Phenothiazine–rhodamine‐based colorimetric and fluorogenic ‘turn‐on' sensor for Zn2+ and bioimaging studies in live cells

A phenothiazine–rhodamine (PTRH) fluorescent dyad was synthesized and its ability to selectively sense Zn²⁺ ions in solution and in in vitro cell lines was tested using various techniques. When compared with other competing metal ions, the PTRH probe showed the high selectivity for Zn²⁺ ions that was supported by electronic and emission spectral analyses. The emission band at 528 nm for the PTRH probe indicated the ring closed form of PTRH, as for Zn²⁺ ion binding to PTRH, the λ_em get shift to 608 nm was accompanied by a pale yellow to pink colour (under visible light) and green to pinkish red fluorescence emission (under UV light) due to ring opening of the spirolactam moiety in the PTRH ligand. Spectral overlap of the donor emission band and the absorption band of the ring opened form of the acceptor moiety contributed towards the fluorescence resonance energy transfer ON mechanism for Zn²⁺ ion detection. The PTRH sensor had the lowest detection limit for Zn²⁺, found to be 2.89 × 10^?8 M. The sensor also demonstrated good sensing application with minimum toxicity for in vitro analyses using HeLa cells.     

Rhodamine-based ‘turn-on’ fluorescent probe for Cu(II) and its fluorescence imaging in living cells

A novel rhodamine spirolactam derivative 3′,6′-Bis(diethylamino)-2-(2-hydroxyethylamino) spiro[isoindoline-1,9′-xanthen]-3-one (RO1) was synthesized, and characterized by high-resolution mass spectrometry (HRMS), X-ray crystallography, Infrared spectroscopy (IR), and ¹H NMR and ¹³C NMR spectroscopy. RO1 exhibited highly sensitive and exclusively selective fluorescence response toward Cu²⁺ over other metal ions with a detection limit of 0.56 ppb in mixed aqueous solution. The fluorescence was pH-independent in the wide range pH 3.1–11.6. The turn-on fluorescence enhancement of the probe is based on Cu²⁺ induced ring-opening mechanism of the rhodamine spirolactam. Moreover, by means of fluorescence microscopy experiments, it was demonstrated that RO1 could monitor trace Cu²⁺ changes by live cell imaging.     

A new rhodamine‐based fluorescent chemodosimeter for mercuric ions in water media

A new rhodamine–ethylenediamine–nitrothiourea conjugate (RT) was synthesized and its sensing property as a fluorescent chemodosimeter toward metal ions was explored in water media. Analytical results from absorption and fluorescence spectra revealed that the addition of Hg²⁺ ions to the aqueous solution of the chemodosimeter RT caused a distinct fluorescence OFF–ON response with a remarkable visual color change from colorless to pink; however, no clear spectral and color changes were observed from other metal ions including: Zn²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Ag⁺, Fe²⁺, Cr³⁺, Co³⁺, Ni²⁺, Ca²⁺, Mg²⁺, K⁺ and Na⁺. The sensing results and the molecular structure suggested that a Hg²⁺‐induced a desulfurization reaction and cyclic guanylation of the thiourea moiety followed by ring‐opening of the rhodamine spirolactam in RT are responsible for a distinct fluorescence turn‐on signal, indicating that RT is a remarkably sensitive and selective chemodosimeter for Hg²⁺ ions in aqueous solution. Hg²⁺ within a concentration range from 0.1 to 25 μM can be determined using RT as a chemodosimeter and a detection limit of 0.04 μM is achieved. Copyright © 2014 John Wiley & Sons, Ltd.     

A triazole‐based fluorescence probe for detecting Hg2+ ions and its biological application

A new compound, ethyl 5‐phenyl‐2‐(p‐tolyl)‐2H‐1,2,3‐triazole‐4‐carboxylate was successfully introduced and synthesized as a novel rhodamine B derivative named REPPC, and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high resolution mass spectrometry (HRMS). It showed an obvious fluorescence and UV–visible light absorption enhancement towards Hg²⁺ ion without interference from common metal ions in N,N‐dimethylformamide–H₂O (pH 7.4). The spirolactam ring moiety of rhodamine in REPPC was converted to the open‐ring form generating a 1:1 complex with the intervention of a mercury ion, verified by electrospray ionization‐mass spectroscopy testing and density functional theory calculation. REPPC was used to visualize the level of mercury ions in living HeLa cells with encouraging results.     

