Novel probes showing specific fluorescence enhancement on binding to a hexahistidine tag

The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel-nitrilotriacetic acid (Ni(2+)-NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal-NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal-NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni(2+) and Co(2+) were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni(2+) and Co(2+) complexes of NTACs in pH 7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)(6)-Tyr-NH(2); Tyr was included to allow convenient concentration measurement) in pH 7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co(2+) complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide.     

An organoselenium-based highly sensitive and selective fluorescent "turn-on" probe for the Hg2+ ion

An organoselenium-based NSe(3) type of tripodal system 2 as a Hg(2+)-selective fluorescence "turn-on" probe is described. The "turn-on" fluorescence behavior of this selenotripod 2 is significant because it depends on Hg-Se bond formation and acts as a reporting unit for this system. The system exhibits immediate response (15 s) with a subnanomolar detection limit (0.1 nM) for the Hg(2+) ion. It efficiently detects both aqueous and nonaqueous Hg(2+) at 2 nM concentration.     

Design and Synthesis of a Selective Chemosensor for Zn^2＋

A hydroxyl substituted phenolic Schiff base 1, used as sensor for detection of Zn^2＋, was synthesized and investigated. It was found that a strong fluorescence emission was observed when 1 bound to Zn^2＋ in acetonitrile, whereas no fluorescence emission was detected when 1 bound to other metal ions （Fe^2＋, Co^2＋, Ni^2＋, Cu^2＋, Cd^2＋, Hg^2＋, Mg^2＋, Pb^2＋, Ca^2＋, Ba^2＋, Sr^2＋） except for Mg^2＋, for which a weak fluorescence emission was detected in the same condition. Competition experiment showed that no obvious interference was observed in its fluorescence while 1 performed the titration with Zn^2＋ in the different mixtures of metal ions. To understand the site where Zn^2＋ coordinated to the ligand and the mechanism of binding, three other hydroxyl substituted phenolic Schiff bases 2-4 were synthesized and their binding reactions with Zn^2＋were also investigated.     

The "off-on" fluorescence detection of alkaline earth metal ions by using 1-naphthylacetanilide derivatives: new cation sensors based on oligo-ethylene oxides connecting 1-naphthylacetanilide at its terminals.

New fluorescent ionophores containing the 1-naphthylacetanilide moiety based on oligo-ethylene oxide (1(4) and 1(5)) have been synthesized, and their photochemical behaviors have been studied. In the absence of a metal ion, the 1-naphthylacetanilide moiety showed a weak fluorescence emission (fluorescence "off state"). However, complexation with Ca(2+) induced a large enhancement effect on the fluorescence intensity of 1(4) and 1(5) from the naphthalene ring (fluorescence "on" state). This behavior can be explained in terms of a similar twisted intramolecular charge-transfer relaxation mechanism. Such "off-on" fluorescence responses of 1(4) and 1(5) provide the digital detection of alkaline earth metal ions.     

Midrange affinity fluorescent Zn(II) sensors of the Zinpyr family: syntheses, characterization, and biological imaging applications

The syntheses and photophysical characterization of ZP9, 2-{2-chloro-6-hydroxy-3-oxo-5-[(2-{[pyridin-2-ylmethyl-(1H-pyrrol-2-ylmethyl)amino]methyl}phenylamino)methyl]-3H-xanthen-9-yl}benzoic acid, and ZP10, 2-{2-chloro-6-hydroxy-5-[(2-{[(1-methyl-1H-pyrrol-2-ylmethyl)pyridin-2-ylmethylamino]methyl}phenylamino)methyl]-3-oxo-3H-xanthen-9-yl}benzoic acid, two asymmetrically derivatized fluorescein-based dyes, are described. These sensors each contain an aniline-based ligand moiety functionalized with a pyridyl-amine-pyrrole group and have dissociation constants for Zn(II) in the sub-micromolar (ZP9) and low-micromolar (ZP10) range, which we define as "midrange". They give approximately 12- (ZP9) and approximately 7-fold (ZP10) fluorescence turn-on immediately following Zn(II) addition at neutral pH and exhibit improved selectivity for Zn(II) compared to the di-(2-picolyl)amine-based Zinpyr (ZP) sensors. Confocal microscopy studies indicate that such asymmetrical fluorescein-based probes are cell permeable and Zn(II) responsive in vivo.     

