Imaging gastrointestinal damage due to acute mercury poisoning using a mitochondria-targeted dual near-infrared fluorescent probe.

Mercury (Hg) is one of the most widespread pollutants that pose serious threats to public health and the environment. People are inevitably exposed to Hg via different routes, such as respiration, dermal contact, drinking or diet. Hg poisoning could cause gingivitis, inflammation, vomiting and diarrhea, respiratory distress or even death. Especially during the developmental stage, there is considerable harm to the brain development of young children, causing serious symptoms such as intellectual disability and motor impairments, and delayed neural development. Therefore, it's of great significance to develop a specific, quick, practical and labor-saving assay for monitoring Hg2+. Herein, a mitochondria-targeted dual (excitation 700 nm and emission 728 nm) near-infrared (NIR) fluorescent probe JZ-1 was synthesized to detect Hg2+, which is a turn-on fluorescent probe designed based on the rhodamine fluorophore thiolactone, with advantages of swift response, great selectivity, and robust anti-interference capability. Cell fluorescence imaging results showed that JZ-1 could selectively target mitochondria in HeLa cells and monitor exogenous Hg2+. More importantly, JZ-1 has been successfully used to monitor gastrointestinal damage of acute mercury poisoning in a drug-induced mouse model, which provided a great method for sensing Hg species in living subjects, as well as for prenatal diagnosis.

Probing Variations of Reduction Activity at the Plasma Membrane Using a Targeted Ratiometric FRET Probe.

Plasma membrane (PM) is the turntable of various reactions that regulate essential functionalities of cells. Among these reactions, the thiol disulfide exchange (TDE) reaction plays an important role in cellular processes. We herein designed a selective probe, called membrane reduction probe (MRP), that is able to report TDE activity at the PM. MRP is based on a green emitting BODIPY PM probe connected to rhodamine through a disulfide bond. MRP is fluorogenic as it is turned off in aqueous media due to aggregation-caused quenching, and once inserted in the PM, it displays a bright red signal due to an efficient fluorescence energy resonance transfer (FRET) between the BODIPY donor and the rhodamine acceptor. In the PM model, the MRP can undergo TDE reaction with external reductive agents as well as with thiolated lipids embedded in the bilayer. Upon TDE reaction, the FRET is turned off and a bright green signal appears allowing a ratiometric readout of this reaction. In cells, the MRP quickly labeled the PM and was able to probe variations of TDE activity using ratiometric imaging. With this tool in hand, we were able to monitor variations of TDE activity at the PM under stress conditions, and we showed that cancer cell lines presented a reduced TDE activity at the PM compared to noncancer cells.

A nucleus targetable fluorescent probe for ratiometric imaging of endogenous NO in living cells and zebrafishes.

Nitric oxide (NO) is an important cellular messenger molecule in the cardiovascular, nervous and immune systems. Real-time monitoring of NO activity in specific organelles of live cells is important to understand its biological function. In this work, a nucleus targetable ratiometric NO probe, Hoe-Rh-NO, is developed by linking Hoechst to rhodamine spirolactam. The Hoechst part conducts nucleus targeting and the rhodamine spirolactam senses NO. The two fluorophores constitute a NO switchable FRET system for ratiometric imaging. This newly developed probe Hoe-Rh-NO displays good nucleus targeting ability, high sensitivity and selectivity towards NO, low cytotoxicity and most importantly detects NO through ratiometric fluorescence imaging. By using Hoe-Rh-NO, we confirmed the presence of NO in the nucleus and detected endogenous NO during inflammation in cells and zebrafishes.

Developmental stages of zebrafish (Danio rerio) embryos and toxicological studies using foldscope microscope.

Zebrafish (Danio rerio), is a well-established vertebrate animal model widely used in developmental biology and toxicological research. In the present study, foldscope is used as an innovative tool to study the developmental stages and toxicological analysis of the zebrafish embryos. Briefly, the developmental stages, such as zygote, cleavage, blastula, gastrula, segmentation, and pharyngula formation are observed and documented using simple foldscope. Toxicological parameters upon exposure to different concentration of ethanol extract of Curcuma longa and its lead compound, ar-turmerone along with rhodamine B (bio-coupler) on zebrafish embryos are analyzed upto 72 hr using foldscopes in live condition. The lethal endpoints, such as coagulation, lack of somite formation, non-detachment of tail, and lack of heartbeat are clearly monitored and documented using foldscope. Bio-evaluation of test compounds with the aid of foldscope confirms that the toxicity is directly proportional to the concentration. Our results conclude that, ethanol extract of C. longa, ar-turmerone and rhodamine B exposed embryos remains healthy up to 96, 48, and 24 µg concentrations, respectively. Embryos exposed to higher concentrations become coagulated, however normal physiological active movement of tail lashing and heartbeat are evident in lower concentration exposed embryos. Except coagulation, no other abnormalities are observed and interestingly, the hatching ability is not delayed, when compared with the control embryos. It is confirmed that the test compounds are not highly toxic to zebrafish embryos. Hence it can be used for further analysis, especially for studying the neural-regeneration and its neuronal development in zebrafish embryos.

