Reaction-based Fluorescent Probe Research Articles

A sensitive PET “turn on” fluorescent probe for specific detection of 4-methoxy-thiophenol

Thiophenols as a series of industrial products are widely used and high toxicity to the organisms and the environment. The fluorescent probe technique offers important insights into thiophenols convenient sensing. Herein, we report a 1,8-naphthalimide/ triphenylamine-based fluorescent probe (TPA-NI-NO2) for the specific detection of 4‑methoxy-thiophenol (MTP). The probe can “turn on” specific detect MTP with the limit of detection (LOD) of 80 nM among various relevant analytes and homologues (p-Cl-C6H4SH, p-NO2-C6H4SH, C6H5SH and o-CH3-C6H5SH). The sensing mechanism was explored by the HPLC experiment and the PET process was determined by density functional theory (DFT) calculations. In addition, the specificity of the probe was also studied by the Gibbs free energy (ΔG) analysis of the reaction between the probe and different thiophenols. The reaction between probe and MTP gives the minimum of the ΔG value (∼ -9.7280 kcal/mol) and that of other thiophenols-mediated is -2.5283 to -5.8911 kcal/mol, which proves the thermodynamic chemical equilibrium can optimize the design of reaction-based fluorescent probes with excellent specificity. At last, TPA-NI-NO2 was successfully applied to Hela cells imaging.

BODIPY- imine based fluorescence “turn on” chemosensor for selective sensing of Hg2+

The detection of mercury has gained considerable attention due to its both environmental and biological impacts. In this study, a BODIPY- imine chemosensor (3) having an imine molecule at the meso position of the BODIPY core was synthesized, and its structure was characterized by spectroscopic and analytical methods as well as single crystal XRD. Chemosensor 3 showed a poor emission band in the DMSO/H2O (9:1 v/v) solvent system (ΦFl < 0.01). Upon the addition of Hg2+, the fluorescence intensity of 3 increased notably with a 0.031 ΦFl value, and the limit of detection for Hg2+ was calculated as 0.90 μM. The sensing mechanism of 3 was investigated 1H NMR titration studies and Hg2+ ion induced hydrolysis of the imine double bond and thus activating the PET OFF proccess. The BODIPY- imine compound (3) can be used as reaction-based fluorescence probe for the sensing of Hg2+ ion.

Ligand reaction-based fluorescent peptide probes for the detection of Cu2+ and glutathione.

Copper is a critical element in both human and animal metabolic processes. Its role includes supporting connective tissue cross-linking, as well as iron and lipid metabolism; at the same time, copper is also a toxic heavy metal that can cause harm to both the environment and human health. Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine combined with sulfhydryl groups. Its properties include acting as an antioxidant and facilitating integrative detoxification. GSH is present in both plant and animal cells and has a fundamental role in maintaining living organisms. GSH is the most abundant thiol antioxidant in the human body. It exists in reduced and oxidized forms within cells and provides significant biochemical functions, such as regulating vitamins such as vitamins D, E, and C, and facilitating detoxification. A fluorescent probe has been developed to detect copper ions selectively, sensitively, and rapidly. This report outlines the successful work on creating a peptide probe, TGN (TPE-Trp-Pro-Gly-Cln-His-NH2 ), with specific Cu2+ detection capabilities, and a significant fluorescence recovery occurred with the addition of GSH. This indicates that the probe can detect Cu2+ and GSH concurrently. The detection limit for Cu2+ in the buffer solution was 264 nM (R2 = 0.9992), and the detection limit for GSH using the TGN-Cu2+ complex was 919 nM (R2 = 0.9917). The probe exhibits high cell permeability and low biotoxicity that make it ideal for live cell imaging in biological conditions. This peptide probe has the capability to detect Cu2+ and GSH in biological cells.

A Reaction-Based ESIPT Fluorescent Probe for the Detection of Hg2+ with Large Stokes Shift.

A novel reaction-based fluorescent probe 1 for Hg2+ was designed and synthesized. 1 was almost nonfluoresent due to inhibition of the ESIPT process between hydroxy group and imid carbonyl oxygen by diphenylphosphinothioate group. After reacting with Hg2+, the fluorescence intensity of 1 exhibited significant enhancement owing to recovery of the ESIPT process via Hg2+-promoted desulfurization-hydrolysis of the diphenylphosphinothioate moiety and cleavage of the P-O bond. 1 not only showed rapid response, high sensitivity, excellent selectivity for Hg2+ over other metal ions, but also could detect Hg2+ with large Stokes shift (165nm), which was attributed to the ESIPT process. Moreover, the reaction mechanism was fully validated by absorption spectra, fluorescence spectra, fluorescence color as well as ESI-MS analysis. 1 is the reaction-based ESIPT fluorescent probe for the detection of Hg2+ with large Stokes shift, rapid response, high sensitivity and selectivity.

