表面等离子体耦合定向发射荧光法
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摘要
表面等离子体耦合定向发射荧光法(SPCDE)是利用激发态的荧光团和金属表面等离子体的耦合近场作用而建立起来的表面增强荧光技术。作为一种新型荧光技术，SPCDE展现出其独特的优点：高度的角度定向发射性、荧光发射偏振性、波长分辨性、有效的背景信号抑制等，其在生物传感、界面分析等领域中显露出广阔的应用前景。本论文致力于构建表面等离子体耦合定向荧光法的实验装置，并在此基础上开展相关研究，进而将其应用于生物传感体系。论文分为六章：
第一章绪论。该章从表面增强荧光技术的简要介绍开始，阐述了表面等离子体共振的原理并以此引出表面等离子体耦合定向荧光法的技术原理、发展及其在生物领域中的应用，最后提出本论文的研究思路。
第二章表面等离子体耦合定向荧光检测的实现。在自制多功能荧光仪上实现SPCDE荧光信号的检测，并且该仪器与激光作为光源的SPCDE仪相比可以进一步获得激发光谱信息。通过实验对SPCDE展开深入的研究。结果证明，纳米金属表面的荧光团与等离子体耦合产生定向发射荧光信号，该信号具有高p-偏振性。理论计算模拟结果与实验相吻合。同时研究表明了SPCDE可以在无需增加光学元件的条件下实现波长分辨，并且具有抑制背景信号的能力。
第三章表面等离子体耦合定向荧光法的小型装置。以改装过的市售激光笔作为光源，自主搭建SPCDE小型实验装置。该装置具有结构简单，操作方便，成本低廉等优点，完全能够满足一般的SPCDE测试要求。并运用该装置进行了表面等离子体耦合定向荧光法和表面等离子体场增强荧光法的光学性质的比较。
第四章基于过渡金属铁的SPCDE研究。首次成功地把表面等离子体耦合定向荧光法研究拓展到重要的过渡金属铁。菲涅耳方程理论计算预测355nm-1088nm范围内的光在固定角度范围内(70°-75°)能够有效耦合25nm光滑铁膜的表面等离子体。实验证明金属铁膜上荧光染料的SPCDE信号具有较高的p-偏振性和定向性，并且定向发射角度都固定在一定范围。通过与金膜为基底的SPCDE信号相比较，证明在纳米铁膜上可以实现固定检测角度监控不同波长的荧光信号。
第五章基于核酸适配子构型转换的SPCDE荧光传感。采用核酸适配子作为识别和报告分子，首次提出结合金属共振能量转移猝灭效应和表面等离子体耦合增强效应来实现对凝血酶简单、快速、特异检测。通过对比不同化学固定方式，选择亲和素-生物素法作为适配子探针的固定方法。通过对荧光信号偏振和信号角度分布情况考察，证明了荧光增强是由表面等离子耦合效应引起的。
第六章结语与展望。总结了本论文研究工作的创新性，并对研究工作的进一步开展进行了展望。
Surface Plasmon Coupled Directional Emission (SPCDE)is a novel surfaceenhanced fluorescence method based on the near-field interaction of excitedfluorophores with surface plasmon on the thin metal films.SPCDE displays uniqueadvantages which include strong directional emission,unique polarization,background rejection and wavelength resolution.Therefore,it has a promising andwide application prospect in the biological detection and interface analysis.Thisdissertation focuses on the design of instrumental systems for SPCDE fluorescencemeasurement,the investigation of relevant theoretical and experimental research andthe applications in biochemistry.The dissertation consists of six chapters.
In the first chapter,the development of surface plasmon coupled directionalemission was reviewed.Surface enhanced fluorescence spectroscopy techniques,theoretical principle and properties of Surface Plasmon Resonance and SPCDE,theresearch progress and application of SPCDE in biochemistry were described in detail,then the plans for the dissertation were put forward.