Polypeptide-rhodamine B probes containing laminin/fibronectin receptor-targeting sequence (YIGSR/RGD) for fluorescent imaging in cancers.

Currently, fluorescent imaging is one of the most promising diagnostic approaches for facile detection of cancers in situ in thanks to a fluorescent probe. Two novel polypeptide-based fluorescent probes for different biomarkers to cancers are reported here. These probes focused on tyrosine-isoleucine-glycine-serine-arginine (YIGSR) and arginine-glycine-aspartic (RGD), which receptors play an important role in the extracellular matrix and are overexpressed in tumor cells and then can be used as tumor-targeting groups in fluorescent imaging. In this work, the pentpeptide-rhodamine B derivative (YIGSR-RhB) and tripeptide-rhodamine B derivative (RGD-RhB) were synthesized respectively by using the solid phase synthesis methods. These derivatives were further characterized by 1HNMR, MS, UV and IR, etc. Their fluorescent and biocompatibility properties, such as the cell cytotoxicity, cell uptake and fluorescent imaging of tumor cells, and fluorescent imaging in BALB/c female mice with 4T1 tumors and C57 mice with B16F10 tumor in vivo, were also measured. Experiment results demonstrated that YIGSR-RhB and RGD-RhB possessed the low cell cytotoxicity, good tumor-targeting property and fluorescent properties similar to rhodamine B. Moreover, YIGSR-RhB and RGD-RhB can be taken up highly by the B16F10 melanoma cells and 4T1 breast cancer cells, and then achieve the good fluorescent imaging in these tumor cells in vitro and tumors of mice in vivo. Therefore, YIGSR-RhB and RGD-RhB can be used as the potential tumor-targeting probes for fluorescent imaging. They can directly attach the cell membrane and specifically target to the tumor cells.

A novel cell-penetrating Janus nanoprobe for ratiometric fluorescence detection of pH in living cells.

pH regulates the function of many organelles and plays a pivotal role in requiring multitud cellular behaviors. Compared with single fluorescent probes, ratio fluorescent probes have higher sensitivity and immunity to interference. Herein, a novel Janus ratio nanoprobe was developed for intracellular pH detection. Modified rhodamine B probe and fluorescein isothiocyanate (FITC) were individually encapsulated in the independent hemispheres of Janus microparticles fabricated via Pickering emulsion. Moreover, it exhibits a satasified ratiometric detection of pH compared to the previous core-shell structure and organic small molecule probe. Accordingly, the Janus nanoprobe possesses many important features as an attractive sensor, including high anti-jamming capability, excellent stability, good reversibility and low cytotoxicity. Variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor, which can respond to wide range of pH values from 3 to 8. To be more specific, with a single excitation wavelength of 488 nm, there are dual emission bands centered at 538 nm and 590 nm. Also the Janus nanoprobe displays a excellent linear relationship in the physiologically relevant pH range of 4.0-6.0. Consequently, detecting of pH and imaging was successfully achieved in living cells, which provides a simple and reliable method for detecting intracelluar pH and other similar substances.

Cell-penetrating peptide-modified quantum dots as a ratiometric nanobiosensor for the simultaneous sensing and imaging of lysosomes and extracellular pH.

The selective measurement of cellular pH at specific regions is significant for the in-depth study of cell functions and pathological processes. Herein, we combined our previously developed rhodamine B-labeled cell-penetrating peptide spirolactam derivative with quantum dots to create a ratiometric nanobiosensor for the simultaneous sensing and imaging of lysosomes and extracellular pH.

Engineering dithiobenzoic acid lactone-decorated Si-rhodamine as a highly selective near-infrared HOCl fluorescent probe for imaging drug-induced acute nephrotoxicity.

A highly selective lysosome-targeting NIR fluorescent probe (Lyso-SiR-2S) for HOCl was constructed based on Si-rhodamine B spirodithiolactone. This probe was very effectively employed to sense HOCl produced in various living cells and to visualize fluctuations of endogenous HOCl resulting from GEN-induced acute kidney injury in vivo for the first time.

A FRET ratiometric fluorescent probe for detection of Hg2+ based on an imidazo[1,2-a]pyridine-rhodamine system.

