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Mitochondria play a crucial role in maintaining cellular homeostasis, and the depolarization of mitochondrial membrane potential (MMP) is an important signal of apoptosis. Additionally, protein misfolding and aggregation are closely related to diseases including neurodegenerative diseases, diabetes, and cancers. However, the interaction between MMP changes and disease-related protein aggregation was rarely studied. Herein, we report a novel "turn-on" fluorescent probe MitoRhB that specifically targets to mitochondria for Cu2+ detection in situ. The fluorescence lifetime (τ) of MitoRhB exhibits a positive correlation with MMP changes, allowing us to quantitatively determine the relative MMP during SOD1 (A4 V) protein aggregation. Finally, we found that (1) the increasing concentrations of copper will accelerate the depolarization of mitochondria and reduce MMP; (2) the depolarization of mitochondria can intensify the degree of protein aggregation, suggesting a new routine of copper-induced cell death mediated through abnormal MMP depolarization and protein aggregation.
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Cobre , Colorantes Fluorescentes , Potencial de la Membrana Mitocondrial , Agregado de Proteínas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Cobre/química , Cobre/metabolismo , Humanos , Colorantes Fluorescentes/química , Mitocondrias/metabolismo , Mitocondrias/química , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/química , Células HeLaRESUMEN
Aggrephagy describes lysosomal transport and degradation of protein aggregates via cellular macroautophagy, a key mechanism to prevent neurodegenerative diseases. Here, we develop a dual-probe method to visualize the aggrephagy process and resolve its viscosity heterogeneity using fluorescence lifetime imaging (FLIM). The dual-probe system consists of (1) a near-infrared lysosomal targeting FLIM probe (Lyso-P1) that is derived from a rhodamine scaffold with a tailored pKa value to accommodate an acidic lysosomal environment and (2) a green BODIPY-based FLIM probe (Agg-P2) that reports on degradation of cellular aggregates via HaloTag. Both probes exhibit acid-resistant, viscosity-dependent fluorescence intensity and lifetime (τ) responses, which are ready for intensity- and FLIM-based imaging. Photochemical, theoretical, and biochemical characterizations reveal the probes' mechanism-of-actions. In cells, we exploit Lyso-P1 and Agg-P2 to simultaneously quantify both lysosomal and protein aggegates' viscosity changes upon the aggrephagy process via FLIM. We reveal orthogonal changes in microenvironmental viscosities and morphological heterogeneity upon various cellular stresses. Overall, we provide an imaging toolset to quantitatively study aggrephay, which may benefit screening of aggrephay modulators for disease intervention.
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Colorantes Fluorescentes , Lisosomas , Imagen Óptica , Viscosidad , Colorantes Fluorescentes/química , Humanos , Lisosomas/química , Lisosomas/metabolismo , Agregado de Proteínas , Células HeLa , Compuestos de Boro/química , Rodaminas/químicaRESUMEN
Fluorescence lifetime imaging has been a powerful tool for biomedical research. Recently, fluorescence lifetime-based multiplexing imaging has expanded imaging channels by using probes that harbor the same spectral channels and distinct excited state lifetime. While it is desirable to control the excited state lifetime of any given fluorescent probes, the rational control of fluorescence lifetimes remains a challenge. Herein, we chose boron dipyrromethene (BODIPY) as a model system and provided chemical strategies to regulate the fluorescence lifetime of its derivatives with varying spectral features. We find electronegativity of structural substituents at the 8' and 5' positions is important to control the lifetime for the green-emitting and red-emitting BODIPY scaffolds. Mechanistically, such influences are exerted via the photo-induced electron transfer and the intramolecular charge transfer processes for the 8' and 5' positions of BODIPY, respectively. Based on these principles, we have generated a group of BODIPY probes that enable imaging experiments to separate multiple targets using fluorescence lifetime as a signal. In addition to BODIPY, we envision modulation of electronegativity of chemical substituents could serve as a feasible strategy to achieve rational control of fluorescence lifetime for a variety of small molecule fluorophores.
