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1.
Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.  相似文献   

2.
The incorporation of gold nanoparticles (Au NPs) as quencher modules in fluorescent probes for DNA damage caused by intracellular hydroxyl radicals (HO*) is reported. Au NPs of 15 nm diameter were decorated with DNA oligomers terminating in thiol functions in their 3' positions and possessing 5' fluorophore modifications. The Au NPs, which have high extinction coefficients, functioned as excellent fluorescent quenchers in the fluorophore-Au NP composites. FRET is switched off as a factor of HO*-induced strand breakage in the single-stranded DNAs, restoring the fluorescence of the quenched fluorophores, which can be followed by spectrofluorimetry. In vitro assays with HO*-generating Fenton reagent demonstrated increases in fluorescence intensity with a linear range from 8.0 nM to 1.0 microM and a detection limit as low as 2.4 nM. Confocal microscopic imaging of macrophages and HepG2 revealed that the probe is cell-permeable and intracellular HO*-responsive. The unique combination of good selectivity and high sensitivity establishes the potential value of the probe for facilitating investigations of HO*-mediated cellular homeostasis and injury.  相似文献   

3.
We have developed a novel design of optical nanothermometers that can measure the surrounding temperature in the range of 20–85 °C. The nanothermometers comprise two organic fluorophores encapsulated in a crosslinked polymethacrylate nanoshell. The role of the nanocapsule shell around the fluorophores is to form a well‐defined and stable microenvironment to prevent other factors besides temperature from affecting the dyes’ fluorescence. The two fluorophores feature different temperature‐dependent emission profiles; a fluorophore with relatively insensitive fluorescence (rhodamine 640) serves as a reference whereas a sensitive fluorophore (indocyanine green) serves as a sensor. The sensitivity of the nanothermometers depends on the type of nanocapsule‐forming lipid and is affected by the phase transition temperature. Both the fluorescence intensity and the fluorescence lifetime can be utilized to measure the temperature.  相似文献   

4.
The fluorescence emission of the dual‐fluorophore Ca2+ ion sensor molecule, calcium‐green 2 (CG‐2), has been characterized using dual‐polarization imaging at the single‐molecule level. By comparing the fluorescence intensity of individual CG‐2 molecules in two mutually orthogonal polarization image channels, information about the relative orientation of the two constituent fluorophores in the molecule is obtained. Experimental results from polarization measurements are compared with those predicted from a geometric model based on coupled‐fluorophores that are randomly distributed in space. The results confirm previous optical spectroscopy‐based predictions of the orientation of CG‐2′s fluorophores, and the general applications of this dual‐polarization imaging approach for characterizing the optical properties of molecules containing multiple fluorophores is discussed.  相似文献   

5.
Polyamidoamine (PAMAM) dendrimers of generations zero (G0) to four (G4), and a hyperbranched polyurea (HB‐PU), were functionalized with 1,5‐dansyl (1,5‐D), 2,5‐dansyl (2,5‐D), 2,6‐dansyl (2,6‐D) and nitrobenzofurazan (NBD) fluorophores that change their fluorescence emission wavelength in response to chemical environment, and the resulting dendritic polymers were characterized by MALDI‐TOF mass spectrometry, 1H NMR, 13C NMR, and fluorescence spectroscopy. Fluorophore‐functionalized dendritic polymers were then reacted further with 3‐acryloxypropyldimethoxymethylsilane (AOP‐DMOMS) at various fluorophore to DMOMS substitution ratios. The resulting materials were cast onto glass slides, and cured into robust nanostructured coatings. Coatings with 50% fluorophore–50% DMOMS substitution showed the strongest fluorescence and the best physical properties. Coated coupons were tested against a wide range of analytes including the chemical warfare agent simulants dimethyl methylphosphonate (DMMP) and chloroethylethylsulfide (CEES), and the water‐methanol‐ethanol series. It was found that the ability of the coatings to distinguish between analytes decreased with increasing cross‐link density for both dendrimer and hyperbranched polymer‐based coatings. It was also found that the percent fluorophore substitution and the type of dendritic polymer carrying the fluorophore had no significant effect upon fluorescence emission wavelength, but fluorescence emission wavelength became less dependent upon solvent with increasing dendrimer generation and molecular mass. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5101–5115, 2009  相似文献   

