A bright two-photon fluorescent probe for real-time monitoring autophagy in living cells

A novel donor-acceptor(D-A) type of two-photon(TP) fluorescent probe,i.e.Lyso-OSC,based on the lysosome-targeting morpholine group was developed.The polarity sensing coumarin group was functionalized as the acceptor and the 1-vinyl-4-methoxybenzene group was engineered as the donor.The fluorescence intensity and emission maximum wavelength of Lyso-OSC are highly sensitive to the polarity changes of solvent.The two-photon absorption cross-section and tissue penetration depth are up to 254 GM and 150 μm,respectively.The strong fluorescence,high sensitivity to polarity,low cytotoxicity,and accurate lysosome-targeting ability entail Lyso-OSC the excellent performance in detecting the polarity changes ofcellular environment.To this end,a bright,real-time imaging autophagy of living cells has been achieved.     

一种基于萘酰亚胺的检测细胞内pH值的荧光探针及其生物成像应用

细胞内溶酶体的pH值对细胞自噬、吞噬、酶加工等各项生命活动有着重要影响.细胞核是真核细胞中最大的细胞器,控制着生物体内的遗传和代谢过程,参与代谢过程的酶对pH值的变化很敏感.因此,研究细胞体内的pH值变化至关重要.我们设计并以简单的两步反应合成了一种新型荧光探针NpH-1.该探针以萘酰亚胺作为荧光团,以吗啉基团作为对p...     

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

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

A simple,water-soluble,Fe3+-selective fluorescent probe and its application in bioimaging

A simple,water-soluble,Fe³⁺-selective fluorescent probe,derived from rhodamine B,was synthesized and characterized.The probe exhibits a fluorescence response toward Fe³⁺ with acceptable sensitivity and selectivity and even facilitates visual or naked-eye detection of Fe³⁺.The experiment results show that the response of the probe to Fe³⁺ is pH-independent over a wide range of 4.0-10.0.In addition,fluorescence microscopic imaging experiments have proven that the probe is cell permeable and can be used for monitoring intracellular Fe³⁺ in living cells.     

Fluorescein-derived fluorescent probe for cellular hydrogen sulfide imaging

In this work, a fluorescein-derived fluorescent probe for H2 S based on the thiolysis of dinitrophenyl ether is reported. This probe exhibits turn-on fluorescence imaging of H2 S in living cells and bulk solutions with excellent selectivity. The reaction mechanism was explained by means of absorption, fluorescence and HPLC–MS.     

A benzothiazole-based fluorescent probe for thiol bioimaging

This study reports a benzothiazole-based fluorescent probe with simple structure for thiols. This probe exhibited high on/off signal ratios and good selectivity toward thiols over other analytes, and was successfully applied to the imaging of thiols in living cells.     

A ratiometric fluorescent probe based on carbon dots assembly for intracellular lysosomal polarity imaging with wide range response

Lysosomal polarity is considered a key indicator of lysosomal function due to its significant impact on membrane fluidity and enzymatic reactions in lysosomes. Monitoring lysosomal polarity can gain insight into the related physiological and pathological processes and develop new diagnostic methods. However, current fluorescent probes with lysosomal polarity response suffer from narrow linear range, photobleaching and complicated preparation. Herein, a ratiometric fluorescent probe(r-b CDs) for ...     

用于谷胱甘肽检测的新型罗丹明衍生物荧光探针EI北大核心CSCD

合成了一种反应型近红外荧光探针N-Rh-GSH,该探针以罗丹明衍生物为荧光母体,通过与谷胱甘肽(GSH)作用触发螺环的开关来实现信号的响应,其开环释放的荧光产物具有760 nm的近红外发射波长。细胞实验表明,该探针可实现对活细胞中GSH的成像。     

缺氧响应荧光探针的成像及治疗应用

由于肿瘤内部细胞远离血管, 其氧气消耗量远远超出血液供应量, 因此容易导致肿瘤缺氧. 肿瘤缺氧会引发肿瘤扩散加速、 诱导某些基因过表达及产生药物抗药性等问题. 基于此, 发展性能优异的缺氧响应荧光探针对肿瘤的诊断和治疗具有重要意义. 本文对缺氧响应荧光探针在成像及治疗方面的应用进展进行了综合评述, 介绍了硝基、 偶氮键和醌3种常用的缺氧响应基团, 并探讨了它们在缺氧微环境下的识别机理; 介绍了缺氧响应荧光探针的构建及其在生物成像方面的最新研究成果; 总结了缺氧响应荧光探针在基因治疗、 光动力学治疗、 化学治疗及协同治疗方面的研究进展; 展望了缺氧响应荧光探针在临床诊断和治疗方面的应用前景.     