A novel imidazo[1,2-a]pyridine-rhodamine ratiometric fluorescent probe IP-Hg for Hg2+ based on a fluorescence resonance energy transfer mechanism has been developed. The probe has been proved to show high sensitivity and high selectivity toward Hg2+. Furthermore, it could be used for imaging Hg2+ in cells and in polluted water.

An in cellulo-activated multicolor cell labeling approach used to image dying cell clearance.

Dying cell clearance is critical for myriad biological processes such as tissue homeostasis. We herein report an enzyme-activated fluorescence cell labeling approach and its use for multicolor imaging of dying cell clearance. Diacetylated 4-hydroxymandelic acid (DHA)-conjugated dyes give rise to reactive quinone methides upon deacetylation in live cells, which in turn covalently labels cellular proteins. With partner cells tagged with distinct fluorescence, apoptotic cell clearance by Raw 264.7 macrophages and epithelial HeLa cells was captured by confocal microscopy, showing the potential of DHA-based cell labeling for investigating cell-cell interactions.

A structure optimized fluorescent probe for highly sensitive monitoring drug induced lysosomal pH value changes.

Lysosomes generally maintain the weak acidic microenvironment, to ensure highly efficient activity and functions of hydrolytic enzymes and proteins. Aberrations of the lysosomal pH may result in cellular functional changes and influence human physiology, possibly causing serious diseases. Small-molecular fluorescent probes based imaging techniques capable of providing information on target locations are considerably appreciated. Herein, by reducing the size of the typical lysosome targetable group 4-(2-aminoethyl) morpholine, we rationally designed a rhodamine analogue probe Ly-HN2AM with N-Aminomorpholine as the ring-closed switch and the lysosome targetable moiety for visualizing lysosomal pH changes. With the benefit of constructing multi-pentacyclic intramolecular hydrogen bond when binding with the H+, Ly-HN2AM gives a highly sensitive response towards pH values ranging from 4.79 to 6.07, with a remarkable higher pKa 5.35 over the typical 4-(2-aminoethyl) morpholine modified probes. The new probe was successfully applied to visualize pH value changes in lysosome-associated physiological and pathological processes with excellent photostability and low cytotoxicity, indicating the potential applications of lysosome specific bioimaging.

A simple highly specific fluorescent probe for simultaneous discrimination of cysteine/homocysteine and glutathione/hydrogen sulfide in living cells and zebrafish using two separated fluorescence channels under single wavelength excitation.

Biothiols such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) are widely found in mammalian cells. They are closely related to the production and metabolic pathways and play very important roles in physiological and pathological activities. Therefore, the quantitative detection of these biothiols is of great significance. Although many fluorescent probes have been successfully used to track biothiols in biological samples, the fluorescence method for simultaneously detecting these biothiols using separated fluorescence emission channels under single wavelength excitation is still immature. In this work, we prepared the conjugate of seminaphthorhodafluor (SNARF) dye and 7-nitro-1,2,3-benzoxadiazole (NBD) using as a simple long-wavelength fluorescent probe SNARF-NBD for specific detection of biothiols. Cys/Hcy and GSH/H2S were identified by two separated fluorescence emission channels under single wavelength excitation, which showed good selectivity and sensitivity. In addition, SNARF-NBD has low cytotoxicity and shows good imaging ability in living cells and zebrafish.

Ratiometric enhanced fluorometric determination and imaging of intracellular microRNA-155 by using carbon dots, gold nanoparticles and rhodamine B for signal amplification.

An ultrasensitive and highly reliable ratiometric assay is described for the determination of microRNA-155. It works at the attomolar concentration level and has high selectivity which warrants its potential application in cancer biomarker tracking. The excellent performance of this method results from (a) the use of a hybrid conjugate prepared from Rhodamine B (RhB), carbon dots (CDs) and probe-microRNA, and (b) from the measurement of fluorescence resonance energy transfer (FRET) that is observed in the AuNP/target-microRNA system as a result of RNA hybridization. The dye RhB (emission peak at 580 nm) serves as an internal reference. The sensitivity of this assay is increased by about 30% because of the broad emissions of CDs (489 nm and 665 nm) through a sequential FRET phenomenon. RhB-CDs were covalently bio-conjugated to probe microRNA. In the presence of AuNPs, the fluorescence of the CDs is quenched, while in the presence of microRNA-155, the ratio of fluorescences at 489 and 665 nm (I489/I665) is enhanced again. A linear relationship exists between the ratio of fluorescence and the concentration of microRNA-155 in the range from 1 aM to 0.1 µM, and the detection limit is 0.3 aM. The assay was applied to quantitative studies of target microRNA-155 in multiple pathways associated with cancer progression in biological fluids include human serum samples and cancer cells. The nanoprobe also deliver clear signal to microRNA target in fixed and lived MDA-MB-231 cells. Graphical abstract A ratiometric FRET sensing method used for microRNA-155 detection at aM concentration level using CDs and AuNPs as donor-acceptor respectively and Rhodamine B as amplification reagent. The application of assay for imaging of microRNA-155 in fixed and live MDA-MB-231 cells is demonstrated.