6.
Photoluminescent energy transfer was investigated in conjugated polymer‐fluorophore blended thin films. A pentiptycene‐containing poly(phenyleneethynylene) was used as the energy donor, and 13 fluorophores were used as energy acceptors. The efficiency of energy transfer was measured by monitoring both the quenching of the polymer emission and the enhancement of the fluorophore emission. Near‐infrared emitting squaraines and terrylenes were identified as excellent energy acceptors. These results, where a new fluorescent signal occurs in the near‐infrared region on a completely dark background, offer substantial possibilities for designing highly sensitive turn‐on sensors. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3382–3391, 2010  相似文献   

7.
Hydrophilic ionic polyurethanes with 4‐chloromethylphenylcarbamoyl‐1‐oxymethylpyrene located on the quaternary ammonium structure from a polymer based on poly(ethylene glycol), isophorone diisocyanate, and N‐methyldiethanolamine were prepared by a quaternization reaction, in which the amount of pyrene covalently attached to the polymeric backbone ranged from 1.14 to 19.82 mmol of fluorophore/100 g of polymer. It was interesting to compare the photoluminescence of the pyrene polyurethane carrying a few mole percent of pyrene moieties with that of a third polymer resulting from its subsequent quaternization with benzyl chloride up to a concentration of ionic groups as in the latter (quaternization degree = 14.15%). The process of excimer formation between the pyrene molecules attached to the ionic polyurethane was investigated in tetrahydrofuran (THF), dimethylformamide, film, and THF/H2O to illustrate the expected differences in the polymer behavior compared with that of the starting pyrene derivative. The formation of aggregates or core–shell micelles was sustained by the fluorescence data, which indicated the existence of pyrene units in the ground state of the molecule, giving rise thus to an explanation for the high excimer‐to‐monomer intensity ratio. The fluorescence decay of pyrene polyurethanes in the presence of various concentrations of nitrobenzene used as a quencher was analyzed too when the fluorescence quenching in the polymer solution normally followed Stern–Volmer kinetics. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3945–3956, 2005  相似文献   

8.
This paper reports a reversible dual fluorescence switch for the detection of a proton target and 2,4,6‐trinitrotoluene (TNT) with opposite‐response results, based on fluorophore derivatization of silica nanoparticles. Fluorescent silica nanoparticles were synthesized through modification of the surface with a nitrobenzoxadiazole (NBD) fluorophore and an organic amine to form a hybrid monolayer of fluorophores and amino ligands; the resultant nanoparticles showed different fluorescence responses to the proton target and TNT. Protonation of the amino ligands leads to fluorescence enhancement due to inhibition of photoinduced electron transfer (PET) between the amine and fluorophore. By contrast, addition of TNT results in fluorescence quenching because a fluorescence resonance energy transfer (FRET) happens between the NBD fluorophore and the formed TNT–amine complex. The fluorescence signal is reversible through washing with the proper solvents and the nanoparticles can be reused after centrifugal separation. Furthermore, these nanoparticles were assembled into chips on an etched silicon wafer for the detection of TNT and the proton target. The assembled chip can be used as a convenient indicator of herbicide (2,4‐dichlorophenoxyacetic acid) and TNT residues with the use of only 10 μL of sample. The simple NBD‐grafted silica nanoparticles reported here show a reversible signal and good assembly flexibility; thus, they can be applied in multianalyte detection.  相似文献   

9.
Two new FRET pairs, warfarin (WF)–curcumin (CUR) and curcumin–rhodamine B (RhB), are explored by using surfactant‐based self‐assembled soft systems as scaffolds. The study is extended to design a two‐step concurrent FRET system based on these three fluorophores, which is an important mechanism to devise artificial light‐harvesting/antenna systems. Surfactant systems of varying nature (cationic, anionic, nonionic, and zwitterionic) are exploited to modulate the energy transfer in different FRET systems. Interestingly, micelle/water interfacial‐charge‐responsive FRET is observed owing to selective solubilization of the fluorophores during co‐solubilization. The step‐one FRET (WF→CUR) is switched on in cationic and zwitterionic media but switched off in anionic/nonionic media, whereas the step‐two FRET from CUR to RhB is switched on in anionic/nonionic and zwitterionic media. However, both the FRET steps (WF→CUR→RhB) are observed to be active only in zwitterionic medium. Co‐solubilized, appropriately mixed fluorophores having multistep FRET possibilities can be switched on/off selectively as and when required and energy efficiency can be tuned to an optimal level by varying the nature and geometry of the micellar scaffold. Thus, the two FRET pairs selectively acknowledge all types of media for their anticipated applications in biological systems, as structural tools, and for the development of artificial light‐harvesting/antenna systems and lasers.  相似文献   

