共查询到20条相似文献,搜索用时 15 毫秒
1.
Takakusa H Kikuchi K Urano Y Kojima H Nagano T 《Chemistry (Weinheim an der Bergstrasse, Germany)》2003,9(7):1479-1485
A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor. 相似文献
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Xu K Liu X Tang B Yang G Yang Y An L 《Chemistry (Weinheim an der Bergstrasse, Germany)》2007,13(5):1411-1416
3',6'-Bis(diphenylphosphinyl)fluorescein (PF-1) was synthesized as a highly selective and sensitive fluorescent probe for imaging O(2) (.-) in living cells. The design strategy for the probe was based on the nucleophilic mechanism of O(2) (.-) to mediate deprotection of this probe to give fluorescein. Upon reaction with O(2) (.-), the probe exhibits a strong fluorescence response and high selectivity for O(2) (.-) over other reactive oxygen species and some biological compounds. The phosphinate-based probe, as a new fluorescent reagent, is cell-permeable and can detect micromolar changes of O(2) (.-) concentrations by using confocal microscopy in living cells. The unique combination of good selectivity, high sensitivity, good water solubility, and rapid reactivity establishes the potential value of the probe for facilitating investigations of the generation, metabolism, and mechanisms of superoxide-mediated cellular homeostasis and injury. 相似文献
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We report a simple fluorescence method for detection of cyanide sensitively and selectively based on the dissolution of polymer-coated gold nanoparticles by cyanide. The lowest concentration for quantification of cyanide ions was 3.0×10(-7) M, and other common anions nearly have no influence. Furthermore, several real water samples spiked with cyanide, including local groundwater, tap water, boiled water, and lake water, were analyzed, and the experimental results demonstrated that our sensing system worked well in the above water samples, with a good linear correlation. 相似文献
4.
BODIPY‐Based Fluorescent Thermometer as a Lysosome‐Targetable Probe: How the Oligo(ethylene glycols) Compete Photoinduced Electron Transfer
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Hua Wang Dr. Yongquan Wu Yanlin Shi Pan Tao Prof. Dr. Xing Fan Prof. Dr. Xinyan Su Prof. Dr. Gui‐Chao Kuang 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(8):3219-3223
A novel BODIPY‐based fluorescent thermometer, which shows a lysosome‐targeting property, was successfully prepared. Due to the electron‐donating ability of the oligo(ethylene glycols), the photoinduced electron‐transfer pathway from morpholine to BODIPY dye is blocked. The fluorescence of the thermometer quenched by intramolecular rotation at room temperature was progressively enhanced during heating due to the increased microviscosity around the fluorophore. 相似文献
5.
The sub-millisecond protonation dynamics of the chromophore in S65T mutant form of the green fluorescent protein (GFP) was
tracked after a rapid pH jump following laser-induced proton release from the caged photolabile compoundo-nitrobenzaldehyde. Following a jump in pH from 8 to 5 (which is achieved within 2 μs), the fluorescence of S65T GFP decreased
as a single exponential with a time constant of ∼90 μs. This decay is interpreted as the conversion of the deprotonated fluorescent
GFP chromophore to a protonated non-fluorescent species. The protonation kinetics showed dependence on the bulk viscosity
of the solvent, and therefore implicates bulk solvent-controlled protein dynamics in the protonation process. The protonation
is proposed to be a sequential process involving two steps: (a) proton transfer from solvent to the chromophore, and (b) internal
structural rearrangements to stabilize a protonated chromophore. The possible implications of these observations to protein
dynamics in general is discussed 相似文献
6.
Charier S Ruel O Baudin JB Alcor D Allemand JF Meglio A Jullien L Valeur B 《Chemistry (Weinheim an der Bergstrasse, Germany)》2006,12(4):1097-1113
This paper evaluates the 5-aryl-2-pyridyloxazole backbone to engineer donor-acceptor fluorescent pH probes after one- or two-photon absorption. Parent fluorophores, as well as derivatives that can be used to label biomolecules, can be easily obtained in good yields. These molecules exhibit a large one-photon absorption in the near-UV range, and a strong fluorescence emission that covers the whole visible domain. The 5-aryl-2-pyridyloxazole derivatives also possess significant cross sections for two-photon absorption. Upon pyridine protonation, large shifts were observed in the absorption spectra after one- and two-photon excitation, as well as in the emission spectra. This feature was used to measure the pK(a) of the investigated compounds that range between 2 and 8. In most of the investigated derivatives, the pK(a) increased upon light excitation and protonation exchanges took place during the lifetime of the excited state, as shown by phase-modulation fluorometry analysis. Several 5-aryl-2-pyridyloxazole derivatives are suggested as efficient probes to reliably measure the pH of aqueous solutions by means of ratiometric methods that are dependent on fluorescence emission. 相似文献
7.