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Phase separation is a common biological phenomenon in the liquid environment of organisms. Phase separation has been shown to be a key cause of many existing incurable diseases, such as the protein aggregates formed by phase separation of Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, etc. Tracking the occurrence of phase separation in vivo is critical to many disease detection methods and solving many treatment problems. Its physicochemical properties and visual detection methods have flourished in the last few years in chemical biology, among which the fluorogenic toolbox has great application potential compared to the traditional detection methods that cannot visualize the phase separation process intuitively, but just show some parameters indirectly. This paper reviews the mechanism and disease correlation proven in recent years for phase separation and analyzes the detection methods for phase separation, including functional microscope imaging techniques, turbidity monitoring, macromolecule congestion sensing, in silico analysis, etc. It is worth mentioning that the qualitative and quantitative analysis of aggregates formed by phase separation using in vitro parameters has successfully provided basic physical and chemical properties for phase separation aggregates, and is an important cornerstone for researchers to carry forward the past and break through the existing technical shackles to create new in vivo monitoring methods such as fluorescence methodology. Crucially, fluorescence methods for cell microenvironment imaging based on different mechanisms are discussed, such as AIE-based probes, TICT-based probes and FRET-based probes, etc.
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Enfermedad de Alzheimer , Humanos , BiologíaRESUMEN
Degenerative diseases are closely related to the changes of protein conformation beyond the steady state. The development of feasible tools for quantitative detection of changes in the cellular environment is crucial for investigating the process of protein conformational variations. Here, we have developed a near-infrared AIE probe based on the rhodamine fluorophore, which exhibits dual responses of fluorescence intensity and lifetime to local viscosity changes. Notably, computational analysis reveals that NRhFluors fluorescence activation is due to inhibition of the RACI mechanism in viscous environment. In the chemical regulation of rhodamine fluorophores, we found that variations of electron density distribution can effectively regulate CI states and achieve fluorescence sensitivity of NRhFluors. In addition, combined with the AggTag method, the lifetime of probe A9-Halo exhibits a positive correlation with viscosity changes. This analytical capacity allows us to quantitatively monitor protein conformational changes using fluorescence lifetime imaging (FLIM) and demonstrate that mitochondrial dysfunction leads to reduced protein expression in HEK293 cells. In summary, this work developed a set of near-infrared AIE probes activated by the RACI mechanism, which can quantitatively detect cell viscosity and protein aggregation formation, providing a versatile tool for exploring disease-related biological processes and therapeutic approaches.
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In this study, we synthesized an amorphous metal-organic framework by adjusting the concentration of precursors, and established a two-enzyme system consisting of lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH), which successfully achieved coenzyme recycling, and applied it to the synthesis of D-phenyllactic acid (D-PLA). The prepared two-enzyme-MOF hybrid material was characterized using XRD, SEM/EDS, XPS, FT-IR, TGA, CLSM, etc. In addition, reaction kinetic studies indicated that the MOF-encapsulated two-enzyme system exhibited faster initial reaction velocities than free enzymes due to its amorphous ZIF-generated mesoporous structure. Furthermore, the pH stability and temperature stability of the biocatalyst were evaluated, and the results indicated a significant improvement compared to the free enzymes. Moreover, the amorphous structure of the mesopores still maintained the shielding effect and protected the enzyme structure from damage by proteinase K and organic solvents. Finally, the remaining activity of the biocatalyst for the synthesis of D-PLA reached 77% after 6 cycles of use, and the coenzyme regeneration still maintained at 63%, while the biocatalyst also retained 70% and 68% residual activity for the synthesis of D-PLA after 12 days of storage at 4 °C and 25 °C, respectively. This study provides a reference for the design of MOF-based multi-enzyme biocatalysts.
