Speciality: Solid state Physics Code:

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1 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY INSTITUTE OF PHYSICS PHAM MINH TAN SYNTHESIS AND OPTICAL PROPERTIES OF DYE- DOPED SILICA NANOPARTICLES FOR BIOMEDICAL APPLICATIONS Speciality: Solid state Physics Code: THE SUMMARY OF DOCTOR OF PHILOSOPHY IN PHYSICS HA NOI, 2015

2 The work was completed at the Center for Quantum Electronics, Institute of Physics, Vietnam Academy of Science and Technology Supervisor: 1. Assoc. Prof. Dr. Tran Hong Nhung 2. Assoc. Prof. Dr. Tong Kim Thuan Examiner 1: Assoc. Prof. Dr. Pham Van Hoi, Institute of Materials Science, Vietnam Academy of Science and Technology Examiner 2: Assoc. Prof. Dr. Le Anh Tuan, Advanced Institute for Science and Technology, Hanoi University of Science and Technology Examiner 3: Assoc. Prof. Dr. Mai Anh Tuan, International Training Institute for Materials Science, Hanoi University of Science and Technology The thesis will be defended at the evaluating council (institutional level) Place of defending: Institute of Physics, Viet Nam Academy of Science and Technology (VAST). No. 10, Dao Tan, Ba Dinh, Hanoi Time: at..day.. month Thesis can be found at: - National Library of Hanoi - Library of Institute of Physics, VAST

3 1 FOREWORDS Dye-doped silica nanoparticles (NPs) are nanoporous SiO 2 microparticles containing a large number of organic dye molecules in a single silica particle. Silica-based NPs also are stable structure, non-toxic and potential in high biological compatibility. If the methods and processes are used appropriately, a large number of dyes can be inserted into a single silica nanoparticle (from dozens to thousands of pigment molecules). Therefore, brightness and optical signal amplification of dyes contained in silica nanoparticles are many times higher than those of individual colored molecules. The brightness of the fluorescence signal of silica nanoparticles able to be controlled by number of dye molecules in each particle with the greatest dye density is limited only by the fluorescence quenching. If bioanalytical applications are selected appropriately, silica nanoparticles can generate significant improvements in sensitivity analyses. Moreover, due to the fact that detained in silica-based NPs, dyes are protected from environmental impacts. Moreover, due to silica-based nanoparticles containing very little of free oxygen, optical decomposition are minimized. High optical durability allowing silica NPs used in applications that require powerful stimulating intensity for long periods. Moreover, silica particles with the (- OH) group on the surface may join a chemical reaction to form functional groups capable of specific link with biomolecules such as the amino group (-NH2), the carboxyl group (- COOH) or the thiol group (- SH). By adjusting the synthesized parameters, it can control particle sizes, dye-doped amount in particles as well as dye-doped types inserted into them. So, it can synthesize a large group of luminescent particles with diversified optical properties used in marker. Silica itself is an environmental friendly biological substance. Therefore, they can be multi-

4 2 functional particles: both detection of diseases and attachment of medications. Therefore, the silica particles are under the generation of novel biological markers, which promise to be widely used in the analysis and biomarkers. Therefore, the topic of the thesis selected is Synthesis and optical properties of dye-doped silica nanoparticles for biomedical applications The objectives of research contents of the thesis: (i) To synthesize organic dye-doped silica NPs oriented for applications of biomarkers. (ii) To apply synthesized silica nanoparticles as markers for detection E. coli O157: H7 and breast cancer cells by immunofluorescence method. Key Research Contents of the thesis: Research of the thesis is conducted by the experimental method with the following contents: (i) Study and synthesis dye-doped silica nanoparticles with functional groups on the particle surface by sol-gel method. (ii) Survey the chemical structure from the infrared absorption spectrum. (iii) Investigate the optical properties of synthesized NPs in order to evaluate the advance characteristics. (iv) Apply the nanoparticles in biology and medicine such as labeling E. coli O157: H7, breast cancer cells BT-474 and KPL-4; establishing optical methods to quantify bacterial cells E. coli O157: H7 and breast cancer cells KPL-4. The scientific meaning and practice of the thesis: The thesis is a basic research work oriented applications. The object of the thesis is optical nanomaterial applied in analysis of biology and medicine. The results of the thesis promise interesting additional information to studies in organic dyedoped silica NPs. For the first time in Vietnam, the quantity of bacteria and cancer cells was determined by spectroscopy methods using nanoparticles. These results are for the studies on cancer detection and treatment.

