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荧光共振能量转移及相关分析方法

荧光共振能量转移及相关分析方法荧光共振能量转移（Fluorescence Resonance Energy Transfer，FRET）是一种通过分子间非辐射能量转移过程来研究分子间相互作用的技术。

它广泛应用于生物医学领域，用于研究生物大分子（如蛋白质和核酸）的相互作用、结构和功能。

在FRET过程中，一个分子的荧光就能转移到另一个分子上，既不发出荧光也不带来辐射能量损失。

这种能量转移的效率与两个分子之间的距离密切相关，通常要求两个分子的距离在1至10纳米之间。

FRET的原理基于荧光分子的特性。

荧光分子（受体）吸收光子能量后进入激发态，然后通过非辐射能量转移的方式将能量传递给一个接受体分子。

接受体分子可以是一个发生荧光的分子，也可以是一个荧光猝灭剂。

如果接受体分子是一个发生荧光的分子，它将重新辐射有相同或不同波长的光子。

如果接受体分子是一个猝灭剂，它将通过非辐射机制将能量降低为热或振动能。

荧光共振能量转移具有很多优点。

首先，由于非辐射能量转移的效率与分子间距离的六次方成反比关系，所以FRET可以提供亚纳米尺度的分辨率。

其次，FRET技术操作简单，不需要复杂的分析设备，可以在大多数实验室中实施。

此外，FRET可以在单个分子水平上进行研究，提供了对生物大分子的高灵敏度分析。

因此，FRET广泛应用于生物医学研究中各个方面，例如蛋白质相互作用、信号传导、酶活性和膜蛋白结构的研究。

在FRET研究中，有几种常用的分析方法。

首先是通过荧光显微镜观察FRET信号。

荧光显微镜可以实现对单个分子或细胞的FRET信号实时监测和定量分析。

其次是可以借助分点突变的手段，通过改变受体分子上的特定氨基酸残基，来研究荧光共振能量转移的机制和效率。

此外，还可以利用光谱和时间分辨荧光光谱法来研究FRET现象。

这些方法能够提供更加详细和准确的FRET分析结果。

除了上述基本的FRET分析方法，还有一些改进和扩展的技术被用于更加复杂和精确的研究。

第九章分子荧光光谱法Molecular-fluorescence-spectroscopy

测量荧光的仪器主要由四个部分组成：激发光源、样品池、双单色器系统、检测器。
特殊点：有两个单色器，光源与检测器通常成直角。基本流程如图：单色器：选择激发光波长的第一单色器和选择发射光(测量)波长的第二单色器；光源：灯和高压汞灯，染料激光器(可见与紫外区) 检测器：光电倍增管。
仪器框图
该型仪器可进行荧光、磷光的发光分析；
同步扫描技术
根据激发和发射单色器在扫描过程中彼此间所保持的关系，同步扫描可分为固定波长差(Δλ)和固定能量差及可变波长三种；
辐射复合发光过程：
1. 自由激子复合(X)； 2. 导带电子—中性受主复合
(e，A0)； 3. 施主—受主对复合
(D0，A0)； 4. 束缚于中性施主上的——
激子复合 (D0，X)； 5. 中性施主——价带空穴的复合(D0，h)；
中性受主、电离施主或受主上的和等电子杂质上的束缚激子复合而发光。
3.激发光谱与发射光谱的关系
（4）取代基效应：芳环上有供电基，使荧光增强。
3.内滤光作用和自吸现象
内滤光作用：溶液中含有能吸收激发光或荧光物质发射的荧光，如色胺酸中的重铬酸钾；
自吸现象：化合物的荧光发射光谱的短波长端与其吸收光谱的长波长端重叠，产生自吸收；如蒽化合物。
4、溶液荧光的猝灭
碰撞猝灭：氧的熄灭作用等。
四、仪器结构流程
2. 激发态分子的失活: 激发态分子不稳定，它要以辐射
或无辐射跃迁的方式回到基态。
λ1
λ2
λ2/
λ3
λ4
无辐射跃迁:
(1) 振动弛豫：激发态分子由同一电子能级中的较高振动能级转至较低振动能级的过程，其效率较高。 (2) 内转换：相同多重态的两个电子能级间，电子由高能级回到低能级的分子内过程。 (3) 系间窜越：激发态分子的电子自旋发生倒转而使分子的多重态发生变化的过程。 (3) 外转换：激发态分子与溶剂或其它溶质相互作用、能量转换而使荧光（或磷光）减弱甚至消失的过程。荧光强度的

