Time gating improves sensitivity in energy transfer assays with terbium chelate/dark quencher oligonucleotide probes

Lanthanides are attractive as biolabels because their long luminescence decay rates allow time-gated detection, which separates background scattering and fluorescence from the lanthanide emission. A stable and highly luminescent terbium complex based on a tetraisophthalamide (TIAM) chelate is paired with a polyaromatic-azo dark quencher (referred to as a Black Hole Quencher or BHQ) to prepare a series of 5'TIAM(Tb)/3'BHQ dual-labeled oligonucleotide probes with no secondary structure. Luminescence quenching efficiency within terbium/BHQ probes is very dependent on the terbium-BHQ distance. In an intact probe, the average terbium-BHQ distance is short, and Tb --> BHQ energy transfer is efficient, decreasing both the terbium emission intensity and lifetime. Upon hybridization or nuclease digestion, which spatially separate the Tb and BHQ moieties, the Tb luminescence intensity and lifetime increase. As a result, time-gated detection increases the emission intensity ratio of the unquenched probe/quenched probe due to the shorter lifetime of the quenched species. A 40-mer probe that has a 3-fold increase in steady-state luminescence upon digestion has a 50-fold increase when gated detection is used. This study demonstrates that time gating with lanthanide/dark quencher probes in energy transfer assays is an effective means of improving sensitivity.     

A new approach for the detection of carbon-centered radicals in enzymatic processes using prefluorescent probes

In this communication we propose a novel application for prefluorescent probes in the detection of free carbon-centered radicals in enzymatic processes. Prefluorescent probes combine a fluorescent moiety tethered to a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of carbon-centered radicals and can be used for the quantitative evaluation of yields and kinetics. As a test system we used horseradish peroxidase, an oxidoreductase that is widely accepted to operate by a radical-mediated mechanism. We used the prefluorescent probe (quinoline-TEMPO), where a quinoline moiety has been tethered to 2,2,6,6-tetramethylpiperidin-1-oxyl.     

A DNA oligonucleotide-hemin complex cleaves t-butyl hydroperoxide through a homolytic mechanism

Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex ( approximately 35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of approximately 250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of < or =5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from beta-scission of the primary tert-butyloxyl radical product.     

Surface Modification and Functionalization of Microporous Hybrid Material for Luminescence Sensing

We report here on the preparation of novel luminescent core‐shell material by initial coating with polyelectrolytes and subsequent with a silica shell on the lanthanide complexes loaded zeolite L microcrystals. Lanthanide complexes loaded zeolite L was prepared by insertion of 2‐thenoyltrifluoroacetone (TTA) into the nanochannels of zeolite crystals by gas diffusion of TTA to Eu³⁺ exchanged zeolite L, coating a silica shell on the lanthanide complexes loaded zeolite L resulted to the novel luminescent core‐shell material. The luminescent core‐shell material was further functionalized with silylated terbium(III) complex and the obtained material was used as the luminescence sensing of dipicolinic acid (DPA), which is a major constituent of many pathogenic spore‐forming bacteria.     

稀土铕、铽配合物在温度发光传感领域的研究进展

稀土发光配合物材料基于其独特的4f-4f电子跃迁表现出优异的发光性能,特别是铕和铽配合物材料,发光波长在可见光区范围内,发射谱带狭窄且尖锐(半峰宽通常小于10 nm),非常适合应用于显示设备和传感装置.同时,具备温度依赖发光性能的铕和铽配合物能够实现高灵敏度、高效的温度传感过程,使其有望用于流体动力学、航空航天、环境工...     

Luminescent nanoparticles prepared by encapsulating lanthanide chelates to silica sphere

The luminescent nanoparticles were prepared by encapsulating the [LnL₄]^? (Ln = Eu, Tb; L = BTFA, HFAA, TTFA, TFAA) complexes anion into the silicon framework. We firstly synthesized a series of novel siloxy-bearing lanthanide complex precursor, and then encapsulated them into the silica sphere by a modified Stöber process. As a result, four europium and two terbium tetrakis β-diketonate complexes functionalized silica sphere nanoparticles were obtained and characterized in detail using Fourier transform infrared spectra, X-ray diffraction, scanning electronic microscope, thermogravimetric analysis, luminescence excitation and emission spectroscopy, luminescence lifetime measurements, and diffuse reflectance UV–Vis spectroscopy. The result shows that these luminescent nanoparticles maintain the distinctive luminescence character of lanthanide chelate including broad excitation spectra, line-like emission spectra, high quantum efficiency, and long luminescent lifetime, which makes them great potential application in the synthesis of luminescent nanoparticle.     

