铕-吡哌酸时间分辨荧光法检测DNA含量

在pH值为7.2的Tris-HCl缓冲溶液中,铕与吡哌酸反应形成配合物。该体系中加入鲱鱼精DNA分子作用后荧光强度显著增强,并且在一定浓度范围内,DNA浓度与其荧光强度呈良好的线性关系,据此建立了一种简单的测定DNA的时间分辨荧光分析新方法。考查了体系的时间分辨荧光光谱,通过与普通荧光光谱的对比突显了采用时间分辨荧光法的优势,并对反应条件进行了优化。该方法对DNA的检测限为0.03 mg·L-1(3σ),对浓度为4.0 mg·L-1的DNA进行11次平行测定,其相对标准偏差为0.3％。DNA的浓度在0.1～6.0 mg·L-1范围内与荧光强度呈良好的线性关系,线性方程为：ΔI=89.58c(mg·L-1)+0.920 5,线性相关系数r=0.999 6。此方法已应用于合成样品中DNA的测定,结果和加标回收率令人满意。     

Synthesis of AS1411-Aptamer-Conjugated CdTe Quantum Dots with High Fluorescence Strength for Probe Labeling Tumor Cells

In this paper, we report microwave-assisted, one-stage synthesis of high-quality functionalized water-soluble cadmium telluride (CdTe) quantum dots (QDs). By selecting sodium tellurite as the Te source, cadmium chloride as the Cd source, mercaptosuccinic acid (MSA) as the capping agent, and a borate-acetic acid buffer solution with a pH range of 5–8, CdTe nanocrystals with four colors (blue to orange) were conveniently prepared at 100 °C under microwave irradiation in less than one hour (reaction time: 10–60 min). The influence of parameters such as the pH, Cd:Te molar ratio, and reaction time on the emission range and quantum yield percentage (QY%) was investigated. The structures and compositions of the prepared CdTe QDs were characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, selective area electron diffraction, and X-ray powder diffraction experiments. The formation mechanism of the QDs is discussed in this paper. Furthermore, AS1141-aptamer-conjugated CdTe QDs in the U87MG glioblastoma cell line were assessed with a fluorescence microscope. The obtained results showed that the best conditions for obtaining a high QY of approximately 87 % are a pH of 6, a Cd:Te molar ratio of 5:1, and a 30-min reaction time at 100 °C under microwave irradiation. The results showed that AS1141-aptamer-conjugated CdTe QDs could enter tumor cells efficiently. It could be concluded that a facile high-fluorescence-strength QD conjugated with a DNA aptamer, AS1411, which can recognize the extracellular matrix protein nucleolin, can specifically target U87MG human glioblastoma cells. The qualified AS1411-aptamer-conjugated QDs prepared in this study showed excellent capabilities as nanoprobes for cancer targeting and molecular imaging.     

Monitoring Local Unfolding of Bovine Serum Albumin During Denaturation Using Steady-State and Time-Resolved Fluorescence Spectroscopy

In a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropiclly induced denaturation of Bovine Serum Albumin (BSA) using the fluorescence decay kinetics at different stages in the denaturation of BSA by guanidinium hydrochloride (GuHCl). In this work, we gain a more detailed insight into the BSA denaturation process by investigating the thermodynamics of the process. Structural changes were monitored spectrophotometrically via the intrinsic protein fluorescence from tryptophan residues, and the extrinsic fluorescence from 1,8-anilinonaphthalene sulphonate (ANS). ANS tends to locate in a variety of binding sites in BSA which are located in different domains, and these can be selectively populated using different, 1:1 and 1:10 molar ratios of BSA to ANS. The data from steady-state and time-resolved fluorescence spectroscopy were analyzed using thermodynamic two-state and three-state models and the lifetime data clearly indicated the presence of an intermediate state during denaturation. A global analysis using non-linear regression gave a DG_H2O,D⁰ = 6.7  \textkcal.\textmo\textl ^{- 1} \Delta G_{{H_2}O,D}^0 = 6.7\;{\text{kcal}}.{\text{mo}}{{\text{l}}^{ - 1}} for the complete unfolding of the BSA-ANS complexes, and a DG_H2O,I⁰ = 0.9  \textkcal.\textmo\textl ^{- 1} \Delta G_{{H_2}O,I}^0 = 0.9\;{\text{kcal}}.{\text{mo}}{{\text{l}}^{ - 1}} for the first step to the intermediate. Therefore, the unfolding energy of the intermediate, which appears mostly at intermediate GuHCl concentrations (1.0 to 1.5 M), to the denatured state, is 5.8 kcal.mol⁻¹. The lifetime analysis of the BSA-ANS complexes also shows clearly that there are differences in stability of the BSA domains, with domain III unfolding first at low GuHCl concentrations (<1.5 M).     