In the second chapter,an instrumental system for surface plasmon coupleddirectional emission technique was developed in a laboratory-constructedspectrofluorimeter.This setup can get more information from excitation spectrum,ascompared SPCDE system with laser light source.The theoretical calculation andexperimental results showed that SPCDE was the coupling of the surface plasmonsand excited fluorophores on a thin metal film,resulting in a radiation of surfaceplasmons into the higher refractive index media,which showed highly directional andpolarized emission.The properties of background rejection and wavelength resolutionby selecting the observation angle were demonstrated.
In the third chapter,a micro-spectrometer of SPCDE was designed with modifiedcommercial laser pointer as light source.The experimental results revealed theinstrument had good advantages,such as simple construction,convenient operationand low cost.In this study,the different properties of two surface enhancedfluorescence methods between surface plasmon field-enhanced fluorescence and surface plasmon coupled directional emission were compared.
In the fourth chapter,SPCDE studies have been firstly successfully extended toiron which was an important transition metal.Theoretical Fresnel calculationsperformed in the ultraviolet to near-infrared spectral range (355-1088nm)predictedthe near-field generation of surface plasmons in 25nm iron thin films.The use of ironfilms in SPCDE to generate highly polarized and directional emission wasdemonstrated by experiments.Both theoretical calculations and experimental resultsshowed that coupled fluorescence through iron films occurred over a narrow range ofangles.This suggested that compared with gold films,where there was a strongdependence of emission angle with coupling wavelength,“fixed angle”geometriescould be used over a broad range of wavelength applications on iron films.
In the fifth chapter,surface plasmon coupled directional emission based onconformational switching signaling aptamer was studied.Using the aptamer as bothrecognition and signaling elements,we firstly proposed the combination of theenhancement of SPCDE and the quenching by metal to detect thrombin.Bycomparing different chemical methods for the immobilization of aptamer,biotin-avidin method was finally chosen.The enhanced directional fluorescenceemission of signaling aptamer was observed due to the binding of a target thrombinwith aptamer which induces conformational switching.The polarization and angulardistribution of the observed emission demonstrated that the fluorescence enhancementwas due to surface plasmon.
In the sixth chapter,the innovation of the study was concluded and the prospectof this research was given.
第一章绪论。该章从表面增强荧光技术的简要介绍开始，阐述了表面等离子体共振的原理并以此引出表面等离子体耦合定向荧光法的技术原理、发展及其在生物领域中的应用，最后提出本论文的研究思路。
第二章表面等离子体耦合定向荧光检测的实现。在自制多功能荧光仪上实现SPCDE荧光信号的检测，并且该仪器与激光作为光源的SPCDE仪相比可以进一步获得激发光谱信息。通过实验对SPCDE展开深入的研究。结果证明，纳米金属表面的荧光团与等离子体耦合产生定向发射荧光信号，该信号具有高p-偏振性。理论计算模拟结果与实验相吻合。同时研究表明了SPCDE可以在无需增加光学元件的条件下实现波长分辨，并且具有抑制背景信号的能力。
第三章表面等离子体耦合定向荧光法的小型装置。以改装过的市售激光笔作为光源，自主搭建SPCDE小型实验装置。该装置具有结构简单，操作方便，成本低廉等优点，完全能够满足一般的SPCDE测试要求。并运用该装置进行了表面等离子体耦合定向荧光法和表面等离子体场增强荧光法的光学性质的比较。
第四章基于过渡金属铁的SPCDE研究。首次成功地把表面等离子体耦合定向荧光法研究拓展到重要的过渡金属铁。菲涅耳方程理论计算预测355nm-1088nm范围内的光在固定角度范围内(70°-75°)能够有效耦合25nm光滑铁膜的表面等离子体。实验证明金属铁膜上荧光染料的SPCDE信号具有较高的p-偏振性和定向性，并且定向发射角度都固定在一定范围。通过与金膜为基底的SPCDE信号相比较，证明在纳米铁膜上可以实现固定检测角度监控不同波长的荧光信号。
第五章基于核酸适配子构型转换的SPCDE荧光传感。采用核酸适配子作为识别和报告分子，首次提出结合金属共振能量转移猝灭效应和表面等离子体耦合增强效应来实现对凝血酶简单、快速、特异检测。通过对比不同化学固定方式，选择亲和素-生物素法作为适配子探针的固定方法。通过对荧光信号偏振和信号角度分布情况考察，证明了荧光增强是由表面等离子耦合效应引起的。
第六章结语与展望。总结了本论文研究工作的创新性，并对研究工作的进一步开展进行了展望。
Surface Plasmon Coupled Directional Emission (SPCDE)is a novel surfaceenhanced fluorescence method based on the near-field interaction of excitedfluorophores with surface plasmon on the thin metal films.SPCDE displays uniqueadvantages which include strong directional emission,unique polarization,background rejection and wavelength resolution.Therefore,it has a promising andwide application prospect in the biological detection and interface analysis.Thisdissertation focuses on the design of instrumental systems for SPCDE fluorescencemeasurement,the investigation of relevant theoretical and experimental research andthe applications in biochemistry.The dissertation consists of six chapters.
