A highly sensitive ratiometric fluorescence probe for sensing and imaging sulfite in food samples and living cells.

In this work, a ratiometric and chromogenic fluorescent probe 1 was synthesized for the detection of SO32-. The probe 1 at PBS (10 mM, pH = 7.4) presented a marked emission band at 661 nm. Upon addition of SO32- ions, a highly emissive adduct with a marked fluorescence at 471 nm were obtained through a Michael addition. The probe 1 displayed a noticeable fluorescence ratiometric response with a large shift (190 nm) in emission wavelength. The probe can quantitatively detect SO32- with high specificity, fast response (within 130 s) as well as low detection limit (13 nM), and a large Stokes shift (139 nm). Fluorescence imaging of HeLa cells indicated that 1 could be used for monitoring the intrinsically generated intracellular SO32- in living cells by ratiometric fluorescence imaging. Furthermore, 1 could be application in real water and sugar samples with high sensitivity and good recoveries.

A Biomimetic Upconversion Nanobait-Based Near Infrared Light Guided Photodynamic Therapy Alleviates Alzheimer's Disease by Inhibiting ß-Amyloid Aggregation.

Aberrant ß-amyloid (Aß) fibrillation is the key event in Alzheimer's disease (AD), the inhibition and degradation of which are recognized as a promising therapeutic strategy to alleviate the nerve damage of AD. Photodynamic therapy (PDT) holds great potential for modulation of Aß self-assembly, which is nevertheless limited by the inefficient utilization of reactive oxygen species (ROS). Herein, an erythrocyte membrane (EM)-modified core-shell upconversion nanoparticle (UCNP/Cur@EM) is designed and fabricated as a biomimetic nanobait to improve the PDT efficiency in AD. The UCNP with the outlayer of mesoporous silica is synthesized to load a high amount of the photosensitizer (curcumin), the unique optical feature of which can trigger curcumin to generate ROS upon near-infrared light (NIR) irradiation. Integration of EM enables the biomimetic nanobait to attract Aß peptides trapped in the phospholipid bilayer, restraining the growth of Aß monomers to form aggregates and improving the utilization rate of ROS to degrade the preformed Aß aggregates. In vivo studies demonstrate that UCNP/Cur@EM irradiated by NIR enables to decrease Aß deposits, ameliorates memory deficits, and rescues cognitive functions in the APP/PS1 transgenic mouse model. A biocompatible and controllable way is provided here to inhibit the amyloid protein-associated pathological process of AD.

A New Lysosome-Targeted NIR Fluorescent Probe for Specific Detection of Cysteine over Homocysteine and Glutathione.

In this paper, by modifying the thioxanthene-benzothiozolium fluorophore, BCy-Cys, a lysosome-targeted near-infrared (NIR) fluorescent probe was synthesized for the detection of cysteine (Cys) from homocysteine (Hcy)/glutathione (GSH). As expected, BCy-Cys exhibited high selectivity and high sensitivity for detection of Cys over Hcy/GSH, with an extremely low limit of detection at 0.31 µM, marked by obvious color changes. HRMS was conducted to confirm that the fluorescence intensity at 795 nm was significantly enhanced by the enhancement of intramolecular charge transfer (ICT). Importantly, BCy-Cys could be used to visualize both exogenous and endogenous lysosomal Cys, signifying its potential application in complex organismal systems.

A New Phenylazo-Based Fluorescent Probe for Sensitive Detection of Hypochlorous Acid in Aqueous Solution.

In this paper, we designed and synthesized a novel phenylazo-based fluorescent probe (RHN) for the sensing and imaging of hypochlorous acid (HClO) in mitochondria in living cells. In this process, HClO promoted the oxidation of the phenylazo group to generate a free Rhodol fluorophore moiety, which in turn restored strong fluorescence and realized the detection of HClO. As expected, RHN exhibited high selectivity, high sensitivity and rapid response, with detection limits as low as 22 nM (1.155 ng/mL). Importantly, the results of the cell imaging experiments indicated that RHN has the ability to image and sense HClO in mitochondria, which is of great significance for exploration of the specific role of HClO in both the immune system and diseases.