5 3 Structure of the thesis: The dissertation scientific main results were discussed in 5 articles published by domestic and international specialized journals. The thesis consists of 147 pages divided into 4 chapters. CHAPTER 1. LITERATURE REVIEW 1.1. Organic dyes Chemical structure Organic dyes (fluorescence organic compounds) have been used in biochemical and biophysical assays, biological and medicine studies named as fluorescence techniques. Dyes used for bio-labelling are organic dyes containing double-conjugated bonds and strongly absorb light excited from ultraviolet region to the near-infrared region. Their chemical structures are characterized by containing of benzene rings, pyridine, azine, pyron, etc. situated in the same plane The structure of the energy levels and optical transitions S 2 τ n ps T 2 S 1 ν τ i ns Absorption Fluorescenc e τ F ns T 1 S 0 ν Phosphorescence τ F µs Figure 1.3. The energy level structure and optical transitions of dye

6 4 molecules The energy structure of dye molecules is the combination of electronic states, vibrational and rotational states of the molecule. Based on Bohr model, Jablonski propose a simplification structure reflecting mainly important characteristics of the quantum migration in dye molecules. (Figure 1.3). Of which, singlet states of electron are S 0, S 1, S 2, etc. and triple states of electron are T 1, T 2, etc. Total spin quantum numbers of singlet and triple states are S = 0 and S = 1 respectively. In a singlet state, two electrons will have antiparallel spins. Inversely, when two electrons in the same triplet state, they will have other parallel spins Organic dye-doped silica nanoparticles Latex and silica nanoparticles Latex and silica NPs (polymer NPs) are dye-doped NPs (i) Dyes can be mounted on the surface or inserted into nanoparticles.(ii) In comparison with original organic dyes, the optical durability of these NPs is higher. (iii) The brightness of fluorescence signal of silica and latex NPs can be controlled by numbers of dye molecules in each particle with the greatest dye density limited only by the fluorescence quenching. A single NPs can contain thousands dyed molecules. Therefore, fluorescence signal is an average one and it is not blingking Silica/ormosil nanoparticles Despite of mentioned advantages above, silica NPs are still have some disadvantages such as (i) optical homogeneity is not high because of numberous porous structures, (ii) Dye molecules can escape from the silica base. To overcome this shortcoming, organically modified silicates (ormosils) was used. It is the sol-gel glass containing organic components linked to the Si atom by stability - covalent bonds. This ormosil type is

7 5 prepared by the sol-gel method from alkoxysilic containing an organic group linked to Si atom by a strong Si-C bond without separation of the hydrogenolysis. The silica NPs used in medical - biological applications are often functionalized with the amino, carboxyl and thiol functional groups. The functionalization is processed by using precursors with the organic groups such as the amino group (NH 2 ), the carboxyl group (COOH) or the thiol group (SH) which are biocompatible Synthesized methods Organic dye-doped silica NPs are often synthesized by three methods: Stöber method, the micelle method and the reverse micelle method The physicochemical characteristics Matrix Materials Silica matrix of the NPs has two roles: first, generating pores which disperse dye-doped. Second, protecting the dye-doped from the external environment i.e from optical decomposition. Since the free oxygen concentration in silica matrix is three order lower than that of normal solvent, the optical durability of dye molecules is expected to be improved when they are inside the matrix. Therefore, the quality of matrix is an important factor affecting the quality of the silica NPs when they are used as fluorescent markers The luminance and optical durability (i) The silica NPs have a very high luminance because they contain a large amount of dye molecules. Thus, the fluorescence intensity of a silica nanoparticle can be generally a thousand times greater than the fluorescence intensity of a dye molecule in the same solvent substance. The luminance can be controlled by changing numbers of dye molecules in a