A highly selective colorimetric and “Off-On” fluorescence sensor for CN~- based on Zn(salphenazine) complex

The development of sensors for selective detection of cyanide ion(CN~-) is an important mission to accomplish because of the versatility and toxicity of CN~-. In the present work, an "ensemble"-based colorimetric and fluorescent sensor(L2-Zn~(2+)) for CN~-ion has been developed. The addition of cyanide ions removed Zn~(2+) from the ensemble(L2-Zn~(2+)) in aqueous medium, resulting in a color change of the solution from red to buff and a "turn-on" fluorescent response. Also, the sensitivity of both the fluorescenceand colorimetric-based assay is below the maximum allowable level of cyanide ions in drinking water set by the World Health Organization. In addition, test strips, which served as convenient and efficient CN~- test kits, were fabricated based on the sensor.Notably, the selective detection of cyanide with L2-Zn~(2+) for practical application was also performed in sprouting potatoes.     

邻羟基萘甲醛缩对二甲氨基苯甲酰腙的合成及其对锌离子的识别

设计并合成了用于识别锌离子的荧光传感分子——2-羟基-1-萘甲醛缩-4-二甲氨基苯甲酰腙(1),其结构经1H NMR,IR和MS表征。利用荧光光谱研究了在乙腈中1对过渡金属离子(Zn2+,Cd2+,Cu2+,Hg2+,Pb2+和N i2+)的识别能力。结果表明:1表现出对Zn2+的良好选择性,Zn2+的加入导致1的长波长荧光增强449倍。Job曲线确定1与Zn2+形成1∶1型配合物。     

Three rhodamine-based "off-on" chemosensors with high selectivity and sensitivity for Fe3+ imaging in living cells

Three new rhodamine-based probes Y1-Y3 were synthesized as "off-on" chemosensors for Fe(3+) imaging in living cells. The recognizing behaviors were investigated both experimentally and computationally. The crystal structure of the complex Y3-Fe(3+) revealed that Fe(3+) preferred to coordinate with the N atom of benzothiazole moiety rather than the O atom of carboxyl group.     

A new "turn-on" naphthalenedimide-based chemosensor for mercury ions with high selectivity: successful utilization of the mechanism of twisted intramolecular charge transfer, near-IR fluorescence, and cell images

For the first time, a new near-IR "turn-on" fluorescent chemosensor with high selectivity for Hg(2+) ions was designed according to the twisted intramolecular charge transfer (TICT) mechanism. The selective fluorescence enhancement effect can be optimized by modulating the solvent systems. And this naphthalenedimide-based sensor with long wavelength absorption and emission can be used to image intracellular Hg(2+) ions in living Hela cells.     

Oxidative cyclization of thiosemicarbazone: an optical and turn-on fluorescent chemodosimeter for Cu(II)

A weakly fluorescent thiosemicabazone (L(1)H) was found to be a selective optical and "turn-on" fluorescent chemodosimeter for Cu(2+) ion in aqueous medium. A significant fluorescence enhancement along with change in color was only observed for Cu(2+) ion; among the other tested metal ions (viz. Na(+), K(+), Mg(2+), Ca(2+), Cr(3+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), Ag(+), Ni(2+), Co(2+), Fe(3+) and Mn(2+)). The Cu(2+) selectivity resulted from an oxidative cyclization of the weak fluorescent L(1)H into highly fluorescent rigid 4,5-dihydro-5,5-dimethyl-4-(naphthalen-5-yl)-1,2,4-triazole-3-thione (L(2)). The signaling mechanism has been confirmed by independent synthesis with detail characterization of L(2).     

Solvent dependent fluorescent properties of a 1,2,3-triazole linked 8-hydroxyquinoline chemosensor: tunable detection from zinc(II) to iron(III) in the CH3CN/H2O system