Reactive oxygen species produced upon photoexcitation of sunscreens containing titanium dioxide (an EPR study)

Commercial sunscreen products containing titanium dioxide were irradiated with lambda>300 nm and the formation of oxygen- (.OH, O2.-/.OOH) and carbon-centered radicals was monitored by EPR spectroscopy and spin trapping technique using 5,5-dimethyl-1-pyrroline N-oxide, alpha-phenyl-N-tert-butylnitrone (PBN), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin traps, and free nitroxide radical 4-hydroxy-2,2,6,6-tetramethylpiperidine N-oxyl. The photoinduced production of singlet oxygen was shown by 4-hydroxy-2,2,6,6-piperidine. The generation of reactive oxygen radical species upon irradiation of sunscreens significantly depends on their composition, as the additives present (antioxidants, radical-scavengers, solvents) can transform the reactive radicals formed to less harmful products. The continuous in situ irradiation of titanium dioxide powder, recommended for cosmetic application, investigated in different solvents (water, dimethyl sulfoxide, isopropyl myristate) resulted in the generation of oxygen-centered reactive radical species (superoxide anion radical, hydroxyl and alkoxyl radicals).     

自旋标记荧光探针表征生物活性分子的自由基损伤

生命过程中产生的经基自由基(^˙OH)已引起广泛关注.目前,对于^˙OH的研究主要集中在直接对^˙OH进行定量表征^[1~3]和问接检测^˙OH诱导损伤生物大分了的损伤产物.^˙OH诱导损伤生物大分了,能够产生大量的碳中心自由基^[4,5].     

EPR investigation of persistent radicals produced from the photolysis of dibenzyl ketones adsorbed on ZSM-5 zeolites

Photolysis of ketones (1, 1-oMe, 2, 2-oMe, 3, and 4) adsorbed on ZSM-5 zeolites produces persistent carbon-centered radicals that can be readily observed by conventional steady-state EPR spectroscopy. The radicals are persistent for time periods of seconds to many hours depending on the supramolecular structure of the initial radical@zeolite complex and the diffusion and reaction dynamics of radicals produced by photolysis. The structures of the persistent radicals responsible for the observed EPR spectra are determined by a combination of alternate methods of generation of the same radical, by deuterium substitution, and by spectral simulation. A clear requirement for persistence is that the radicals produced by photolysis must either separate and diffuse from the external to the internal surface or be generated within the internal surface and separate and diffuse apart. The persistence of radicals located on the internal surface is the result of inhibition of radical-radical reactions. Radicals that are produced on the external surface and whose molecular structure prevents diffusion into the internal surface are transient because radical-radical reactions occur rapidly on the external surface. The reactions of the persistent radicals with oxygen and nitric oxide were directly studied in situ by EPR analysis. In the case of reaction with oxygen, persistent peroxy radicals are formed in high yield. The addition of nitric oxide scavenges persistent radicals and leads initially to a diamagnetic nitroso compound, which is transformed into a persistent nitroxide radical by further photolysis. The influence of variation of radical structure on transience/persistence is discussed and correlated with supramolecular structure and reactivity of the radicals and their parent ketones.     

Identification of emissive lanthanide complexes suitable for cellular imaging that resist quenching by endogenous anti-oxidants

Excited state quenching by urate and ascorbate of selected europium and terbium(III) macrocyclic complexes has been assessed and related to the ease of complex visualisation by optical microscopy inside various living cells, e.g. CHO, COS and NIH 3T3. It is the relative insensitivity of certain sterically encumbered complexes to dynamic quenching by urate that favours their usage for in cellulo applications. Non-covalent binding of the complex by protein also shields the excited lanthanide(III) ion from collisional quenching; this effect is most marked for a cationic triamide complex, [Ln.1](3+), consistent with its ease of visualisation by luminescence microscopy.     

Photochromic Terbium Phosphonates with Photomodulated Luminescence and Metal Ion Sensitive Detection

Rational selection and modification of rare earth metal centers and photoactive organic linkers enables designable multiphotofunctionality to come to fruition in new hybrid coordination polymer materials. By using a viologen‐functionalized diphosphonate linker, two terbium phosphonate compounds ( Tb‐1 and Tb‐2 ) have been constructed, which display reversible photochromic reactions in response to UV light and soft X‐ray irradiation. In addition, the photo‐induced electron‐transfer reaction can modulate the luminescent emission to thus realize photoluminescence switching behavior. Furthermore, both terbium phosphonates can serve as highly sensitive sensors to probe Cu²⁺ in solution through their luminescence. Thus, they represent the first photochromic examples of lanthanide phosphonate‐based materials with photomodulated luminescence and sensitive detection of metal ions.     

