1,2-diaminocyclohexane scaffold

By appending a pair of carboxamidoquinoline pendants onto 1,2-diaminocyclohexane scaffold via N-alkylation, multifunctionalized ACAQ was designed and synthesized as a water soluble fluorescent ratiometric chemosensor for Zn²⁺. In 50% aqueous methanol buffer pH 7.4 solution, upon excitation at 316 nm, ACAQ (5 μM) displayed a selective ratiometric fluorescence changes with a shift from 410 to 490 nm in response to the interaction with Zn²⁺. After binding with 1 equiv of Zn²⁺, ACAQ exhibited a 12-fold enhancement in I₄₉₀/I₄₁₀ characterized by a clear isoemissive point at 440 nm. The metal sensor binding mode was established by Job’s plot and the combined fluorescence and ¹H NMR spectroscopic method. The selectivity of the probe toward biological relevant cations and transition metal ions was proven to be good. In addition, the interference caused by Cu²⁺ and Cd²⁺ in the quantitation of Zn²⁺ can be completely eliminated by the use of diethyldithiocarbamate as the screening agent. Exploitation of ACAQ as the sensing probe, ratiometric determination of Zn²⁺ with the limit of detection (LOD) at 28.3 nm can be realized. In addition, the unique responsive properties of the probe toward Fe³⁺ and Zn²⁺ were used to construct a fluorescent switch. The membrane permeability of ACAQ to living cells and bio-imaging of Zn²⁺ were demonstrated.     

Ratiometric Fluorescence Imaging of Cellular Polarity: Decrease in Mitochondrial Polarity in Cancer Cells

Mitochondrial polarity strongly influences the intracellular transportation of proteins and interactions between biomacromolecules. The first fluorescent probe capable of the ratiometric imaging of mitochondrial polarity is reported. The probe, termed BOB, has two absorption maxima (λ_abs=426 and 561 nm) and two emission maxima—a strong green emission (λ_em=467 nm) and a weak red emission (642 nm in methanol)—when excited at 405 nm. However, only the green emission is markedly sensitive to polarity changes, thus providing a ratiometric fluorescence response with a good linear relationship in both extensive and narrow ranges of solution polarity. BOB possesses high specificity to mitochondria (R_r=0.96) that is independent of the mitochondrial membrane potential. The mitochondrial polarity in cancer cells was found to be lower than that of normal cells by ratiometric fluorescence imaging with BOB. The difference in mitochondrial polarity might be used to distinguish cancer cells from normal cells.     

A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems

The specific detection of cysteine (Cys) over homocysteine (Hcy), glutathione (GSH) and other amino acids is of great significance for studying its biological functions as well as for the diagnosis of related diseases. Chloroacetyl group was often used as a reaction site for cysteine fluorescent probes for its sensitivity and selectivity. However, high background fluorescence and low stability are common problems encountered by such probes. Here, four chloroacetyl group based fluorescent probes (C1, C2, C3, and H4) was synthesized for a comparative study. We found that the inefficient quenching ability of chloroacetyl group turned into an advantage when connected with a ratiometric fluorophore. With the modification of chloroacetyl group, probe H4 displayed excellent ratiometric property and great selectivity for Cys, the stability was also improved. Additionally, the probe was successfully applied for quantitative detection of Cys in fetal bovine serum and real-time imaging in living HeLa cells with low toxicity.     

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⁺.     

Quantitative Fluorescence Ratio Imaging of Intralysosomal Chloride Ions with Single Excitation/Dual Maximum Emission

Fluorescence ratio imaging is currently being used to quantitatively detect biologically active molecules in biosystems; however, two excitations of most existing fluorescent ratiometric probes account for cumbersome operating conditions for imaging. Thus, a fluorescent ratiometric probe, 6‐methoxyquinolinium–dansyl (MQ‐DS), for Cl^? with single excitation/dual maximum emission has been developed. MQ‐DS can preferably localize into lysosomes and display excellent photostability. Upon excitation at a single wavelength, it responds precisely and instantaneously to changes in Cl^? concentrations, and it can be conveniently utilized to implement real‐time fluorescence ratio imaging to quantitatively track alterations in Cl^? levels inside cells treated under various pH conditions, and also in zebrafish with acute wounds. The successful application of the new probe in bioimaging may greatly facilitate a complete understanding of the physiological functions of Cl^?.     

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.     

基于1,8-萘酰亚胺与罗丹明B间荧光共振能量转移的高选择性Hg2+比率荧光探针

以罗丹明B与1,8-萘二甲酰亚胺反应合成了1个高选择性Hg²⁺比率荧光探针(RN). 在甲醇/乙腈/4-羟基哌嗪乙磺酸缓冲溶液(pH=7.2, 体积比8:1:1)中, RN对Hg²⁺具有比色和比率荧光双重响应. 加入Hg²⁺后, RN的紫外-可见光谱在约556 nm处产生强吸收, 溶液由浅绿色变为橙色, 其它金属离子对RN的紫外-可见光谱几乎无影响. 无Hg²⁺存在时, RN的荧光光谱在540 nm处出现萘二甲酰亚胺荧光团的特征峰; 加入Hg²⁺后, 540 nm处的发射带逐渐消失, 同时在580 nm附近产生强荧光, 荧光颜色从绿色变为橙色. 这归因于从萘酰亚胺到开环罗丹明B的荧光共振能量转移(FRET), 探针RN对Hg²⁺的比率荧光响应具有高选择性, 不受其它共存金属离子的干扰.     