Precise Electron-Withdrawing Strength Modulation of ESIPT Probes for Ultrasensitive and Specific Fluorescence Sensing.

The precise regulation of the electron-withdrawing/electron-donating strength in a probe is of great significance for the design of reaction-based fluorescent probes with specific functionalities. Here, a family of excited-state intramolecular proton transfer (ESIPT)-based probes with fluorescence turn-on sensing properties toward KMnO4 was designed by precisely modulating the electron-withdrawing strength of the substituents located at the para-position of the recognition group. It is found that -F, -CHO, and -H as the electron-withdrawing groups bound at the probe can specifically recognize KMnO4, which ensures a blue emission displayed by the reaction products. Especially with -CHO as the electron-withdrawing group, the reaction product shows the most stable fluorescence. The probe 2-(benzo[d]oxazol-2-yl)-4-formylphenyl acrylate (BOPA-CHO) demonstrated a more superior sensing performance toward KMnO4, including a low limit of detection (LOD, 0.96 nM), a rapid response (<3 s), and a rather good selectivity even in the presence of 21 interferents. Moreover, the practicality of the probe was further verified by a test pen comprising a BOPA-CHO-embedded sponge, which is capable of detecting KMnO4 solid with a naked-eye LOD of 11.62 ng. The present probe design and modulation strategy would open up a new path for the design of high-performance fluorescent probes.

Arylidene Meldrum's Acid: A Versatile Structural Motif for the Design of Enzyme-Responsive "Covalent-Assembly" Fluorescent Probes with Tailor-Made Properties.

Latent cyclic C-nucleophiles are recently proving their value in the field of reaction-based fluorescent probes, far beyond their primary utility in organic synthesis. They are typically used to introduce a Michael acceptor moiety acting as a recognition/reaction site for analyte to be detected or as a kinetic promoter of fluorogenic cascade reactions triggered by a reactive species. C-nucleophiles bearing a further reactive handle offer an additional opportunity for tuning the physicochemical/targeting properties or providing drug-releasing capabilities to these probes, through the covalent attachment of ad hoc chemical moiety. In order to implement such strategy to fluorogenic enzyme substrates based on the "covalent-assembly" principle, we have explored the potential of some functionalized derivatives of barbituric acid, piperidine-2,4-dione and Meldrum's acid. Our investigations based on the rational design and validations of enzyme-responsive caged precursors of fluorescent pyronin dyes and 7-(diethylamino)coumarin-3-carboxylic acid, led to identify a versatile candidate suitable for this late-stage structural optimization approach. This Meldrum's acid derivative enables to either enhance water solubility or achieve the reversible conjugation of a targeting ligand, while promoting in situ formation of fluorophore upon enzymatic activation. This study opens the way to novel multifunctional fluorescence imaging probes and optically modulated small conjugate-based theranostics.

All-Nontoxic Fluorescent Probe for Biothiols and Its Clinical Applications for Real-Time Glioblastoma Visualization.

Fluorescence-guided surgery (FSG) is a surgical method to selectively visualize the tumor site using fluorescent materials with instrumental setups in the operation rooms. It has been widely used in the surgery of brain tumors, such as glioblastoma (GBM), which is difficult to distinguish from normal tissue. Although FSG is crucial for GBM surgery, the commercially available fluorescent materials for FSG have shown serious adverse effects. To satisfy the clinical demand, we recently reported reaction-based fluorescent probes based on a 4-chloro-7-nitrobenzofurazan (NBD) fluorophore that can detect cysteine (Cys) and homocysteine (Hcy), a biomarker of GBM, and their applications for the GBM diagnosis and FSG. However, our probes have cellular toxicity issues arising from the leaving group (LG) that is generated after the reaction of the fluorescent probe and the analytes. In this study, we disclosed a nontoxic fluorescent probe for sensing biothiols and their clinical applications for real-time human glioblastoma visualization. Systematic toxicity analysis of several LGs was conducted on several cell lines. Among the LGs, 2-hydroxy-pyridine showed negligible toxicity, and its fluorescent probe derivative (named NPO-o-Pyr) showed high specificity and sensitivity (LOD: 0.071 ppm for Cys; 0.189 ppm for Hcy), a fast response time (<5 min) to Cys and Hcy, and high biocompatibility. In addition, NPO-o-Pyr can significantly detect the GBM site both in actual clinical samples as well as in the GBM-xenografted mouse model. We are confident that NPO-o-Pyr will become a new substitute in FSG due to its capability to overcome the limitations of the current fluorescent probes.