分子光谱分析Chapter01

0.3 Differences between emission and absorption of radiation 吸光：基态→电子各激发态跃迁吸光：基态→电子各激发态跃迁; 发射： υ 发射：S1(υ=0)→S0(υ=i)的辐射跃迁 υ 的辐射跃迁;
分别携带被观察物体的激发态或基态信息信息，分别携带被观察物体的激发态或基态信息，可以从不同侧面激发态了解物质的内部结构。了解物质的内部结构。
发光概述?chp1荧光分析?principleoffluorimetry荧光的原理?thefluorescencemechanism荧光方法?characteristicsoffluorescencespectrum荧光光谱特性?fluorescencedecayandlifetime荧光衰减和寿命?quantumyield量子产率?fluorescenceintensity荧光强度01发光现象luminescentphenomena煤气燃烧蓝色火焰炽热铁丝黄色火焰煤气燃烧蓝色火焰炽热铁丝黄色火焰发光介绍introductiontoluminescence荧光灯管电激发发光白炽灯泡发光汞灯365nm3brcarbazole奇特磷光奇特磷光ex363nm光棒化学反应发光汞灯365nmfonkos菌悬浮液takenfromdrternuraevenmanysinglecelledorganismsarebioluminescent
二、发光的类型 (Type of luminescence )？ • Photoluminescence (光致发光光致发光): 光致发光 Fluorescence/Fluorimetry; Phosphorescence/Phosphorimetry; • Chemiluminescence (化学发光化学发光); 化学发光 • Bioluminescence (生物发光生物发光); 生物发光 • Radioluminescence (辐射发光辐射发光); 辐射发光 • Electroluminescence(电致发光电致发光); 电致发光 • Sonoluminescence(声致发光声致发光); 声致发光

光谱法研究药物小分子与蛋白质大分子的相互作用的英文

Spectroscopic Study of the Interaction between Small Molecules and Large Proteins1. IntroductionThe study of drug-protein interactions is of great importance in drug discovery and development. Understanding how small molecules interact with proteins at the molecular level is crucial for the design of new and more effective drugs. Spectroscopic techniques have proven to be valuable tools in the investigation of these interactions, providing det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding.2. Spectroscopic Techniques2.1. Fluorescence SpectroscopyFluorescence spectroscopy is widely used in the study of drug-protein interactions due to its high sensitivity and selectivity. By monitoring the changes in the fluorescence emission of either the drug or the protein upon binding, valuable information about the binding affinity and the binding site can be obt本人ned. Additionally, fluorescence quenching studies can provide insights into the proximity and accessibility of specific amino acid residues in the protein's binding site.2.2. UV-Visible SpectroscopyUV-Visible spectroscopy is another powerful tool for the investigation of drug-protein interactions. This technique can be used to monitor changes in the absorption spectra of either the drug or the protein upon binding, providing information about the binding affinity and the stoichiometry of the interaction. Moreover, UV-Visible spectroscopy can be used to study the conformational changes that occur in the protein upon binding to the drug.2.3. Circular Dichroism SpectroscopyCircular dichroism spectroscopy is widely used to investigate the secondary structure of proteins and to monitor conformational changes upon ligand binding. By analyzing the changes in the CD spectra of the protein in the presence of the drug, valuable information about the structural changes induced by the binding can be obt本人ned.2.4. Nuclear Magnetic Resonance SpectroscopyNMR spectroscopy is a powerful technique for the investigation of drug-protein interactions at the atomic level. By analyzing the chemical shifts and the NOE signals of the protein in thepresence of the drug, det本人led information about the binding site and the mode of binding can be obt本人ned. Additionally, NMR can provide insights into the dynamics of the protein upon binding to the drug.3. Applications3.1. Drug DiscoverySpectroscopic studies of drug-protein interactions play a crucial role in drug discovery, providing valuable information about the binding affinity, selectivity, and mode of action of potential drug candidates. By understanding how small molecules interact with their target proteins, researchers can design more potent and specific drugs with fewer side effects.3.2. Protein EngineeringSpectroscopic techniques can also be used to study the effects of mutations and modifications on the binding affinity and specificity of proteins. By analyzing the binding of small molecules to wild-type and mutant proteins, valuable insights into the structure-function relationship of proteins can be obt本人ned.3.3. Biophysical StudiesSpectroscopic studies of drug-protein interactions are also valuable for the characterization of protein-ligandplexes, providing insights into the thermodynamics and kinetics of the binding process. Additionally, these studies can be used to investigate the effects of environmental factors, such as pH, temperature, and ionic strength, on the stability and binding affinity of theplexes.4. Challenges and Future DirectionsWhile spectroscopic techniques have greatly contributed to our understanding of drug-protein interactions, there are still challenges that need to be addressed. For instance, the study of membrane proteins and protein-protein interactions using spectroscopic techniques rem本人ns challenging due to theplexity and heterogeneity of these systems. Additionally, the development of new spectroscopic methods and the integration of spectroscopy with other biophysical andputational approaches will further advance our understanding of drug-protein interactions.In conclusion, spectroscopic studies of drug-protein interactions have greatly contributed to our understanding of how small molecules interact with proteins at the molecular level. Byproviding det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding, spectroscopic techniques have be valuable tools in drug discovery, protein engineering, and biophysical studies. As technology continues to advance, spectroscopy will play an increasingly important role in the study of drug-protein interactions, leading to the development of more effective and targeted therapeutics.。