A novel fluorescent probe based on rhodamine hydrazone derivatives bearing a thiophene group for Al3+

In the present work, a novel 5‐methyl‐thiophene‐carbaldehyde‐functionalized rhodamine 6G Schiff base (RA) was designed and easily prepared as an Al³⁺ fluorescent and colorimetric probe, which could selectively and sensitively detect Al³⁺ by showing enhanced fluorescence emission. Meanwhile distinct color variation from colorless to pink also provided ‘naked eye’ detection of Al³⁺, due to the ring spirolactam opening of the rhodamine derivative. Other metal ions (including K⁺, Mg²⁺, Na⁺, Ba²⁺, Mn²⁺, Cd²⁺, Fe²⁺, Ni²⁺, Pb²⁺, Zn²⁺, Hg²⁺, Co²⁺, Li⁺, Sr²⁺ and Cu²⁺) could only induce limited interference. The detection limit of the fluorescent probe was estimated to be 4.17 × 10^?6 M, the binding constant of the RA–Al³⁺ complex was 1.4 × 10⁶ M^?1. Moreover, this fluorescent probe RA possessed high reversibility. As aluminum is a ubiquitous metal in nature and plays vital roles in many biological processes, this chemosensor could be explored for biological study applications. Copyright © 2015 John Wiley & Sons, Ltd.     

‘Turn‐on’ fluorescent chemosensors based on naphthaldehyde‐2‐pyridinehydrazone compounds for the detection of zinc ion in water at neutral pH

A series of naphthaldehyde‐2‐pyridinehydrazone derivatives were discovered to display interesting ‘turn‐on’ fluorescence response to Zn²⁺ in 99% water/DMSO (v/v) at pH 7.0. Mechanism study indicated that different substituent groups in the naphthaldehyde moiety exhibited significant influence on the detection of Zn²⁺. The electron rich group resulted in longer fluorescence wavelengths but smaller fluorescence enhancement for Zn²⁺. Among these compounds, 1 showed the highest fluorescence enhancement of 19‐fold with the lowest detection limit of 0.17 μmol/L toward Zn²⁺. The corresponding linear range was at least from 0.6 to 6.0 μmol/L. Significantly, 1 showed an excellent selectivity toward Zn²⁺ over other metal ions including Cd²⁺.     

A tricorn‐rhodamine fluorescent chemosensor for detection of Co2+ ions

A novel chemosensor TrisRh based on tris(2‐aminoethyl)amine and rhodamine 6G is designed and synthesized as a fluorescence turn‐on probe for Co²⁺ ions that is paramagnetic with a property of quenching fluorescence. Rhodamine spirolactam forms are nonfluorescent, whereas, ring‐opening of corresponding spirocyclic induced by Co²⁺ results in strong fluorescence emission. Upon the addition of Co²⁺ ions, TrisRh can display significant enhancements in absorbance and fluorescence intensity as well as evident colorific transformation, which can be perceived by the naked eye. The association stoichiometry of TrisRh to Co²⁺ ions was inferred to be 1:1 through Job's plot and electrospray ionization mass spectrometry analysis. The binding model was speculated from Fourier transform infrared spectra and ¹H–nuclear magnetic resonance technologies. Significantly, the limit of detection was determined to be as low as 1.22 nmol. Furthermore, TrisRh can exhibit robust anti‐jamming ability against other interference metal ions.     

A rhodamine‐triazole fluorescent chemodosimeter for Cu2+ detection and its application in bioimaging

A rhodamine‐based fluorescent chemodosimeter rhodamine hydrazide‐triazole (RHT) tethered with a triazole moiety was developed for Cu²⁺ detection. In aqueous medium, the RHT probe exhibited high selectivity and sensitivity toward Cu²⁺ among other metal ions. The addition of Cu²⁺ triggered a fluorescence emission of RHT by 384‐fold (Φ = 0.33) based on a ring‐opening process and a subsequent hydrolysis reaction. Moreover, RHT also showed a selective colorimetric response toward Cu²⁺ from colorless solution to pink, readily observed with the naked eye. The limit of detection of RHT for Cu²⁺ was calculated to be 1 nM (0.06 ppb). RHT was successfully demonstrated to detect Cu²⁺ in Chang liver cells by confocal fluorescence microscopy.     

An integrated system of pyrene and rhodamine-6G for selective colorimetric and fluorometric sensing of mercury(II)

A pyrene and rhodamine-6G functionalized simple chemosensor L is studied toward sensing of metal ions in solution extensively. L shows selective color change from colorless to pink in the presence of Hg²⁺ in acetonitrile and the UV-Vis study shows peak at 525 nm with a ε value of 5.2 × 10⁴ M⁻¹ cm⁻¹ due to selective ring opening of rhodamine spirolactam moiety. The selective sensing of Hg²⁺ by L in the presence of other metal ions and reversible nature of “OFF-ON-OFF” functionality of L by Hg²⁺ and EDTA, respectively, are also established. The fluorescence study of L in the presence of Hg²⁺ shows emission at 550 nm when excited at 525 nm (ring opened rhodamine wavelength) or 340 nm (pyrene wavelength) in dry CH₃CN. Thus L acts as a selective colorimetric and fluorometric probe (dual probe) for the Hg²⁺ in solution. Metal ion sensing ability of L is also carried out in water as well as in aqueous Hepes buffer. These studies suggest that the fluorescence output of L in presence of Hg²⁺ in aqueous environment is apparently due to the generation of acid upon addition of Hg²⁺ salt in water.     