A highly selective fluorescent probe for detection of H2S based-on benzothiazole and its application

Hydrogen sulfide plays an essential roles in physiological and pathophysiological process. It is still a challenge to design a simple and reasonable H2S-specfic fluorescent probe. Herein, we develop a simple reaction-based fluorescent probe for detecting H2S in DMF/Tris-HCl buffer system. Fluorescence emission spectra were used to investigate the recognition performance of probe L towards hydrogen sulfide. The results shown that probe L has a specific recognition towards hydrogen sulfide over other tested metal cations and anions with a rapid response. The probe L combined with H2S in a 1: 1 manner with a detection limit of 0.372 μM. The appropriate pH range for detecting H2S was 4 ∼ 8. The proposed recognition mechanism was studied by fluorescence titration spectrum, Job’s plot, 1H NMR titration experiment and mass spectrometry analysis. Moreover, probe L was applied in real water samples, test strips and fluorescent films to detect H2S successfully.

Evolving a high-performance bio-imaging tool derived from a compact fluorophore as well as creating a reaction-based fluorescent probe for precise determination of Ag+

Commonly, emission of red-region fluorescence is inherently in need of substantial conjugating segments and fused rings, which inescapably burdens fluorescent molecules in their size and molecular weight, impairing their membrane permeability and biocompatibility. Herein, a novel dicyanocoumarin 2-(7-(N,N-diethylamino)-4-(trifluoromethyl)–2H-chromen-2-ylidene)malononitrile (CouMal) has been evolved from traditional coumarin core for the first time. Without enlarged π-conjugation system, the low-molecular-weight CouMal unexpectedly possesses red-region fluorescence both in the solid and solution states with considerable brightness. Based on its compact structure and long-wavelength emission, the CouMal exhibits great cytomembrane penetrability and reduced cytotoxicity, enabling its promising application for intracellular imaging and in vivo imaging. Innovatively, enlightened by the flexible synthetic procedure of CouMal, a reaction-based sensing system named TC-Mal-Et3N, has been freshly designed for the precise detection of silver ion (Ag+). The limit of detection of this sensing system is as low as 60.9 nM. This Ag+-triggered composite probe also exhibits superb selectivity and short reaction time, which has been applied for the accurate quantification of both Ag+ and silver nanoparticles (AgNPs) in real-world representative samples.

Reaction-based fluorescent and chemiluminescent probes for formaldehyde detection and imaging.

Formaldehyde (FA), a reactive carbonyl species, is classified as Group 1 carcinogen by International Agency for Research on Cancer (IARC) in 2004. In addition, clinical studies have implicated that elevated levels of FA have been associated with different kinds of diseases, such as neurodegenerative diseases, diabetes, and chronic liver and heart disorders. However, in addition to the direct inhalation of FA in the environment, most organisms can also produce FA endogenously by demethylases and oxidases during the metabolism of amino acids and xenobiotics. Since FA plays an important role in physiological and pathological processes, developing reliable and efficient methods to monitor FA levels in biological samples is crucial. Reaction-based fluorescent/chemiluminescent probes have provided robust methods for FA detection and real-time visualization in living organisms. In this highlight, we will summarize the major developments in the structure design and applications of FA probes in recent years. Three main strategies for designing FA probes have been discussed and grouped by different reaction mechanisms. In addition, some miscellaneous reaction mechanisms have also been discussed. We also highlight novel applications of these probes in biological systems, which offer powerful tools to discover the diverse functions of FA in physiology and pathology processes.

A novel xanthene-based fluorescence turn-on probe for highly selective detection of Hg2+ in water samples and living cells

Mercury ions are highly toxic to living organisms and environment, hence Monitoring and quantification of mercury ions remain great significance. Herein, we have designed and synthesized a novel reaction-based fluorescence probe RANS for detection of Hg2+ by using a xanthene derivate as fluorophore and a thiosemicarbazide moiety as the recognition site. By applying mercury-promoted desulfurization reactions, probe RANS exhibited excellent capability of detecting Hg2+, such as rapid response (120 s), high sensitivity (0.019 μM) and good selectivity. Upon reaction with Hg2+, the thiosemicarbazide group of probe RANS formed 1,3,4-oxadiazole along with a significant color change from colorless to light green. Importantly, RANS has been successfully applied to selectively imaging Hg2+ in living cells with low cytotoxicity and quantitative detection of Hg2+ in real water samples.