fluorescence

Fluorescence spectroscopyFluorescence spectroscopy –why ? (3)This spectroscopy looks for changes in various “aspects of fluorescence”on the interaction between a drug and its receptor. These “aspects”may include the following•Emission spectra•Excitation spectra•Quantum yield ~ quenching•Polarisation•Fluorescent lifetimeNatural (Intrinsic) and Imposed (Extrinsic) Fluorescence (4) Natural (intrinsic) fluorescence in biological macromolecules is limited.In proteins certain residues have the ability to impart fluorescence when in a special environment (in order of importance)•Tryptophan(W)•Tyrosine(Y)•Phenylalanine(F)Imposed (Extrinsic) fluorescence may be introduced when a protein is modified with a fluorescent reagent known as a fluorophore(fluor)this is useful as long as the fluor•may be introduced in a unique location•adequately responds to the D-R interaction•does not affect the binding affinity of the drug with the receptorλλmaxEM ..λEXThis fluor is less accessible to the quenching agent This fluor isAdvantages of Fluorescence spectroscopy (14)Highly sensitive techniqueUp to 1,000 times more sensitive than UV / visible spectroscopy. Therefore often used in drug or drug metabolite determinations by HPLC with fluorimetric detector. Non-fluorescing compounds can be made fluorescent –derivitisation.Selective versatile techniqueSince excitation and emission wavelengths are utilised, gives selectivity to an assay compared to UV / visible spectroscopy. Differing modes of spectroscopy yield wide versatility.。

荧光共振能量转移技术在生命科学中的应用及研究进展

荧光共振能量转移技术在生命科学中的应用及研究进展一、本文概述荧光共振能量转移（Fluorescence Resonance Energy Transfer，FRET）是一种在分子尺度上测量距离和相互作用的强大技术，广泛应用于生命科学领域。

FRET依赖于两个荧光分子间的非辐射能量转移，当两个荧光分子足够接近时，一个荧光分子（称为供体）可以通过偶极-偶极相互作用将其激发态能量转移给另一个荧光分子（称为受体）。

由于能量转移效率与供体和受体之间的距离紧密相关，因此，FRET可以被用作一种灵敏的分子尺度的距离探测器。

本文将对荧光共振能量转移技术在生命科学中的应用及其研究进展进行全面的探讨，旨在展现这一技术在生物学、医学等领域中的重要作用和潜在价值。

二、FRET技术的基本原理荧光共振能量转移（FRET）是一种非辐射性的能量转移过程，它发生在两个荧光分子之间，其中一个分子（称为供体）在激发状态下，能够将能量转移给另一个邻近的且激发态能量较低的荧光分子（称为受体）。