Time-Resolved Fluorescent Imaging of Glucose

A method for the fluorescent imaging of glucose is described that is based on the detection of enzymatically produced hydrogen peroxide, using the europium(III) tetracycline complex as the fluorescent probe incorporated into a hydrophilic polymer layer. Coadsorption of glucose oxidase (GOx) makes these sensor layers respond to the hydrogen peroxide produced by the GOx-assisted oxidation of glucose. The hydrogel layers are integrated into a 96-microwell plate for a parallel and simultaneous detection of various samples. Glucose is visualized by means of time resolved luminescence lifetime imaging. Unlike in previous methods, the determination of H₂O₂ does not require the addition of peroxidase or a catalyst to form a fluorescent product. The lifetime-based images obtained are compared with conventional fluorescence intensity-based methods with respect to sensitivity and the dynamic range of the sensor layer. The main advantages provided by this sensing scheme for H₂O₂ include reversibility, applicability at neutral pH, and the straightforwardness of the transducer system and the imaging device.     

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.     

葡萄球菌核酸酶蛋白的时间分辨荧光与热力学特性

葡萄球菌核酸酶(SNase)是一种小型球状蛋白,其变体常用来研究蛋白质的折叠过程。不同于之前报道的研究方法和技术手段,采用时间相关单光子计数(TCSPC)及飞秒荧光上转换技术,结合紫外吸收谱和稳态荧光光谱,研究了SNase蛋白变体Δ+PHS和Δ+PHS+I92A的荧光动力学,以及不同温度下蛋白结构与热稳定性的关系,证明蛋白质内色氨酸残基可作为一种内源性探针对蛋白变体的结构折叠和热稳定性进行印证和研究。衰减相关光谱(DAS)表明了两种变体随温度变化的不同趋势,在此基础上进一步分析了这两种变体的结构折叠及热稳定性的差异。皮秒时间分辨发射光谱(TRES)显示色氨酸残基存在0.5 ns的连续光谱弛豫过程,而光谱移动量可作为SNase变体蛋白结构紧密程度的判断依据。飞秒上转换数据分析结果中,0.5 ps的DAS在光谱蓝端为正、红端为负,表明了色氨酸残基受到弛豫效应的影响。200 ps的寿命则说明色氨酸残基与周围猝灭基团之间存在电子转移过程。时间分辨荧光各向异性(anisotropy)的分析结果则说明了色氨酸残基在蛋白质体系内具有独立的局部运动,且其强弱与变体的热稳定性和热运动的整体效果有关。测量和分析色氨酸残基的时间分辨荧光性质为深入研究SNase蛋白的结构和功能提供了新的思路。     

LDS751染料分子的时间分辨荧光谱

本文介绍用飞秒时间分辨荧光凹陷探测技术,获得激发态分子时间分辨荧光光谱的方法。文中描述了实验方法及其特点,给出了ＬＤＳ７５１染料分子的飞秒时间分辨荧光光谱。     

固相时间分辨荧光免疫标记技术研究

通过固相时间分辨荧光免疫分析双功能螯合剂4,7-二氯磺基苯-1,10菲罗啉-2,9-二羧酸标记抗-乙型肝炎表面抗体(HBsAb)IgG实验,对于BCPDA标记蛋白质的方法进行了研究。结果表明：BCPDA在相对温和条件下能与蛋白质反应,反应后蛋白质的相对生物活性高于78％,标记比为23～55,蛋白回收率达60％以上。在一定条件下与铕离子形成稳定的BCPDA-Eu^3 (HBsAb)IgG标记物。利用自建的分析方法,测定了标记过程的有关参数。并对标记物的某些光学特性进行了研究。     