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Estructuras Metalorgánicas , Estructuras Metalorgánicas/química , Cinética , Lactato Deshidrogenasas/metabolismo , Glucosa Deshidrogenasas/metabolismo , Biocatálisis , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Cysteine (Cys) is a vital amino acid in the body, and its abnormal expression level is associated with many diseases. In this study, a novel fluorescent probe ACHB was synthesized, showing high selectivity, anti-interference ability and achieving accurate detection of cysteine. Different from most previous off-on probes, ACHB showed an on-off fluorescence response to Cys. Acrylic ester was used as a recognizer while green fluorescence protein (GFP) chromophore derivative 4-hydroxybenzylidene-imidazolinone (HBI) was used as the fluorophore. The addition of Cys leads to the hydrolysis of the red-emitting probe (613 nm), releasing a precursor with a lower fluorescent signal and showing an on-off spectral signal, which was ideal for obtaining sensitive detection with high specificity. Furthermore, the probe was successfully applied for simultaneous determination of cysteine (Cys) in living cells and biological sample (mouse serum). In conclusion, probe ACHB is a promising tool to display the intracellular cysteine concentration level, providing a good visualization method for clinical diagnosis and scientific basic research.
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Cisteína , Colorantes Fluorescentes , Humanos , Ratones , Animales , Cisteína/química , Colorantes Fluorescentes/química , Fluorescencia , Proteínas Fluorescentes Verdes , Células HeLaRESUMEN
The accumulation of pesticide residues may cause harm to the human body and the environment. Traditional chromatographic methods are limited by stringent testing conditions, so it is necessary to develop convenient and efficient methods for pesticide residue detection. Fluorescence assays have great potential in the development of portable detection tools due to their fast response and intuitive visualization. In this paper, we reviewed nanomaterial-based fluorescent probes for pesticide residue detection that have been reported in recent years, including small molecule probes, metal-organic framework fluorescent probes, fluorescent quantum dot probes, and nanocluster probes. In addition, we describe the design strategy, detection mechanism, and practical application of these probes in detail. The latest progress and application strategies of fluorescent probe detection methods based on nanomaterials are comprehensively discussed, and prospects are proposed.
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Nanoestructuras , Residuos de Plaguicidas , Puntos Cuánticos , Colorantes Fluorescentes/química , Humanos , Residuos de Plaguicidas/análisisRESUMEN
In this study, we synthesized a novel biocatalyst by encapsulating lactate dehydrogenase (LDH) in the metal-organic framework ZIF-90 by one-pot embedding. It showed strong biological activity for efficient synthesis of D-phenyllactic acid (D-PLA). The morphology and structure of LDH@ZIF-90 was systematically characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, confocal laser scanning microscopy (CLSM) and gas sorption. According to thermogravimetric analysis (TGA), the enzyme loading of the biocatalyst was 3 %. The Michaelis-Menten constant (Km) and maximal reaction rate (Vmax) of LDH@ZIF-90 were similar to those of free LDH, which proved that ZIF-90 had good biocompatibility to encapsulate LDH. At the same time, LDH@ZIF-90 exhibited enhanced tolerance to temperature, pH and organic solvents, and its reusability was greatly improved with 68 % of initial enzyme activity remaining after 7 rounds of recylcing. Overall, LDH encapsulated in ZIF-90 may be an economically competitive and environmentally friendly novel biocatalyst for the synthesis of D-PLA.
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L-Lactato Deshidrogenasa , Estructuras Metalorgánicas , Lactatos , Estructuras Metalorgánicas/química , Poliésteres , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Cancer is a disease that seriously threatens human health. Over the past few decades, researchers have continued to find ways to cure cancer. Currently, the most commonly used clinical techniques are surgery, chemotherapy, radiotherapy and so on. Among them, photodynamic therapy (PDT) has received extensive attention due to its better therapeutic effect and lower side effects. However, the inherent microenvironmental hypoxia of tumor tissue leads to unsatisfactory therapeutic effects. Therefore, researchers have conducted in-depth research on the hypoxia problem in PDT therapy. This review classified photodynamic therapy according to the response mechanism and summarized the strategies developed to overcome tumor hypoxia in recent years. Among them, research strategies can be divided into five types: type I PDT photosensitizers, introducing exogenous oxygen, O2 carriers using nanomaterials, generating endogenous oxygen by catalytic reactions, and combination with prodrugs that inhibit the consumption of endogenous oxygen. Finally, we also list some studies using combination therapy, such as microbes, photothermal therapy, etc. It can be guaranteed that the review can provide theoretical guidance for the development of anti-hypoxic PDT tools.
