Preparation,Characterization and Application of Fluorescent Terbium Complex-Doped Zirconia Nanoparticles

Novel zirconia-based fluorescent terbium nanoparticles have been prepared as a fluorescent nanoprobe for time-resolved fluorescence bioassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion consisting of a strongly fluorescent Tb³⁺ complex, N,N,N¹, N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-phenylpyridine]tetrakis(acetate)-Tb³⁺(BPTA-Tb³⁺), Triton X-100, hexanol, and cyclohexane by controlling co-condensation of Zr(OCH₂CH₃)₄ and ZrOCl_2. The characterizations by transmission electron microscopy and fluorometric methods indicate that the nanoparticles are uniform in size, 33± 4 nm in diameter, and have a fluorescence quantum yield of 8.9% and a long fluorescence lifetime of 2.0 ms. The zirconia-based fluorescent terbium nanoparticles show high stability against basic dissolution in a high pH aqueous buffer compared to the silica-based nanoparticles. A surface modification and bioconjugation method for the fluorescent nanoparticles was developed, and the nanoparticle-conjugated streptavidin (SA) was used for time-resolved floroimmunoassy (TR-FIA) of human prostate specific antigen (PSA). The result shows that the zirconia-based fluorescent terbium nanoparticles are useful as a fluorescent nanoprobe for time-resolved fluorescence bioassay.     

时间分辨激光荧光光谱技术在免疫分析中的应用

本研究以时间分辨激发荧光光谱分析技术为基础 ,进行稀土离子标记的激光激发的时间分辨荧光免疫分析 (TRFIA)研究。实验以自行合成的二乙三胺五醋酸酐 (DTPAA)为双功能螯合剂。用 Eu3+标记兔抗人 (RAH) Ig G抗体 ,依据解离增强原理 (DEL FIA) ,研究了 Eu3+ -β萘甲酰三氟丙酮 (β- NTA)的荧光分辨体系 ,测定了荧光光谱和荧光寿命 ,建立了铕离子分析检出方法 ,其工作曲线范围为 1× 10 - 7～ 1× 10 - 1 1g· m L- 1 ,检测限为 1× 10 - 1 3g· m L- 1 ,相对标准偏差为 6 .4%。结合 TRFIA方法学研究 ,进行了人血清丙型肝炎病毒抗体 (Anti- HCV)检测。并同酶联免疫法 (EL ISA)对比。取得 TRFIA法阳性检测率明显高于EL ISA法的结果。     

多环芳烃时间分辨荧光光谱特性研究

利用时间分辨荧光光谱技术,研究了菲、荧蒽、芴、蒽、芘等五种多环芳烃的荧光时间分辨发射光谱特性。以289 nm受激拉曼光作为激发光源,研究了289 nm激发光作用下五种多环芳烃的延时特性和门宽特性。并以多环芳烃随延时时间的荧光峰强度衰减关系曲线,得到菲、荧蒽、芴、芘的荧光寿命分别为37.0, 32.7, 10.9, 147.0 ns。不同荧光物质具有特定的荧光光谱特性,多环芳烃时间分辨荧光光谱特性的研究可以为复杂水体中不同种类多环芳烃的诊断提供依据。     

Effect of Polystyrene Microsphere Surface to Fluorescence Lifetime Under Two-Photon Excitation

Molecular assays such as immunoassays are often performed using solid carriers and fluorescent labels. In such an assay format a question can be raised on how much the fluorescence of the label is influenced by the bio-affinity binding events and the solid carrier surface. Since changes in fluorescence intensity as labels bind to surfaces are notoriously difficult to quantify other approaches are preferred. A good indicator, independent of the fluorescence intensity of the label, is the fluorescence lifetime of the marker fluorophore. Changes in fluorescence lifetime reliably indicate the presence of dynamic quenching, energy transfer or other de-excitation processes. A microsphere based assay system is studied under two-photon excitation. Changes in fluorescence lifetime are studied as labeled protein conjugates bind on microsphere surfaces – both direct on the surface and with a few nanometer distance from the surface. Fluorescence signal is measured from individual polystyrene microspheres and the fluorescence lifetime histogram is simultaneously recorded. The results indicate that self-quenching and quenching by the polystyrene surface are both present in such a system. However, the effect of the surface can be avoided by increasing the distance between the surface and the label. Typical distances achieved by a standard sandwich type of assay, are already sufficient to overcome the surface induced quenching in fluorescence detection.     