8 6 silica nanoparticle with the greatest dye density limited only by the fluorescence quenching. (ii) Optical durability is a particularly parameter indicator for use of nanoparticles in biological applications. Measurements and observations in biology often takes place in an extended period of time. Silica matrix acts as a dye protective shell from the direct effect of light stimulus. Therefore, optical durability of dye-doped silica-base particles is larger than that of the traditional dye corresponding in the same condition. This makes dye-doped silica NPs suitable for applications that require high signal to noise ratio Silica NPs of bio-functionalization Requirements of silica NPs of bio-functionalization NPs in general and silica NPs in particular can be applied in biology, they must meet some following fundamental requirements: (i) disperse in water; (ii) having appropriate functional groups and surface chemistry; (iii) having protective shell; (iv) do not affect the identifying functions of biomolecules and the ability to link to receptor molecules; (v) does not affect the morphology and structure of biological molecules. Therefore, the size of NPs must be smaller than a certain limit value Conjugation of silica NPs with biomolecules The silica NPs are linked with biological molecules in two main ways: direct and indirect bindings. Direct binding: Biological molecules adsorb onto the surface of nanoparticles via non-covalent bonds. This binding is nonspecific. Indirect binding: using an other substance (proteins, antibodies, peptides, etc.) to serve as the linking bridges between biomolecules and nanoparticles. This substance consists of two active heads: one head

9 7 associated with NPs and an other head attached to biomolecules. CHAPTER 2. EXPERIMENTS Sol-gel method is used to synthesize organic dye-doped silica NPs (RB and FITC). The surfaces of NPs are functionalized with biocompatible functional groups such as amine, carboxyl or thiol. The copolymer reaction with TEOS or methyltriethoxysilane MTEOS is processed by using the organic precursors with the organic groups such as the amino group (NH 2 ), the carboxyl group (COOH) or the thiol group (SH) affiliated with Si. The silica NPs are also functionalized by coating a shell of poly ethylene glycol (PEG), protein as bovine serum albumin (BSA) or streptavidin (SA). This shell having the effect of protection and also act as biocompatible functional groups. The chemical structure was investigated by analyzing their infrared absorption spectrum. Particle morphology and size are assessed through images SEM, TEM, and DLS and FCS measurements. The optical properties of materials are studied by absorption spectrum (UV-Vis), fluorescence spectrum and luminescent lifetime measurements. The methods of medical and biological applications are surveyed through the flow cytometry device and the optical microscope. CHAPTER 3. SYNTHESIZED NPs AND THEIR OPTICAL PROPERTIES 3.1. Results of synthesizing of silica NPs with various functional groups on the particle surface using the micelle method The silica nanoparticles are synthesized by the normal micelle method with functional groups (-OH, - NH 2, - SH) through the polymerization reaction with MTEOS. Simultaneously, particles are coated by a PEG layer

10 8 and BSA and SA protein layers to both synthesize functional groups and participle protection in the biological environment Particle shape and size: Silica NPs synthesized are spherical like shape and has a diameter in range nm Chemical structures: Chemical structures of particles are examined by the characteristic lines for O-Si-O, Si-CH 3, Si-C bonds, etc. in the matrix network and the characterized oscillation lines of functional groups on the particle surface The optical properties Absorption and fluorescence Absorption Normalized :4SBA30 2:4SBA30DT 3:4SBT30 4:4SBA30PEG 5:4SBO30 6:RB/H2O Wavelength (nm) Fluorescence Normalized : 4SBA30 2: 4SBA30DT 3: 4SBT30 4: 4SBA30PEG 5: 4SBO30 6: RB/H2O Wavelength (nm) Figure 3.8 and 3.9. Normalized absorption and fluorescence spectra of silica NPs with different functional groups The absorption and fluorescence spectra of silica NPs with various functional groups on the particle surface are similar to that of free RB dye in water but a slight blue-shift. It means that there is the interaction between doped dye molecules and the matrix network. However, this interaction is weak and does not affect on the optical properties of the dye. From the absorption and fluorescence spectra, parameters and optical properties of RB doped silica nanoparticles such as the quantum efficiency,

11 9 the fluorescence lifetime, the radiative relaxation rate and non-radiative relaxation rate, with different functional groups are calculated The quantum efficiency and fluorescence lifetime The quantum efficiency (QE) Q and fluorescence lifetime τ of this sample array are all increase comparing with these of free RB molecules in water. The results also demonstrate that key the main reason of QE increase is the increase of radiative relaxation rate Γ r while the τ increase mainly due to the decrease of non-radiative relaxation rate Γ nr (Figure 3.10). These results are consistent with the behavior of dye molecules in solutions. This demonstrates that the functionalization does not have strong influence on the optical properties of dye molecules. Lifetime (ns) Quantum efficiency Q Q Fit line Speed of non-radiative relaxation rate (x10 8 s -1 ) Γ r /Γ nr Figure The relationship between τ and Γ nr ; QE and Γ r /Γ nr 3.2. Results of synthesis of silica NPs with various sizes by the normal micelle method The absorption and fluorescence spectra of silica NPs doped with RB represent the absorption and fluorescence spectra of free RB in water. However, spectrum absorption peaks shift from 1 nm to 6 nm and fluorescence spectral peak shift from 4 nm to 10 nm towards the longer