An N-nitrosation reaction-based fluorescent probe for detecting nitric oxide in living cells and inflammatory zebrafish

Nitric oxide (NO), an essential biological messenger molecule, participates in various physiological and pathological processes. The sensitive and specific detection of NO is of great significance for understanding the biological function of NO. Here, we synthesized a fluorescent probe (Rho-NO) for highly selective detection of NO both in vitro and in vivo. The high selectivity of Rho-NO is attributed to the fact that NO is easily replaced by electron donor amino group to form N-nitrosation products, causing rhodamine spiro ring open and fluorescence emit. Rho-NO showed a good linear response to NO (0–100 µM) with a low detection limit (0.06 µM). Importantly, it exhibited excellent specificity for NO detection in human serum and was also applied for imaging NO in living cells and inflammatory model of zebrafish. This work proves the potential of Rho-NO in pathological research and disease diagnosis.

A novel selective probe for detecting glutathione from other biothiols based on the concept of Fluorescence Fusion

Biological thiols importantly regulate the intracellular redox activity and metabolic level, but many of the developed probes for biothiols are facing difficulty in effectively distinguishing GSH from Cys/Hcy due to the similarity in mechanism. In this work, despite the previous pattern of “Logic Gate”, we reported the concept of “Fluorescence Fusion” for the first time to achieve only one excitation-emission process. The exploited the probe, MZ-NBD, could quickly measure GSH in 10 min with a large Stokes shift (130 nm). Though the reacting mechanism was similar, only GSH could cause the “Fluorescence Fusion” with only one strong fluorescence response while Cys/Hcy caused two peaks. Adjusting the excitation wavelength could hardly split the fused peak into two. Though image recognition by artificial intelligence could easily distinguish the patterns of peaks, here we used the signal-treating method to realize the high selectivity towards GSH. Moreover, MZ-NBD could be utilized for rapid detection of GSH in living MCF-7 cells, which was more suitable for GSH than using the “Logic Gate” strategy. More than introducing a novel probe with the new concept, this work was meaningful as the linker of traditional reaction-based fluorescent probes and potential image recognition by artificial intelligence, thus led to various future researches in inter-disciplines.

Elegant cooperation of AIE, ESIPT and ICT effects into tetraarylimidazole-based fluorophore and its tuning for ratiometric detection of carbon monoxide

Carbon monoxide (CO) is an important gasotransmitter to regulate various physiological processes. Visualization of CO in situ would be conducive to uncover its physiological/pathological role. Unfortunately, most current CO fluorescent probes display aggregation-caused quenching (ACQ) characters. Herein, we reported a new fluorophore (MTPIM-HBT) coupling with aggregation-induced emission (AIE), excited-state intramolecular proton transfer (ESIPT), and intramolecular charge transfer (ICT) characteristics by attaching 2-(2′-hydroxyphenyl)benzothiazole (HBT) on methoxyl-substituted tetraarylimidazole derivative. Because of the synergistic effects, this fluorophore presented an all-purpose platform for developing ratiometric fluorescent probes with promising optimized properties. Hence, two reaction-based fluorescent probes (MTIPIM-AF-CO and MTPIM-AE-CO) for ratiometric detection and imaging of CO were rationally designed based on these unique scaffolds via one-step facilely synthesis. These probes have the merits of superior selectivity and excellent sensitivity with detection limit 47.5 nM and 32.2 nM, respectively. Significantly, the practicability of probes for visual detection of CO in air and ratiometric detection of exogenous and endogenous CO in living cells were successfully elucidated as well.

Reaction-based fluorescent silk probes with high sensitivity and selectivity to Hg2+and Ag+ions

Fluorescent functionalized silks have been prepared. These fluorescent silk probes exhibit obvious fluorescence quenching effects only to Hg2+or Ag+.

Development of a novel chromophore reaction-based fluorescent probe for biogenic amines detection.

Biogenic amines (BAs) are important biomarkers to monitor meat spoilage. However, the design of efficient BA fluorescent probes with distinct colorimetric and ratiometric fluorescent dual-channels is still a critical challenge because of similar chemical properties and basicity between BAs and other amines. Herein, pyrrolopyrrole cyanine (PPCy-1) is reported to display distinctly high reactivity toward BAs through an ultrasensitive irreversible chromophore reaction for the first time. The reaction mechanism is ascribed to synergistic aza-Michael addition and B-N detachment, followed by hydrolysis to produce low-conjugated diketopyrrolopyrrole and heteroaromatic acetonitrile compounds. As a result, colorimetric and ratiometric fluorescent dual-channel (Δλab = 188 nm and Δλem = 151 nm) signals and a limit of detection up to 62.1 nM level for BA solution are acquired. In addition, the colorimetric detection of volatile amine vapor using the PPCy-1-loaded filter paper, showing a color change from green to yellow, is feasible. A simple and cost-effective fluorescence "turn on" method using the filter paper or the CAD-40 resin loaded with PPCy-1 to detect TVB (total volatile bases) originating from shrimp spoilage is further demonstrated.

