The objectives of research contents of the thesis: (i) to synthesize and investigate the optical properties of gold nanostructures spherical, rod and core/shell SiO2/Au shape with controlled sizes; (ii) to try application of synthesized gold nanostructures in imaging and in photothermal.
VIETNAM ACCADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY INTITUTE OF PHYSICS *** DO THI HUE SYNTHESIS AND OPTICAL PROPERTIES OF GOLD NANOSTRUCTURES SPHERICAL, ROD AND CORE/SHELL SIO2/Au SHAPE FOR BIOMEDICAL APPLICATIONS Speciality : Solid State Physics Code : 944 01 04 THESIS SUMARIZATION Ha Noi – 2018 The work was completed at the Center for Quantum Electronics, Institute of Physics, Vietnam Academy of Science and Technology Supervisor: Dr Nghiem Thi Ha Lien Assoc Prof Dr Tran Hong Nhung Referee 1: Dr.Nguyen Cao Khang Referee 2: Prof Dr Nguyen Nang Dinh Referee 3: Dr Nguyen Tran Thuat The thesis will be presented and defended at the Scientific Committee of Institute of Physics held in: at The thesis can be found at the library: - National Library of Hanoi - Library of Institute of Physics, VAST Origin of thesis title The gold nano structures are one in the nano physical types used more in the biomedical applications for increasing sensitivity of the diagnostic and the targeted therapy These are the research directions that many labs in the world and in Viet Nam are interested in developing Gold nanospheres show an extinction sccross-section (absorption and scattering) 4-5 orders higher than conventional absorbing dyes In particular, the plasmon resonance of the gold or silver nanostructures can be tuned to specific wavelengths across the visible and infrared range of the electromagnetic spectrum, for applications ranging from the construction of photonic crystals to biophotonics Moreover, the superior properties of gold nanostructures are stability structural, non-toxic, highly biocompatible, and they are easily surface function to bind to biomolecules such as amino acids, enzymes, DNA and drug molecules through the -SH group With these unique surface-chemistry properties, the applied studies of gold nanostructures are more and more developed and promising great achievements in biomedical applications For example: (i) gold nanoparticles are capable of carring drugs, delivering drugs and photoluminescence in tissue; (ii) core / shell nanostructures with strong light scattering from visible to near infrared (NIR) are applied to in vivo imaging in the body (10 cm) to present cancer cells At the same time, with the ability to absorb light intensively in the near-infrared region, gold nanoshells are used to destroy cancer cells by phototherapy without compromising healthy cells also not affect the genetic factors Based on the actual exigency and the ability to response those exigencies of the gold nanostructures, as well as from the research situation in the world and in Viet Nam, we chose and study the topic: “Synthesis and optical properties of gold nanostructures spherical, rod and core/shell SiO2/Au shape for biomedical applications” The objectives of research contents of the thesis: (i) to synthesize and investigate the optical properties of gold nanostructures spherical, rod and core/shell SiO2/Au shape with controlled sizes; (ii) to try application of synthesized gold nanostructures in imaging and in photothermal Usefulness of the thesis: The thesis is a basic research to orientate application of gold nanostructures that are and will be promising many applications in nanotechnology, especially bioapplications The thesis has found a simple process for synthesising gold nanoparticles at room temperature by the seeded growth with particle size be controlled in a wide range from 2nm to over 200nm The thesis has controlled minutely the gold film thickness of nanoshells from 10-30 nm by control of seed concentration At the same time, the use of uniform and small size Duff-Baiker