Design and synthesis of a new fluorescent probe for cascade detection of Zn2+ and H2PO4− in water and targeted imaging of living cells

Design and synthesis of new fluorescence probes with good water‐solubility is of great importance to better understanding the significant role of ions which are related to biology and the environment. As important ions, zinc ion (Zn²⁺) and dihydrogen phosphate ion (H₂PO₄^?) display essential roles in living systems, and quantitative detection of these ions in water is still a challenge. In order to consider the significant role of the galactose moiety in the design of a water‐soluble fluorescence sensor, herein, we have developed a novel probe, Gal‐AQTF, for the cascade detection of Zn²⁺ and H₂PO₄^? with excellent selectivity in water. Through the introduction of the galactose moiety onto the sensor AQTF, which has been reported earlier by us, the water‐solubility, cell compatibility and targeting ability were enhanced. Gal‐AQTF has been successfully applied in the imaging of the living cells of HepG2 and A549, and illustrated good selectivity for the HepG2 cells which overly express the asialoglycoprotein (ASGP) receptor.     

A comparison of Zn2+- and Ca2+-triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition

Intracellular Zn²⁺ toxicity is associated with mitochondrial dysfunction. Zn²⁺ depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn²⁺-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn²⁺-induced depolarization with the effects of Ca²⁺ in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca²⁺ caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg²⁺, ADP and cyclosporine A. Zn²⁺ also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn²⁺-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca²⁺ and Zn²⁺ in a calcein-retention assay. Consistent with the well-documented ability of Ca²⁺ to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn²⁺-treated mitochondria. Considered together, our results suggest that Ca²⁺ and Zn²⁺ depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn²⁺-induced depolarization, and that Zn²⁺ is not a particularly potent mitochondrial inhibitor.     

ICPBCZin: a red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

A new fluorescent Zn²⁺ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn²⁺ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3′,2′:3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn²⁺ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn²⁺ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn²⁺ probe.     

A sensitive fluorescent sensor based on the photoinduced electron transfer mechanism for cefixime and ctDNA

Cefixime is a third generation orally administered cephalosporin that is frequently used as a broad spectrum antibiotic against various gram‐negative and gram‐positive bacteria. In this study, a simple and sensitive fluorescent sensor for the determination of the cefixime and ctDNA was established based on the CdTe:Zn²⁺ quantum dots (QDs). The fluorescence of CdTe:Zn²⁺ QDs can be effectively quenched by cefixime in virtue of the surface binding of cefixime on CdTe:Zn²⁺ QDs and the subsequent photoinduced electron transfer process from CdTe:Zn²⁺ QDs to cefixime, in particular, the high sensitivity of QDs fluorescence emission to cefixime at the micromole per liter level, which render the cefixime‐CdTe:Zn²⁺ QDs system into fluorescence “OFF” status, then turn on in the presence of ctDNA. Furthermore, the Fourier transform infrared (FTIR) spectra of characteristic bands of C–N and N–H groups of cefixime endow evidence for the interaction of cefixime with CdTe:Zn²⁺ QDs. The relative electrochemical behavior of the affinity of CdTe:Zn²⁺ QDs for cefixime and ctDNA reveals the potential molecular binding mechanism.     

New Alternately Colored FRET Sensors for Simultaneous Monitoring of Zn2+ in Multiple Cellular Locations

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn²⁺based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²⁺ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²⁺ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²⁺. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²⁺ uptake simultaneously in two compartments, revealing that nuclear Zn²⁺ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²⁺ more slowly and with a delay of 600–700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²⁺ and reveal that nuclear Zn²⁺ is buffered at a higher level than cytosolic Zn²⁺.     

Development of a dual functional luminescent sensor for zinc ion based on a peptidic architecture

A synthetic peptide bearing a lanthanide complex, TbOTZ exhibits a decrease of chromophore fluorescence and a concomitant luminescence enhancement due to sensitized Tb³⁺ upon Zn²⁺ binding. Thus, TbOTZ can be a valuable tool for ratiometric sensing of Zn²⁺ as well as for time-resolved fluorescence detection with a single molecule.     

