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Institutionen för fysik, kemi och biologi Examenarbete Plasmonic Enhanced Fluorescence using Gold Nanorods Ming-Tao Lee Examensarbetet utfört vid Linköping Universitet June 2010 LITH-IFM-A-EX--10/2298

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Institutionen för fysik, kemi och biologi Examenarbete Plasmonic Enhanced Fluorescence using Gold Nanorods Ming-Tao Lee Examensarbetet utfört vid Linköping Universitet June 2010 LITH-IFM-A-EX--10/2298 SE Linköpings universitet Institutionen för fysik, kemi och biologi Linköping Institutionen för fysik, kemi och biologi Plasmonic Enhanced Fluorescence using Gold Nanorods Ming-Tao Lee Examensarbetet utfört vid Linköping Universitet June 2010 Handledare Erik Martinsson Examinator Thomas Ederth Avdelning, institution Division, Department Chemistry Department of Physics, Chemistry and Biology Linköping University Datum Date Språk Language Svenska/Swedish Engelska/English Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport ISBN ISRN: LITH-IFM-A-EX--10/2298--SE Serietitel och serienummer ISSN Title of series, numbering URL för elektronisk version Titel Title Plasmonic Enhanced Fluorescence using Gold Nanorods Författare Author Ming-Tao Lee Sammanfattning Abstract The aims of this study are to first immobilize positively charged gold nanorods to negatively charged cell culture surfaces. Second, to use polyelectrolytes for controlling the distance between gold nanorods and fluorophores. This is used to optimally determine the distance, of which maximum fluorescence enhancement is achieved, between gold nanorods and fluorophores. In order to approach these aims, we use UV/VIS absorption spectroscopy, fluorescence spectroscopy, atomic force microscopy, and ellipsometry. The results show that we could control the immobilization of gold nanorods on plastic microwell plates and create reproducible polyelectrolyte layers, in order to control the distance between the gold nanorods and fluorophores. In addition, the localized surface plasmon resonance wavelength red shifted as the PELs increased. In conclusion, we found that the maximum fluorescence enhancement of the fluorophores (Cy7) is about 2.3 times at a fluorophores-nanoparticles separation of approximately 9-12 nm. This work contributes some research information towards the design of optical biochip platforms based on plasmon-enhanced fluorescence. Nyckelord Keyword Localized Surface Plasmon Resonance, plasmonic gold nanorods, Polyelectrolyte, Layer-by- Layer, Fluorophore, Plasmonic Enhanced Fluorescence, Microwell Plate Contents Abstract 3 Abbreviations and explanations.4 1 Background Introduction Aim of the project Theory Fluorescence Metal Naoparticles Oprical properties of metal nanoparticles Localized surface plasmons resonance Plamonic Gold nanorods Plasmon enhanced fluorescence.11 3 Materials and Methods Overview of the system Synthesis of gold nanorods Materials Substrates Chemicals Polyelectrolyte Fluorophores Surface preparation Gold nanorod preparation and immobilization.18 #$%$& '()(*+,!-./-01203! (+,5555$ $ 6! Microwell plate Polyelelectrolyte build-up Fluorophore conjugation Characterization techniques Atomic Force Microscopy Transmission Electron Microscopy UV/VIS Microscopy Null Ellipsometry Fluorscence plate reade ! ! 4 Results and Discussion Determination of LSPR of Gold Nanorods Colloid GNRs GNRs bound on the microwell plate GNR immobilization reproducibility Adsorption of PELs Thickness measurement Surface coverage LSPR changes upon polyelectrolyte adsorption Fluorescence measurement Cy5 fluorescence measurement Cy7 fluorescence measurement Comparison between Cy5 and Cy Why Cy7 relative fluorescence enhances? Conclusion Future investigations Acknowledgements References !! &! Abstract The aims of this study are to first immobilize positively charged gold nanorods to negatively charged cell culture surfaces. Second, to use polyelectrolytes for controlling the distance between gold nanorods and fluorophores. This is used to optimally determine the distance, of which maximum fluorescence enhancement is achieved, between gold nanorods and fluorophores. In order to approach these aims, we use UV/VIS absorption spectroscopy, fluorescence spectroscopy, atomic force microscopy, and ellipsometry. The results show that we could control the immobilization of gold nanorods on plastic microwell plates and create reproducible polyelectrolyte layers, in order to control the distance between the gold nanorods and fluorophores. In addition, the localized surface plasmon resonance wavelength red shifted as the PELs increased. In conclusion, we found that the maximum fluorescence enhancement of the fluorophores (Cy7) is about 2.3 times at a fluorophores-nanoparticles separation of approximately 9-12 nm. This work contributes some research information towards the design of optical biochip platforms based on plasmon-enhanced fluorescence.!! #! Abbreviations and explanations AFM Arb.u. CTAB CyDye DMSO DNA LSPR MWCO NHS NIR NPs OM PAH PE PELs PSS PEF SE TEM UV/Vis Atomic Force Microscopy Arbitrary Units Hexadecyltrimethylammoniumbromide cyanine dye Dimethyl sulfoxide deoxynucleic acid Localized Surface Plasmon Resonance molecular weight cut off N-Hydroxy-succinimid Near Infrared Nanoparticles Optical Microscope! poly (allylamine hydrochloride) Polyelectrolyte polyelectrolyte layers poly (sodium 4-styrenesulfonate) Plasmonic Enhanced Fluorescence Spontaneous emission Transmission Electron Microscope Ultra Violet/Visible spectroscopy! res Resonance peak wavelength! %! 