Comparison Between Fluorescence Correlation Spectroscopy and Time-Resolved Fluorescence Anisotropy as Illustrated with a Fluorescent Dextran Conjugate

The motional properties of rhodamine green alone and conjugated to 10-kDa dextran have been studied by fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA). With FCS the translational diffusion times of the fluorescent particles can be determined, which are directly proportional to the shear viscosity as shown in aqueous solutions of different sucrose concentrations. With TRFA the rotational correlation times of the fluorescent particles can be determined. TRFA experiments in the case of fluorescent dextran reveal a distinct restricted internal motion of the fluorescent probe independent of the slower overall rotation of the polysaccharide. The fast depolarization is most likely due to internal motion and not to energy transfer between different rhodamine green molecules in the same dextran, since a higher viscosity of the solvent increases the correlation time for internal motion proportionally. FCS and TRFA yield complementary information in the sense that the correlation time for overall dextran rotation can be accurately determined from the translational diffusion coefficient.     

Improvements in the Sensitivity of Time Resolved Fluorescence Energy Transfer Assays

The effect on fluorescence resonance energy transfer (FRET) of multiple labelling of DNA oligonucleotides with donor lanthanide chelate and acceptor CyDye fluors has been investigated. It is shown that using a multiple donor lanthanide chelate with a single acceptor Cy or Cy5 can increase sensitivity and fluorescence output. The enhanced FRET observed in the multiple donor label system has been utilised in two different DNA based assay formats to demonstrate the advantages over a steady state fluorescence assay and a radiometric assay.     

Propidium Iodide and PicoGreen as Dyes for the DNA Fluorescence Correlation Spectroscopy Measurements

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe–nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.     

New Perspectives of Fluorescence Correlation Spectroscopy

The principle of fluorescence correlation spectroscopy is outlined. The technique has been applied to a mutant of the well-known green fluorescent protein. A comparative study has been made with time-resolved fluorescence anisotropy. The latter experiment shows that the fluorophore is rigidly bound inside the protein matrix follows the rotation of the whole protein and does not show any fast restricted motion. It is evident from fluorescence correlation spectroscopy that some excited-state reaction plays a role, since the autocorrelation traces show a significant effect on the incident laser power. Other potential applications of fluorescence correlation spectroscopy are presented as taken from very recent publications.     

The Influence of Solvent Viscosity on the Fluorescence Decay and Time-Resolved Anisotropy of Green Fluorescent Protein

This report describes fluorescence decay and time-resolved anisotropy studies of green fluorescent protein (GFP) in various environments. The addition of glucose and fructose, NaCl, or polyethylene glycol changes the viscosity of the medium surrounding the GFP. Both the time-resolved anisotropy and the fluorescence decay of GFP are measured and it is shown that only the time-resolved anisotropy of GFP is affected by the viscosity, but not its fluorescence decay.     

Steady-State and Time-Resolved Fluorescence Polarization Behavior of Acenaphthene

Steady-state and time-resolved fluorescence polarization studies have been carried out on acenaphthene (ACE) in low-temperature glass solutions and at room temperature. In the low-temperature glass the fluorescence polarization values vary considerably with both emission and excitation wavelength. There is a time dependence (on the nanosecond time scale) of the fluorescence anisotropy, r(t), at 77 K, which has a strong dependence upon the excitation and emission wavelengths. Under these conditions, the time-dependent decay of the anisotropy is not attributable to chromophoric motion. The observations are consistent with emission from two closely lying and interconverting excited states. Rate constants for the photophysical processes involved have been determined by fitting the data using a model proposed by Fleming et. al. The results are discussed with particular reference to the care required in using dynamic fluorescence polarization measurements to determine energy transfer rates in systems containing this chromophore.     

原油样品激光诱导荧光的时间分辨光谱特性研究

为探索激光诱导时间分辨荧光光谱技术应用于海洋悬浮溢油原位探测的可行性,对来自胜利油田六个不同井区不同密度的原油样品的时间分辨荧光光谱进行了探测分析。结果发现,各原油样品荧光发射的持续时间基本相同,从ICCD中数字延时发生器(DDG)的输入延时52 ns开始,到输入延时82 ns左右结束,各原油样品的荧光峰强度随时间变化曲线的半高宽约10 ns;不同原油样品的最强荧光峰位及其衰减寿命不尽相同,并且与样品密度有一定相关性,密度相近的原油具有相近的最强荧光峰位和相似的荧光寿命。对比六种原油样品的时间分辨荧光光谱发现,在荧光增强时,原油荧光光谱峰位不变,当荧光从最大强度开始衰减时,六种原油样品的荧光光谱峰位均出现了不同程度(17~30 nm)的红移现象,这一定程度上反映出原油中各荧光组分的荧光衰减速率存在差异,或者存在荧光组分之间的能量传递。所观测到的原油密度相关的时间分辨光谱信息和荧光峰红移现象可望成为水下悬浮溢油识别的有效特征之一。     