A triazole-containing 8-hydroxyquinoline (8-HQ) ether 2 was efficiently synthesized in two steps from the "click" strategy. Compound 2 gave a strong fluorescence (Φ = 0.21) in nonprotic solvent like CH(3)CN, and a weak fluorescence (Φ = 0.06) in protic solvent like water. In water, a more than 100 nm red shift of the fluorescence maximum was observed for compound 2 in comparison with that in CH(3)CN. This fluorescence difference may be attributed to the intermolecular photoinduced proton transfer (PPT) process involving the protic solvent water molecules. Similarly, this intermolecular PPT process was also observed in the high-water-content CH(3)CN aqueous solution (e.g., CH(3)CN/H(2)O = 5/95, v/v). The water content in the CH(3)CN/H(2)O binary solvent mixture greatly affected the fluorescence intensity (e.g., Φ = 0.06 and 0.25 when CH(3)CN/H(2)O = 5/95 and 95/5, v/v, respectively) and emission wavelength. Using this interesting property, by simple variation of the water content in the CH(3)CN aqueous solution, compound 2 was tuned from a selective "turn-on" fluorescent sensor for Zn(2+) (CH(3)CN/H(2)O = 5/95, v/v) to a ratiometric one for Zn(2+) and a selective "turn-off" one for Fe(3+) (CH(3)CN/H(2)O = 95/5, v/v) over a wide range of pH value. In high-water-content (CH(3)CN/H(2)O = 5/95, v/v) aqueous solution compound 2 shows a selective "turn-on" response toward Zn(2+), with a 10-fold enhancement in the fluorescence intensity at 428 nm and a 62 nm blue shift of the emission maximum (490 to 428 nm) due to the inhibition of intermolecular PPT process upon chelating with Zn(2+). However, in a less polar solvent (CH(3)CN/H(2)O = 95/5, v/v) in which compound 2 has high fluorescence (quantum yield =0.25), it shows a ratiometric response toward Zn(2+), with a continuous decrease of the fluorescence intensity at 399 nm and an increase at 423 nm. More interestingly, in this case, it also exhibits a very sensitive, selective, and ratiometric fluorescence quenching in the presence of Fe(3+), with an 81 nm red shift of the emission maximum (399 to 480 nm) in a wide range of pH through a metal ligand charge transfer (MLCT) effect.     

A highly selective fluorescence-based polymer sensor incorporating an (R,R)-salen moiety for Zn(2+) detection

A chiral polymer incorporating an (R,R)-salen moiety was synthesized by the polymerization of (R,R)-1,2-diaminocyclohexane with 2,5-dibutoxy-1,4-di(salicyclaldehyde)-1,4-diethynyl-benzene by a nucleophilic addition-elimination reaction. The fluorescence responses of the (R,R)-salen-based polymer toward various metal ions were investigated by fluorescence spectra. Compared with other cations, such as Na(+), K(+), Mg(2+), Ca(2+), Mn(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), Ag(+), Cd(2+), Hg(2+), and Pb(2+), Zn(2+) can lead to a pronounced fluorescence enhancement as high as 7.8-fold together with an obvious blue-shift change of the chiral polymer. More importantly, the fluorescent color of the polymer changed to bright blue instead of weak yellow after addition of Zn(2+), which can be easily detected by the naked eye. The results indicate that this kind of chiral polymer, incorporating an (R,R)-salen moiety as a receptor in the main chain backbone, can exhibit high sensitivity and selectivity for Zn(2+) recognition.     

Color-Tunable Light-up Bioorthogonal Probes for In Vivo Two-Photon Fluorescence Imaging

Light-up bioorthogonal probes have attracted increasing attention recently due to their capability to directly image diverse biomolecules in living cells without washing steps. The development of bioorthogonal probes with excellent fluorescent properties suitable for in vivo imaging, such as long excitation/emission wavelength, high fluorescence turn-on ratio, and deep penetration, has been rarely reported. Herein, a series of azide-based light-up bioorthogonal probes with tunable colors based on a weak fluorescent 8-aminoquinoline ( AQ ) scaffold were designed and synthesized. The azido quinoline derivatives are able to induce large fluorescence enhancement (up to 1352-fold) after click reaction with alkynes. In addition, the probes could be engineered to exhibit excellent two-photon properties (δ=542 GM at 780 nm) after further introducing different styryl groups into the AQ scaffold. Subsequent detailed bioimaging experiments demonstrated that these versatile probes can be successfully used for live cell/zebrafish imaging without washing steps. Further in vivo two-photon imaging experiments demonstrated that these light-up biorthogonal probe outperformed conventional fluorophores, for example, high signal-to-noise ratio and deep tissue penetration. The design strategy reported in this study is a useful approach to realize diverse high-performance biorthogonal light-up probes for in vivo studying.     

Study of the absorption and emission spectroscopy of "A-B" type photosensitive compounds including two-photon chromophore and benzophenone moiety

"A-B" type photosensitive compounds including two-photon chromophore and benzophenone moiety have been designed, synthesized and characterized. The UV-vis absorption and fluorescence emission of the compounds have been extensively studied in various solvents. The results show that the absorption of "A-B" type compounds displays obvious double absorption bands, one of which at short-wavelength is related to the benzophenone moiety, the other at long-wavelength is mainly contributed by chromophore. The emission of "A-B" type compounds at 500-700nm shows an "unexpected" blue-shift comparing with that of the sole chromophore. The photosensitive compounds with amino group display strong emission in apolar solvents and have a low fluorescence quantum yields in polar solvents. In contrast, the compounds without amino group exhibit strong fluorescence emission in polar solvents, and low fluorescence quantum yields in apolar solvents. The fluorescence quantum yields of "A-B" type compounds are higher than those of the sole chromophore. The discoveries suggest that charge redistribution induced by the introduction of benzophenone moiety plays a key role on the absorption and emission spectroscopy.     