10.
We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)‐sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET‐conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)‐enhanced emission of 3 and 4 in lysosomes. It was further shown using two‐color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino‐functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.  相似文献   

11.
Chen H  Kenny JE 《The Analyst》2012,137(1):153-162
One of the conventionally accepted requirements for parallel factor analysis (PARAFAC) to handle the fluorescence excitation emission matrices (EEMs) is the independence of each component's absorption and emission spectra in simple mixtures of fluorophores. EEMs of samples in which F?rster resonance energy transfer (FRET) occurs between fluorophores seem to fail to meet this requirement. A rigorous theoretical treatment of the steady-state kinetics in the present work indicates that the fluorescence in the presence of FRET, excited by relatively weak excitation light intensity, can be reasonably separated into additive contributions from three parts: donors, acceptors and FRET. This prediction is for the first time verified experimentally in sodium dodecyl sulfate micellar solutions containing biphenyl as the energy donor and 2,5-diphenyloxazole as the energy acceptor. The experimental EEMs were well fitted to three components as predicted. A well accepted diagnostic test called core consistency (CC), specifically designed for modeling simple mixtures of fluorophores with PARAFAC, was found to be negative for the 3-component model in the present study. The simultaneous occurrence of good model fit and significantly negative CC when modeling fluorophore mixtures by conventional PARAFAC would be indicative of the presence of physical/chemical processes (e.g., FRET) that deviate from the conventional working requirements for PARAFAC. The extent of FRET has been independently measured or calculated by three methods: 1) decrease in steady state fluorescence of donor; 2) lifetime measurements with population analysis; and 3) Poisson statistics based on PARAFAC-determined distribution constants. The results of the three methods are consistent. The normalized scores of the three components found by PARAFAC also agree to within a few percent with relative concentrations in aqueous and micelle phases determined from distribution constants for the solutions prepared with nine different combinations of total donor and acceptor concentrations. Our theoretical treatment also for the first time spells out in detail the relationship between the PARAFAC scores and concentrations of components, in terms of photophysical constants of the components and spectral shape factors.  相似文献   

12.
Two arylenevinylene compounds bearing the cyano group at α‐position ( 6 ) and β‐position ( 9 ) from the dialkoxylphenylene unit were synthesized, in which the molecular termini were functionalized with 3‐bromocarbazole. The Suzuki coupling copolymerization of these compounds with 1,4‐bis[(3′‐bromocarbazole‐9′‐yl)methylene]‐2,5‐didecyloxybenzene and 9,9‐dihexylfluorene‐2,7‐bis(boronic acid) was carried out to obtain copolymers ( cp67 and cp97 ) containing the cyano‐substituted arylenevinylene fluorophore of 7 mol %. Model compounds ( 6 ′ and 9 ′) corresponding to the arylenevinylene fluorophore were also prepared. The UV spectra of copolymers resembled that of homopolymer hp with no arylenevinylene segment in both CHCl3 solution and thin film. The emission maxima of copolymers in CHCl3 (394 nm) agreed with that of homopolymer indicating that the emission bands originated from the carbazole‐fluorene‐carbazole segment. The emission maximum wavelength of copolymer cp67 in thin film (477 nm) indicated fluorescence from the cyano‐substituted arylenevinylene fluorophore because of the occurrence of fluorescence resonance electron transfer. In contrast, copolymer cp97 showed fluorescence at 528 nm to suggest the formation of a new emissive species such as a charge‐transfer complex (exciplex). © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 91–98, 2010  相似文献   