Effect of Oxygen Free Radicals on Myosin in Muscle Fibres. A DSC and EPR study

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence of free radical generating system. Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak- binding state into strong-binding state. DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modulation of luminescence intensity of lanthanide complexes by photoinduced electron transfer and its application to a long-lived protease probe

Luminescent lanthanide complexes (Tb(3+), Eu(3+), etc.) have excellent properties for biological applications, including extraordinarily long lifetimes and large Stokes shifts. However, there have been few reports of lanthanide-based functional probes, because of the difficulty in designing suitable complexes with a luminescent on/off switch. Here, we have synthesized a series of complexes which consist of three moieties: a lanthanide chelate, an antenna, and a luminescence off/on switch. The antenna is an aromatic ring which absorbs light and transmits its energy to the metal, and the switch is a benzene derivative with a different HOMO level. If the HOMO level is higher than a certain threshold, the complex emits no luminescence at all, which indicates that the lanthanide luminescence can be modulated by photoinduced electron transfer (PeT) from the switch to the sensitizer. This approach to control lanthanide luminescence makes possible the rational design of functional lanthanide complexes, in which the luminescence property is altered by a biological reaction. To exemplify the utility of our approach to the design of lanthanide complexes with a switch, we have developed a novel protease probe, which undergoes a significant change in luminescence intensity upon enzymatic cleavage of the substrate peptide. This probe, combined with time-resolved measurements, was confirmed in model experiments to be useful for the screening of inhibitors, as well as for clinical diagnosis.     

Free radical sensor based on CdSe quantum dots with added 4-amino-2,2,6,6-tetramethylpiperidine oxide functionality

The association and resulting fluorescence quenching of CdSe quantum dots by 4-amino-2,2,6,6-tetramethylpiperidine oxide (4-amino-TEMPO), a persistent nitroxide, have been examined using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. EPR data suggest binding constants around (8 +/- 4) x 10(6) M(-1) for green (2.4-2.5 nm) nanoparticles, and the application of Job's method indicates that the preferred mode of binding involves one or two quencher molecules per quantum dot, although more quenchers could bind at high concentrations of 4-amino-TEMPO. Fluorescence quenching by 4-amino-TEMPO is at least 3 orders of magnitude more efficient than by TEMPO itself, reflecting the strong binding confirmed by the EPR data. Stern-Volmer plots are nonlinear and in light of the EPR data probably reflect ready accessibility of the CdSe surface to one or two 4-amino-TEMPO molecules, while additional quenchers can only bind if they displace trioctylphosphine oxide ligands. Quantum dot-4-amino-TEMPO complexes can be used as free radical sensors, since the fluorescence (quenched by the nitroxide) is readily restored when radicals are trapped to form alkoxyamines.     

Lanthanide luminescence quenching as a detection method in ion chromatography. Chromate in surface and drinking water

Dynamic quenching of Eu(III) and Tb(III) luminescence by inorganic anions as a detection method in ion chromatography was investigated. To obtain a high luminescence intensity, lanthanide(III) complexes are formed with ligands which make indirect excitation of the ions possible. Only a few anions (e.g., nitrite, chromate) induce efficient dynamic luminescence quenching. Chromate is an efficient quencher of Tb-acac luminescence. Samples of tap water and surface water, spiked with chromate, were injected into a high-performance liquid chromatographic system with post-column addition of the luminescent complex. In this way, a detection limit of 1.1 . 10(-7) M (13 ppb) of chromate could be obtained.     

近红外发光稀土配合物及杂化材料研究进展

稀土因其特殊的物理和化学性质,在信息技术、能源技术、生物技术等高科技领域及国防建设等方面都起着非常重要的作用,中国作为稀土大国,十分重视对稀土材料的研究和开发。稀土离子近红外发光(750～1700 nm)在激光和光纤通讯、医学诊断、免疫分析等热门领域的潜在应用,受到了科研人员的极大关注。稀土离子本身发光极弱,通过分子内传能有机配体可以敏化稀土离子发光,但稀土配合物常受外界干扰,其稳定性较差,若将其与凝胶、介孔材料、离子液体等无机基质复合,得到具有良好光、热稳定性和化学稳定性的有机/无机杂化材料。总结了近些年来近红外发光稀土配合物及近红外发光稀土杂化材料的研究进展,并对其发展前景进行了展望。     

