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.

A lysosome-targeted near-infrared fluorescent probe for imaging of acid phosphatase in living cells.

Fluorescent probes for the detection of acid phosphatases (ACP) are important in the investigation of the pathology and diagnosis of diseases. We reported a lysosome-targeted near-infrared (NIR) fluorescent probe SHCy-P based on a novel NIR-emitting thioxanthene-indolium dye for the detection of ACP. The probe showed a long wavelength fluorescence emission at λem = 765 nm. Due to the ACP-catalyzed cleavage of the phosphate group in SHCy-P, the probe exhibited high selectivity and sensitivity for the 'turn-on' detection of ACP with a limit of detection as low as 0.48 U L-1. The probe SHCy-P could also be used to detect and image endogenous ACP in lysosomes. In light of these prominent properties, we envision that SHCy-P will be an efficient optical imaging approach for investigating the ACP activity in disease diagnosis.

Novel rhodamine dye with large Stokes shifts by fusing the 1,4-diethylpiperazine moiety and its applications in fast detection of Cu2.

Rhodamine dyes were widely developed for designing probes due to their excellent photophysical properties and biocompatibility. However, traditional rhodamine dyes still bear major drawbacks of short emission wavelengths (<600 nm) and narrow Stokes shifts (<30 nm), which limit their biological imaging applications. Herein, we reported a novel mitochondria-targeted fluorescent dye JRQ with near-infrared (NIR) emission wavelength and improved Stokes shift (63 nm) by tuning the donor-acceptor-donor (D-A-D) character of the rhodamine skeleton. As expected, JRQ exhibited multiple excellent properties and could accumulate in mitochondria, and can therefore be used as a signal reporter for the design of fluorescent probes by taking advantage of the fluorescence controlled mechanism of the ring opening and closing chemical processes of the spirolactone platform. By using JRQ as a precursor, a highly sensitive fluorescent probe JRQN for the fast detection of mitochondrial Cu2+ ions was synthesized based on the Cu2+-triggered specific hydrolysis mechanism because mitochondria are an important reservoir of intracellular Cu2+. We expect that the Stokes shift increase of rhodamine dyes via tuning the donor-acceptor-donor (D-A-D) character of the rhodamine skeleton will provide a novel synthetic approach for the development of rhodamine dyes and expansion of their applications.

A deep-red fluorescent molecular rotor based on donor-two-acceptor modular system for imaging mitochondrial viscosity.

A new donor-two-acceptor modular fluorescence rotor DpCy7 involving a phenolate donor unit and two benzothiazolium acceptor moieties was designed and synthesized. The DpCy7 underwent an internal charge transfer to form a Cy7-like longer conjugated system fluorochrome at a physiological pH. The probe exhibited a strong turn-on (8.5-fold) deep-red emission with a larger Stokes shift in glycerol aqueous solutions with restriction of rotation. Both the fluorescence intensity and fluorescence lifetime displayed the linear relationship of viscosity changes in the logarithmic plots. Furthermore, the HeLa cell imaging experiments of DpCy7 indicated that the rotor could be used to monitor the mitochondrial viscosity in living cells. This new type of deep-red fluorescence rotor provides a potential platform for determining viscosity at subcellular levels.

A near-infrared fluorescent probe with an improved Stokes shift achieved by tuning the donor-acceptor-donor character of the rhodamine skeleton and its applications.

In this paper, we report a novel near-infrared (NIR) mitochondrion-targeted fluorescent probe, RQS, with an improved Stokes shift (96 nm) for the specific detection of mitochondrial mercury ion (Hg2+) because mitochondrion is one of the main targeted organelles of Hg2+. For the preparation of the probe, a novel asymmetrical fluorescent xanthene dye RQ was first synthesized by tuning the donor-acceptor-donor (D-A-D) character of the rhodamine skeleton, and then the probe RQS was constructed by the mechanism of mercury-promoted ring-opening reaction. As expected, RQS could be used for the specific detection of Hg2+ with high selectivity, high sensitivity, and a detection limit down to the nanomolar range (2 nM). Importantly, RQS is capable of specifically distributing in mitochondria, and thus detect Hg2+ in real-time and provided a potential tool for studying the cytotoxic mechanisms of Hg2+.