gold particles creates a thinner, less rugged gold film, which is better than today's Simultaneously, we have created small nanoshells less than 100 nm with plasmon absorption peaks about 700 nm Thesis structure The thesis consists of 158 pages arranged into chapters Chapter 1: OVERVIEW OF RELATED MATTERS AND THEORY 1.1 Optical properties of metallic nanoparticles 1.1.1 Surface plasmon resonance (SPR) Surface plasmon polaritons are electromagnetic excitations propagating at the interface between a dielectric and a conductor Metals as Au, Ag and Cu have plasmon frequencies in the visible light region, so their nanostructures have color effects The colour of the colloidal solutions is the result of the scattering and absorbing of light by the surface plasmon The optical properties of metallic nanostructures are explained by the theory of Mie and Gans 1.1.2 The Mie theory - the dependence of optical properties on particle size From the Mie theory can calculate scattering sca and the absorption cross section abs of a particle as follows: sca 512 − m = k R + m abs = 32πkR3Im[ 𝜀−𝜖𝑚 ] 𝜀+2𝜖𝑚 Where k is the number of waves, R is the radius of the particle The above formulas show that when the particle size is small, the scattering efficiency is smaller than the absorption efficiency Absorption A of a sample of dispersed nanoparticles is given by: 𝐼0 𝜆 𝐴(𝜆) = 𝑙𝑜𝑔 𝐼𝜆 = 2.303 𝑁 𝜎𝑒𝑥𝑡 𝑙 𝜆 where ext is the extinction cross section of the sample at wavelength and N number of particles in a liter, l is the thickness of the absorbing medium (cm) 1.1.3 Optical characteristics of gold nanostructures As a result of the Mie theory, it is possible to see the plasmon absorption spectra of the metal nanoparticles depending on the particle size 1.1.3.1 Gold nanoparticle Optical properties depend on the size of the particle: particles less than 20 nm in diameter scatter negligible As the particle diameter increases, the contribution of surface plasmon scattering increases significantly So large particles are suitable for imaging applications based on scattering of light Depending on the purpose of application, gold nanoparticles with suitable dimensions will be selected 1.1.3.2 Gold nanorod Figure The distribution of charge on a nanorod below the excitation of the light The optical properties of the gold nanorods depend on the ratio of the aspect ratio of gold nanorods, particularly, the longitudinal surface plasmon resonance (LSPR) red – shifts when the aspect ratio increases 1.1.3.3 Nano core/shell SiO2/Au Relative thickness of core-to-shell layer is sensitive towards the position of the SPR band Figure Variation in SPR band with shell thickness Figure Hybridization model describing interaction between sphere and cavity plasmons to give rise to nanoshell plasmon Plasmon excitation from nanoshell particles can be viewed as an interaction between plasmon response from a nanosphere and a nanocavity (fig 3) The frequencies of these modes (bonding and antibonding) can be expressed as 𝜔𝑛± = 𝜔𝑝 𝑅1 √1 + 4𝑛(𝑛 + 1)( )2𝑛+1 ] [1 ± 2𝑛 + 𝑅2 where, R1 is the inner radius of the shell, R2 the outer radius of the shell, n the order of spherical harmonics, p the bulk plasmon frequency, n+ the antisymmetric plasmon and n- the symmetric plasmon 1.2 Methods for synthesising of gold nanostructures 1.2.1 Seeded growh method This method has the advantage of synthesizing at room temperature, easy to control particle size, uniform size and less byproducts However, to obtain the results that this method requires: - Creating seeds are single dispersed, uniform in shape and size - Controlling pH of growth solution 1.2.2 Methods for making gold nanoparticles 1.2.3 Synthesis of gold nanorods 1.2.4 Synthesis of nano core/shell SiO2/Au 1.3 Application of gold nanostructures 1.3.1 Bio-marking and imaging 1.3.2 Photothermal 1.4 Characterization techniques Chapter 2: Synthesis and optical properties of gold nanospheres 2.1 