A Ratiometric and near-Infrared Fluorescent Probe for Imaging Cu<Superscript>2+</Superscript> in Living Cells and Animals

We have rationally constructed a novel ratiometric and near-infrared Cu²⁺ fluorescent probe based on a tricarbocyanine chromophore. The new probe NIR-Cu showed a ratiometric fluorescent response to Cu²⁺ with a large emission wavelength shift (up to 142 nm) in the far-red to near-infrared region. The probe also displayed a large variation in the fluorescence ratio (I₆₃₆/I₇₇₈) to Cu²⁺ species with high sensitivity and selectivity. Additionally, the developed probe NIR-Cu was suitable for fluorescence imaging of Cu²⁺ in living cells and mice.     

Simultaneous registration of Ca2+ transients and volume changes in rat mesangial cells: Evaluation of the effects of protein kinase C down-regulation

The intracellular free Ca²⁺ concentration ([Ca²⁺]_i) could be correlated with the contractile response in rat mesangial cells using an apparatus which measured both biochemical processes simultaneously. Long-term pretreatment of mesangial cells with 12-O-tetradecanoly-phorbol 13-acetate (24 h, 500 nM) increased the (20 nM) angiotensin II-induced mobilization of Ca²⁺ and led to an enhanced and sustained contraction of the cells. The contractile response was delayed by approximately 3.5 s with respect to the intracellular increase in Ca²⁺ concentration. The simultaneous registration of Ca²⁺ transients and cell contractions confirms that [Ca²⁺]_i is the major determinant of the angiotensin II-mediated mesangial cell contraction.Dedicated to Professor Horst H. A. Linde on the occasion of his 60th birthday.     

The Dependence of Benzo-15-Crown-5 Ether-Containing Oligo Paraphenylene Vinylene (CE-OPV) Emission Upon Complexation with Metal Ions in Solution

The effects of metal salts (NaCl, CaCl₂·2H₂O, EuCl₃·6H₂O and Eu(OAc)₃) on the steady state and time resolved fluorescence behaviour of poly(para phenylene vinylene) oligomers containing benzo-15-crown-5 ether units (CE-OPV) have been investigated. The presence of EuCl₃ causes a significant (8–9 fold) increase in the fluorescence emission intensity of the OPV segments, as compared to pure CE-OPV, in 99:1 methanol/chloroform solution and a small (9 nm) red shift in the emission maximum. The presence of Na⁺ or Ca²⁺ results in less marked increases in fluorescence intensity compared to Eu³⁺. In the presence of Eu³⁺ and Na⁺, the fluorescence intensity increases approximately linearly with metal ion concentration up to a metal ion/CE-OPV molar ratio of 10. The emission enhancement is not related to a simple 1:1 (CE-OPV:metal ion) complex formation process. In contrast, in acetonitrile, CE-OPV shows complex fluorescence quenching behaviour as a function of EuCl₃ concentration. This solvent dependence suggests that the emission changes with metal concentration are related to the formation of charge-transferred complexes. The marked changes in fluorescence quantum yield of the PPV backbone due to complexation with metal ions makes CE-OPV a sensitive fluorescent probe for metal ions, or may be exploited for improving the quantum yield of PPV-based devices.     

Luminescent Fluorene-Based Bis-Pyrazolyl Aniline Ligand for Aluminum Detection

The design, synthesis, and photophysical properties of a new fluorene-based fluorescent chemosensor, 4-((E)-2-(2-(benzo[d]thiazol-2-yl)-9,9-diethyl-9H-fluoren-7-yl)vinyl)-N,N-bis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzenamine (AXF-Al), is described for the detection of Al³⁺. AXF-Al exhibited absorption at 382 nm and strong fluorescence emission at 542 nm (fluorescence quantum yield, Φ _F, of 0.80). The capture of Al³⁺ by the pyrazolyl aniline receptor resulted in nominal change in the linear absorption (372 nm) but a large hypsochromic shift of 161 nm in the fluorescence spectrum (542 to 433 nm, Φ _F?=?0.88), from which Al³⁺ was detected both ratiometrically and colorimetrically. The addition of other metal ions, namely Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg ²⁺ and Pb²⁺, produced only minimal changes in the optical properties of this probe. The emission band of this probe was also accessed by two-photon excitation in the near-IR, as two-photon absorption (2PA) is important for potential applications in two-photon fluorescence microscopy (2PFM) imaging. The 2PA cross section of the free fluorenyl ligand AXF-Al was 220 GM at 810 nm and 235 GM at 810 nm for the Al-ligand complex, practically useful properties for 2PFM.     