这一过程的发生需要供体和受体之间的距离足够近，通常在10纳米以内。

当供体被光激发后，它的电子会从基态跃迁到激发态，如果这个激发态的能量高于受体的基态与激发态之间的能量差，那么供体就可以通过偶极-偶极相互作用将能量传递给受体，使其从基态跃迁到激发态。

受体随后会以发射荧光的形式释放能量，返回到基态。

FRET技术的关键参数包括能量转移效率、供体与受体之间的距离以及供体和受体的相对光谱重叠程度。

能量转移效率通常与供体和受体之间的距离的六次方成反比，这意味着当两者之间的距离稍有增加时，能量转移效率会迅速下降。

因此，FRET对距离的变化非常敏感，使得它成为一种强大的工具，能够用于研究分子间的相互作用、蛋白质构象变化以及生物分子间的动态过程。

FRET技术还可以通过比较供体和受体的荧光信号强度来定量测量分子间的距离，从而揭示生物分子间的相互作用机制。

例如，在蛋白质相互作用的研究中，可以通过将供体和受体分别标记在两个不同的蛋白质上，观察它们之间的FRET信号变化来推断蛋白质之间的结合和解离过程。

荧光光谱技术在蛋白质研究中的应用

荧光光谱技术在蛋白质研究中的应用The application of studying fluorescence spectroscopy on protein学生姓名：林亚南院系：理学院专业：应用物理学号：1035006E-mail：rabbitnone@摘要荧光光谱法对研究蛋白质结构及其构象变化是很重要的。

描述了荧光光谱的概念、发光机理及特点，介绍了荧光光谱仪的仪器原理和结构，记述了荧光光谱技术在检测蛋白质的构象变化、蛋白质的含量和酶活性方面的具有应用。

关键词：荧光光谱法; 蛋白质; 构象AbstractFluorescence spectroscopy is very important for studying protein structure and conformation changes. The concept and principle of fluorescence spectroscopy are introduced at firstthen the application of studying fluorescence spectroscopy on protein is explained.Key words: fluorescence spectroscopy; protein; conformation目录引言 (4)主要内容 (5)一、荧光光谱技术 (5)1.荧光发光机制 (5)2.常用荧光参数 (5)二、荧光光谱仪的原理及结构 (6)三、蛋白质的内源荧光与荧光探针 (7)1. 蛋白质的内源荧光 (8)2. 蛋白质的外源荧光 (8)四、荧光光谱法在蛋白质研究中的应用 (8)1. 利用蛋白质的天然荧光检测蛋白质的构象变化 (8)2. 利用荧光探针检测蛋白质的构象变化 (9)3. 测定蛋白质的含量 (9)4. 测定酶的活性 (9)5. 研究小分子与蛋白质间的相互作用 (10)五、蛋白质荧光光谱研究的一些新方法 (10)1. 同步荧光光谱 (10)2.三维荧光光谱法 (11)3.荧光共振能量传递分析 (12)结论 (13)参考文献(References) : (14)引言16世纪，西班牙科学家Nicholas Monardes观察到，贮放在由菲律宾紫檀木制成的杯中的水会发出一种神奇而迷人的蓝光。

Fluorescence spectroscopy and multi-way techniques PARAFA

Received 12th July 2013 Accepted 9th September 2013 DOI: 10.1039/c3ay41160e /methods
Introduction
a
University of New South Wales, Water Research Centre, Sydney, Australia. E-mail: krm@.au; Fax: +61 2 9313 8624; Tel: +61 2 9385 4601 Technical University of Denmark, National Institute for Aquatic Resources, Charlottenlund, Denmark. E-mail: cost@aqua.dtu.dk
PARAllel FACtor analysis (PARAFAC) is used in the chemical sciences to decompose trilinear multi-way data arrays and facilitate the identication and quantication of independent underlying signals, termed ‘components’. In 2011–2012, 334 Scopus-indexed journal and conference papers were published with keywords “PARAFAC” or “parallel factor analysis”. In the subset of papers where PARAFAC was used primarily as a tool for data interpretation (n ¼ 238, thus excluding 96 papers concerned primarily with developing or comparing algorithms, tools or statistical methodologies), PARAFAC was applied across research elds (medical, pharmaceutical, food, environmental, social, and information science) and to a wide range of data