Materials 2.2 Synthesis of Duff-Baiker gold Figure 4: Transmission electron microscopy images of Au seed particles – scale 20 nm (left); absorption spectra of seed gold colloid TEM image shows that gold nanoparticles are formed with an average size of nm The absorption spectra of the seed solution is characterized spectra of small gold nanoparticles (less than 10 nm): broad spectrum with resonant peak in the range of 505 nm - 510 nm 2.3 Synthesis of gold nanospheres by seed growth method 2.3.1 Role of Gold plating solutions (GPS) in the growth of Au seeds Figure Absorption spectra of the solutions with varied pH values We chose to use a pH 9.4 hydroxyde solution as a seeded growth solution 2.3.2 Effect of Seed Concentration Figure Effect of seed concentration Transmission electron microscopy images of Au particles obtained using different concentrations of seed while keeping the amount of Au precursor added constant The results showed that the larger the [Au3+] / [seed Au ), the larger the seed was and when the ratio was greater than 12.5, the particles lost symmetry This can be explained by the La Mer mechanism 2.3.3 Seeded Growth Synthesis of Au NPs of Up to 200 nm 2.3.3.1 Using the Duff-Baiker gold particles 2.3.3.2 Using the citrate gold particles Figure Seeded growth with the dilution of the seed solution Transmission electron microscopy images of nanoparrticles obtained after different growth steps, scale 100 nm 10 Dimensions obtained from calculations, TEM images, and DLS measurements are relatively consistent This again shows that by using the seeded growth method with the pH 9,4 growth solution, we can completely controlled synthesis the good quality gold nanoparticles at the room temperature Figure Plasmon resonance spectra of small gold nanoparticles obtained from step to step (A), normalized plasmon resonance spectra of solutions obtained from step to step 16 (B) and plasmon resonance spectra of large gold nanoparticles obtained from step 17 to step22 (C) After each step of the growth, the gold nanoparticles are larger, the plasmon absorption peak shift toward the long wave At the same time, it is also possible to see that the spectral widths of these spectra are much narrower than that of small particle sizes (less than 10nm) and in this size range the 11 spectral width increases with the increase in particle size This is the result of the interaction between electromagnetic waves and large metal nanoparticles Chapter 3: Synthesis and optical properties of gold nanoshell SiO2/Au 3.1 Materials and methods 3.2 Synthesis of amino-functionalized silica nanoparticles Figure TEM images of silica NPs prepared by sol–gel with the increasing amounts of ammonia and corresponding sizes of (a) 40 ± nm, (b) 65 ± nm,(c) 110 ± nm, (d )130 ± nm, and (e) 150 ± nm, before functionalization with APTES, and f– j the respective silica NPs obtained after functionalization with APTES The same scale bar (100 nm) applies to all images The sizes of the spherical particles were regulated by the amount of ammonia used during the synthesis: a corresponding increase in the average diameter from 40 to 150 nm was obtained with the increasing amounts of ammonia from 0,9 ml to 1,3 ml Results of the zeta potential and infrared absorption spectra showed that silica nanoparticles functioned successfully with APTES molecules 3.3 Seed particles: THPC–gold-decorated APTES- functionalized silica NPs 12 Figure 10 TEM images of THPC–gold-decorated on (a) bare silica NPs, in citrate buffer solution, (b) APTES-functionalized silica NPs, in citrate buffer solution Scale bars are 100 nm for all images TEM images show that gold nanoparticles adsorpt very little and uneven on the surface silica nanoparticles are only groups -OH Whereas, the DuffBaiker gold nanoparticles adsorpt uniformly on the surface of the silica nanoparticles are –NH2 groups due to the electrostatic interaction between the amino - NH2 