1 Background 1.1 Introduction In recent years, the interest in using metallic nanoparticles (NPs) has increased significantly, and they have been used in a wide range of applications in physics, chemistry and biology. These nanoscale metallic particles are promising, especially noble metals such as gold and silver, because of their unique optical properties such as surface plasmon. Localized surface plasmon resonance (LSPR) is a collective electron charge oscillation in nanometer-sized metallic particles when excited by light with a certain frequency [1]. Since the LSPR takes place on the boundary of metal and external medium, these oscillations are very sensitive to any change to this boundary, such as changing shape, size or surrounding environment to the metal surface [2]. Fluorescent probe has become one of the dominant sensing technologies in medical diagnostics over the past decade. Although it is a very sensitive technique, it suffers from several disadvantages, such as particularly poor photostability, low emission intensities, and low quantum yield of the fluorophores. Its applications in advanced biomolecule studies are limited. Therefore, it is crucial to create a capable set of fluorescent probe with improved photostability and higher emission rates to advance studies of single biomolecule function [3]. During the interactions of the excited-state fluorophores with noble metallic surface plasmons, coherent electron oscillations, which give favorable effects to the fluorophores, emit higher rate of radiative emission. This method is called the Plasmonic Enhanced Fluorescence (PEF), a promising technique that can be used to increase sensitivity and detection limits for fluorescent probe sensing [4]. Since the last decade, the synthesis of metal nanoparticles (NPs) has become more mature in controlling the shape, and the size. In this thesis, gold nanorods (GNRs) have been used to facilitate the fluorescence process by increasing radiative decay rate during the process. When the distance between fluorophores and the metal is above 5nm, an interesting coupling interaction between the fluorophores and the enhanced electrical field nearby the metal structure occurs, resulting in enhancing both excitation and radiative emission rates. These optical enhancements improve fluorescence detection and imaging schemes [5]. However, when the distance between fluorophores and the metal is too close (less than 5 nm), the fluorescence is quenched [6]. To study how the distance between fluorophores and GNRs optimizes the fluorescence enhancement, two opposite charged polyelectrolytes were used to separate the GNRs from the fluorophores. A negatively charged poly-sodium-4-styrenesulfonate (PSS), and a positively charged poly-allylamine hydrochloride (PAH) are used as the spacer between fluorophores and GNRs. Layer-by-layer (LBL) assembly of each polyelectrolyte gives rise to nanoscale control over the GNRs-based architecture [7].! 7! 1.2 Aim of the project The aim of the project is to determine the optimal distance between plasmonic GNRs and fluorophores, when the maximum fluorescence enhancement is achieved. Furthermore, by using different fluorophores (Cy5, Cy7) to determine, which gives a better match to the GNRs' LSPR, and hence, influences the PEF effect.! 8! 2 Theory 2.1 Fluorescence Fluorescence is the emission of electromagnetic radiation lit by a substance that has absorbed radiation of a certain wavelength. This wavelength is never the same as the emission wavelength. In most cases, the energy of the absorption light is larger than that of the emission of light with a longer wavelength. The different energy between the absorbed and emitted photons is due to the non-radiative decay, such as heat. Fig. 2.1 is an electronic-state diagram illustrating the fluorescence process: Figure 2.1: the energy level diagram illustrating the three processes of fluorescence emission! 