Luminescence detection of cysteine based on Ag-mediated conformational change of terbium ion-promoted G-quadruplex

In this work, we developed a simple and sensitive method for the detection of cysteine (Cys) by employing terbium ion (Tb³⁺)-promoted G-qudraplex (G4/Tb) as a luminescent probe, which is based on Ag⁺-mediated conformational change of G4/Tb. Due to Ag⁺ is able to compete with Tb³⁺ to bind guanine at G4, the presence of Ag⁺ can lead to the formation of G4/Tb–Ag⁺ complex and disrupt the structure of G4/Tb. Meanwhile, the binding of Ag⁺ with G4/Tb will also cause the alteration of the excited state of G4 and more efficient energy transfer from G4 to Tb³⁺, enhancing the luminescence of G4/Tb. However, upon the addition of Cys, Ag⁺ will be released from G4/Tb–Ag⁺ complex because of the high affinity of Cys to Ag⁺. This results in the re-formation of the conformation of G4/Tb and the decrease of the luminescence of G4/Tb. So, Ag⁺-enhanced luminescence of G4/Tb is associated with its conformational transformation. As a luminescent probe for Cys, G4/Tb not only shows excellent selectivity and sensitivity with a detection limit of 20 nM, but also possesses the features of simple preparation, easy reproducibility, and eliminating the interferences from background fluorescence. We envision that the presented strategy might provide new insight into the biosensing applications of lanthanide complex.     

Intramolecular Hydrogen Bonding: The Case of β‐Phosphorylated Nitroxide (= Aminoxyl) Radical

Alkoxyamines and persistent nitroxide (= aminoxyl) radicals are important regulators of nitroxide‐mediated radical polymerization. Since polymerization times decrease with the increasing homolysis rate constant of the C? ON bond homolysis between the polymer chain and the aminooxy moiety, the factors influencing the cleavage rate constant are of considerable interest. It has already been shown that the value of the homolysis rate constant k_d is very sensitive to the stabilization of both released radical species. X‐Ray, EPR, and kinetic data showed that the intramolecular H‐bonding radical in the 1‐(diethoxyphosphoryl)‐2,2‐dimethylpropyl 2‐hydroxy‐1,1‐dimethylethyl nitroxide ( 3a ) (homologue of 2‐hydroxy‐1,1‐dimethylethyl 1‐phenyl‐2‐methylpropyl nitroxide ( 2a )) did not occur with the nitroxide moiety as expected but with the phosphoryl group. However, the polymerization rate of styrene (= ethenylbenzene) was significantly enhanced.     

Photophysical properties of the prefluorescent nitroxide probes QT and C343T

The photophysical properties of the nitroxide prefluorescent probes 4-(3-hydroxy-2-methyl-4-quinolinoyloxy)-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (QT) and 2,3,4,6,7,8-hexahydro-quinolizino [1,10,9-gh] coumarin-3-carboxylic acid (1-oxyl-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (C343T) were evaluated as a function of pH and solvent properties. The absorbance of QT showed high pH sensitivity. The pKa values for the different ionization forms involved in the acid-base equilibrium of the quinoline chromophore were determined in the ground and excited states. The fluorescence lifetimes of QT, and N-hydroxylamine (QTH) and quinoline methyl ester (QMe) derivatives, showed that the intramolecular quenching efficiency by the nitroxide moiety is independent of the quinoline ionization form. The fluorescence and absorbance of C343T were highly sensitive to solvent polarity in agreement with a charged transfer excited state of the chromophore. However, we noted a decrease in the intramolecular fluorescence quenching efficiency by the nitroxide moiety when changing the polarity of the solvent from hexane to water. This behavior has been attributed to a suppression of an energy transfer mechanism in the nitroxide quenching process in very polar solvents. The results obtained in micelles allow us to propose QT and C343T as sensors for pH and micropolarity, respectively, in addition to their role as monitors for free radicals or hydrogen transfer from phenols.     

Rapid exchange luminescence: nitroxide quenching and implications for sensor applications

The quenching of terbium emission in the sensitized complex Tb3+-cs124-DTPA by nitroxide radical TEMPO derivatives in aqueous solutions has been studied with time-averaged and time-resolved methods. The time-resolved results show more quenching than the time-averaged values, opposite to the behavior expected for static quenching. A rapid exchange model with a slightly fluorescent fluorophore/quencher complex is proposed. Due to the long time scale of Tb3+ emission, dynamical averaging must be considered in the interpretation of experiments. The rapid exchange limit is shown to be consistent with the present results. The utility of these observations in the design of sensors that are not limited by a background level is noted.