Principles of Fluorescence Spectroscopy

For fluorophore
r 0.4,
20040200
P 0.5
I I11,
r P0
XMUPFS01-ITF02
Polarization and anisotropy
荧光偏振与荧光各向异性可通过以下公式相互转换：
3r P 2r
2P r 3 P
当体系中存在多种荧光体时，所测得的荧光各向异性是各种荧光体荧光各向异性的平均值：
Three dimension spectra
ex / nm
em / nm
引自林竹光等人的论文
20040200
XMUPFS01-ITF02
1.5.5 Fluorescence lifetime
Definition Lifetime for Single molecule: the time the molecule spends in the excited state prior to return to the ground state. Average lifetime: the average time the molecule spends in the excited state prior to return to the ground state.
20040200
XMUPFS01-ITF02
Definition
polarization z x
激发偏振器
I II I P I II I
y I I
发射偏振器
anisotropy
检测器
I II I r I II 2 I
I 0, r P 1.0
Excitation and emission spectra

紫外荧光光谱法英语

紫外荧光光谱法英语Ultraviolet Fluorescence Spectroscopy.Ultraviolet fluorescence spectroscopy is an analytical technique that employs the fluorescence emitted by molecules excited by ultraviolet (UV) light to characterize chemical species. This method has found widespread applications in various fields, including chemistry, biochemistry, pharmacology, and environmental science.Principles of UV Fluorescence Spectroscopy.The principle of UV fluorescence spectroscopy lies in the absorption of UV light by molecules, which then emit light at longer wavelengths, known as fluorescence. This emission occurs when the absorbed energy causes electronsin the molecules to transition from a lower energy state to an excited state. As the electrons relax back to the lower energy state, they emit radiation in the form of light. The wavelength and intensity of this emitted light arecharacteristic of the specific molecular structure and can be used for identification and quantification.Instrumentation.UV fluorescence spectroscopy requires specialized instrumentation, primarily a UV-Vis spectrophotometer with a fluorescence detector. These instruments typically consist of a light source, a monochromator to select a specific wavelength of UV light, a sample compartment, and a detector to measure the emitted fluorescence. Modern spectrophotometers often incorporate advanced features such as multi-wavelength excitation and emission scanning, which provide richer spectral information.Applications of UV Fluorescence Spectroscopy.1. Biochemical Analysis: UV fluorescence spectroscopyis widely used in biochemistry to study protein-ligand interactions, protein conformational changes, and nucleic acid structure. Fluorescent probes can be attached to specific sites on proteins or nucleic acids, allowing theirbehavior to be monitored under different conditions.2. Drug Discovery and Pharmacology: This technique is employed in drug discovery to screen potential drugs for their binding affinity to biological targets. By monitoring the changes in fluorescence upon drug binding, researchers can assess the affinity and selectivity of drugs.3. Environmental Science: UV fluorescence spectroscopy has been used to monitor pollutants in water and air. Fluorescent tracers can be used to trace the fate and transport of pollutants, providing insights into environmental contamination and remediation.4. Materials Science: In materials science, UV fluorescence spectroscopy is used to study the optical properties of materials, such as quantum dots and fluorescent dyes. This technique can provide information about the energy levels and electronic states of these materials, which is crucial for their applications in optoelectronic devices.Advantages and Limitations.Advantages:High Sensitivity: UV fluorescence spectroscopy can detect very low concentrations of fluorescent species, making it suitable for trace analysis.Selectivity: By choosing specific excitation and emission wavelengths, UV fluorescence spectroscopy can provide information about specific components in complex mixtures.Non-Destructive: This technique does not require the destruction of samples, allowing multiple measurements to be performed on the same sample.Limitations:Fluorescent Probe Dependence: The application of UV fluorescence spectroscopy often relies on the availability of suitable fluorescent probes or dyes. Not all moleculesexhibit strong fluorescence, limiting the scope of this technique.Interference from Background Fluorescence: The presence of background fluorescence from the sample matrixor solvents can interfere with the measurement, affecting the accuracy and reliability of results.Instrument Cost and Maintenance: Specialized UV-Vis spectrophotometers with fluorescence detection capabilities can be costly, and regular maintenance is required toensure accurate measurements.Conclusion.UV fluorescence spectroscopy is a powerful analytical tool that has found widespread applications in various fields. Its ability to provide sensitive and selective information about molecular structure and interactions has made it a valuable resource for researchers in biochemistry, pharmacology, environmental science, and materials science. Despite its limitations, UV fluorescence spectroscopycontinues to evolve and improve, providing new insights into the behavior and properties of chemical species.。