Salicylaldehyde Phenylhydrazone: A New Highly Selective Fluorescent Lead (II) Probe

The fluorescence intensity of salicylaldehyde phenylhydrazone (L), in 1:1 (v/v) CH₃OH:H₂O was enhanced by ca. 100 times with a blue shift in emission maximum, on interaction with Pb²⁺ ion. No enhancement in fluorescent intensity of L was observed on interaction with metal ions - Na⁺, K⁺, Ca²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Cd²⁺ and Hg²⁺. This signal transduction was found to occur via photoinduced electron transfer (PET) mechanism. A 1:1 complexation between Pb²⁺ and L with log β?=?7.86 has been proved from fluorescent and UV/Visible spectroscopic data. The detection limit of Pb²⁺ was calculated to be 6.3?×?10^?7?M.     

Conformational Changes of the Troponin—Tropomyosin Complex on F-Actin Observed by Fluorescence Resonance Energy Transfer Measurements

Contraction of vertebrate striated muscle is regulated by the strong Ca²⁺-dependent interaction among troponin (Tn), tropomyosin (Tm), and actin on the thin filament. Using fluorescence resonance energy transfer (FRET), the interactions between Tm and the Tn complex or between Tm and the Tn subunit, TnI or TnC, with or without other troponin subunits, were characterized in the presence or absence of F-actin and Ca²⁺ ions. Cys-190 of Tm was selectively labeled with the acceptor probe, 4-dimethylaminophenylazophenyl 4-maleimide. Troponin was selectively labeled at position 9 or 133 of TnI and position 98 of TnC with a donor probe, 5-(2-iodoacetylaminoethyl)aminonaphtha lene 1-sulfonic acid. FRET measurements indicate that the interaction between TnI and Tm alone is very weak, but that in the presence of F-actin, TnI binds to the proper binding site on Tm even in the absence of TnT. The distances between Cys-190 of Tm on F-actin and Cys-9 or Cys-133 of Tnl or Cys-98 of TnC in the reconstituted Tn were determined to be 52.8, 53.7, Å and 56.5 Å, respectively, in the absence of Ca²⁺, indicating that the Tnl—TnC complex, the globular portion of Tn, is located near Cys-190 of Tm on the reconstituted thin filaments. Upon binding of Ca²⁺ to TnC, these distances increased by 5.6 and 1.4 Å or decreased by 5.4 Å, respectively. These Ca²⁺-induced changes in Tn—Tm seem to occur only when F-actin is present, suggesting that the stable complex formation of TnI with the outer domain of F-actin upon removal of Ca²⁺ is a very important event during inhibition.     

A Highly Selective and Sensitive Fluorescent Chemosensor for Aluminum Ions Based on Schiff Base

An efficient “off–on” type fluorescent chemosensor, (E)-N′-(4-(diethylamino)-2-hydroxybenzylidene)-2-hydroxybenzohydrazide (H ₂ L), based on Schiff base for the determination of Al³⁺ has been designed, synthesized, and evaluated. Upon treated with Al³⁺, the fluorescence of H ₂ L was enhanced 45-fold due to the chelation-enhanced fluorescence (CHEF) effect based on the formation of a 1:1 complex between the chemosensor and Al³⁺. Other metal ions, such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Ga³⁺, Zn²⁺, Cr³⁺, Cd²⁺, Ag⁺, Fe³⁺, In³⁺, Mn²⁺, Pb²⁺, Co²⁺, and Ni²⁺ had little effect on the fluorescence. The results demonstrate that the chemosensor H ₂ L has stronger affinity with Al³⁺ than other metal ions. The detection limit of H ₂ L for sensing Al³⁺ is 3.60 × 10^?6 M in EtOH–H₂O (3:7, v/v) solution. And the recognizing behavior has been investigated both experimentally and computationally.     