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Neoplasias , Fotoquimioterapia , Biología , Línea Celular Tumoral , Humanos , Hipoxia , Neoplasias/tratamiento farmacológico , Oxígeno , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Microambiente TumoralRESUMEN
Photodynamic therapy (PDT) is a promising noninvasive treatment that has drawn great attention. However, the hypoxic environment in tumors seriously limits the therapeutic effect of oxygen-dependent chemicals and PDT. Herein, a versatile nanocomposite DF-BODIPY@ZIF-8 with oxygen-generating ability was developed based on zeolitic imidazolate framework-8 (ZIF-8) by loading the near-infrared photosensitizer DF-BODIPY to overcome hypoxia-induced drug resistance in cancer therapy. ZIF-8 can catalyze the decomposition of hydrogen peroxide in tumors and increase the dissolved oxygen concentration, resulting in a significant improvement in PDT efficacy. Additionally, we found that enhancing the electronegativity of substituents can effectively reduce the energy level difference (ΔEst) between the minimum singlet state (S1) and the lowest triplet state (T1), leading to the enhancement of the singlet oxygen quantum yield. In vitro experiments suggested that DF-BODIPY@ZIF-8 indeed had a higher singlet oxygen quantum yield and better tumor cell phototoxicity than free DF-BODIPY. In vivo experiments also demonstrated that DF-BODIPY@ZIF-8 could effectively eliminate 4T1 tumors under light irradiation. Thus, we conclude that increasing the electronegativity of substituents and introducing a ZIF-8 material can effectively improve the singlet oxygen quantum yield and overcome the hypoxia limitations for high-efficiency PDT.
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Estructuras Metalorgánicas , Nanocompuestos , Neoplasias , Fotoquimioterapia , Zeolitas , Humanos , Fármacos Fotosensibilizantes/química , Fotoquimioterapia/métodos , Estructuras Metalorgánicas/química , Oxígeno Singlete/química , Peróxido de Hidrógeno , Hipoxia/tratamiento farmacológico , Oxígeno , Neoplasias/tratamiento farmacológicoRESUMEN
Herein, a lysosomal targeting LysFP@ZIF-8 metal-organic framework (MOF) was fabricated using fluorescent protein chromophore-based probe (LysFP) for selectively detection of carboxylesterase 1 (CES1) in living cells. Unlike the regular small molecule fluorescent probes, LysFP@ZIF-8 showed wide range pH tolerabiligy, high selectivity and sensitivity to CES1 in bio-samples, and was successfully applied to achieve the visual monitoring of CES1 activity in living cells. Low detection limit and high fluorescence quantum yield was calculated as 79 ng/mL and 0.76 for LysFP@ZIF-8, respectively. Furthermore, LysFP@ZIF-8 can also serve as a fluorescence indicator of organophosphates pesticide exposure in the way of hydrolyzing the carboxylic acid ester group in LysFP. This type of probe can inspire the development of fluorescent tools for further explore many pathological processes.
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Estructuras Metalorgánicas , Hidrolasas de Éster Carboxílico , Colorantes Fluorescentes , Lisosomas , OrganofosfatosRESUMEN
Glycans and glycoconjugates in nature include macromolecules with important biological activities and widely distributed in all living organisms. These oligosaccharides and polysaccharides play important roles in a variety of normal physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation, and immune response. However, the heterogeneity of naturally occurring glycans and glycoconjugates complicates detailed structure-activity relationship studies resulting in an incomplete understanding of their mechanisms of action and hindering further applications. Therefore, the synthesis of homogeneous, or nearly homogeneous, structurally defined glycans is of great significance for the development of carbohydrate-based drugs. One-pot synthesis represents the fastest strategy to assemble oligosaccharides and polysaccharides, although unfortunately, typically relies on random assembly. In this review, we examine the progress that has been made in the controlled one-pot synthesis of homogeneous or nearly homogeneous oligosaccharides and polysaccharides providing a broad spectrum of options to access size-controlled glycan products.