分子荧光光谱法

仪器框图
该型仪器可进行荧光、磷光和发光分析；
分子荧光光谱法
同步扫描技术
根据激发和发射单色器在扫描过程中彼此间所保持的关系，同步扫描可分为固定波长差()和固定能量差及可变波长三种；
同步扫描技术可简化光谱，谱带变窄，减少光谱重叠，提高分辨率；如图。
合适的可减少光谱重叠；酪氨酸和色氨酸的荧光激发光谱相似，发射光谱严重重叠，但 <15nm的同步光谱只显示酪氨酸特征光谱； >60nm时，只显示色氨酸的特征光谱，实现分别测定。
分子荧光光谱法
3.内滤光作用和自吸现象内滤光作用：溶液中含有能吸收激发光或荧光物质发射的荧光，如色胺酸中的重铬酸钾；自吸现象：化合物的荧光发射光谱的短波长端与其吸收
光谱的长波长端重叠，产生自吸收；如蒽化合物。
分子荧光光谱法
4、溶液荧光的猝灭碰撞猝灭；氧的熄灭作用等。
分子荧光光谱法
四、仪器结构流程
（多为 S1→ S0跃迁），发射波长为 ‘2的荧光； 10-
7～10 -9 s 。由图可见，发射荧光的能量比分子吸收的能量小，波
长长； ‘2 > 2 > 1 ；
分子荧光光谱法
二、激发光谱与荧光(磷光)光谱
1.荧光(磷光)的激发光谱曲线固定测量波长(选最大发射波长),化合物发射的荧光(磷
测量荧光的仪器主要由四个部分组成：激发光源、样品池、双单色器系统、检测器。
特殊点：有两个单色器，光源与检测器通常成直角。基本流程如图：单色器：选择激发光波长的第一单色器和选择发射光(测量)波长的第二单色器；光源：灯和高压汞灯，染料激光器(可见与紫外区) 检测器：光电倍增管。
分子荧光光谱法
200 260 320 380 440 500 560 620 室温下菲的乙醇溶液荧（磷）光光谱

荧光共振能量转移的原理与应用

荧光共振能量转移的原理与应用荧光共振能量转移，简写FRET（Fluorescence Resonance Energy Transfer），是一种生物技术应用。

它是通过特定的物质之间的相互作用来转移激发能量。

这种物质之间相互作用通常指分子之间的能量转移，即一种荧光物质通过电子共振与另一种感受荧光物质之间的非辐射能量转移。

FRET技术已经在生物医学研究中得到了广泛应用，例如信号转导、DNA测序、药物筛选和基因调控，还可以研究蛋白-蛋白相互作用或酶的激活，或确定单个分子中某些区域的组成和构象。

一. 荧光共振能量转移的原理荧光共振能量转移机理是一种非辐射退激，通过近距离内分子之间的相互作用实现能量的转移。

这里的丝氨酸和双氧核苷酸可以理解为应用FRET技术需要的两种物质，发射的荧光是制定位于丝氨酸上的标记分子（donor）的产物，被感受均相分子（acceptor）受体吸收而部分消耗。

因此，在这样的情况下，当donor分子和acceptor分子之间的距离在2-10nm之间时，donor的发射被acceptor分子吸收而不被发射，这意味着能量已经在它们之间转移。

公式表示如下：E= R0^6 / (R+R0^6)其中E代表FRET的效率，R代表donor和acceptor间的距离，R0代表生命距离，通常使用样品中donor和acceptor的重叠互补荧光光谱来测定grass探测荧光的荧光强度减少（quenching）。

二. 荧光共振能量转移的应用1. 信号转导和电子传输研究荧光共振能量转移技术已经成功地应用于许多重要的交互分子信号通路中，包括G蛋白偶联受体、酪氨酸激酶、信号分子、核酸和细胞质蛋白复合物，来研究它们之间的交互和信号传递。