functional groups on the nano silica and -COO groups on Duff-Baiker gold nanoparticles 3.4 Gold–silica core–shell formation 3.4.1 Effect of HCHO concentration The minimum HCHO required for the reduction reaction to form the core / shell structure is determined to be a molar ratio of HCHO to Au3+ in the growth solution to 2.5 3.4.2 Gold nanoshell SiO2/Au 3.4.2.1 SiO2/ Au nanoshells with varying shell thickness 13 Figure 11 HTEM and corresponding EDX analysis image of silica core in diameter following the gold-plating process Yellow corresponds to Au, red corresponds to O; Green corresponds to Si; scale bar of 100 nm 3.4.2.2 Synthesis nanoshell on the silica core with a diameter of 40-150 nm 14 Figure 12 TEM images of silica NPs with varying sizes of a 40 ± nm, b 65 ± nm, c 100 ± nm, d 110 ± nm, e 130 ± nm, and f 150 ± nm coated with gold shells Each frame shows the development of the nanoshells at different stages of the deposition process: (i) bare APTESfunctionalized silica, (ii) gold-decorated APTES-functionalized silica, (iii) partial gold shell growth, and (iv) complete gold shell formation 15 Figure 13 Normalized plasmon resonance spectra of SiO2/ Au with the same shell thickness and varying core diameters Chapter 4: Synthesis and optical properties of gold nanorods (GNRs) 4.1 Materials and methods Gold nanorods are synthesised by seeded growth methods This method consists of two stages: preparation of the gold seed and preparation of gold nanorod of various aspect ratios By analyzing the role of forming factors, we found that many factors influence the formation and development of gold nanorods, such as: Ag+, CTAB, AA, Au3+ concentration 4.2 Synthesizing of GNRs 4.2.1 Effect of Ag+ concentration In order to consider role of Ag+ in the formation of GNRs, different samples of GNRs were prepared Firstly, 150 µl HAuCl4 0.023 mM was put into 10 ml of the mixture CTAB 0.1M and BDAC 0.00 5M in different reactions Then, various volumes of AgNO3 corresponding to the Ag+ concentrations of 0.0 mM; 0.024 mM; 0.040 mM; 0.048 mM; 0.064 mM; 16 0.080 mM; 0.107 mM; 0.134 mM were added in the above reaction solutions Next, 25 µl AA 0.25M reductant reaction agent was added Finally, 100 µl of the gold seed solution was put into each reaction solution under vigorous stirring at room temperature The growth process of GNRs was maintained in hours 4.2.3 Effect of Ag+ concentration The amount of 0.25 M AA was varied from - 40 μl and the ratio of concentration [AA] to [Au3+] was observed from insufficient 0,92 to excess 2.45 4.3 Results 4.3.1 The shape and composition of the gold nanorods Figure 14 XRD spectra of gold nanorods 4.3.2 The influence of factors on the formation and development of the GNRs 4.3.2.1 Ratio of molar concentration [Ag+] to [Au3+] 17 Figure 15 TEM images of the GNR solutions upon changing of the Ag+ concentrations at scale bare of 20 nm Figure 15 shows TEM images of the GNRs prepared under different Ag+ concentrations as: mM; 0.04 mM; 0.064 mM; 0.08 mM; 0.107 mM, and 0.134 mM We can easily see that the yield of the rod and their uniformity increase while the diameter of the particles decreases as the Ag+ concentration increases from 0.04 mM to 0.107 mM.Without Ag+ ions, TEM image indicates that the obtained sample contains different shapes, including spherical, triangle and a few high aspect ratio rod-like particles This is consistent with results published in the ref Figure 16 The absorption spectra of the GNR solutions prepared under different Ag+ concentrations 18 4.3.2.2 Effect of AA concentration Figure 17 The absorption spectra of the GNR solutions prepared under different AA concentrations The ratio of molar concentration [AA]/[Au3+] is less than 0.92, gold nanorod are not formed When this ratio is bigger than 1.22, the absorption spectra of the solutions have features of the typical gold nanorods 4.4 Effect of the refractive index of the medium 4.4.1 Effect