A Michael addition reaction-based fluorescent probe for malononitrile detection and its applications in aqueous solution, living cells and zebrafish.

It is highly desirable to detect malononitrile in organisms and human bodies owning to its inherent toxicity. With dicyanovinyl as the recognition site, a Michael addition reaction-based fluorescent probe Hcy-DCV was developed for malononitrile detection. A notable advantage of this probe is that it responds quickly to malononitrile without any additive to speed the sensing reaction. It has a good water solubility and the detection limit was determined to be 6.92 ppb in 100% aqueous solution. In particular, Hcy-DCV exhibited good selectivity towards malononitrile over other interfering substances including hydrazine and other active methylene compounds. The probe was applied successfully to quantitate malononitrile in pure water with satisfying recovery and relative standard deviation. Additionally, the ability of visualizing malononitrile by using probe-coated strip papers was displayed, which may facilitate the on-site detection of malononitrile. Moreover, the bioimaging of malononitrile in living H1975 cells and zebrafish larvae was also demonstrated. All the experimental results suggested the potential of Hcy-DCV for practical detection of malononitrile in both environmental and biological samples.

N-Alkylation of 2-methoxy-10H-phenothiazine revisited. A facile entry to diversely N-substituted phenothiazine-coumarin hybrid dyes

N-Alkylation of 2-methoxy-10H-phenothiazine, a valuable building block for the synthesis of bioactive compounds and reaction-based fluorescent probes, has been revisited aimed at introducing a substituent easily convertible into cationic or zwitterionic side chains. We focused our attention on the 3-dimethylaminopropyl group since its derivatization through reactions with various alkyl halides or sultones is a well-established and effective way to enhance polarity of diverse hydrophobic molecular scaffolds. This two-step functionalization approach was applied to the synthesis of novel phenothiazine-coumarin hybrid dyes whose spectral features, especially their NIR-I emission, have been determined in aqueous media with the ultimate goal of identifying novel fluorescent markers for bioanalytical applications, including fluorogenic detection of reactive oxygen species (ROS) through selective S-oxidation reaction of phenothiazine scaffold.

A NIR fluorescence probe having significant fluorescence turn-on signal at 700 nm and large Stokes shift for rapid detection of HOCl in vivo

With a hybrid coumarin-dicyanoisophorone as report unit and dimethylthiocarbamate as response site, a novel reaction-based fluorescence probe (CDCI-HClO) was synthesized herein for rapid detection of hypochlorous acid (HOCl). CDCI-HClO can respond to HOCl quickly (almost in seconds), selectively, and sensitively, and give an obviously enhanced signal of near-infrared fluorescence at 700 nm. The detection limit of CDCI-HClO for HOCl is about 4 nM. Moreover, with the merit of a large Stokes shift (190 nm), CDCI-HClO was successfully applied to the imaging of HOCl in live cell, zebrafish, and living mice. All results demonstrated that CDCI-HClO is a valuable new NIR fluorescence imaging tool to detect hypochlorous acid in living systems.

Reaction-based highly selective and sensitive monomer/polymer probes with Schiff base groups for the detection of Hg2+ and Fe3+ ions.

It is urgent and important to detect heavy metals in environments. In this work, novel reaction-based fluorescent probes were obtained by Schiff base reaction. The probes with Schiff base moiety (-C=N-) undergo irreversible hydrolysis in the presence of Hg2+ and Fe3+. They exhibit perfect high selectivity and sensitivity to Hg2+and Fe3+ ions. Upon the addition of Hg2+and Fe3+, fluorescence intensity of the probes increased notably. And the color of the probe changes from brown to bright green under UV light, which can realize "naked eye" detection. In addition, Schiff base group was introduced into polyurethane chain through condensation polymerization reaction. As expected, the fluorescent polyurethane probe (P2) maintained the detection performance of its original small molecules (BSD). Even more P2 showed a more sensitive detection effect than BSD, and the detection limits of P2 for Hg2+ and Fe3+ reach 0.19μM and 0.21μM, respectively. It indicates that Reaction-based probes could be a useful tool for the detection of Hg2+ and Fe3+.