A cis-Diol/pH Dual-Responsive Upconversion Nanoplatform: Synthesis, Characterization, and In Vitro Demonstration.

Integrating the functions of bioimaging, targeting and controlled release of therapeutic agents into a single nanoparticle is of great interests in nanomedicine and nanobiology. Herein, a cis -diol/pH dual-responsive upconversion nanoparticle (UCNP)-based theranostic platform has been developed for delivery of the anticancer drug to cancer cells. This nanoplatform is based on the strategic design of targetable hyaluronan modified UCNPs (HA-UCNPs) that are coupled with aminobenzeneboronic acid (APBA) to obtain APBA-UCNPs, having favorable tumor selectivity as well as the capacity for capturing cis-diol-containing therapeutics. The controlled release function is then achieved through the self-assembly of hydroxycamptothecin derivative ligands onto the surfaces of APBA-UCNPs, which is controllable in a stimuli-dependent manner. The UCNP-based theranostic probe taken up by tumor cells via receptor-mediated endocytosis liberates drugs triggered by competitive glucose at low pH in endosomes/lysosomes, resulting in cell apoptosis. The dual-responsive mechanism of boronate ester bonds gives a chemoselective strategy for controlled release of drug within tumor cells, establishing an alternative approach to treat a broad spectrum of diseases exploiting similar boronic acid-involved therapeutics.

A mitochondria-targeted near-infrared fluorescent probe with a large Stokes shift for real-time detection of hypochlorous acid.

Hypochlorous acid (HOCl) has been known to be intertwined with various pathophysiological processes. In this paper, a novel mitochondria-targeted near-infrared (NIR) fluorescent probe L based on a chromenylium-phenothiazine conjugate has been designed and synthesized for the detection of hypochlorous acid. Due to the HOCl-promoted intramolecular charge transfer processes, the probe L shows near-infrared fluorescence emission at 672 nm, large Stokes shifts of 97 nm, fast response, and high selectivity and sensitivity. Furthermore, the probe L is biocompatible, cell-membrane permeable and mitochondria-targetable, and can be used for endogenous HOCl imaging in living cells.

Novel π-extended hybrid xanthene dyes with two spirolactone rings for optoelectronic and biological applications.

The design, synthesis, and photophysical properties of organic fluorophores have attracted considerable research interest due to the utility of these compounds for various optoelectronic, analytical, and biological applications. In this study, we synthesized two novel π-extended red-emitting hybrid xanthene dyes, each of which has two spirolactone rings and combines a seminaphthofluorescein moiety and a seminaphthorhodafluor moiety in a single molecule. The photophysical properties of the dyes in methanol in the presence of acid, base, and metal cations were investigated. Mono-ring-opened seminaphthofluorescein and seminaphthorhodafluor forms of the dyes could be obtained by the addition of OH- or H+, respectively. Owing to the changes in the absorbance spectra of the mono-ring-opened forms induced by addition of H+ and OH-, the dyes could perform simultaneously the functions of an XOR gate and an INHIBIT gate, with the absorbances at 510 and 560 nm as outputs, respectively, and could act as half-subtractors with H+ and OH- as inputs. Furthermore, stepwise ring-opening could be induced by Hg2+ ions in methanol. In water, the dyes existed in double-ring-opened forms that emitted deep-red fluorescence and were mitochondria-targetable, suggesting that these chromophores might be useful as fluorescence tracers in biological applications. Because the absorption and fluorescence properties of these fluorophores can be regulated via their two spirolactone rings, we expect that these compounds will find utility in various optoelectronic, analytical, and biological applications.

Synthesis of near-infrared fluorescent rhodamines via an SNArH reaction and their biological applications.