New Application of a Known Molecule: Rhodamine B 8-hydroxy-2-quinolinecarboxaldehyde Schiff Base as a Colorimetric and Fluorescent “Off-On” Probe for Copper (II)

Rhodamine B 8-hydroxy-2-quinolinecarboxaldehyde Schiff base (1) has been investigated as a colorimetric and fluorescent “off-on” probe for the recognition of Cu²⁺ in aqueous solution. Probe 1 was synthesized by condensation of rhodamine B hydrazide and 8-hydroxy-2-quinolinecarboxaldehyde, which exhibited good selectivity for Cu²⁺ among a range of biologically and environmentally important metal ions. The Cu²⁺ recognition event undergoes a Cu²⁺ promoted hydrolysis of probe 1 to release rhodamine B and the recognition process is barely interfered by other coexisting metal ions.

A Dual-Target Fluorescent Probe with Response-Time Dependent Selectivity for Cd<Superscript>2+</Superscript> and Cu<Superscript>2+</Superscript>

A novel fluorescent probe (NT) was developed by merging 2-hydrazinylbenzothiazole with 2-hydroxy-1-naphthaldehyde for the detection of Cd²⁺ and Cu²⁺. The probe alone is almost nonfluorescent due to the isomerization of C=N in the excited state. The addition of Cd²⁺ can cause an immediate strong green fluorescence owing to the suppression of C=N isomerization by Cd²⁺-coordination. Furthermore, NT gives a delayed turn-on fluorescence response to Cu²⁺ although it is a vigorous fluorescence quencher, which was thanks to the inhibition of the electron transfer between excited fluorophore and paramagnetic Cu²⁺ by sulfur donor. Based on fluorescence spectra and ESI-MS analysis, the binding modes between NT and Cd²⁺/Cu²⁺ were proposed.     

A Sensitive Schiff-Base Fluorescent Chemosensor for the Selective Detection of Zn2+

A Schiff-base fluorescent probe – N, N^/-bis(salicylidene) trans 1, 2 – diaminocyclohexane (H ₂ L) was synthesized and evaluated as a chemoselective Zn²⁺ sensor. Upon treatment with Zn²⁺, the complexation of H ₂ L with Zn²⁺ resulted in a bathochromic shift with a pronounced enhancement in the fluorescence intensity in ethanol solution. Moreover, other common alkali, alkaline earth and transition metal ions failed to induce response or minimal spectral changes. Notably, this chemosensor could distinguish clearly Zn²⁺ from Cd²⁺. The stoichiometric ratio and association constant were evaluated using Benesi – Hildebrand relation giving 1:1 stoichiometry. This further corroborated 1:1 complex formation based on Job’s plot analyses.     

Quinoline group grafted carbon nanotube fluorescent sensor for detection of Cu ion

A carbon nanotube-based fluorescent chemosensor MWNTs-glycine-N-8-quinolylamide (MWNTs-GNQ) has been designed and synthesized. Steady-state fluorescence emission studies showed that this material displays high selectivity and sensitivity for the Cu²⁺ ion over other cations such as Zn²⁺, Cd²⁺, Mg²⁺, Ca²⁺, and Ni²⁺.     

A Dansyl-Rhodamine Ratiometric Fluorescent Probe for Hg<Superscript>2+</Superscript> Based on FRET Mechanism

Based on resonance energy transfer (FRET) from dansyl to rhodamine 101, a new fluorescent probe (compound 1) containing rhodamine 101 and a dansyl unit was synthesized for detecting Hg²⁺ through ratiometric sensing in DMSO aqueous solutions. This probe shows a fast, reversible and selective response toward Hg²⁺ in a wide pH range. Hg²⁺ induced ring-opening reactions of the spirolactam rhodamine moiety of 1, leading to the formation of fluorescent derivatives that can serve as the FRET acceptors. Very large stokes shift (220 nm) was observed in this case. About 97-fold increase in fluorescence intensity ratio was observed upon its binding with Hg²⁺.     