A highly selective and sensitive red-emitting fluorescent probe for visualization of endogenous peroxynitrite in living cells and zebrafish.

Peroxynitrite (ONOO-) has been proven to participate in various physiological and pathological processes, and may also be a contributing factor in many diseases. In view of this, there is a need to develop detection tools for unambiguously tracking a small amount of endogenous ONOO- to reveal its exact mechanisms. In this paper, a colorimetric and red-emitting fluorescent probe Red-PN, based on a rhodamine-type fluorophore and hydrazide reactive site is described. The probe Red-PN possesses the advantages of rapid response (within 5 s), visual color change (from colorless to pink), preeminent sensitivity (detection limit = 4.3 nM) and selectivity. Because of these outstanding performances, it was possible to accurately detect endogenous ONOO-. It was encouraging that the probe Red-PN could be used effectively for tracking the relatively low levels of endogenous and exogenous ONOO- in living cells and zebrafish. Thus, it is envisioned that the probe Red-PN would have promising prospects in applications for imaging ONOO- in a variety of biological settings.

A Doubly-Quenched Fluorescent Probe for Low-Background Detection of Mitochondrial H2O2.

Hydrogen peroxide (H2O2) is an important product of oxygen metabolism and plays a crucial role in regulating a variety of cellular functions. Fluorescent probes have made a great contribution to our understanding of the biological role of endogenous H2O2. However, fluorescent probes for H2O2 featuring aryl boronates can suffer from moderate turn-on fluorescence responses. Strategies that can reduce the background fluorescence of these boronate-masked probes would significantly improve the sensitivity of endogenous H2O2 detection. In this work, we propose a general and reliable double-quenching concept for the design of fluorescent probes with low background fluorescence. A new fluorescent probe was developed for the detection of endogenous H2O2 in mitochondria of live cancer cells. This probe exploits a boronate-driven lactam formation and an eliminable quenching moiety simultaneously (i.e., the double-quenching effect) to reduce the background fluorescence, which ultimately results in the achievement of a >50-fold fluorescence turn-on. A linear concentration range of response between 1 and 60 µM and a detection limit of 0.025 µM can be obtained. This study not only presents a highly sensitive fluorescent probe for the detection of H2O2 but also provides a new concept for the design of fluorescent probes with a previously unachievable fluorescence off-on response ratio for other types of ROS and many other biologically relevant analytes.

Dual-binding pyridine and rhodamine B conjugate derivatives as fluorescent chemosensors for ferric ions in aqueous media and living cells.

Two new pyridine-type rhodamine B chemosensors (RBPO and RBPF) used to detect Fe3+ have been designed and synthesized, and the sensing behavior towards various metal ions was evaluated via UV-vis and fluorescence spectroscopic techniques. Both RBPO and RBPF not only have good spectral responses to Fe3+ in an EtOH/H2O solution (3 : 1, v/v, HEPES, 0.5 mM, pH = 7.33) with low detection limits and high binding constants, but also suffer from less interference from common metal cations. The two chemosensors are further proven to be practical in sensitively monitoring trace Fe3+ in real water specimens. Intracellular imaging applications demonstrated that RBPO and RBPF can be used as two fluorescent chemosensors for the detection of Fe3+ in living human breast adenocarcinoma (MCF-7) cells.

A Highly Selective "Turn-on" Fluorescent Probe for Detection of Fe3+ in Cells.

A new "turn-on" fluorescent probe Py based on rhodamine and piperonaldehyde was designed and synthesized for detecting Fe3+ in cells. The free probe Py was non-fluorescent. While only upon addition of Fe3+, the significant increase of the fluorescence and color were observed which could be visible directly by "naked-eye". The probe Py shows high selectivity and sensitivity for Fe3+ over other common metal ions in EtOH-H2O (3/2, v/v) mixed solution. The association constant and the detection limit were calculated to be 4.81 × 104 M-1 and 1.18 × 10-8 mol/L respectively. The introduction of piperonaldehyde unit could increase probe rigidity which could enhance its optical properties. Meanwhile, the binding mode between Py and Fe3+ was found to be a 1:1 complex formation. The density functional theory (DFT) calculations were performed which would further confirm the recognition mechanism between probe Py and Fe3+. In addition, the probe has been proved to be reversible for detecting Fe3+. Moreover, the probe Py was used to detect Fe3+ in cells successfully.