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Carbohidratos/química , Química Farmacéutica/tendencias , Glicoconjugados/química , Oligosacáridos/química , Polisacáridos/química , Secuencia de Carbohidratos , Técnicas de Química Sintética , Química Farmacéutica/métodos , Glicosilación , Humanos , Inflamación , Metástasis de la Neoplasia , Tamaño de la Partícula , PolímerosRESUMEN
Designing fluorescent probe for detecting carboxylesterase 1 is remains challenging. Herein, a red emission human carboxylesterase 1 (CES1) probe (CAE-FP) was synthesized based on fluorescent protein chromophore. Probe CAE-FP can specific detect human CES1 with high selectively. The fluorescence quantum yield was calucated as 0.19. The carboxylic acid ester in CAE-FP could be easily hydrolyzed by CES1 under physiological conditions, and this process could induce the obvious fluorescence signal in red emission region. The detection limit of CES1 was calculated as 84.5 ng/mL. Due to the biological detoxification mechanism of carboxylesterase and the obvious inhibitory effect of pesticides on its activity, CAE-FP was applied to detect carbamate pesticide and have achieved good application results. Since fluorescent protein chromophore has excellent biocompatibility, probe CAE-FP with good cell membrane permeable and was successfully applied to monitor the real activities of CES1 in living cells. In summary, this is one of the few reported fluorescent probes that can specific detect the real-time activity of CES1 in biological samples. Besides, we first applied the fluorescent protein chromophore to construct the specific target enzyme probe. This work would contribute to further investigate CES1-associated physiological and pathological processe.
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Carboxilesterasa , Plaguicidas , Carbamatos , Hidrolasas de Éster Carboxílico , Ésteres , Colorantes Fluorescentes , HumanosRESUMEN
In this work, a red emission fluorescent probe CBZ-BOD@zeolitic imidazolate framework-8 (ZIF-8) was fabricated based on metal-organic frameworks (MOFs) for detecting carboxylesterase 1 (CES1). The small molecule probe CBZ-BOD was first synthesized and then used to prepare the functionalized MOF material. ZIF-8 was chosen as an encapsulation shell to improve the detection properties of CBZ-BOD. Using this unique porous materials, ultrasensitive quantification of CES1 and chlorpyrifos was successfully realized. The low detection limit and high fluorescence quantum yield were calculated as 1.15 ng/mL and 0.65 for CBZ-BOD@ZIF-8, respectively. CBZ-BOD@ZIF-8 has good biocompatibility and was successfully applied to monitor the activity of CES1 in living cells. A molecular docking study was used to explore the binding of CES1 and CBZ-BOD, finding that CES1 can bind with the probe before and after hydrolysis. This type of materialized probe can inspire the development of fluorescent tools for further exploration of many pathological processes.
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Compuestos de Boro/química , Hidrolasas de Éster Carboxílico/análisis , Cloropirifos/análisis , Colorantes Fluorescentes/química , Estructuras Metalorgánicas/química , Zeolitas/química , Colorantes Fluorescentes/síntesis química , Humanos , Estructuras Metalorgánicas/síntesis química , Simulación del Acoplamiento Molecular , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The low-molecular weight biothiols like glutathione (GSH) and cysteine (Cys) play many important roles in various biological processes, the imbalance of biothiols level will lead to many diseases. However, methods that can selectively detect the GSH and Cys was rarely reported because of the similar reactivity and structure. Here, a fluorogenic method was presented to selectively detect the GSH and Cys in vitro using probe ML-FP based on fluorescent protein mimics. Probe ML-FP is a fluorescence turn on probe with lysosome targeting capacity. 2,4-dinitrobenzenesulfonyl serves as fluorescence quench and detection group in probe ML-FP. Differentiating the GSH and Cys was realized benefit from the different reaction time as well as fluorescence response between probe and target species. Low detection limit (4.98â¯nM for Cys and 4.39â¯nM for GSH) as well as fast response time was founded in this work. Probe ML-FP possess excellent biocompatibility due to fluorescent protein chromophore and was successfully used for bioimaging in living cells.