A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform

A ratiometric fluorescent probe for H₂S was developed based on a coumarin– benzopyrylium platform. The ratiometric sensing is realized by a selective conversion of acyl azide to the corresponding amide, which subsequently undergoes an intramolecular spirocyclization to alter the large π-conjugated system of CB fluorophore. Compared with the traditional azide-based H₂S probes, the proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid response (∼1 min) towards H₂S, which is superior to most of the azide-based H₂S probes. Preliminary fluorescence imaging experiments show that probe 1 has potential to track H₂S in living cells.     

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.     

High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2′-Bipyridine Scaffold for Endogenous Zn2+ and Its Application to Living Cells

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn²⁺, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn²⁺ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (K_d = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn²⁺. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn²⁺ in living systems.     

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...     

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.     

Fluorescent carbazolyldithiane as a highly selective chemodosimeter via protection/deprotection functional groups: a ratiometric fluorescent probe for Cd(II)

A carbazole-based bis-dithiane (1) was rationally constructed in a straightforward manner for the selective and ratiometric fluorescent detection of Cd²⁺ with the concept of aldehyde group protection/deprotection. The probe showed a ratiometric fluorescent response to Cd²⁺ with a large emission wavelength shift (>50 nm) and displayed high selectivity for Cd²⁺ over other metal ions due to distinct deprotection conditions. In addition, a Cd²⁺-promoted dethioacetalization mechanism was proposed.     

Fluorescent azophenol-quinazoline dyad as multichannel reversible pH indicator in aqueous media: an innovative concept on diazo based dyads

A novel azophenol-quinazoline dyad 1 has been designed, synthesized and demonstrated as an efficient reversible multichannel pH indicator through distinct signalling in aqueous media. Owing to the competence between highly fluorescent quinazoline moiety and a well known fluorescence quencher diazo group, dyad 1 is moderately fluorescent in nature. Under acidic conditions 1 displays diverse fluorogenic changes (blue emission at pH 4.25; green at pH 1.80) while under basic condition (pH 11.80) chromogenic changes were observed.     

A ratiometric fluorescent probe for fluoride ion employing the excited-state intramolecular proton transfer

A ratiometric fluorescent probe 1 for fluoride ion was developed based on modulation of the excited-state intramolecular proton transfer (ESIPT) process of 2-(2′-hydroxyphenyl)benzimidazole (HPBI) through the hydroxyl group protection/deprotection reaction. The probe 1 was readily prepared by the reaction of HPBI with tert-butyldimethylsilyl chloride (TBS-Cl) and shows only fluorescence emission maximum at 360 nm. Upon treatment with fluoride in aqueous DMF solution, the TBS protective group of probe 1 was removed readily and ESIPT of the probe was switched on, which resulted in a decrease of the emission band at 360 nm and an increase of a new fluorescence peak around 454 nm. The fluorescent intensity ratio at 454 and 360 nm (I₄₅₄/I₃₆₀) increases linearly with fluoride ion concentration in the range 0.3-8.0 μmol L⁻¹ and the detection limit is 0.19 μmol L⁻¹. The proposed probe shows excellent selectivity toward fluoride ion over other common anions. The method has been successfully applied to the fluoride determination in toothpaste and tap water samples.     

Reaction-based fluorescent probe for detecting of sulfur dioxide derivatives and hydrazine via distinct emission signals

Recently, the construction of multiple analytes responsive fluorescent probes with distinct emission signals has attracted widely attention. Thus, we have designed and synthesized a new fluorescent probe, 2-(2-hydroxyphenyl)benzothiazole dye skeleton (HBT-1), for the detection of sulfur dioxide and hydrazine. Significant fluorescence enhancements in two distinct emission bands (λ_em?=?464?nm and 498?nm) were generated when HBT-1 reacted with sulfur dioxide derivatives or hydrazine, respectively. Furthermore, the probe HBT-1 response can be saturated surpurisingly at the low concentration (100?μM), shorter reaction time for sulfur dioxide derivatives, while a longer reaction time and greater concentration (400?μM) for hydrazine. In other words, the probe HBT-1 can detect sulfur dioxide derivatives without hydrazine interference at low analyte concentrations.     

Ratiometric fluorescence chemosensors for copper(II) and mercury(II) based on FRET systems

A system based on FRET mechanism, comprising a coumarin donor and a rhodamine acceptor, was developed for the selective and quantitative detection of metal ions. Fluorescent chemosensors RCs, linked by 1,2-diethylamine, exhibit significant fluorescence enhancement and excellent selectivity toward Cu²⁺. Fluorescent probes CRB and CR6G, linked by hydrazide, function as ratiometric receptors for Cu²⁺ chromogentically and fluorogentically in organic-aqueous media. Furthermore, the characteristic rhodamine-based fluorescence response of CRB (excitation at 550 nm) exhibits high selectivity for Hg(II). The construction of this kind of universal FRET system opens a broader prospect for future design of ratiometric fluorescent probes.     

Enhanced oxidase-like activity of g-C3N4 nanosheets supported Pd nanosheets for ratiometric fluorescence detection of acetylcholinesterase activity and its inhibitor

The undesirable enzymatic activity of nanozymes under near neutral p H condition and the traditional single signal output always restrict the analytical application of nanozyme-based biosensors.Herein,graphitic carbon nitride nanosheets supported palladium nanosheets composite （Pd/g-C₃N₄） with both oxidase-like activity and fluorescent property is synthesized.Notably,Pd/g-C₃N₄exhibits enhanced oxidase-like activity compared to Pd NSs under p H 7.4.By c...