In the first chapter,the development of surface plasmon coupled directionalemission was reviewed.Surface enhanced fluorescence spectroscopy techniques,theoretical principle and properties of Surface Plasmon Resonance and SPCDE,theresearch progress and application of SPCDE in biochemistry were described in detail,then the plans for the dissertation were put forward.
In the second chapter,an instrumental system for surface plasmon coupleddirectional emission technique was developed in a laboratory-constructedspectrofluorimeter.This setup can get more information from excitation spectrum,ascompared SPCDE system with laser light source.The theoretical calculation andexperimental results showed that SPCDE was the coupling of the surface plasmonsand excited fluorophores on a thin metal film,resulting in a radiation of surfaceplasmons into the higher refractive index media,which showed highly directional andpolarized emission.The properties of background rejection and wavelength resolutionby selecting the observation angle were demonstrated.
In the third chapter,a micro-spectrometer of SPCDE was designed with modifiedcommercial laser pointer as light source.The experimental results revealed theinstrument had good advantages,such as simple construction,convenient operationand low cost.In this study,the different properties of two surface enhancedfluorescence methods between surface plasmon field-enhanced fluorescence and surface plasmon coupled directional emission were compared.
In the fourth chapter,SPCDE studies have been firstly successfully extended toiron which was an important transition metal.Theoretical Fresnel calculationsperformed in the ultraviolet to near-infrared spectral range (355-1088nm)predictedthe near-field generation of surface plasmons in 25nm iron thin films.The use of ironfilms in SPCDE to generate highly polarized and directional emission wasdemonstrated by experiments.Both theoretical calculations and experimental resultsshowed that coupled fluorescence through iron films occurred over a narrow range ofangles.This suggested that compared with gold films,where there was a strongdependence of emission angle with coupling wavelength,“fixed angle”geometriescould be used over a broad range of wavelength applications on iron films.
In the fifth chapter,surface plasmon coupled directional emission based onconformational switching signaling aptamer was studied.Using the aptamer as bothrecognition and signaling elements,we firstly proposed the combination of theenhancement of SPCDE and the quenching by metal to detect thrombin.Bycomparing different chemical methods for the immobilization of aptamer,biotin-avidin method was finally chosen.The enhanced directional fluorescenceemission of signaling aptamer was observed due to the binding of a target thrombinwith aptamer which induces conformational switching.The polarization and angulardistribution of the observed emission demonstrated that the fluorescence enhancementwas due to surface plasmon.
In the sixth chapter,the innovation of the study was concluded and the prospectof this research was given.
引文
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16. Malicka J, Gryczynski I, Lakowicz JR. DNA hybridization assays using metal-enhanced fluorescence. Biochemical and Biophysical Research Communications 2003;306:213-218.