of CTAB concentration 4.4.2 Effect of surface molecules Chapter 5: Application Experiment 5.1 Characteristics of PEG, BSA, GSH, IgG -HER2 and BT-474 5.2 Binding with biocompatible molecules: BSA, PEG, GSH and IgG HER2 antibody 5.2.1 Coherence principle 19 5.2.2 Some results mount Figure 18 The FTIR of the BSA and Au @ BSA 5.3 Results of using gold nanoparticles in cellular images Figure 19 Dark-field microscope image of (A) BT-474, (B)BT-474 cancer cell is labeled with IgG -HER2 antibody on Au @ IgG-HER2 complex and (C)BT-474 cancer cells are incubated with Au @ BSA Dark-field microscopy reveals the role of SiO2/Au in cell marking by scattering the light, which increases the image contrast of the observed cell 5.4 Photothermal of gold nanostructures on meat tissue Gold nanoshells and nanorods absorbing strongly the light in the near infrared region are used in this effect The results showed that with the same lighting conditions, the temperature of the samples containing gold 20 nanoparticles was in the range of 49 ÷ 510C for sample mm thick, 45 ÷ 460C for sample mm thick, 40 ÷ 430C for sample mm thick and 33 ÷ 350C for sample mm thick We calculated that the optical conversion efficiency η for SiO2/Au was 22% and the gold nanorod was 24% CONCLUSION The thesis reachs aims about studying, fabricating and investigating the optical properties of different gold nanostructures, such as spherical, rod and core/shell shapes Simultaneously, fabricated materials are applied in cellular imaging and investigating the photothermal effects on meat tissue The results as well as the new contributions of the thesis are: Synthesis Using of seed – mediated method to synthesize gold nanostructures at room temperature with controlled size: Monodispersed spherical gold nanoparticles with varying diameters from 5.5 nm to 200 4.5 nm are synthesized by seed – mediated method The seeds 1-3 nm in diameter (Duff-Baiker gold nanoparticles) and 19 nm in diameters (citrate gold nanoparticles) are grown in the solution containing gold ions complex [AuClx(OH) 4-x]- Core /shell nanoparticles SiO2/Au with a core diameter of 40-150 nm, and layer thicknesses varying between 12 nm to 22 nm are synthesized by 4step seed – mediated method The dependence of the plasmon resonance spectra on the ratio of core diameter to shell thickness is investigated The results show that when the seeds are large enough to cover the surface of the core, the plasmons resonance have two resonance peaks and can translate to the long wave (800nm-900nm) Small gold nanorods with the ratio aspects between to 4.5 and plasmon resonance peaks at 700 nm - 900 nm are synthesized on the basis of surveying the influence of factors on the anisotropic development of the seeds in the solution At the same time, study the dependence of the optical 21 properties of the gold nanorods on the refractive index of the environment surrounding them 2.Application The thesis presents the results of application of gold nanostructures in two directions: diagnosis and treatment of diseases The results show that, due to the scattering strongly of light in the visible region, SiO2/Au give good results in cell imaging At the same time, due to the strong absorption of the near-infrared light, the gold nanorods and SiO2/Au make to increase the ambient temperature from 4°C to 12°C when illuminated by laser 808 nm Recommendations The results obtained from this thesis show the potential of gold nanoparticles in diagnosis and treatment of diseases In preparation for this treatment, preclinical studies, in vivo studies, should be conducted carefully At the same time, it is possible to study the formation of multifunctional gold nanoparticles to meet a variety of applications 22 LIST OF RESEARCH WORKS USED IN THE THESIS Tran Hong Nhung, Nghiem Thi Ha Lien, Emmanuel Fort, Le Quang Huan, Nguyen Thi Quy, Do Quang Hoa, Vu Thi Thuy Dương, La Thi Huyen, Nguyen Thanh