A turn-on fluorescent probe for Au3+ based on rodamine derivative and its bioimaging application

A new fluorescent probe RY was synthesized for the detection of Au³⁺ ions based on a rhodamine B derivative. The fluorescent probe showed good selectivity and sensitivity to Au³⁺ ions. Obvious color and fluorescence changes could be observed with the naked eye while the fluorescent probe reacted with the Au³⁺ ions. The detection limit of the probe was determined to be 36 ppb by the fluorescence titration; the excellent linear relationship suggests that the probe is potentially useful for quantitative detection of Au³⁺ in vitro. We also demonstrated its bioimaging application in both living cells and mice; this was the first time that a fluorescent probe was successfully applied to imaging Au³⁺ in living animals.     

A fluorescence turn-on probe for cysteine and homocysteine based on thiol-triggered benzothiazolidine ring formation

We synthesized a new coumarin-based probe TP, containing a disulfide moiety, to detect biothiols in cells. A fluorescence turn-on response is induced by the thiol–disulfide exchange of the probe, with subsequent intramolecular benzothiazolidine ring formation giving rise to a fluorescent product. The probe exhibits an excellent selectivity for cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) and other amino acids. The fluorescent probe also exhibits a highly sensitive fluorescence turn-on response to Cys and Hcy with detection limits of 0.8 μM for Cys and 0.5 μM for Hcy. In addition, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrates that the probe TP could be an efficient fluorescent detector for thiols in living cells.     

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 red-emitting water-soluble fluorescent probe for biothiol detection with a large Stokes shift

A highly water soluble fluorescent probe was developed for sensitive and selective detection of biothiols with a red emission and a large Stokes shift. The probe was successfully applied to detect biothiols both in aqueous solution and in living cells.     

Dual-functional gadolinium-based copper(II) probe for selective magnetic resonance imaging and fluorescence sensing

A unique gadolinium complex, Nap-DO3A-Gd, comprising a naphthylamine luminescent moiety, a di-2-picolylamine (DPA) binding chelator, and a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) moiety has been designed and synthesized as a dual-functional probe for selective magnetic resonance imaging and fluorescent sensing of copper(II) in living cells. Nap-DO3A-Gd exhibited a turn-on manner of relaxivity changes and a fluorescent quenching toward Cu(2+). Through the introduction of naphthalamide into the Gd(3+) contrast agent platform to restrict the coordination ability of the DPA chelator and with Gd(3+) coordinating to the DPA moiety to turn away the interferences of other metal cations from Cu(2+) detection, the probe featured selective relaxivity changes toward Cu(2+) over other metal ions and brought unique Cu(2+)-specific luminescent responses. The probe was water-soluble with the luminescent detection limit established at 6 ppb and was successfully used for luminescence imaging detection of copper(II) in living cells. The results demonstrated the efficiency and advantage of our approach in the development of a dual-modality image.     

A New Highly Selective and Sensitive Assay for Fluorescence Imaging of .OH in Living Cells: Effectively Avoiding the Interference of Peroxynitrite

A new nonredox fluorescent probe to realize the imaging of hydroxyl radicals (^.OH) in living cells was designed and synthesized. The structure comprised the fluorescent dye boron dipyrromethene (BDP) and a 2,2,6,6‐tetramethyl‐1‐piperidinoxyl (TEMPO) unit. This probe could rapidly respond to ^.OH with a detection limit of 18 pM , and it possessed superior photostability and pH insensitivity. Other reactive oxygen species (ROS) and relevant intracellular components did not interfere. In particular, the important problem of ONOO^? interference was efficiently avoided. An MTT assay proved that the probe was not very cytotoxic. The probe could penetrate into intact cell membranes to selectively detect intracellular ^.OH without causing cellular damage in living mice macrophages, normal human liver cells. and human hepatoma cells. These advantageous characteristics make the fluorescent probe potentially useful as a new candidate to detect ^.OH in broad biosystems.     