Lysosomes-targeting near-infrared fluorescent probe for the detection of pH in living cells.

A new near-infrared fluorescent probe Qcy-OH with a turn-on mechanism based on a acceptor-donor-acceptor (ADA) π-electron system that can undergo an internal charge transfer to form new fluorochromes was developed for monitoring pH fluctuations in biological systems. With the pH value increases, Qcy-OH exhibited a strong pH-sensitive response from 1.1 to 9.0 (pKa = 4.41) and a good linear response from pH 3.0 to 5.5 in aqueous solution and in living cells. In addition, based on the phenolic and ketonic structure interconversions of the fluorescent backbone, Qcy-OH showed rapidly and reversibly response to pH with high selectivity. Because the introduce of two benzothiazolium units in the backbone of the probe as the electron-withdrawing group to enhance the selectivity for intracellular lysosomes, the probe had been applied successfully for tracking lysosomal pH changes and the fluorescence changes showed a good linear enhancement from pH 3.0 to 7.4 in living cells. We believe that this sulfur-driving lysosomes-targeting ability of the probe affords a guarantee for achieving long-term monitoring of lysosomal pH biology by the elimination of harmful protonating effects of the probe.

Mitochondria-targeted fluorescent probe for visualization of exogenous and endogenous methylglyoxal in living cells.

An activatable mitochondria-targeted fluorescent probe Hcy-OPD was synthesized for the detection of methylglyoxal (MGO). For the introduction of a preorganized isopropylamino group on the aromatic o-diamine framework to regulate the hindrance effect, Hcy-OPD showed high selectivity and sensitivity (0.22 µM) for monitoring MGO. The probe can be applied successfully in the imaging of exogenous and endogenous MGO in living cells.

Rational design of a large Stokes shift xanthene-benzothiozolium dyad for probing cysteine in mitochondria.

Xanthene-modified cyanine dyes are considered to be an effective means to extend the emission wavelength and improve the photo-stability of cyanine dyes. However, the fluorophores obtained by this strategy generally have narrow Stokes shifts, which severely limits their application in biological imaging. Herein, a reasonable design strategy is adopted to provide an effective strategy to commendably improve the Stokes shift of xanthene-benzothiozolium fluorophores via the simultaneous expansion of a molecular π-conjugated system and heteroatomic substitution. Combined with density functional theory calculation guidance, the O atom is replaced with the S atom in the xanthene moiety and a π-conjugated benzene ring is introduced in the benzothiozolium moiety. Surprisingly, the results of the spectroscopic experiment showed that the fluorescence emission wavelength of PhCy-OH was extended to 803 nm, and the Stokes shift was improved to 68 nm. PhCy-Cys can effectively distinguish Cys from homocysteine (Hcy) and glutathione (GSH) with an extremely low detection limit of 0.166 µM. Importantly, PhCy-Cys has the ability to image endogenous Cys in mitochondria, providing the potential for exploring the specific function and mechanism of Cys in regulating oxidative stress in mitochondria.

A highly sensitive sensor for colorimetric detection of palladium(II) in lysosomes and its applications.

Considering the scarcity of palladium ion probes with subcellular organelle targeting, especially probes with near-infrared (NIR) emission wavelength fluorophores, our group has been working to overcome this problem and looking forward to providing potential practical tools for exploring the toxicity of palladium ions at the subcellular level. In this paper, a novel colorimetric and NIR fluorescent probe, BHCy-Pd, for the specific detection of palladium ions (Pd2+) in lysosomes via an internal charge-transfer (ICT) mechanism was designed and synthesized. As expected, BHCy-Pd exhibited a rapid, selective, and sensitive response for palladium with an ultralow limit of detection at 5.9 nM, accompanied by a distinct color change from purple to blue. Furthermore, BHCy-Pd can be made into a simple test strip for rapid and easy detection of Pd2+ in practical applications. Importantly, BHCy-Pd is capable of specific distribution in lysosomes, and thus can detect Pd2+ in real-time, thereby providing a potential tool for studying the cytotoxicity of Pd2+ ions at the subcellular level.