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60. Weisenberg M, Asian K, Hortle E, Geddes CD. Directional surface plasmon coupled chemiluminescence from nickel thin films: Fixed angle observation. Chemical Physics Letters 2009;473:120-125.
61. Ray K, Chowdhury MH, Lakowicz JR. Observation of surface plasmon-coupled emission using thin platinum films. Chemical Physics Letters 2008;465:92-95.
62. Chowdhury MH, Ray K, Geddes CD, Lakowicz JR. Use of silver nanoparticles to enhance surface plasmon-coupled emission (SPCE). Chemical Physics Letters 2008;452:162-167.
63. Asian K, McDonald K, Previte MJR, Zhang YX, Geddes CD. Silver island nanodeposits to enhance surface plasmon coupled fluorescence from copper thin films. Chemical Physics Letters 2008;464:216-219.
64. Previte MJR, Asian K, Zhang YX, Geddes CD. Surface plasmon coupled phosphorescence (SPCP). Chemical Physics Letters 2006;432:610-615.
65. Zhang J, Gryczynski Z, Lakowicz JR. First observation of surface plasmon-coupled electrochemiluminescence. Chemical Physics Letters 2004;393:483-487.
66. Chowdhury MH, Malyn SN, Asian K, Lakowicz JR, Geddes CD. First observation of surface plasmon-coupled chemiluminescence (SPCC). Chemical Physics Letters 2007;435:114-118.
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68. Borejdo J, Calander N, Gryczynski Z, Gryczynski I. Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope. Optics Express 2006;14:7878-7888.
69. Borejdo J, Gryczynski Z, Calander N, Muthu P, Gryczynski I. Application of surface plasmon coupled emission to study of muscle. Biophysical Journal 2006;91:2626-2635.
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81. Matveeva E, Gryczynski Z, Gryczynski I, Malicka J, Lakowicz JR. Myoglobin immunoassay utilizing directional surface plasmon-coupled emission. Analytical Chemistry 2004;76:6287-6292.
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84. Smith DS, Kostov Y, Rao G. SPCE-based sensors: Ultrafast oxygen sensing using surface plasmon-coupled emission from ruthenium probes. Sensors and Actuators B-Chemical 2007; 127:432-440.
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12. Smith DS, Kostov Y, Rao G. SPCE-based sensors: Ultrafast oxygen sensing using surface plasmon-coupled emission from ruthenium probes. Sensors and Actuators B-Chemical 2007; 127:432-440.
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14. Geddes CD, Gryczynski I, Malicka J, Gryczynski Z, Lakowicz JR. Directional surface plasmon coupled emission. Journal of Fluorescence 2004; 14:119-123.
15. Matveeva E, Gryczynski Z, Gryczynski I, Lakowicz JR. Immunoassays based on directional surface plasmon-coupled emission. Journal of Immunological Methods 2004;286:133-140.
16. Malicka J, Gryczynski I, Gryczynski Z, Lakowicz JR. DNA hybridization using surface plasmon-coupled emission. Analytical Chemistry 2003;75:6629-6633.
17. Smith DS, Kostov Y, Rao G, Gryczynski I, Malicka J, Gryczynski Z, Lakowicz JR. First observation of surface plasmon-coupled emission due to LED excitation. Journal of Fluorescence 2005;15:895-900.
18. Gryczynski I, Malicka J, Gryczynski Z, Nowaczyk K, Lakowicz JR. Ultraviolet surface plasmon-coupled emission using thin aluminum films. Analytical Chemistry 2004;76:4076-4081.