Near-infrared (NIR) dyes are of great interest in biomedicine due to diminished interfering absorption and fluorescence from biological samples, reduced scattering, and enhanced tissue penetration depth. In this context, we report the synthesis of rectilinearly π-extended rhodamine dyes using a unique intramolecular nucleophilic substitution of aromatic hydrogen (SNArH) strategy. The strategy makes use of an SNArH reaction between a preorganized aromatic amino nitrogen and an electron-deficient carbon in the xanthylium ion. The SNArH reaction presented herein can be performed under mild conditions without a transition metal catalyst and can be expected to enable the preparation of a wide variety of π-extended near-infrared fluorescent rhodamine dyes. Using this strategy, seven rectilinearly π-extended rhodamines (RE1-RE7) that had fluorescence emission wavelengths in the near-infrared region were synthesized. RE1, RE3, and RE4 were lysosome targetable and showed good photostabilities. In addition, using dye RE1 as a precursor, we constructed a novel NIR fluorescent turn-on probe (RE1-Cu), which can be used for detecting Cu2+ in living cells, demonstrating the value of our NIR functional fluorescent dyes.

A rhodamine-based fast and selective fluorescent probe for monitoring exogenous and endogenous nitric oxide in live cells.

Nitric oxide, an important cellular messenger molecule, plays a wide range of physiological roles in neurotransmission, vascular relaxation, platelet activation, and immune response. In this work, we synthesized a selective and sensitive cationic fluorescent probe (ROPD) for monitoring exogenous and endogenous nitric oxide in live cells by grafting a NO-trapper o-phenylenediamine (OPD), via one of the amino groups of OPD, onto a rhodamine fluorophore at the C-3 position. The probe exhibited high selectivity and sensitivity towards NO with fast response in the water phase over a wide pH range. At the same time, the probe ROPD is cell-membrane permeable and can be used for the real-time visualization of exogenous NO in both L929 and HeLa cells, and endogenous NO in stressed RAW 264.7 cells.

A unique rectilinearly π-extended rhodamine dye with large Stokes shift and near-infrared fluorescence for bioimaging.

The synthesis of a novel rectilinearly π-extended rhodamine dye (TJ730) has been described. It exhibits NIR fluorescence emission with a Stokes shift of >110 nm. TJ730 and its ester derivative are cell permeable, have low cytotoxicity, are biocompatible, and can be directly used as NIR tracers for lysosome staining. Using TJ730 as a precursor, two probes have been prepared for Cu2+.

A highly selective and sensitive fluorescent probe for hypochlorous acid and its lysosome-targetable biological applications.

A novel lysosome-targetable fluorescence probe PT-1 based on a photoinduced electron transfer (PET) mechanism has been designed and synthesized. For comparison, the probe PT-2 without morpholine moiety as a lysosome-directing group was also synthesized and investigated. Probes PT-1 and PT-2 exhibited high selectivity, high sensitivity (with the detection limits down to the 10-10M range) and response in real time (within 10s) toward HOCl over other reactive oxygen species (ROS). Both PT-1 and PT-2 were cell permeable and enabled them to be used for monitoring of HOCl in living cells. Meanwhile, the probe PT-1 demonstrated an accurately lysosome-targeting ability, and was successfully applied to image of exogenous, endogenous produced HOCl in living cells. The success of subcellular imaging suggested that the probe PT-1 could be used in further applications for the investigation of biological functions and pathological roles of HOCl at organelle levels.

A highly selective and sensitive fluorescent probe for Cu2+ based on a novel naphthalimide-rhodamine platform and its application in live cell imaging.

Copper plays important roles in a variety of fundamental physiological processes. At the cell organelle level, aberrant copper homeostasis in lysosomes can lead to various serious diseases. Herein, a bifluorophore-based, lysosome-targetable Cu2+-selective ratiometric fluorescent probe (V) has been synthesized by reasonable design. The probe V shows high selectivity toward Cu2+ ions over other cations and exhibits high sensitivity (1.45 nM) for the detection of Cu2+ ions. Meanwhile, the probe is cell permeable and suitable for ratiometric visualization of lysosomal Cu2+ in the living cell.