A new near-infrared fluorescent probe for sensing extreme acidity and bioimaging in lysosome.

Since the intracellular pH plays an important role in the physiological and pathological processes, however, the probes that can be used for monitoring pH fluctuation under extreme acidic conditions are currently rare, so it is necessary to construct fluorescent probes for sensing pH less than 4. In this work, we developed a new near-infrared (NIR) fluorescent probeCy-SNNfor sensing pH fluctuation under extremely acidic conditions. For the preparation of this probe, benzothiozolium moiety was chosen as lysosomal targeting unit and NIR fluorophore, and barbituric acid moiety was fused in the polymethine chain of probe to introduce protonation center. Surprisingly, on the basis of the balance of quaternary ammonium salts and free amines, the pkavalue ofCy-SNNwas calculated as low as 2.96, implying thatCy-SNNcan be used in acidic conditions with pH < 4. Moreover,Cy-SNNexhibited highly selective response to H+over diverse analytes in real-time with dependable reversibility. Importantly,Cy-SNNcan be used to specifically target lysosome, providing potential tools for monitoring the function of lysosome in autophagy process.

Novel near-infrared spectroscopic probe for visualizing hydrogen sulfide in lysosomes.

Considering the scarcity of hydrogen sulfide (H2S) probes with subcellular organelle targeting, especially probes with near-infrared (NIR) emission wavelengths fluorophores, our group has been working to overcome this problem and looking forward to providing potential practical tools for exploring the relationship between the physiology and pathology of H2S at subcellular level. In this paper, a novel colorimetric and NIR fluorescent probe SHCy-H2S for the specific detection of H2S in lysosome over other biological thiols was designed and synthesized. The xanthene-benzothiozolium fluorophore was chosen to provide fluorescence emission maxima over 735 nm, and 2,4-dinitrophenyl group was chosen as fluorescence quenching group and specific H2S response site. Impressively, SHCy-H2S exhibited high selectivity, fast response and detection limit as low as 0.116 µM for H2S, marked obvious color changes in naked-eye and fluorescence. Specially, SHCy-H2S was capable of specifically imaging endogenous lysosomal hydrogen sulfide, providing a potential tool for exploring the function of H2S at subcellular level.

Novel lysosome-targeted fluorescent molecular rotors based on a cyanine-like modular system and their application in living cells.

Two novel fluorescence molecular rotors DpIn and NaIn were designed and synthesized involving of indolium units linked with meta-diphenol or ortha-naphthalenediol moiety, respectively. They underwent intramolecular charge transfer to form a cyanine-like modular system at a physiological pH. In glycerol aqueous solutions, the probe DpIn exhibited NIR strong emission (3-fold) at ca. 700 nm, while the probe NaIn displayed a turn-on emission (8-fold) with a larger Stokes shift (⊿λ ≈ 97 nm). The HeLa cell imaging experiments indicated probe DpIn and NaIn both exhibited excellent selectivity for staining intracellular lysosomes instead of mitochondria. 1H NMR spectra revealed that more electrons were accumulated around benzene ring of indolium groups, which could be the evidence for its basic character leading to the lysosomes targeted staining. Furthermore, the probe NaIn proved to be an ideal lysosome-targeting tracer for monitor the changes of viscosity caused by stimuli in living cells.

An NADH-selective and sensitive fluorescence probe to evaluate living cell hypoxic stress.

Cellular disease and senescence are often accompanied by an imbalance in the local oxygen supply. Under hypoxia, mitochondrial NADH and FADH2 cannot be oxidized by the mitochondrial electron transport chain, which leads to the accumulation of reducing equivalents and subsequent reduction stress. Detecting changes in intracellular NADH levels is expected to allow an assessment of stress. We synthesized a red fluorescent probe, DPMQL1, with high selectivity and sensitivity for detecting NADH in living cells. The probe DPMQL1 has strong anti-interference abilities toward various potential biological interferences, such as metal ions, anions, redox species, and other biomolecules. In addition, its detection limit can reach the nanomolar level, meaning it can display small changes in NADH levels in living cells, so as to realize the evaluation of cell-based hypoxic stress.