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Cisteína/metabolismo , Colorantes Fluorescentes/farmacología , Glutatión/metabolismo , Proteínas Fluorescentes Verdes/farmacología , Lisosomas/metabolismo , Línea Celular Tumoral , Fluorescencia , HumanosRESUMEN
Hypochlorous acid/hypochlorite (HOCl/OCl-), as one of the most important reactive oxygen species (ROS), plays an important role in various physiological and pathological processes. Nonproperly located or abnormal concentration of OCl-, however, is associated with many diseases. Thus, developing the fluorescent probe for detecting OCl- is of great significance. To this end, in last decade, many fluorescent probes have been developed and applied for detecting HOCl/OCl- in vitro and in vivo. Despite a great progress has achieved, the development and application of near-infrared fluorescent HOCl/OCl- probe still have some challenges. For example, highly specific and sensitive NIR fluorescent HOCl/OCl- probes applied in endogenous OCl- detection and subcellular organelle bioimaging. In this review, we summarized the representative cases of HOCl/OCl- probes with properties that mentioned above. The discussion contains design strategies, detection mechanisms, as well as applications in bioimaging.
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Colorantes Fluorescentes , Ácido HipoclorosoRESUMEN
Microenvironment-related parameters like viscosity, polarity, and pH play important roles in controlling the physical or chemical behaviors of local molecules, which determine the physical or chemical behaviors of surrounding molecules. In general, changes of the internal microenvironment will usually lead to cellular malfunction or the occurrence of relevant diseases. In the last few decades, the field of chemicobiology has received great attention. Also, remarkable progress has been made in developing viscosity-sensitive fluorescent probes. These probes were particularly efficient for imaging viscosity in biomembranes as well as lighting up specific organelles, such as mitochondria and lysosome. Besides, there are some fluorescent probes that can be used to quantify intracellular viscosity when combined with fluorescence lifetime (FLIM) and ratiometric imaging under water-free conditions. In this review, we summarized the majority of viscosity-sensitive chemosensors that have been reported thus far.
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Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Animales , Humanos , Lisosomas/química , Microscopía Fluorescente/métodos , Mitocondrias/química , Imagen Óptica/métodos , Espectrometría de Fluorescencia/métodos , ViscosidadRESUMEN
A BODIPY disulfonate BODIPY-diONs with two-photon fluorescent turn-on effect was developed as fluorescence probe for selective detection of glutathione over cysteine and homocysteine. BODIPY-diONs is weakly fluorescent due to the 2,4-dinitrobenzenesulfonyl quencher group. When GSH was added, a SNAr substitution reaction was triggered. The red emission of the BODIPY fluorophore at 675 nm was switched on, with a 27-fold emission enhancement in fluorescence intensity. The color of the solution changed from blue to green together with fluorescence appeared within 5 s. The absorbance and emission maxima of the probe BODIPY-diONs were achieved at 650 nm and 675 nm, respectively (quantum yield: 0.11). Interestingly, under the sapphire pulsed laser's 800 nm irradiation, in presence of GSH, the two-photon excited fluorescence (TPEF) of probe BODIPY-diONs was turned on, affording an OFF-ON response signal and a strong emission band at 682 nm. Furthermore, for detection of GSH, the chemodosimeter BODIPY-diONs exhibits high sensitivity and excellent anti-interference with low detection limit of 0.17 µM, and it works effectively within a wide pH range. Furthermore, the imaging studies proved that the probe BODIPY-diONs is suitable for the detection of GSH in complete physiological media.