Fluorescence probe for hypochlorous acid in water and its applications for highly lysosome-targetable live cell imaging.

Hypochloric acid (HOCl) plays important roles in cell signaling and homeostasis, such as anti-inflammation and immune regulation, pathogen response and so on. Accordingly, direct detection of HOCl at the organelle level is important for investigation of the complex contributions of HOCl to human health. In the present study, a water soluble lysosome-targeting fluorescent probe Lyso-1 bearing a hydrazone moiety as a HOCl-responsive site and a morpholine unit as a lysosomal-targeting group has been synthesized and evaluated for its ability to image lysosomal HOCl. The probe Lyso-1, based on a novel HOCl-promoted hydrazone oxidation strategy, showed a highly selective fluorescent off/on response to HOCl with the various reactive oxygen species in water. With increasing amount of ClO- from 0.5 to 2.5 µM, a linear correlation between the fluorescence intensity (570 nm) of Lyso-1 and [ClO-] was found, and the regression equation was y = 96.65 + 110.2068[ClO-] with a linear coefficient R of 0.9920. The detection limit is determined to be 60 nM. Lyso-1 demonstrated a perfect lysosomal targetable ability, and was successfully applied to image of exogenous, endogenous produced lysosomal HOCl in live L929 cells. The success of subcellular imaging indicated that the lysosome-targetable probe Lyso-1 could be used in further applications for the investigation of biological functions and pathological roles of HClO at organelle levels.

A highly selective and sensitive photoinduced electron transfer (PET) based HOCl fluorescent probe in water and its endogenous imaging in living cells.

A probe based on the phenothiazine-acridine orange conjugate (Ptz-AO) has been designed and synthesized for the sensitive and selective detection of HOCl. Ptz-AO has excellent properties, including pH-independence of fluorescence, high resistance to photobleaching, and response in real time. The value of Ptz-AO was confirmed by exogenous, endogenous and the real-time imaging of HOCl in vitro using a fluorescence microscope.

A Novel Styryldehydropyridocolinium Homodimer: Synthesis and Fluorescence Properties Upon Interaction with DNA.

A novel homodimer of the styryldehydropyridocolinium dye (TPTP) has been synthesized and characterized. Free TPTP exhibited low fluorescence quantum yield and large Stokes shift (over 160 nm) in water. However, it showed a significant fluorescence turn-on effect upon intercalation into DNA base pairs. Meanwhile, the fluorescence intensity of the intercalated structures formed by TPTP and DNA decreased quickly upon addition of deoxyribonuclease I, indicating that the dye can be used to monitor deoxyribonuclease I activity and DNA hydrolysis. Electrophoresis analysis revealed that the dye had intercalative binding to DNA and can potentially be used for DNA staining in electrophoresis. Thus, the innate nature of large Stokes shift and excellent fluorescence turn on effect upon interaction with DNA endue the dye with a wide range of applications.

Molecular design and synthesis of a pH independent and cell permeant fluorescent dye and its applications.

Fluorescent dyes have played crucial roles in the field of molecular imaging as fluorescent fluorophores. In this work, a novel water-soluble and pH-independent fluorescent xanthene dye, a hydroxyl regioisomeric 3',4'-benzorhodol, has been designed and synthesized. Compared with those of rhodol dyes, the absorption (ca. 570 nm) and maximum emission (ca. 620 nm) of the dye are largely red-shifted. Due to its ring-opened zwitterion structure in water media, the dye showed good membrane permeability and distributed in the whole cell cytoplasm upon incubation with live cells. Meanwhile, the dye could be easily modified to probes. The hydrazide derivative of the dye exhibited an excellent Hg(2+) selectivity over other relevant metal ions with a detection limit down to 3 nM. Thus, the excellent fluorescence properties and chemical properties of the dye allow it to be designed as a fluorescent chemosensor and biomarker for biological applications.