Bis-Benzimidazolyl Diamide Based Fluorescent Probe for Copper(II): Synthesis, Structural and Fluorescence Studies

A new fluorescent probe based on a bis-benzimidazole diamide N ²,N ^2′-bis[(1-ethyl-benzimidazol-2-yl)methyl]biphenyl-2,2′-dicarboxamide ligand L ₁ with a biphenyl spacer group and a Copper(II) trinuclear metallacycle has been synthesized and characterized by X-ray single crystallography, elemental and spectral (FT-IR, ¹H & ¹³C NMR, UV-Visible) analysis. The fluorescence spectra of L ₁ in MeOH show an emission band centered at 300 nm. This band arises due to benzimidazolyl moiety in the ligating system. The diamide L ₁ in the presence of Cu²⁺ show the simultaneous ‘quenching’ of (300 nm) and ‘enhancement’ of (375 nm) emission band. Similar fluorescence behavior was found in water–methanol mixture (9:1). The new emission band at 375 nm is attributed to intra ligand π–π* transition of the biphenyl moiety. L ₁ exhibited high selectivity and sensitivity towards Cu²⁺ in both the medium over other common metal ions like Ni²⁺, Co²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Pb²⁺ and Hg²⁺. The binding constant with Cu²⁺ was calculated by the Benesi-Hildebrand equation. Selective “off-on-off” behavior of L ₁ in methanol has also been studied. The fluorescent intensity of 375 nm bands in L ₁ enhances (turns-on) upon addition of Cu²⁺ and quenches (turn-off) upon addition of Na₂-EDTA.     

A New On-fluorescent Probe for Manganese (II) Ion

A new fluorescent probe for Mn²⁺ ion, (6E)-N-((E)-1,2-diphenyl-2-(pyridin-2-ylimino)ethylidene)pyridin-2-amine (L), has been synthesized from benzil and 2-amino pyridine and characterized. In 1:1 (v/v) CH₃CN:H₂O (pH 4.0, universal buffer) L exhibits fluorescent intensity with emission peak at λ_max 360 nm on excitation with photons of 310 nm. Fluorescent intensity of L increases distinguishingly on interaction with Mn²⁺ ion compared to metal ions—Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺ and Ag⁺ individually or all together. The enhancement in fluorescent intensity is due to snapping of photoinduced electron transfer (PET) prevailed in free L. Fluorescence and UV/visible spectral data analysis shows that binding stoichiometry between Mn²⁺ and L is 1:1 with log β?≈?3.0. Both L and its Mn²⁺ complex were optimised using density functional theory (DFT) and vibrational frequency calculations confirm that both are at local minima on the potential energy surfaces.     

A New Piperidine Derivatized-Schiff Base Based “Turn-on” Cu<Superscript>2+</Superscript>Chemo-Sensor

In this study, we report the synthesis of new Schiff base E-1-(((1-benzylpiperidin-4-yl)imino)methyl)naphthalenee-2-ol (L) and evaluation of its fluorescence response toward Cu²⁺ ion. Preliminary, solvent effect, metal selectivity and metal ligand ratio were analyzed through UV-Visible study. Fluorescence response toward Cu²⁺ was carried to assess the fluorescent property of synthesized Schiff base. The probe exhibited a higher fluorescence enhancement in the presence of Cu²⁺ over other metal ions (Ni²⁺, Zn²⁺, Hg²⁺, Co²⁺, Cd²⁺, Al³⁺, Fe²⁺, and Pb²⁺). The binding stoichiometry between L and Cu²⁺ has been investigated using Job’s plot and Benesi-Hildebrand equation and it was found that ligand L can form 1:1 L-Cu²⁺ complex with binding constant (K _a) of 4?×?10⁴ LM^?1.     