Ulrich G Ziessel R Harriman A 《Angewandte Chemie (International ed. in English)》2008,47(7):1184-1201
The world of organic luminophores has been confined for a long time to fairly standard biological labeling applications and to certain analytical tests. Recently, however, the field has undergone a major change of direction, driven by the dual needs to develop novel organic electronic materials and to fuel the rapidly emerging nanotechnologies. Among the many diverse fluorescent molecules, the Bodipy family, first developed as luminescent tags and laser dyes, has become a cornerstone for these new applications. The near future looks extremely bright for "porphyrin's little sister". 相似文献
8.
Fluorogenic probes dual-labeled with reporter and quencher dyes use a change in fluorescence to monitor biochemical events (e.g., substrate binding or enzyme digestion). Such events change the reporter-quencher distance, which affects fluorescence. Recently, it is has been shown that static quenching through intramolecular dimers is an important mechanism that can sometimes be more efficient than F?rster resonance energy transfer (FRET). 相似文献
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Vladimir M. Mekler Alexander Z. Averbakh Andrew B. Sudarikov Olga V. Kharitonova 《Journal of photochemistry and photobiology. B, Biology》1997,40(3):278-287
A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates. 相似文献
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Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish
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Xiaofeng Wu Dr. Lihong Li Prof. Dr. Wen Shi Dr. Qiuyu Gong Prof. Dr. Huimin Ma 《Angewandte Chemie (International ed. in English)》2016,55(47):14728-14732
Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems. 相似文献
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Das AK Hasegawa JY Miyahara T Ehara M Nakatsuji H 《Journal of computational chemistry》2003,24(12):1421-1431
Two ground-state protonation forms causing different absorption peaks of the green fluorescent protein chromophore were investigated by the quantum mechanical SAC/SAC-CI method with regard to the excitation energy, fluorescence energy, and ground-state stability. The environmental effect was taken into account by a continuum spherical cavity model. The first excited state, HOMO-LUMO excitation, has the largest transition moment and thus is thought to be the source of the absorption. The neutral and anionic forms were assigned to the protonation states for the experimental A- and B-forms, respectively. The present results support the previous experimental observations. 相似文献
17.
The ‘leading coordinate’ approach to computing an approximate reaction pathway, with subsequent determination of the true minimum energy profile, is applied to a two-proton chain transfer model based on the chromophore and its surrounding moieties within the green fluorescent protein (GFP). Using an ab initio quantum chemical method, a number of different relaxed energy profiles are found for several plausible guesses at leading coordinates. The results obtained for different trial leading coordinates are rationalized through the calculation of a two-dimensional relaxed potential energy surface (PES) for the system. Analysis of the 2-D relaxed PES reveals that two of the trial pathways are entirely spurious, while two others contain useful information and can be used to furnish starting points for successful saddle-point searches. Implications for selection of trial leading coordinates in this class of proton chain transfer reactions are discussed, and a simple diagnostic function is proposed for revealing whether or not a relaxed pathway based on a trial leading coordinate is likely to furnish useful information. 相似文献
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By using distributed computing techniques and a supercluster of more than 20,000 processors we simulated folding of a 20-residue Trp Cage miniprotein in atomistic detail with implicit GB/SA solvent at a variety of solvent viscosities (gamma). This allowed us to analyze the dependence of folding rates on viscosity. In particular, we focused on the low-viscosity regime (values below the viscosity of water). In accordance with Kramers' theory, we observe approximately linear dependence of the folding rate on 1/gamma for values from 1-10(-1)x that of water viscosity. However, for the regime between 10(-4)-10(-1)x that of water viscosity we observe power-law dependence of the form k approximately gamma(-1/5). These results suggest that estimating folding rates from molecular simulations run at low viscosity under the assumption of linear dependence of rate on inverse viscosity may lead to erroneous results. 相似文献
20.
Therése Klingstedt Dr. Hamid Shirani Dr. K. O. Andreas Åslund Dr. Nigel J. Cairns Dr. Christina J. Sigurdson Prof. Michel Goedert Dr. K. Peter R. Nilsson 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(31):10179-10192
Protein misfolding diseases are characterized by deposition of protein aggregates, and optical ligands for molecular characterization of these disease‐associated structures are important for understanding their potential role in the pathogenesis of the disease. Luminescent conjugated oligothiophenes (LCOs) have proven useful for optical identification of a broader subset of disease‐associated protein aggregates than conventional ligands, such as thioflavin T and Congo red. Herein, the molecular requirements for achieving LCOs able to detect nonthioflavinophilic Aβ aggregates or non‐congophilic prion aggregates, as well as spectrally discriminate Aβ and tau aggregates, were investigated. An anionic pentameric LCO was subjected to chemical engineering by: 1) replacing thiophene units with selenophene or phenylene moieties, or 2) alternating the anionic substituents along the thiophene backbone. In addition, two asymmetric tetrameric ligands were generated. Overall, the results from this study identified conformational freedom and extended conjugation of the conjugated backbone as crucial determinants for obtaining superior thiophene‐based optical ligands for sensitive detection and spectral assignment of disease‐associated protein aggregates. 相似文献