Bright fluorescent chemosensor platforms for imaging endogenous pools of neuronal zinc

A series of new fluorescent Zinpyr (ZP) chemosensors based on the fluorescein platform have been prepared and evaluated for imaging neuronal Zn(2+). A systematic synthetic survey of electronegative substitution patterns on a homologous ZP scaffold provides a basis for tuning the fluorescence responses of "off-on" photoinduced electron transfer (PET) probes by controlling fluorophore pK(a) values and attendant proton-induced interfering fluorescence of the metal-free (apo) probes at physiological pH. We further establish the value of these improved optical tools for interrogating the metalloneurochemistry of Zn(2+); the novel ZP3 fluorophore images endogenous stores of Zn(2+) in live hippocampal neurons and slices, including the first fluorescence detection of Zn(2+) in isolated dentate gyrus cultures. Our findings reveal that careful control of fluorophore pK(a) can minimize proton-induced fluorescence of the apo probes and that electronegative substitution offers a general strategy for tuning PET chemosensors for cellular studies. In addition to providing improved optical tools for Zn(2+) in the neurosciences, these results afford a rational starting point for creating superior fluorescent probes for biological applications.     

Carbohydrate assisted fluorescence turn-on gluco-imino-anthracenyl conjugate as a Hg(II) sensor in milk and blood serum milieu

A new anthracenyl-imino-glucosyl conjugate (L) selectively senses Hg(2+) by turn-on fluorescence with a 13 ± 1 fold enhancement by forming a 2?:?1 complex in pH 5 to 10 even in the presence of several biologically and ecologically relevant metal ions, with a 25 ± 2% fluorescence enhancement at the EPA limit of 2 ppb. L is equally sensitive towards Hg(2+) in the presence of albumin proteins and in blood serum and milk.     

The development of a fluorescence turn-on sensor for cysteine, glutathione and other biothiols. A kinetic study

Two fluorescence probes for the detection of cysteine (Cys), glutathione (GSH) and other biothiols, such as homocysteine (Hcy) and cysteinyl-glycine (Cys-Gly), were developed. These molecular probes are coumarin-based derivatives containing a chalcone-like moiety that reacts with biothiols through a Michael addition reaction, leading to strong fluorescence enhancements. The reactivity of the tested biothiols toward both probes (ChC1 and ChC2) follows the order Cys > GSH > Hcy > Cys-Gly, ChC1 being less reactive than ChC2. Possible interference with other amino acids was assessed. ChC1 and ChC2 display a highly selective fluorescence enhancement with thiols, allowing these probes to be used for fluorimetric thiol determination in SH-SY5Y cells.     

Solvent controlled sugar-rhodamine fluorescence sensor for Cu(2+) detection

A "turn-on" fluorescence probe for Cu(2+) detection has been reported according to a Cu(2+) triggered spirolactam ring-opening reaction. The probe is a double-responsive fluorescent and colorimetric Cu(2+)-specific sensor in aqueous solution containing 20% of acetonitrile with high selectivity and excellent sensitivity (limit of detection is 12 μg L(-1)). Furthermore, the significant color changes visible to the naked eye at the concentration of 3 μM (ca. 0.20 mg L(-1)) are about ten times lower than the WHO (World Health Organization) recommended level (2.0 mg L(-1)) for Cu(2+) ions in drinking water.     

A highly selective turn-on colorimetric, red fluorescent sensor for detecting mobile zinc in living cells

We describe ZRL1, a turn-on colorimetric and red fluorescent zinc ion sensor. The Zn(2+)-promoted ring opening of the rhodamine spirolactam ring in ZRL1 evokes a 220-fold fluorescence turn-on response. In aqueous media, ZRL1 turn-on luminescence is highly selective for Zn(2+) ions, with no significant response to other competitive cations, including Na(+), K(+), Ca(2+), Mg(2+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Cd(2+), or Hg(2+). In addition to these characteristics, preliminary results indicate that ZRL1 can be delivered to living cells and can be used to monitor changes in intracellular Zn(2+) levels.     

A new fluorescent and colorimetric probe for Cu2+ in live cells

Based on a change in structure between spirocyclic (non-fluorescent) and ring-open (fluorescent) forms of rhodamine-based dyes, a new fluorescent and colorimetric Cu(2+) probe was designed and synthesized. Upon treatment with Cu(2+), the weakly fluorescent probe exhibited a strong fluorescence response with high selectivity. In addition, the turn-on fluorescent probe upon the addition of Cu(2+) was applied in live cell imaging.