使用荧光共振能量转移技术可以方便地进行单分子分析实验，并且不需要任何其它荧光组分，节省了成本。

2. DNA测序在通过荧光共振能量转移技术进行脱氧核苷酸测序方法中，鉴别探针必须吸收donor生物分子的能量，这一过程称为能量转移。

分子荧光光谱法(原理和方法)

1. 激发
在室温下物质分子大部分处于基态的最低振动能级且电子自旋配对为单重
态．当吸收一定频率的电磁辐射发生能级跃迁时，可上升到不同激发态
的各振动能级，其中多数分子上升至第一激发单重态这一过程约需１０-
15秒.
激发
2 去活化过程
激发态分子的失活: 激发态分子不稳定，它要以辐射或无辐射跃迁的方式回到基态

kf
k f ki kec kic
ห้องสมุดไป่ตู้
凡是使荧光速率常数kf增大而使其他失活过程（系间窜越、外转换、内转换）
的速率常数减小的因素都可使荧光增强。
根据朗伯-比尔定律
Ia=I0-I=I0(1-10-εbc)
则F=ΦI0（1-10-εbc)=φI0(1-e-2.303εbc) 又因
e-2.303εbc=1-2.303 εbc-(-2.303 εbc)2/2!-(-2.303 εbc)3/3!
对于很稀的溶液，投射到样品溶液上的被吸收的激发光不到2%时，即εbc＜=0.05时，上式的第二项后的各项可以忽略不计。则
F = φI0[1-(1-2.303 εbc)]=2.303 φ I0 εbc
当I0一定时并且浓度C很小时,荧光强度与荧光物质浓度成正比
F = K·C
(K = 2.303 φ I0 εb)
荧光团杂化纳米二氧化硅微球
Molecular fluorescence spectroscopy
概述
分子荧光光谱法(Molecular fluorescence spectroscopy )又称
为荧光光谱法或荧光分析法．是以物质所发射的荧光强度与浓度之间的线性关系为依据进行的定量分析，以荧光光谱的形状和荧光峰对应的波长进行行的定性分析．

荧光光谱法英文

荧光光谱法英文Fluorescence SpectroscopyFluorescence spectroscopy is a powerful analytical technique that has found widespread applications in various fields, including chemistry, biology, materials science, and environmental studies. This analytical method is based on the measurement of the emission of light by a substance that has been excited by the absorption of light or other forms of energy. The process of fluorescence involves the absorption of energy by molecules or atoms, followed by the subsequent emission of light at a longer wavelength than the absorbed light.The fundamental principle of fluorescence spectroscopy is that when a molecule or atom is exposed to light, it can absorb the energy of the incoming photons, causing electrons within the molecule or atom to be excited to higher energy levels. This excitation is a temporary state, and the electrons will eventually return to their ground state, releasing the excess energy in the form of a photon. The energy of the emitted photon is typically lower than the energy of the absorbed photon, resulting in a shift in the wavelength of the emitted light compared to the absorbed light. This wavelength shift is known as the Stokes shift, and it is a key characteristic offluorescence.The intensity and wavelength of the emitted light are influenced by various factors, such as the chemical structure of the fluorescent molecule, the solvent environment, temperature, and the presence of other compounds that can interact with the excited molecules. By analyzing the characteristics of the emitted light, researchers can gain valuable insights into the properties and behavior of the sample under investigation.Fluorescence spectroscopy has a wide range of applications in various fields. In chemistry, it is used for the identification and quantification of organic and inorganic compounds, as well as the study of reaction kinetics and molecular interactions. In biology, fluorescence spectroscopy is employed for the investigation of protein structure and dynamics, the detection and quantification of biomolecules, and the study of cellular processes. In materials science, this technique is used to characterize the properties of polymers, semiconductors, and nanomaterials, among others.One of the key advantages of fluorescence spectroscopy is its high sensitivity, which allows for the detection and quantification of analytes at very low concentrations. Additionally, the technique is non-invasive and can be performed in real-time, making it a valuable tool for in-situ and online monitoring applications. Furthermore, thedevelopment of advanced fluorescent probes and labeling techniques has expanded the versatility of fluorescence spectroscopy, enabling the visualization and tracking of specific molecules or cellular components in complex biological systems.Despite its many benefits, fluorescence spectroscopy also faces some limitations. The presence of interfering compounds, quenching effects, and the potential for photobleaching of the fluorescent molecules can challenge the reliability and accuracy of the measurements. Researchers are constantly working to address these challenges through the development of new instrumentation, data analysis methods, and sample preparation techniques.In conclusion, fluorescence spectroscopy is a powerful and versatile analytical tool that has made significant contributions to various scientific disciplines. As technology continues to advance, the applications of this technique are expected to expand further, providing researchers with new opportunities to gain a deeper understanding of the world around us.。