9! Excited electronic state is initiated by optical absorption (process1), and fluorescence emission occurs when an orbital electron of a molecule, atom or nanostructure relaxes to its ground state by emitting a photon after being excited to a higher energy state (process3). It can be written as: Excitation: S 0 + hv ex! S 1 (2.1) Fluorescence (emission): S 1! S 0 + hv em + heat + (2.2) hv is photon energy with h = Plank constant and! = frequency of light. State S 0 is called the ground state of the atom, and S 1 is called the first excited state. Photon energy hv ex is provided by an external light source that excites the electron to the S 1 state. There are different energy dissipations from excited state to ground state. They can be briefly distinguished into radiative relaxation (r), like fluorescence, and non-radiative relaxation (nr), such as heat (vibrations or rotation). Excited electrons stay in the excited state S 1 and exist during a limited time, which is called lifetime. After this period of time, part of the energy relaxes by non-radiative relaxation (nr) to a lower energy level of excited state S 1 (process2). From state S 1 is when the emission starts to occur. Photon energy hv em is emitted so that the fluorophore atom returns to its initial energy state S 0. The energy of the emitted photon is usually lower than the excitation energy. Therefore, the emitted photon wavelength is longer than the excitation source. The energy difference is called the Stokes shift, and it is determined by (hv ex hv em ). In addition, the fluorescence quantum yield is the ratio between the number of fluorescent photons emitted and the number of photons absorbed. The fluorescence lifetime refers to the average time the molecule stays in its excited state before emitting a photon (process2 in figure 2.1). Fluorescence typically follows first-order kinetics: [S 1 ]=[S 1 ] 0 e! t (2.3) Where [S 1 ] is the concentration of excited state atoms at time t, [S 1 ] 0 is the initial concentration, and is the decay rate of the fluorescence lifetime. This is an exponential decay. Different radiative and non-radiative processes can decrease the excited state concentration. The total decay rate is the sum of two different decay rates:! tot =! rad +! nrad Where tot is the total decay rate, rad is the radiative decay rate, and nrad is the non-radiative decay rate. According to (2.3), the shorter the lifetime t, the larger the total decay rate! tot. The quantum efficiency (QE) reflects a competition between radiative decay and non-radiative processes. It is defined as below: (2.4) (2.5)! 6! 2.2 Metal Nanoparticles Optical properties of metal nanoparticles The free electrons in metal (d electrons in silver and gold) are free to move through the material. The mean free path in gold and silver is about 50 nm [8], and in particle size that is smaller than this, surface is the dominator of the particle. Thus, all opto-electoral interactions happen on the surface. Furthermore, as long as the external electromagnetic field carries certain frequency, resonance oscillation takes place with the electrons Localized Surface Plasmon Resonance Since the resonance oscillation happens on the surface, it is referred to as surface plasmon resonance (SPR). SPR is usually further categorized in two different plasmon modes; propagating surface plasmons that take place on planar metal films and localized surface plasmons (LSPR) that refers to collective electron charge oscillations in metallic NPs with excited light. In practice, the SPR sensor needs a sensing area of at least 10#10 µm. On the other hand, a sensor based on LSPR can have thousands of sensing elements on the same area, which significantly increases the sensing resolution [9]. This work only focuses on the LSPR sensor Plasmonic Gold Nanorods The aspect ratio (long axis/short axis) of metal nanorod affects optical properties. There are two different modes of plasmons resonance in metal nanorods. They are the transverse and longitudinal mode. The transverse surface plasmon is an electronic oscillation across the width of the rod (see figure 2.2). The absorption maximum position is located at ~ 520 nm. The second adsorption maximum is usually larger than 600 nm. It is due to the longitudinal plasmon oscillations in the long side of the rod, and can be tuned by varying the length of the nanorods [10]. The longitudinal resonance is more surface-sensitive than the transverse mode, and the resonance wavelength red shifts when the dielectric constant of the surrounding environment increases [11]. The magnitude of both the longitudinal and the transverse resonance peak increases when the dielectric constant of the surrounding media is increased [12, 13].! :! Figure 2.2 Transverse oscillations and longitudinal oscillations of GNRs LSPR! ; 2.3 Plasmon Enhanced Fluorescence Plasmon enhanced fluorescence [14-16] is a phenomenon that occurs when a fluorescent molecule interacts with a metal nanoparticle or a structured metal surface. Due to the occurrence of LSPR in metal NPs or structured metal surface, the photon emission from the fluorophore is enhanced. The enhancement effect is influenced by several different parameters such as metal, particle size and shape, distance between fluorophore and metal NPs (which will be referred to as the metal from now on). Fluorophores that are close to the metal are exposed to a nearby strong local field. However, one drawback with exciting fluorescence molecules near the metal is quenching, which leads to a reduction in the fluorescence intensity. Quenching means that the fluorescent energy from the excited