13.
在本文中,我们研制了一种基于T-T碱基错配特异性键合汞离子的荧光传感器用于汞离子的检测。该传感器由两条分别标记了荧光基团(F)和淬灭基团(Q)的DNA探针组成,并且含有两对用于结合汞离子的T-T错配碱基。当汞离子存在时,两条探针之间形成T-Hg2+-T结构,作用力增强,从而拉近了荧光基团与淬灭基团之间的距离,发生能量转移,使荧光信号在一定程度上被淬灭。在优化的条件下,我们使用该传感器对汞离子进行检测,动力学响应范围为50nM到1000nM,线性相关方程为y= 5281.13 - 1650.56 lg[Hg2+] ( R2 = 0.985),检测下限为79nM。此外,我们还考察了该传感器的选择性,当用其它干扰离子(浓度都为1.0µM)代替待测离子进行实验时,没有发生明显的荧光淬灭,说明该传感器具有较高的选择性。该传感器的构建为汞离子的检测提供了一条快速、简便的新途径。  相似文献   

14.
Fluorescent polymer particles incorporating pyrene and alkynylpyrenes were prepared by miniemulsion polymerization of styrene. The pyrenes were simply dissolved in styrene monomer and then added to the reaction mixture in a vessel, followed by ultrasonication and the polymerization. The pyrenes were incorporated in the polystyrene particles and gave monomer fluorescence emission. The fluorescent particles incorporating the alkynylpyrenes, which have high fluorescence quantum yield and short fluorescence lifetime, showed the same fluorescence emission intensity under air and nitrogen. The fluorescence of the alkynylpyrenes in the polymer particles will not be quenched by molecular dioxygen in contrast to that in organic solvents. Incorporation of fluorophore into polymer particles will protect the fluorophore from its undesirable interaction with oxygen, affording the particles highly fluorescent even under air. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 1470–1475, 2008  相似文献   

15.
Mono‐, bis‐, and trismaleimides (MM‐2, BM‐4, and TM‐6, respectively) bearing electron‐donating chromophores generally displayed strong fluorescence quenching. The quenching mechanism of fluorescence was revealed by electrochemical studies. On the basis of the electrochemical data, an energy level diagram was established for these maleimides. The electron‐donating ability increased in the order of TM‐6 < BM‐4 < MM‐2. The electron‐accepting ability increased in the opposite order, that is, from MM‐2 to BM‐4 to TM‐6. The copolymerization of multimaleimides with diamine could be well monitored by fluorescence spectroscopy. Compared with fluorescence probe techniques, the fluorescence monitoring could directly reflect the consumption of C?C bonds. Furthermore, a novel trismaleimide resin was preliminarily studied, in which TM‐6 was a dual‐purpose functional monomer acting not only as a crosslinker but also as an intrinsic fluorophore to monitor its crosslinking or cure process. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 304–313, 2006  相似文献   

16.
《中国化学快报》2020,31(5):1083-1086
Au or other metal nanostructures have the ability to strongly quench the fluorescence of fluorophores.This feature has made AuNP-conjugates attractive for the construction of platforms for various bioanalytes to overcome the limitations of small molecule fluorophores(poor solubility,long reaction time).In this paper,an ultrafast "Turn-On" fluo rescent sensor for biothiols was constructed.The sensor is based on the fluorescent resonance energy transfer(FRET) effect between the fluorophore(PN) and AuNPs,which effectively quenches the fluorescence of the fluorophore.In the presence of thiols,PN is displaced and released from AuNP surfaces,and thus,the fluorescence is rapidly restored.The sensor features appreciable water solubility and ultrafast response time(a few seconds for Cys).In addition,it exhibits high selectivity and a detection limit as low as 12 nmol/L for Hcy.Moreover,the sensor presents good biocompatibility and has been successfully applied for imaging biothiols in living cells.  相似文献   

17.
Single‐molecule fluorescence resonance energy transfer (sm‐FRET) has become a widely used tool to reveal dynamic processes and molecule mechanisms hidden under ensemble measurements. However, the upper limit of fluorescent species used in sm‐FRET is still orders of magnitude lower than the association affinity of many biological processes under physiological conditions. Herein, we introduce single‐molecule photoactivation FRET (sm‐PAFRET), a general approach to break the concentration barrier by using photoactivatable fluorophores as donors. We demonstrate sm‐PAFRET by capturing transient FRET states and revealing new reaction pathways during translation using μm fluorophore labeled species, which is 2–3 orders of magnitude higher than commonly used in sm‐FRET measurements. sm‐PAFRET serves as an easy‐to‐implement tool to lift the concentration barrier and discover new molecular dynamic processes and mechanisms under physiological concentrations.  相似文献   