A near-infrared fluorescent probe based on photostable Si-rhodamine for imaging hypochlorous acid during lysosome-involved inflammatory response.

Hypochloric acid (HClO) is mainly distributed in acidic lysosomes of phagocytes and closely associated with numerous physiological and pathological processes, especially inflammatory response. Fluorescent probe has become an important tool for imaging HClO in lysosomes, but suffered from interference from autofluorescence in vivo, phototoxicity to biosamples and photobleaching phenomenon due to their short-wavelength excitation and emission. Unfortunately, up to now, no near-infrared (NIR) lysosome-targetable fluorescent probe has been reported for imaging HClO. In this paper, a near-infrared fluorescent probe Lyso-NIR-HClO for imaging lysosomal HClO was reported for the first time. Lyso-NIR-HClO based on Si-rhodamine is consisted of a morpholine unit as a lysosome-targetable group and a HClO-mediated cyclization reaction site as a response group, which was applied for highly selective and sensitive detection and imaging for endogenous and exogenous HClO in lysosomes, with a linear range from 5.0 × 10-8 to 1.0 × 10-5 M and a detection limit of 2.0 × 10-8 M in vitro. Attributed to NIR emission and excellent photostability of Si-rhodamine, Lyso-NIR-HClO exhibits excellent performances in vivo, such as low interference from intracellular autofluorescence, stable and persistent fluorescence signal and good tissue penetration, which are in favor of accurate, time-lapse and long-term imaging for HClO. Finally, we applied the probe Lyso-NIR-HClO to visualize endogenous HClO during lysosome-involved inflammatory response including bacteria-infected cells and inflamed mouse model with satisfactory results. The above results proved that Lyso-NIR-HClO would be a potentially useful tool for the study of biological functions and pathological roles of HClO in lysosomes, especially role of lysosome in the inflammatory response.

Organic Fluorophore Coated Polycrystalline Ceramic LSO:Ce Scintillators for X-ray Bioimaging.

The current effort demonstrates that lutetium oxyorthosilicate doped with 1-10% cerium (Lu2SiO5:Ce, LSO:Ce) radioluminescent particles can be coated with a single dye or multiple dyes and generate an effective energy transfer between the core and dye(s) when excited via X-rays. LSO:Ce particles were surface modified with an alkyne modified naphthalimide (6-piperidin-1-yl-2-prop-2-yn-1-yl-1 H-benzo[ de]isoquinoline-1,3-(2 H)-dione, AlNap) and alkyne modified rhodamine B ( N-(6-diethylamino)-9-{2-[(prop-2-yn-1-yloxy)carbonyl]phenyl}-3 H-xanthen-3-ylidene)- N-ethylethanaminium, AlRhod) derivatives to tune the X-ray excited optical luminescence from blue to green to red using Förster Resonance Energy Transfer (FRET). As X-rays penetrate tissue much more effectively than UV/visible light, the fluorophore modified phosphors may have applications as bioimaging agents. To that end, the phosphors were incubated with rat cortical neurons and imaged after 24 h. The LSO:Ce surface modified with AlNap was able to be successfully imaged in vitro with a low-output X-ray tube. To use the LSO:Ce fluorophore modified particles as imaging agents, they must not induce cytotoxicity. Neither LSO:Ce nor LSO:Ce modified with AlNap showed any cytotoxicity toward normal human dermal fibroblast cells or mouse cortical neurons, respectively.

Rhodamine B in spices determined by a sensitive UPLC-MS/MS method.

Rhodamine B (RhB) is a banned food additive and has been classified as illegal colourant. Therefore, the risk of RhB contamination should be strictly monitored. In this study, a sensitive UPLC-MS/MS method was applied to monitor RhB in 292 various spices such as chilli, pepper and tomato products. The results showed 22.7% of chilli powder samples, 18.5% of pepper powder samples, 11.1% of chilli oil samples and 9.1% of pepper oil samples were contaminated with RhB. Chilli powder contained RhB up to 44,935 µg/kg with an average of 743 µg/kg, pepper powder up to 65.9 µg/kg with an average of 4.1 µg/kg, chilli oil up to 14.6 µg/kg with an average of 1.0 µg/kg and pepper oil up to 1.1 µg/kg with an average of 0.2 µg/kg, respectively. Considering the common consumption of chilli products and pepper products by so many consumers, RhB exposure is significant and should be decreased.