355 nm纳秒激光作用下荧蒽的时间分辨荧光光谱特征

多环芳烃由于具有三致(致癌、致畸、致突变)特性,其在环境中的检测受到人们广泛关注。利用时间分辨光谱技术,研究了荧蒽乙醇溶液的荧光光谱随延时时间和门宽改变的特性。研究了不同浓度荧蒽的时间分辨荧光光谱特性,以原始浓度的荧蒽为初始溶液,通过逐级稀释的方式,最终将原始溶液稀释16倍,拟合了不同稀释倍数下的荧蒽荧光强度衰减随延时时间变化的动力学曲线,得到了不同浓度荧蒽的拟合荧光寿命。研究结果表明,荧蒽的荧光光谱特性与光谱仪探测器延时时间和门宽宽度密切相关。固定延时时间,随着光谱仪门宽宽度的变化,荧蒽的荧光强度随着门宽的增大而逐渐增强。固定门宽,改变延时时间的过程中,荧蒽的荧光强度随延时时间呈现先增大,后减小的趋势。荧蒽的荧光强度随延时时间的衰减过程符合指数衰减过程,将荧蒽乙醇溶液进行逐级稀释,荧蒽荧光强度与延时时间的衰减进行指数拟合后,得到不同稀释倍数的荧蒽乙醇溶液的衰减动力学参数,随着稀释倍数的增大,拟合得到的荧蒽荧光寿命增大。多环芳烃时间分辨光谱特征的研究,可以为环境中多环芳烃的检测提供技术基础,由于不同荧光物质具有特征的荧光寿命,因此,可以利用多环芳烃与环境中其他荧光物质的不同荧光寿命特性,准确识别环境中的多环芳烃污染物。     

单壁碳纳米管吸附联苯类有机物的荧光光谱分析

利用芳香族化合物对单壁碳纳米管进行了化学修饰,并利用荧光光谱以及时间分辨光谱对修饰后的单壁碳纳米管进行了表征分析.实验发现吸附对三联苯后有1个荧光峰位置发生了蓝移,这说明吸附过程使对三联苯的一些能级分布发生了变化.测量吸附前后对三联苯和蒽甲醇的荧光寿命,发现吸附后的荧光衰减曲线下降趋势更加明显,对曲线进行多指数拟合得出的荧光寿命及其数目发生了变化,分析了可能导致该现象的原因.     

Fluorescence Properties of Frog Skeletal Muscles Treated with Lanthanide Ions

Laser-induced fluorescence from frog skeletal muscle fibers treated with lanthanides, Eu³⁺ and Tb³⁺, was recorded. The fluorescence was weak and overlapped with the Raman scattering by the Ringer solution when the muscle fibers were illuminated with an argon-ion laser. The fluorescence decay rate of the lanthanide in Ringer's solution was 2–3 times larger than that of the lanthanide bound to the muscle fiber. The number of water molecules coordinated to the lanthanide bound to the muscle fiber was determined to be about three. This suggests that lanthanide ions bind superficially to the outer membrane of the muscle fiber.     

Hydrophilic Labeling Reagents of Dipyrrylmethene-BF2 Dyes for Two-Photon Excited Fluorometry: Syntheses and Photophysical Characterization

Recently introduced bioaffinity assay technology, ArcDia TPX, is based on two-photon excited fluorescence (TPE) and it enables separation-free ultra-sensitive immunoassays from microvolumes. Here we present syntheses of novel two-photon excitable fluorescent labeling reagents which have been specially designed to be used as label molecules in the ArcDia TPX assay technique. The labeling reagents are based on dipyrrylmetheneboron difluoride (dipyrrylmethene-BF2) chromophore, which have been substituted with aryl, heteroaryl or arylalkenyl chemical groups to extend the pi-electron conjugation. These substitutions results in a series of dipyrrylmethene-BF2 fluorophores with different photophysical properties. Dipyrrylmethene-BF2 fluorophores have been further substituted with a dipeptide linker unit and finally activated as succinimidyl esters to enable specific coupling with primary amino groups. The dipeptide linker serves as a spacer arm between the label and a target, and enhances the solubility of the label in aqueous solutions. Study of the chemical and photophysical performance of the new labeling reagents is described. The new labeling reagents exhibit high fluorescence quantum yields, and molar absorption coefficients. The results show that the new labels with the hydrophilic dipeptide linker unit provide large two-photon excitation cross-sections, high fluorescence quantum efficiency and good solubility in aqueous solutions. The results suggest that the novel dipyrrylmethene-BF2 labels are highly applicable to bioaffinity assays based on two-photon excitation of fluorescence.     