A lysosome-targeted fluorescent probe for the specific detection and imaging of formaldehyde in living cells.

We presented herien the rational design, synthesis, and photophysical property studies of the lysosome-targeted fluorescence FA probe NP-Lyso, an isopropyl group modified ortho-diaminonaphthalimide derivative. After the reaction of FA and ortho-phenylenediamine modified with the isopropyl group in NP-Lyso, the probe exhibited favorable features such as a large fluorescence enhancement, specific selectivity and high sensitivity for the detection of FA. More importantly, NP-Lyso could be used to detect and image endogenous FA in lysosomes. In light of these prominent properties, we envision that NP-Lyso will be an efficient optical imaging approach for investigating the biofunctions of FA in living systems.

A lysosome-targeting viscosity-sensitive fluorescent probe based on a novel functionalised near-infrared xanthene-indolium dye and its application in living cells.

The viscosity of lysosomes plays a significant role in modulating biological processes and reflects the status and function of this kind of organelle, e.g., locations, morphologies, and components. Herein, we constructed a novel near-infrared (NIR) lysosome-targeting viscosity probe, Lyso-cy, for monitoring viscosity changes in biological systems. The Lyso-cy probe showed very strong fluorescence emission at around 710 nm in viscous media. The fluorescence intensity of Lyso-cy increased 122-fold from when in water to when in 95% glycerol. Moreover, Lyso-cy proved to be an ideal lysosome-targeting tracer for monitoring fluctuations in the viscosity of a living cell with high spatial and temporal resolution under laser confocal microscopy.

Mitochondria-targeted fluorescent probe for imaging endogenous hydrogen sulfide in cellular antioxidant stress.

Hydrogen sulfide (H2S) is believed to play an important role in maintaining cellular redox homeostasis and avoiding oxidative damage caused by abnormally raised ROS levels. Highly selective and sensitive fluorescent probes for the detection and imaging of endogenous H2S in living cells over other biological thiols are desirable. Herein, we developed a mitochondria-targeted fluorescent probe L, a thioxanthene-benzo[e]indolium derivative, for the discrimination of H2S from other chemically similar biothiols. Based on the nucleophilic addition of H2S and the charged electron-deficient C[double bond, length as m-dash]N double bond within the benzo[e]indolium moiety, the generation of L-HS with a shortened π-conjugated system led to significant spectral changes in the visible region. Importantly, the probe L with mitochondria-targeting ability has been successfully used for imaging the endogenous H2S biosynthesized from Cys and GSH and in cellular antioxidant stress.

Novel near-infrared fluorescent probe with a large Stokes shift for sensing hypochlorous acid in mitochondria.

Hypochlorous acid (HOCl) plays a crucial role in various physiological and pathological processes. However, it is still a challenge to design a xanthene-based near-infrared (NIR) fluorescent probe with a large Stokes shift for sensing HOCl. In this work, a novel mitochondria-targeted fluorescent probe, MXS, with a large Stokes shift based on a xanthene-hemicyanine dyad structure, has been successfully designed and synthesized for the specific detection of HOCl. Gratifyingly, the peak-to-peak Stokes shift of MXS was found to be 130 nm, which was obviously larger than those of conventional rhodamine dyes and most reported xanthene-based hypochlorous acid probes. As expected, MXS exhibited high selectivity, high sensitivity, and fast response time (30 s) for the detection of HOCl via a specific HOCl-promoted intramolecular charge transfer process. The detection limit of MXS for HOCl is calculated to be as low as 72 nM, enabling its use within the physiological concentration range of HOCl (5-25 µM). Importantly, MXS is able to permeate cell membranes and accumulate in the mitochondria, which is convenient for monitoring the variation of hypochlorous acid concentration in the mitochondria of living cells.

A novel near-infrared fluorescent probe with large stokes shifts for sensing extreme acidity and its application in bioimaging.