胞内自组装四苯乙烯-生物素纳米粒子形成聚集诱导发光实现肿瘤细胞靶向显像

本文基于“聚集诱导发光”原理设计并合成了一种生物素化的可激活探针Cys(StBu)-Lys(biotin)-Lys(TPE)-CBT(1)用于肿瘤细胞靶向显像研究. 探针1能够被生物素受体高度表达的肿瘤细胞主动摄取,并在细胞内谷胱甘肽的还原作用下自组装形成纳米粒子结构导致“聚集诱导发光”. 基于这种“智能”的策略,本文成功地将探针1应用于肿瘤细胞的靶向显像研究. 这种生物素化的“智能”探针有望进一步发展应用于常规临床中的肿瘤显像.     

Study of Organized Media Using Time-Resolved Fluorescence Spectroscopy

Many photophysical processes which occur on an ultrafast time scale in ordinary liquids become significantly retarded in organized assemblies, by two to three orders of magnitude. Recently many groups have applied ultrafast laser spectroscopy and theoretical methods to elucidate this dramatic phenomenon. Although the implications of this phenomenon in biology and chemistry are not yet fully understood, it has been demonstrated that ultrafast time-resolved fluorescence spectroscopy is a very powerful tool to study the microscopic properties of the organized assemblies and that water or other liquids confined inside these assemblies are fundamentally different from the corresponding liquid in bulk.     

Study of Macromolecular Chain Dynamics in Polymer Complexes by Time-Resolved Fluorescence Spectroscopy

Poly(methyl methacrylate)s labeled with the anthracene fluorophore were prepared by free radical, anionic, and coordination polymerization yielding atactic and syndiotactic polymers. Unlabeled isotactic poly(methyl methacrylate) was prepared by anionic polymerization. Time-resolved fluorescence spectroscopy was used to study polymer association in solution. The time-dependent decays of fluorescence anisotropy show that stereocomplexation causes an increase in rotational correlation times of anthracene fluorophores both embedded in the polymer backbone and attached at the end of the side chain of polymer molecules. The rotational correlation time of anthracene fluorophore in dimethylformamide as a part of stereocomplex is 11.9 and 30 ns in the side chain and embedded in the polymer backbone, respectively, and shorter than 3 ns in noncomplexing solvent.     

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。     

Solvation and Solvent Relaxation in Swellable Copolymers as Studied by Time-Resolved Fluorescence Spectroscopy

The fluorescent probe dimethylaminonaphthylsulfonamide is covalently bound to the ends of the pol(ethylene glycol) chains of the swellable block copolymers poly(ethylene glycol)–polystyrene (PEG-PS) and poly(ethylene glycol)–poly(ethylene imine) (PEG-PEI) to investigate the molecular mobility inside the polymers, swollen by different liquids. Steady-state and time-resolved studies of the Stokes shift between absorption and fluorescence spectra reveal that the probe is solvated by both the polymer matrix and the liquid phase. The extent of solvation by the liquid and the mobility of the microenvironment of the probe depend on both the swelling volume of the polymer and the solubility of the probe in this liquid. Steady-state and time-resolved fluorescence depolarisation measurements show that the polymer matrix forms a very rigid solvent cage, which almost completely immobilizes the probe. Upon solvation of the probe by the liquid, the mobility of the probe increases. In PEG-PEI swollen by polar solvents, the mobilities of the probe itself and of its microenvironment, although not reaching the values observed in homogeneous solution, are significantly higher than in PEG-PS, due to the hydrophilic nature of the polymeric backbone in PEG-PEI.     

Pitfalls and Their Remedies in Time-Resolved Fluorescence Spectroscopy and Microscopy

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of time- and frequency-domain equipment as well as practical remedies against common error conditions, elucidated with several graphs to aid the researcher in visual inspection and quality-control of collected data. A range of artifacts due to sample preparation or to optical and electronic pitfalls are discussed, as are remedies against them. Also recommended data analysis strategies are described.     