荧光分子层析中的全时间分辨图像重建法

在荧光分子层析(Fluorescence molecular tomography,FMT)中.全时间分辨(Time Resolved.TR)测量包含了最多的光子传输信息.基于有限元一有限差分扩散方程的正向模型和Newtown-Raphson的逆向模型,将全时间分辨方法用于时域荧光分子层析中.用模拟数据对算法在空间分辨率、定量性、重建尺寸和灰度的保真度以及噪声稳健性等方面进行了验证.结果表明,此方法能够实时重建荧光产率和荧光寿命图像.与以前发展的基于广义脉冲谱技术(Generalized pulse spectrum technique,GPST)的特征数据法进行图像重建相比较.整体上优于广义脉冲谱技术.     

Silver Fractal-Like Structures for Metal-Enhanced Fluorescence: Enhanced Fluorescence Intensities and Increased Probe Photostabilities

Substantial increases in fluorescence emission from fluorophore-protein–coated fractal-like silver structures have been observed. We review two methods for silver fractal structure preparation, which have been employed and studied. The first, a roughened silver electrode, typically yielded a 100-fold increase in fluorophore emission, and the second, silver fractal-like structures grown on glass between two silver electrodes, produced a 500-fold increase. In addition, significant increases in probe photostability were observed for probes coated on the silver fractal like structures. These results further serve to compliment our recent work on the effects of nobel metal particles with fluorophores, a relatively new phenomenon in fluorescence we have termed both metal-enhanced fluorescence [1] and radiative decay engineering [2,3]. These results are explained by the metallic surfaces modifying the radiative decay rate () of the fluorescent labels. We believe that this new silver-surface preparation, which results in ultrabright and photostable fluorophores, offers a new generic technology platform for increased fluorescence signal levels, with widespread potential applications to the analytical sciences, imaging, and medical diagnostics.     

Probing Si and Ti Based Sol-Gel Matrices by Fluorescence Techniques

The photophysical behavior of several probes incorporated in sol-gel–derived matrices (both monoliths and thin films) has been studied using steady-state and time-resolved fluorescence, along with fluorescence anisotropy to study the matrix structure and to elucidate probe-matrix interactions. The probes studied include laser and solvatochromic dyes along with porphyrins and phthalocyanines. It was found that spectral shifts, time-resolved decays, and quantum yields depend on the type of matrix and its preparation conditions combined with the drying time and the nature of retained solvent, which can be added to act as an anticracking agent. The differences between the results in the TiO₂ matrix, where electron transfer is most probably present, and SiO₂ are shown.     

Fluorescence and electrochemical recognition of nucleosides and DNA by a novel luminescent bioprobe Eu(III)-TNB

The luminescence arising from lanthanide cations offers several advantages over organic fluorescent molecules: sharp, distinctive emission bands allow for easy resolution between multiple lanthanide signals; long emission lifetimes (μs –ms) make them excellent candidates for time-resolved measurements; and high resistance to photo bleaching allow for long or repeated experiments. A method is presented for determination of nucleosides using the effect of enhancement of fluorescence of the easily accessible europium(III)-TNB in presence of different nucleosides. The latter coordinates to Eu(III) -TNB and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. A similar method for the determination of DNA based on the quenching of Eu(III)-TNB has been established. The interaction of Eu(III)-4,4,4 trifluoro-1-(2-naphthyl)1,3-butanedione (TNB) complex with nucleosides (NS) (guanosine, adenosine, cytidine , inosine) and DNA has been studied using normal and time-resolved luminescence techniques. Binding constants were determined at 293 K, 298 K, 303 K, 308 K and 313 K by using Benesi-Hildebrand equation. A thermodynamic analysis showed that the reaction is spontaneous with ΔG being negative. The enthalpy ΔH and the entropy ΔS of reactions were all determined. The formation of binary and ternary complexes of Eu (III) with nucleosides and TNB has been studied potentiometrically at (25.0 ± 0.1) °C and ionic strength I = 0.1 mol.dm⁻³ (KNO₃) . The formation of the 1:1 binary and 1:1:1 ternary complexes are inferred from the corresponding titration curves. Initial estimates of the formation constants of the resulting species and the protonation constants of the different ligands used have been refined with the HYPERQUAD computer program. Electrochemical investigations for the systems under investigations have been carried out using cyclic voltammetry (CV), differential pulse polarography (DPP), and square wave voltammetry (SWV) on a glassy carbon electrode in I = 0.1 mol/L p-toluenesulfonate as supporting electrolyte.     

镧系配合物的荧光光谱

对镧系配合物荧光光谱的研究进展进行了评述,并对其光物理、光化学进行了归纳、分类,特别是对目前研究非常活跃的镧系离子荧光探针法在配合物和生物分子体系结构探测中的应用情况进行了总结。