Dual role of Zn in maintaining structural integrity and suppressing deacetylase activity of SIRT1

Zn²⁺ directly participates in catalysis of histone deacetylase (HDAC) Classes I, II, IV enzymes while its role in HDAC Class III activity is not well established. Herein we investigated the effects of Zn²⁺ on the deacetylase activity of sirtuin 1 (silent mating type information regulation 2 homolog 1, SIRT1). We found that the inherent Zn²⁺ at the zinc-finger motif of SIRT1 is essential for the structural integrity and the deacetylase activity of SIRT1, whereas the exogenous Zn²⁺ strongly inhibits the deacetylase activity with an IC₅₀ of 0.82 μM for Zn(Gly)₂. SIRT1 activity suppressed by the exogenous Zn²⁺ can be fully recovered by the metal chelator EDTA but not by the activator resveratrol. We also identified Zn²⁺ as a noncompetitive inhibitor for the substrates of NAD⁺ and the acetyl peptide P53-AMC. The 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence titration experiments and site-directed mutagenesis study suggested that the exogenous Zn²⁺ binds to SIRT1 but not at the zinc-finger motif. These results indicate that Zn²⁺ plays a dual role in SIRT1 activity. Inherent Zn²⁺ at the zinc-finger motif is structurally related and essential for SIRT1 activity. On the other hand, Zn²⁺ may also bind to another site different from the zinc-finger motif or the binding sites for the substrates or resveratrol and act as a potent inhibitor of SIRT1.     

Release of intracellular Zn<Superscript>2+</Superscript> in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn²⁺ release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn²⁺ release and subsequent cell death. Cortical neurons were loaded with the Zn²⁺ selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn²⁺ levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn²⁺. Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn²⁺ induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn²⁺ release appeared to peak at 1 μM spermine NONOate (123.8 ± 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn²⁺ levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn²⁺ in cortical neurons. The possibility that release of intracellular Zn²⁺ in response to NO could play a role in intracellular signaling is discussed.     

Synthesis and evaluation of a novel ‘off-on’ chemical sensor based on rhodamine B and the 2,5-pyrrolidinedione moiety for selective discrimination of glutathione and its bioimaging in living cells

A new “turn-on” fluorescent probe, RDMBM, based on the rhodamine B dye and the 2,5-pyrrolidinedione moiety was synthesized and characterized. Its sensing behavior toward various amino acids was evaluated via UV–vis and fluorescence spectroscopic techniques. The observed spectral changes showed that RDMBM displays high selectivity and sensitivity toward GSH in MeOH/H₂O (1:2, v/v, pH 7.40, Tris-HCl buffer, 1?mM) solution and that it undergoes 1:1 covalent binding with GSH. More importantly, the hydrogenation and ring-opening of the nitrogen atom in the spirane structure of rhodamine B derivatives were tightly bound to the induction effects of different groups. Furthermore, fluorescence imaging applications demonstrated that RDMBM can be successfully used for the detection of GSH in human breast cancer cells MCF-7.     

In Solution Cation-Induced Secondary and Tertiary Structure Alterations of Human Calprotectin

Calprotectin (CP) is widely considered to have diverse roles including growth inhibitory and apoptosis induction in a number of tumor cell lines and antimicrobial activities. As CP has been proposed to bind metal ions with high affinity, we have studied its functional and primarily its structural behavior upon Zn²⁺ and Mn²⁺ chelation solely and along with Ca²⁺. We employed fluorescence spectroscopy and circular dichroism to determine the resulting modifications. Based upon our findings it is clear that treating CP with ions effectively weakened its natural growth inhibitory activity. Moreover, structural analysis of Zn²⁺ and Mn²⁺-treated CPs indicated remarkable alterations in the regular secondary structures in favor of irregular structures while Zn²⁺ and Mn²⁺ treatment of CP after incubation with Ca²⁺ displayed no remarkable shifts. Tertiary structure investigation using fluorescence spectroscopy showed that CP undergoes conformational changes upon Zn²⁺ and Mn²⁺ treatment whereby Trp residues of protein is slightly exposed to the hydrophilic environment, compactness of CP is compromised, whereas in Ca²⁺-treated CP, the tertiary structure integrity is intact upon Zn²⁺ and Mn²⁺ chelation. Interestingly, CP structural modifications upon Zn²⁺ and Mn²⁺ treatment was significantly comparable, probably due to similar radii and charges of ions. Taken all together, we have concluded that CP maintains its normal nature in Ca²⁺-loaded state when treated with Zn²⁺ and Mn²⁺ ions. It can be suggested that Ca²⁺ not only stabilize CP structure but also helps CP to keep its structure upon metal ions chelation which is involved in host organism defense system.