光热催化原位表征

光热催化原位表征
光热催化是一种利用光能和热能协同作用的催化过程，通过光照激发催化剂表面的电子和振动激发态，从而促进化学反应的进行。

原位表征是指在反应过程中对催化剂表面的结构、组成和活性等进行实时监测和分析。

原位表征技术可以提供关于光热催化过程中催化剂表面物种的变化、反应中间体的生成和消失、表面活性位点的形成和变化等信息。

常用的原位表征技术包括：
1. 原位红外光谱（Infrared Spectroscopy）：通过红外光谱技术可以监测催化剂表面吸附物种的变化，如吸附态中间体、反应产物等。

2. 原位拉曼光谱（Raman Spectroscopy）：拉曼光谱技术可以提供催化剂表面振动模式的信息，从而揭示催化剂表面结构的变化。

3. 原位X射线吸收光谱（X-ray Absorption Spectroscopy）：X射线吸收光谱技术可以用来研究催化剂表面的元素组成、化学价态和配位环境等信息。

4. 原位透射电子显微镜（Transmission Electron Microscopy，TEM）：TEM技术可以提供催化剂表面形貌和结构的高分辨率图像，揭示催化剂纳米结构的演变和催化反应中的动态过程。

5. 原位质谱（Mass Spectrometry）：质谱技术可以监测催化反应中产物的生成和消失，从而了解催化剂的活性和选择性。

通过原位表征技术，可以深入了解光热催化过程中催化剂表面的变化和反应机理，为催化剂设计和优化提供重要的指导和理论基础。

荧光分光光度法

F= 2.303 I0cl 当入射光强度I0一定时，
F= Kc
（3-5）（3-6）
注意：上式在一定旳浓度范围内合用。
荧光物质旳最大浓度为c max 0.05/l。当浓度较大时，即它旳吸光度不小于0.05时，荧光强度与其浓
度旳线性关系将会发生偏离。
在浓度较高时，产生这种偏离旳原因可能是激
发分子间相互碰撞而失去能量（本身猝灭），或者
（7）荧光寿命它是研究分子构造时要求旳参数。定义：荧光强度衰减到1/e所需旳时间，用表达。
任意时间（t）旳荧光强度 If =If0e-t/=If0e-kt
式中：If —移去激发光源后任一时间t时旳荧光强度；
If0—激发时最大旳荧光强度； k —仪器衰减常数； —激发态旳平均寿命。
（8）荧光分析旳敏捷度 —对整个发射光谱而言； /H —对部分发射光谱而言，即对所测到旳不
定其荧光值，然后将减去试剂空白荧光值旳原则溶液荧光值与其相应浓度作图，即得其工作曲线。
根据试液及试液空白荧光值，在此曲线上即可找到试液旳浓度。同步根据工作曲线旳线性情况，能够拟定试液测定旳最高浓度。
c. 内标法
设Cx、Cs分别为试样和标样浓度，Fx、Fx0分别为试样和试样本底旳荧光值；Fs+x为试样加标样旳混合溶液旳荧光值。
基态跃迁到能级较高旳第一电子激发态或第二电子激发态，然后经过无辐射跃迁返回到第一电子激发态旳最低振动能级上，再从该能级降落至基态旳各个不同旳振动能级上，同步释放出相应能量旳分子荧光，最终以无辐射跃迁形式回到基态旳最低振动能级。
需要注意旳是：（1）整个过程是在单线态之间进行旳；
（2）产生荧光旳过程极快，约在10-8 秒左右内完毕；
3.3 荧光分析旳措施及影响原因