In this work, we reported a novel near-infrared (NIR) fluorescent probe RQNN with large Stokes shift (98 nm) for monitoring pH under extremely acidic conditions. For the preparation of this probe, a 1,4-diethylpiperazine moiety was introduced in rhodamine scaffold to tune the electron-donating character, and an o-phenylenediamine was introduced in spironolactone to provide larger steric hindrance. The deprotonated-protonated equilibrium between RQNN, RQNN-H+ and RQNN-H++ were evaluated in different pH by absorption and emission spectra. As expected, RQNN exhibited lower pka values (pka1 = 4.83, pka2 = 2.99), indicating that the probe can be used in extremely acidic pH. Moreover, RQNN possessed highly selective response to H+ over essential metal ions and biologically related redox molecules, high photo-stability, rapid response time, and excellent reversibility. Importantly, the probe had excellent cell membrane permeability and was further applied successfully to monitor pH fluctuations in live cells.

A novel near-infrared fluorescent probe with an improved Stokes shift for specific detection of Hg2+ in mitochondria.

The mercury ion (Hg2+), one of the most notorious heavy metal ions, not only causes environmental pollution, but also endangers human health. There is evidence that Hg2+ tends to accumulate in the mitochondria and to induce apoptosis. However, mitochondria-targeted near-infrared (NIR) fluorescent probes with large Stokes shifts are still scarcely described for the specific detection of Hg2+. In this work, a novel near-infrared fluorescent probe JRQNS with a large Stokes shift (78 nm) was reported, and applied for sensitive and specific detection of Hg2+ in mitochondria by incorporating an additional amine group with fused rings to rhodamine dyes to enhance the electron donating ability of amine groups. As expected, the probe exhibited high selectivity and sensitivity to Hg2+ with a detection limit as low as 1.5 nM and fast response times (3 min), revealing that JRQNS could be used as a practical probe for quantitative detection of Hg2+ in real-time. Importantly, JRQNS can be used as an efficient organelle-targeting probe for imaging Hg2+ in the mitochondria of living cells, and thus detect Hg2+ in real-time there. The application of the probe for its selective localization in mitochondria along with the nanomolar level of limit of detection to Hg2+ ions provided a potential tool for studying the cytotoxic mechanisms of Hg2+.

A lysosome-targeted near-infrared fluorescent probe for imaging endogenous cysteine (Cys) in living cells.

Cysteine (Cys) is one of the most important essential biothiols in lysosomes. Highly selective probes for specific detection and imaging of lysosomal Cys over other biological thiols are rare. Herein, we developed a lysosome-targeted near-infrared fluorescent probe SHCy-C based on a novel NIR-emitting thioxanthene-indolium dye. Due to the turn-on fluorescence response elicited by the intramolecular charge transfer (ICT) processes before and after the reaction with Cys, probe SHCy-C exhibits high selectivity and sensitivity (16 nM) for the detection of Cys. More importantly, probe SHCy-C is found to precisely target lysosomes and achieves the "turn-on" detection and imaging of endogenous Cys in lysosomes.

A near-infrared fluorescent probe based on a novel rectilinearly π-extended rhodamine derivative and its applications.

We designed and synthesized a novel near-infrared (NIR) mitochondria-targeted fluorescent probe RQNA for the specific detection of mitochondrial Cu2+ because mitochondria are important reservoirs of intracellular copper. For the preparation of this probe, a novel π-extended fluorescent xanthene dye RQN was firstly synthesized via an intramolecular nucleophilic substitution of aromatic hydrogen (SNArH) strategy. Then, probe RQNA was prepared by the reaction of RQN and hydrazine hydrate, followed by treatment with acetone. RQNA exhibited selectivity, sensitivity (22 nM), and fast response time (20 s) for the detection of Cu2+via a specific Cu2+-triggered ring-opening and hydrolysis cascade reaction. RQNA is cell-membrane permeable and mitochondria-targetable, and can be used for monitoring mitochondrial Cu2+ in living cells.