A Novel Hg<Superscript>2+</Superscript> Selective Ratiometric Fluorescent Chemodosimeter Based on an Intramolecular FRET Mechanism

A irreversible Hg²⁺ selective ratiometric fluorescence probe FR, a fluorescein fluorophore linked to a rhodamine B hydrazide by a thiourea spacer, was designed and synthesized. The developed probe FR exhibited great ratiometric fluorescence enhancement and remarkable yellow-magenta color change toward Hg²⁺ with excellent selectivity in aqueous acetone solution, and the ratiometric fluorescence response to Hg²⁺ was not interfered by other metal cations including Fe³⁺, Co²⁺, Ni²⁺, Cr³⁺, Zn²⁺, Pb²⁺, Cd²⁺, Ca²⁺, Mg²⁺, Ba²⁺ and Mn²⁺. The linear range and the detection limit of this supposed ratiometric fluorescence method for Hg²⁺ were 0.0–10.0 × 10⁻⁶ and 5 × 10⁻⁸ M, respectively.     

Electron paramagnetic resonance of Gd<Superscript>3+</Superscript> aqua complexes in vitrified aqueous solutions and Gd<Superscript>3+</Superscript> aqua ions adsorbed on the capillary surface

Electron paramagnetic resonance (EPR) spectra of Gd³⁺ aqua complexes are measured in dilute aqueous solutions of Gd(NO₃)₃ (C<0.2 M) at room temperature. A partial resolution of the fine structure observed in the spectra is characteristic of solid disordered systems and results in an increase in the effective width of the EPR line with a decrease in the Gd³⁺ concentration. This phenomenon is explained in terms of adsorption of Gd³⁺ aqua ions on the surface of the measuring capillaries. The fine structure is revealed in the EPR spectra of Gd(NO₃)₃ aqueous solutions, namely, the Gd(NO₃)₃ solutions vitrified at a temperature of 77 K (with an addition of 10–15 vol % glycerol) and Gd(NO₃)₃ solutions quasi-vitrified at 298 K (with an addition of 70–90 vol % glycerol). Analysis of the EPR spectra demonstrates that these solutions contain two types of aqua complexes with fine structure parameters D₁=180 G and D₂=580 G. Reasoning from a comparison with x-ray diffraction data, the fine structure parameters D₁ and D₂ are assigned to higher symmetric eight-coordinate and lower symmetric nine-coordinate Gd³⁺ aqua complexes, respectively.     

A New Thiosemicarbazone-Based Fluorescence “Turn-on” Sensor for Zn<Superscript>2+</Superscript> Recognition with a Large Stokes Shift and its Application in Live Cell Imaging

Selective fluorescence turn on Zn²⁺ sensor with long-wavelength emission and a large Stokes shift is highly desirable in Zn²⁺ sensing area. We reported herein the synthesis and Zn²⁺ recognition properties of a new thiosemicarbazone-based fluorescent sensor L. L displays high selectivity and sensitivity toward Zn²⁺ over other metal ions in DMSO-H₂O (1:1, v/v, HEPES 10 mM, pH = 7.4) solution with a long-wavelength emission at 572 nm and a large Stokes shift of 222 nm. Confocal fluorescence microscopy experiments demonstrate that L is cell-permeable and capable of monitoring intracellular Zn²⁺.

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Graphical Abstract We report a new thiosemicarbazone-based fluorescent sensor (L) for selective recognition of Zn²⁺ with a long wavelength emission and a large Stokes shift.

     

Confocal fluorescence lifetime imaging of free calcium in single cells

Ca²⁺ concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for free calcium and exhibit marked changes in their photophysical properties upon binding. The differences in the fluorescent lifetime of the probes can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within the cells. We describe the use of a fast fluorescence lifetime imaging method in combination with a standard confocal laser scanning microscope for the determination of Ca²⁺ concentrations in single rat cardiac myocytes using the intensity probe Calcium Green.