Phuong, Nguyen Thi Tuyen, Do Thi Hue, “Biofunctionalisation of gold nanoparticles for biomedical applications”, IWNA 2011, November 10-12, 2011, Vung Tau, Viet Nam Thi Ha Lien Nghiem, Tuyet Ngan Le, Thi Hue Do, Thi Thuy Duong Vu, Quang Hoa Do, and Hong Nhung Tran “Preparation and characterization of silica - gold core - shell (SiO2 @ Au) nanoparticles”, advances in Optics photonics spectroscopy and applications VII, ISSN 1859-4271 Thi Ha Lien Nghiem, Thi Hue Do, Van Tuyen Nguyen, Thi Hai Nguyen, Thi My An Nguyen, Duong Vu, Quang Hoa,Thi Thuy Duong Vu and Hong Nhung Tran, “Synthesis and biofunctionalization of gold nanoshells for biomedical applications”, advances in Optics photonics spectroscopy and applications VIII, ISSN 1859-4271 Do Thi Hue, Nghiem Thi Ha Lien and Tran Hong Nhung, “Synthesis and characterization of fine colloidal gold nanoparticles”,the second academic conference on natural science for master and phD students from Cambodia, Laos, Malaysia and Viet Nam, 2012 ISBN 978-604-913-088-5 T.H.L.Nghiem, T.N Le, T.H.Do, T.T.D Vu, Q.H.Do, and H TN Tran, “Preparation and characterization of silica–gold core–shell nanoparticles”, J.Nanoparticle Res., vol 15, no 11, Nov 2013 Thi Hue Do, Thi Thuy Nguyen, Thi Ha Lien Nghiem and Hong Nhung Tran “Synthesis and optical characterization of small diameter gold nanoshells for biomedical applications”, The 3rd Academic Conference on Natural Science for Master and PhD Students From Cambodia - Laos Malaysia - Viet Nam, 11-15 November 2013, Phnom Penh, Cambodia, ISBN 978-604-913-088-5 Do Thi Hue, Nguyen Thi Bich Ngoc, Vu Van Son , Vu Thi Thuy Duong, Nguyen Trong Nghia , Đo Quang Hoa, Tran Hong Nhung, and Nghiem Thi 23 Ha Lien, “ Synthesis gold nanorods for biomedical applications” The International Conference on Applied & Engineering Physics, October 2015 in Hanoi, Vietnam Do Thi Hue, Nguyen Thi Bich Ngoc, Vu Duong, Vu Van Son , Nguyen Trong Nghia , Đo Quang Hoa, Tran Hong Nhung, and Nghiem Thi Ha Lien “ Synthesis siliver nanorods for biomedical applications” The International Conference on Applied & Engineering Physics, October 2015 in Hanoi, Vietnam Do Thi Hue, Chu Viet Ha, Nghiem Thi Ha Lien, Tran Hong Nhung,“Synthesis gold nanorods for biomedical applications”, Journal of Science and Technology,155 (10), 2016 10 Trong Nghia Nguyen, Thi Hue Do, Dinh Hoang Nguyen, Duong Vu, Quang Hoa Do, Hong Nhung Tran, and Thi Ha Lien Nghiem, “Enhanced absorption and fluorescence of gold nanoclusters using initial alkali concentrations”, Applied Physics Express 9, 2016 11 Nguyen Van Minh, Do Thi Hue, Nghiem Thi Ha Lien, Vu Thu Hien, Vu Ngoc Hung, Chu Manh Hoang, “Self-assembly of close-packed monolayer of silica nanospheres on silicon substrate with infrared irradiation”, Proceeding of the third international conference on advaned materials and nanotechnology, (2016), ISBN 978-604-95-0010-7 12 Do Thi Hue, Vu Thi Thuy Duong, Nguyen Trong Nghia, Tran Hong Nhung, Nghiem Thi Ha Lien, “Seeded growth synthesis of gold nanorods for photothermal application” Vietnam Journal of Science and Technology 56 (2) 148-157, 2018 13 V.T.T.Duong, Anh D phan, Nghiem T H Lien, Do T Hue, Do Q Hoa, Do T Nga, Tran H Nhung, and Nguyen A Viet “Near – infrared photothermal response of plasmonic gold – coated nanoparticles in tissues” Phys.status solidi A, 2017,1700564 24 ... spherical, rod and core/shell SiO2/Au shape for biomedical applications The objectives of research contents of the thesis: (i) to synthesize and investigate the optical properties of gold nanostructures. .. dispersed, uniform in shape and size - Controlling pH of growth solution 1.2.2 Methods for making gold nanoparticles 1.2.3 Synthesis of gold nanorods 1.2.4 Synthesis of nano core/shell SiO2/Au 1.3... spectra of SiO2/ Au with the same shell thickness and varying core diameters Chapter 4: Synthesis and optical properties of gold nanorods (GNRs) 4.1 Materials and methods Gold nanorods are synthesised