Design of a phosphinate-based fluorescent probe for superoxide detection in mouse peritoneal macrophages

3',6'-Bis(diphenylphosphinyl)fluorescein (PF-1) was synthesized as a highly selective and sensitive fluorescent probe for imaging O(2) (.-) in living cells. The design strategy for the probe was based on the nucleophilic mechanism of O(2) (.-) to mediate deprotection of this probe to give fluorescein. Upon reaction with O(2) (.-), the probe exhibits a strong fluorescence response and high selectivity for O(2) (.-) over other reactive oxygen species and some biological compounds. The phosphinate-based probe, as a new fluorescent reagent, is cell-permeable and can detect micromolar changes of O(2) (.-) concentrations by using confocal microscopy in living cells. The unique combination of good selectivity, high sensitivity, good water solubility, and rapid reactivity establishes the potential value of the probe for facilitating investigations of the generation, metabolism, and mechanisms of superoxide-mediated cellular homeostasis and injury.     

A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery

We sought to develop a sensitive and quantitative technique capable of monitoring the entire flux of autophagy involving fusion of lysosomal membranes. We observed the accumulation inside lysosomal compartments of Keima, a coral-derived acid-stable fluorescent protein that emits different-colored signals at acidic and neutral pHs. The cumulative fluorescent readout can be used to quantify autophagy at a single time point. Remarkably, the technique led us to characterize an autophagy pathway in Atg5-deficient cells, in which conventional LC3-based autophagosome probes are ineffective. Due to the large Stokes shift of Keima, this autophagy probe can be visualized in conjunction with other green-emitting fluorophores. We examined mitophagy as a selective autophagic process; time-lapse imaging of mitochondria-targeted Keima and GFP-Parkin allowed us to observe simultaneously Parkin recruitment to and autophagic degradation of mitochondria after membrane depolarization.     

Mitochondria-targeted fluorescent probe based on vibration-induced emission for real-time monitoring mitophagy-specific viscosity dynamic

Directly monitoring mitophagy-specific viscosity dynamic in living cells is of great significance but remains challenging. Herein, this study reported a novel mitochondria-targeted fluorescent probe DPAC-DY based on vibration-induced emission (VIE) for monitoring viscosity changes during mitochondrial autophagy. This probe contained N,N'-diphenyl- dihydrodibenzo[a,c]phenazine (DPAC) as the VIE core and two positively charged pyridinium moieties for mitochondria anchoring. As the ambient viscosity increased, the vibration of DPAC-DY could be hindered, and subsequently resulting in the enhancement of fluorescence emission. In vitro and intracellular experiments indicated that the probe DPAC-DY showed highly sensitive response to viscosity due to VIE mechanism. Importantly, by virtue of this probe, in situ and real-time visualization of the specific viscosity dynamics during the mitochondrial autophagy process was achieved. Thus, this work provides a novel strategy for VIE-based viscosity response sensors applied to specific organelles and offers a platform for in-depth study of mitochondrial viscosity-related diseases.     

Anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated fluorescent nanoparticles probe for breast cancer imaging

Fluorescent nanoparticles (FNs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. In this study, anti-EGFR antibody conjugated fluorescent nanoparticles (FNs) (anti-EGFR antibody conjugated FNs) probe was used to detect breast cancer cells. FNs with excellent character such as non-toxicity and photostability were first synthesized with a simple, cost-effective and environmentally friendly modified Stőber synthesis method, and then successfully modified with anti-EGFR antibody. This kind of fluorescence probe based on the anti-EGFR antibody conjugated FNs has been used to detect breast cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-EGFR antibody conjugated FNs can effectively recognize breast cancer cells and exhibited good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of breast cancer cells and offer a new method in detecting EGFR.     

Target‐Triggered NIR Emission with a Large Stokes Shift for the Detection and Imaging of Cysteine in Living Cells

Background autofluorescence from biological systems generally reduces the sensitivity of a fluorescent probe for imaging biological targets. Addressing this challenge requires the development of fluorescent probes that produce emission in the near‐infrared region. Herein, we report the design and synthesis of a fluorescent probe that generates an NIR emission with a large Stokes shift upon the selective response to Cys over Hcy and GSH. The probe is designed to consist of two Cys‐sensing sites, an acrylate ester and an aldehyde installed ortho to each other. The reaction of the probe with Cys triggers an excited state intramolecular proton transfer process upon photo‐excitation, thereby producing an NIR emission with a large Stokes shift. Accordingly, this probe hold great promise for the selective detection of Cys in biological systems. We further demonstrate the capacity of this probe for Cys imaging in living cells.