atom is not emitted as a photon, but instead the coupling of different phenomena (such as heat and surface waves). The fluorescence energy loss depends on the distance between the metal, and is characterized by Föster- like quenching with a rate k q [17]. Once the fluorophore is positioned resonance distance to the metal, it will show fluorescence enhancement compared to free fluorophore with an introduced radiative decay rate # m. In the process of the absorption, more fluorophores are excited by localized metal (m) plasmons with E m field, so called the lighting rod effect [18]. This effect gives rise to an effective increase in the fluorescence signal that means more photons will be detected by the detector. The enhancement happens when fluorophores are positioned with a separation from the metal. This separation is used to avoid fluorescence quenching. The term (2.5) quantum efficiency (QE) can be modified in the presence of metal (m):! (2.6) From (2.6) by introducing LSPR, the non-radiative rate # nrad is reduced, and an extra radiative decay rate # m is induced. Therefore, QE m becomes larger than QE in the presence of metal. In Figure 2.3 (a) and (b), the QE and QE m are illustrated by classical Jablonski diagram, which shows the parameters that are participating in metal-assisted fluorescence process.! Figure 2.3: (a) the energy level diagram illustrating the process of fluorescence emission in the absence of metal. Only radiative rate # rad and non-radiative rate # nrad are taking part in the fluorescence process. (b) In the presence of LSPR, metal enhanced excitation field E m, radiative rate # m, and quenching rate k q are additionally involved in the fluorescence process. In summary, the metal acts as an extra excitation source that means that in the presence of the metal fluorophores feel more exciting light compared to free fluorophores. Therefore, fluorophores result in emitting more photons per unit time when they are positioned within the range of LSPR field E m. In addition to distance dependence, the degree of fluorescence enhancement depends on the spectral overlap between the LSPR mode and the dye absorption and emission along with the local field enhancement properties of the metal [19]. Moreover, an additional radiative rate results in an increase in the total radiative decay rate. However, if a dye has a very high quantum yield (approximately 1), the additional radiative decay rate would not dramatically increase the quantum yield. The more interesting case is for low quantum yield fluorophores.! & 3 Materials and Methods 3.1 Overview of the system There are four main procedures in the experimental setup. A polymer-based microwell plate, used for cell culture with a negatively charged surface is used as a substrate. First CTAB-coated GNRs, which has a positive charge due to the amine group in the stabilizing surfactant CTAB, are immobilized in the wells in the plate. Then polyelectrolytes are deposited using Layer-By-Layer methods to control the distance between GNRs and fluorophores. The last step is to deposit a polyelectrolyte layer with conjugated fluorophores as the top layer. How distance influence PEF effect between GNRs and fluorophores by introducing a spacer is investigated, as shown in figure 3.1. Figure 3.1 An Imaginary picture illustrating the system. Only the photo of the 96 well optical bottom plate is taken from real object.! # 3.2 Synthesis of gold nanorods (Dr. Richard Becker, Chemistry, IFM, did all of the GNRs synthesis.) GNRs with aspect ratio of around 2.7 are mainly used in this project. They are synthesized by a simple seed-mediated method. Cationic ammonium surfactants are used as the directing agent in the synthesis of GNRs in aqueous solutions, and the most widely used surfactant is cetyltrimethylammonium bromide (CTAB). However, the CTAB-directed growth mechanism is not yet completely understood. Since both the adsorption of the surfactants to the GNRs surface and the binding of the complex ions to micelles involve the surfactant head group, it is expected that a change of the head group will alter the growth behavior of GNRs [20]. First, a gold nanoparticle seed solution is prepared by adding ml of an ice-cold solution of 0.01 mm sodium borohydride (NaBH 4 ) to 2 ml of 0.5 mm gold chloride solution (HAuCl4) prepared in 2 ml, 0.2 M CTAB solution under vigorous stirring. The yellow color changes immediately to brown, indicating the formation of gold nanoparticle seeds. Stirring continues for 10 more minutes. This seed is used for the synthesis of gold nanorods. Then, the following solutions are added in the following order: 450 ml of 0.2 M CTAB solution and 450 ml 0.5 mm HAuCl 4 put in a water bath (25 ) to dissolve the CTAB. After that, 18 ml 4 mm of silver nitrate solution (AgNO 3 ) is added and 4.95 ml 0.1 M ascorbic acid. Then 1.8 ml of the seed solution is added and kept in water bath without stirring for 2-3 hours. The violet-brown-colored gold nanorod solution prepared w
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