1．曹佩根，姚建林，徐浩元，顾仁敖，田中群．铁电极表面拉曼光谱的初步研究．光散射学报 2000； 12：87-89．
2．Aslan K，Malyn SN，Geddes CD．Microwave-accelerated surface plasmon-coupled directional luminescence：Application to fast and sensitive assays in buffer，human serum and whole blood．Journal of Immunological Methods 2007；323：55-64．
3．Zhang J，Gryczynski Z，Lakowicz JR．First observation of surface plasmon-coupled electrochemiluminescence．Chemical Physics Letters 2004；393：483-487．
4．Malicka J，Gryczynski I，Gryczynski Z，Lakowicz JR．DNA hybridization using surface plasmon-coupled emission．Analytical Chemistry 2003；75：6629-6633．
5．Gryczynski I，Malicka J，Lakowicz JR，Goldys EM，Calander N，Gryczynski Z．Directional two-photon induced surface plasmon-coupled emission．Thin Solid Films 2005；491：173-176．
6．Matveeva E，Gryczynski Z，Gryczynski I，Lakowicz JR．Immunoassays based on directional surface plasmon-coupled emission．Journal of Immunological Methods 2004；286：133-140．
7．Previte MJR，Zhang YX，Aslan K，Geddes CD．Surface plasmon coupled fluorescence from copper substrates．Applied Physics Letters 2007；91：-
8．Aslan K，McDonald K，Previte MJR，Zhang YX，Geddes CD．Silver island nanodeposits to enhance surface plasmon coupled fluorescence from copper thin films．Chemical Physics Letters 2008；464：216-219．
9．Gryczynski I，Malicka J，Gryczynski Z，Nowaczyk K，Lakowicz JR．Ultraviolet surface plasmon-coupled emission using thin aluminum films．Analytical Chemistry 2004；76：4076-4081．
10．Malicka J，Gryczynski I，Gryczynski Z，Lakowicz JR．Surface plasmon-coupled ultraviolet emission of 2，5-diphenyl-1，3，4-oxadiazole． Journal of Physical Chemistry B 2004；108：19114-19118．
11．Gryczynski I，Malicka J，Lukomska J，Gryczynski Z，Lakowicz JR．Surface plasmon-coupled polarized emission of N-acetyl-L-tryptophanamide．Photochemistry and Photobiology 2004；80：482-485．
12．Asian K，Previte MJR，Zhang YX，Geddes CD．Surface plasmon coupled fluorescence in the ultraviolet and visible spectral regions using zinc thin films Analytical Chemistry 2008；80：7304-7312．
13．Aslan K，Geddes CD．Surface plasmon coupled chemiluminescence from zinc substrates：Directional chemiluminescence．Applied Physics Letters 2009；94：-．
14．Aslan K，Zhang YX，Geddes CD．Surface Plasmon Coupled Fluorescence in the Visible to Near-Infrared Spectral Regions using Thin Nickel Films：Application to Whole Blood Assays．Analytical Chemistry 2009；81：3801-3808．
15．Weisenberg M，Aslan K，Hortle E，Geddes CD．Directional surface plasmon coupled chemiluminescence from nickel thin films：Fixed angle observation．Chemical Physics Letters 2009；473：120-125．
16．Lakowicz JR．Radiative decay engineering 3．Surface plasmon-coupled directional emission．Analytical Biochemistry 2004；324：153-169．
17．杨志林，吴德印，任斌，周海光，田中群．铑电极在紫外区的表面增强拉曼散射机理．光谱学与光谱分析2004；24：682-685．
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21．万惠霖．固体表面物理化学若干研究前沿．厦门：厦门大学出版社，2006：685pp．
22．杨志林，吴德印，姚建林，胡建强，任斌，周海光，田中群．镍电极的表面 增强拉曼散射机理初探.科学通报 2002;47:989-992.
23. Salamona Z, Macleodb HA, Gordon Tollina. Surface plasmon resonance spectroscopy as a tool for investigating the biochemical and biophysical properties of membrane protein systems. Ⅰ: Theoretical principles. Biochimica Et Biophysica Acta-Reviews on Biomembranes 1997; 1331:117-129.
24. Nelson BP, Frutos AG, Brockman JM, Corn RM. Near-infrared surface plasmon resonance measurements of ultrathin films. 1. Angle shift and SPR imaging experiments. Analytical Chemistry 1999;71:3928-3934.
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