•Two novel Eu3+ complex-based luminescence probes for detection of HClO were developed.•The probes exhibited fast luminescence responses towards HClO with good selectivity and high sensitivity.•The probes displayed highly specific localizations in mitochondria and lysosomes, respectively.•The probes were successfully used for imaging of HClO in living cells and laboratory animals.Hypochlorous acid (HClO) plays a vital role in the immune system and is involved in various human diseases. To fully understand its biological functions in cellular signaling pathways, apoptosis and human diseases, effective chemical tools for directly tracing HClO at subcellular levels are greatly demanded. Herein, two mitochondria- and lysosome-targetable luminescent β-diketonate–Eu3+ complexes, Mito-BHHBCB-Eu3+ and Lyso-BHHBCB-Eu3+, were developed as probes for the time-gated luminescence detection of HClO inside mitochondria and lysosomes of living cells, respectively. The probes were designed by incorporating a mitochondria-anchoring (triphenylphosphonium) motif or a lysosome-anchoring (morpholine) motif with a strongly luminescent HOCl-responsive β-diketonate–Eu3+ complex, BHHBCB-Eu3+, to ensure the probe molecules to be driven into mitochondria or lysosomes for responding to HOCl therein. Upon exposure to HClO, the probes exhibited a fast luminescence response (within 5 s) towards HClO with good selectivity and high sensitivity (<15 nM). In live cell experiments, both probes, Mito-BHHBCB-Eu3+ and Lyso-BHHBCB-Eu3+, were successfully located in the corresponding organelles as expected, which enabled exogenous and endogenous HClO to be imaged at subcellular levels. Taking advantages of time-gated luminescence bioimaging technique, the uptake of exogenous HClO by Daphnia magna was also successfully imaged by time-gated luminescence microscopy. The results reveal that Mito-BHHBCB-Eu3+ and Lyso-BHHBCB-Eu3+ could serve as useful tools for real-time imaging of HClO at subcellular levels and in vivo with high specificity and contrast.Two novel Eu3+ complex-based probes were developed for time-gated luminescence imaging of HClO in mitochondria and lysosomes of living cells and laboratory animals.Download high-res image (223KB)Download full-size image

Bioresponsive luminescence probes based on lanthanide complexes have shown great utility in a variety of time-gated luminescence bioassays. However, functional lanthanide complexes that can target individual organelles for probing biospecies therein have rarely been investigated. In this work, a unique Eu3+ complex, Mito-NPSTTA-Eu3+, was designed and synthesized as a probe for the time-gated luminescence sensing of HOCl inside the mitochondria of living cells. The probe showed a fast, highly sensitive and selective luminescence response to HOCl with a wide available pH range (pH 3–10), and highly specific mitochondria-localization performance. Taking advantage of time-gated luminescence bioimaging and the excellent properties of the Eu3+ complex, the generation of endogenous HOCl in RAW 264.7 cells and the uptake of exogenous HOCl by zebrafish were successfully imaged, respectively. The results demonstrated the feasibility of Mito-NPSTTA-Eu3+ for the imaging of mitochondrial HOCl, and validated the potential of our strategy for the design of lanthanide complex-based organelle-targeting bioresponsive probes.

We have developed a ratiometric time-gated luminescence sensory system for in vivo imaging of hypochlorous acid (HClO) by preparing a dual-emissive nanoarchitecture of europium- and terbium-complex-modified silica nanoparticles. The design of this nanoarchitecture is based on our new finding that the strong, long-lived luminescence of the β-diketonate–Eu3+ complex can be rapidly and selectively quenched by HClO. Therefore, the β-diketonate–Eu3+ complex was decorated on the surface of the silica nanoparticles for responding to HClO, while a HClO-insensitive luminescent terbium complex was immobilized in the inner solid core of the nanoparticles to serve as an internal standard. This nanosensing probe combines the advantages of both ratiometric and time-gated detection modes to afford high accuracy and sensitivity. Upon exposure to HClO, the nanoprobe displayed a remarkable luminescence color change from red to green, and the intensity ratio of the green over the red luminescence (I539/I607) showed a rapid, sensitive and selective response to HClO. Additionally, the feasibility of using the nanoprobe for intracellular detection of exogenous and endogenous HClO and for real-time mapping of HClO in small laboratory animals has been demonstrated via ratiometric time-gated luminescence imaging microscopy. The results reveal that the constructed nanoarchitecture cloud is a favorable and useful sensing probe for the real-time imaging of HClO in vivo with high specificity and contrast.

Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, TRP-NO, was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in TRP-NO is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a ∼10-fold contrast window for the detection of NO. These unique properties allowed TRP-NO to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of TRP-NO for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of TRP-NO in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of TRP-NO was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that TRP-NO could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.

•1O2 induced by fluoroquinolones and functionalized graphenes in daphnids was visualized and quantified.•ATLI technology was developed to indicate the distribution of 1O2 in living daphnids.•The strongest luminescence signals of 1O2 were observed in the hindgut of daphnids.•Photogeneration of 1O2 by carboxylated/aminated graphenes was confirmed by EPR.Singlet oxygen (1O2) can be photogenerated by photoactive xenobiotics and is capable of causing adverse effects due to its electrophilicity and its high reactivity with biological molecules. Detection of the production and distribution of 1O2 in living organisms is therefore of great importance. In this study, a luminescent probe ATTA-Eu3+ combined with time-gated luminescence imaging was adopted to detect the distribution and temporal variation of 1O2 photoinduced by fluoroquinolone antibiotics and carboxylated/aminated graphenes in Daphnia magna. Results show that the xenobiotics generate 1O2 in living daphnids under simulated sunlight irradiation (SSR). The photogeneration of 1O2 by carboxylated/aminated graphenes was also confirmed by electron paramagnetic resonance spectroscopy. The strongest luminescence signals of 1O2 were observed in the hindgut of daphnids, and the signals in different areas of the daphnids (gut, thoracic legs and post-abdominal claw) displayed a similar trend of enhancement over irradiation time. Mean 1O2 concentrations at different regions of daphnids within one hour of SSR irradiation were estimated to be in the range of 0.5 ∼ 4.8 μM. This study presented an efficient method for visualizing and quantifying the temporal and spatial distribution of 1O2 photogenerated by xenobiotics in living organisms, which can be employed for phototoxicity evaluation of xenobiotics.Download high-res image (135KB)Download full-size image

We have developed a ratiometric time-gated luminescence sensory system for in vivo imaging of hypochlorous acid (HClO) by preparing a dual-emissive nanoarchitecture of europium- and terbium-complex-modified silica nanoparticles. The design of this nanoarchitecture is based on our new finding that the strong, long-lived luminescence of the β-diketonate–Eu3+ complex can be rapidly and selectively quenched by HClO. Therefore, the β-diketonate–Eu3+ complex was decorated on the surface of the silica nanoparticles for responding to HClO, while a HClO-insensitive luminescent terbium complex was immobilized in the inner solid core of the nanoparticles to serve as an internal standard. This nanosensing probe combines the advantages of both ratiometric and time-gated detection modes to afford high accuracy and sensitivity. Upon exposure to HClO, the nanoprobe displayed a remarkable luminescence color change from red to green, and the intensity ratio of the green over the red luminescence (I539/I607) showed a rapid, sensitive and selective response to HClO. Additionally, the feasibility of using the nanoprobe for intracellular detection of exogenous and endogenous HClO and for real-time mapping of HClO in small laboratory animals has been demonstrated via ratiometric time-gated luminescence imaging microscopy. The results reveal that the constructed nanoarchitecture cloud is a favorable and useful sensing probe for the real-time imaging of HClO in vivo with high specificity and contrast.

Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, TRP-NO, was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in TRP-NO is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a ∼10-fold contrast window for the detection of NO. These unique properties allowed TRP-NO to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of TRP-NO for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of TRP-NO in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of TRP-NO was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that TRP-NO could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.

Bioresponsive luminescence probes based on lanthanide complexes have shown great utility in a variety of time-gated luminescence bioassays. However, functional lanthanide complexes that can target individual organelles for probing biospecies therein have rarely been investigated. In this work, a unique Eu3+ complex, Mito-NPSTTA-Eu3+, was designed and synthesized as a probe for the time-gated luminescence sensing of HOCl inside the mitochondria of living cells. The probe showed a fast, highly sensitive and selective luminescence response to HOCl with a wide available pH range (pH 3–10), and highly specific mitochondria-localization performance. Taking advantage of time-gated luminescence bioimaging and the excellent properties of the Eu3+ complex, the generation of endogenous HOCl in RAW 264.7 cells and the uptake of exogenous HOCl by zebrafish were successfully imaged, respectively. The results demonstrated the feasibility of Mito-NPSTTA-Eu3+ for the imaging of mitochondrial HOCl, and validated the potential of our strategy for the design of lanthanide complex-based organelle-targeting bioresponsive probes.