18.
A strategy to extend the detection range of weakly-binding targets is reported that takes advantage of fluorescence resonance energy transfer (FRET)-based bioassays based on molecular beacon aptamers (MBAs) and cationic conjugated polyelectrolytes (CPEs). In comparison to other aptamer-target pairs, the aptamer-based adenosine triphosphate (ATP) detection assays are limited by the relatively weak binding between the two partners. In response, a series of MBAs were designed that have different stem stabilities while keeping the constant ATP-specific aptamer sequence in the loop part. The MBAs are labeled with a fluorophore and a quencher at both termini. In the absence of ATP, the hairpin MBAs can be opened by CPEs via a combination of electrostatic and hydrophobic interactions, showing a FRET-sensitized fluorophore signal. In the presence of ATP, the aptamer forms a G-quadruplex and the FRET signal decreases due to tighter contact between the fluorophore and quencher in the ATP/MBA/CPE triplex structure. The FRET-sensitized signal is inversely proportional to [ATP]. The extension of the detection range is determined by the competition between opening of the ATP/MBA G-quadruplex by CPEs and the composite influence by ATP/aptamer binding and the stem interactions. With increasing stem stability, the weak binding of ATP and its aptamer is successfully compensated to show the resistance to disruption by CPEs, resulting in a substantially broadened detection range (from millimolar up to nanomolar concentrations) and a remarkably improved limit of detection. From a general perspective, this strategy has the potential to be extended to other chemical- and biological-assays with low target binding affinity.  相似文献   

19.
We synthesized hyperbranched poly(phenylene sulfide) (HPPS) in a simple “one‐pot” way by condensation of potassium 2,4‐dichlorlbenzenthiol. The molecular masses (Mw) of the polymers obtained under the conditions of this work were from 6 × 103 to 1 × 105. XRD pattern indicated substantial loss in crystallinity in HPPS. There was a minimum in the relation of intrinsic viscosity of HPPS in tetrahydrofuran (THF), determined by Ubbelohde viscometer, to molecular mass. Thermal analysis revealed that the HPPSs were very stable with the onset degradation temperature above 400 °C, and remaining weight of about 60% at 800 °C in nitrogen. The maximum emission wavelength of HPPS in THF was about 460 nm, which would red‐shift with the increase of molecular mass or concentration. The quenching behavior of the fluorescence in HPPS quenched by Cu2+ obeyed the Stern–Volmer equation, , where F0 and F are the fluorescence intensity at the reference condition free of quencher and at condition with a quencher concentration of CCu2+, respectively, and k is a constant. The quenching efficiency was still as high as about 20% at Cu2+ concentration of about 10 ppm. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 826–831, 2006  相似文献   

20.
We report the facile synthesis of poly(VI‐co‐MAA) superabsorbent polyampholytic hydrogels (VI = N‐vinylimidazole, MAA = methacrylic acid) via plasma‐ignited frontal polymerization (PIFP). On igniting the top surface of the reactants with air plasma, frontal polymerization occurred and poly(VI‐co‐MAA) hydrogels were obtained within minutes. The preparation parameters were investigated, along with swelling capacity, morphology, and chemical structures of poly(VI‐co‐MAA) hydrogels. Interestingly, the hydrogels are superabsorbent in water and show ampholytic characteristic toward pH. Moreover, the hydrogels are able to capture cationic dyes through electrostatic interaction, offering the potential for further development as dye adsorbents for water purification. In addition, nanocomposite hydrogels were obtained by embedding quantum dots (carbon dots or CdS nanocrystals) into the polymer matrix, which endows the nanocomposite hydrogels with favorable fluorescence and potential applications in bioimaging and biosensing. The results indicate that FP can be applied as an alternative means for facile synthesis of multifunctional hydrogels with additional efficiency and energy‐saving. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 912–920  相似文献   

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