Comparative Studies of Two Araceous Lectins by Steady State and Time-Resolved Fluorescence and CD Spectroscopy

Transitions in the tryptophan microenvironment and secondary structure of two monocot lectins from Sauromatum guttatum and Arisaema tortuosum under different denaturing conditions were studied by steady state and time resolved fluorescence and CD spectroscopy. The lectins exist as tetramers with a single tryptophan residue estimated per monomer, present in a polar environment. Quenching with ionic quenchers showed predominantly electropositive environment for tryptophan residues. Acrylamide had maximum quenching effect. A decrease in KI quenching due to lectin denaturation indicated redistribution of charges as a result of possible conformational change. The two values for lifetimes of tryptophanyl population (1.2–1.4 and 6.3–6.4 ns) reduced substantially on quenching or denaturation. Similarly, both the lectins showed a drastic loss of secondary structure in 5 M Gdn-HCl or 6 M Urea or at pH 2.0 and below. For the first time araceous lectins, like legume lectins are shown to bind adenine. The presence of a compact structure at alkaline pH 10.0–12.0 was observed in CD spectra.     

用时间分辨光谱研究红细胞悬液的荧光发射

实验获得了激光照射红细胞悬液的荧光光谱,并分别监测不同荧光峰值波长处强度随时间的衰变过程,测试了其相应的荧光寿命。结果表明,在波长为407nm的激光照射下,红细胞悬液向外发射中心波长分别位于596,628,692nm的荧光光谱,各荧光峰对应衰变过程的平均荧光寿命分别为1.97,13.31,14.58ns。利用荧光强度和吸收率的加和性表示了混合物的总吸收率和总荧光发射强度,通过理论计算获得了红细胞悬液中锌卟啉、原卟啉和其他游离物参与荧光发射的相对含量和相对强度在不同荧光峰位的变化关系,进一步解释了不同峰位处荧光发射强度和平均荧光寿命的变化原因。     

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。     

基于激光诱导时间分辨荧光的原油识别方法研究

在柴油、汽油、重质燃料油等成品油和原油等溢油油源的区分方面,荧光光谱结合模式识别手段得到了广泛的应用。传统的三维荧光光谱分析方法虽然能够获得溢油样品丰富的成分信息,但难以适应现场应用的要求,目前还停留在实验室检测的阶段。发展适用于现场应用的原油识别方法,对于海洋溢油污染的快速响应与处理意义重大。面向激光雷达的需要,发展了一种基于激光诱导时间分辨荧光手段、结合支持向量机（SVM）模型的原油识别方法,从时间和波长两个不同维度出发,通过对时间窗口和波长范围的选取进行优化,获得了理想的油种识别准确率。实验结果表明通过选取ICCD探测延时为54～74 ns可以将分类正确率从全谱线数据的83.3%提高到88.1%。通过选取波长范围为387.00～608.87 nm的谱线数据,可将疑似油种的分类正确率从全谱线数据的84%提高到100%。激光荧光雷达在实际工作中,受波浪、运载平台晃动等因素的影响,探测延时会出现一定的波动。本文介绍的分类识别方法通过时间和波长两个维度的筛选,更加适用于现场探测数据的识别,并进一步凸显了原油时间分辨荧光光谱特征,为疑似油种分类识别过程中数据量的压缩提供了重要依据。     

Study of Solid Transparent Nanocomposite Organic/Inorganic Matrices and Thin Films by Time-Resolved Fluorescence Techniques

This work focuses on the study of nanocomposite organic/inorganic materials, particularly, those made by the sol-gel method, by using time-resolved fluorescence techniques. A model of stretched exponentials is presented and used to fit fluorescence (luminescence) decay profiles for fluorescence quenching reactions obtained by energy-transfer or by diffusion or both. Various types of information on both bulk and thin-film nanocomposite materials can be obtained by such analysis: for example, determination of the percolation threshold for the organic subphase, localization or mobility of incorporated molecular species, and extraction of structural parameters.