12 10 wavelength. This translation means that the interaction between RB and silica matrix is weak and does not affect on the optical properties. We have showed that (i) At a same absorption, a same numbers of dye-doped molecules per a volume unit, the synthesized dye-doped NPs have higher fluorescence intensity than free RB molecules in water. This means that luminescence efficiency of dye molecules in NPs is higher than that of free RB dye molecules in water. The enhancement of fluorescence intensity is explained that dye molecules distributed among the porous holes in the silica matrix can be protected out of the fluorescence quenching by collisions and optical decomposition due to interaction with oxygen. In addition, the fluorescence intensity of free RB molecules in water can be measured for the highest concentration ~ 10-2 mol/l. At high concentration, the fluorescence quenching phenomenon becomes dominant and fluorescence almost completely extinguished. However, the absorption and fluorescence spectra of dye-doped NPs can be observed with high intensity which corresponding to a concentration of 10-2 mol/l of free RB in water. (ii) From the value of Q and τ of NPs with various sizes, radiative relaxation rate Γ r and non-radiative relaxation rate Γ nr are calculated. The results also show that Q and τ of RB molecule in NPs are also higher than that in water. Q increases due to increase of lifetime τ and radiative relaxation rate Γr.

13 11 Absorption normalized :2SBO20 2:4SBO30 3:5SBO40 4:6SBO60 5:Rb/H2O Fluorescence normalized :2SBO20 2:4SBO30 3:5SBO40 4:6SBO60 5:RB/H2O Wavelength (nm) Wavelength (nm) Figure 3.13 and The normalized absorption and fluorescence spectra of samples of silica NPs at different particle sizes Lifetime (ns) Quantum efficiency Q Q Fit line Speed of non-radiative relaxation rate (x10 8 s -1 ) Γ r/γ nr Figure The relationship between FL and radiative relaxation rate; QE and Γ r /Γ nr The radiative relaxation rate of RB molecule caused namely: - The collisions between dye molecules. This channel is minimized when dye molecules located in the porous holes of silica matrix. - The interactions with the surrounding environment. If the environment is water, the fluorescence will decrease due to the interactions of dye molecules and the free oxygen molecules in the watere. Silica matrix contains less free oxygen, three orders than that in water. Therefore, radiative relaxation rate through this channel will be significantly reduced when dye molecules are in silica matrix. On the other hand, there are the hydroxyl group (OH) on the surface of porous holes, if RB molecules interact strongly with silica matrix, the radiative relaxation rate also can

14 12 increase due to the reaction with the hydroxyl group (OH). Another conclusion is that the weaker interactions between RB dye and matrix the better optical properties (QE and FL) of NPs have, and vice versa Results silica NPs coated by proteins of Bovine serum albumin (BSA) and streptavidin (SA) To protect as well as biocompatible functionalize the NPs, dye-doped silica NPs are coated by BSA and SA proteins. The results showed that the protein coating process does not affect optical properties of dye-doped participles. The optimize amount of SA and BSA to coat particles was investigated and discussed by this study. With particle sizes in range of 100nm and concentration of particles per ml, the amount of BSA sufficient to coat particles is from 1.0 to 1.2 mg/ml. The larger amount of BSA will cause excess of BSA affecting on optical homogeneity of the solution. These data of BSA are only true to a type of particle with size and specific concentration Synthesis of silica NPs by reverse micelle method Effects of ethanol content on the reaction of APTES with FITC. From the dependence of the absorbance of the FITC@APTES solution on the ethanol content, we conclude that when ethanol amount is increased, the absorption spectrum is unchanged. However, the absorbance is proportional to ethanol concentration up to a certain volume. After this certain concentration, no more FITC@APTES are generated. Absorbance (a.u) FITC@APTES1 FITC@APTES3 FITC@APTES5 FITC@APTES7 FITC@APTES10 FITC/Ethanol Wavelength (nm) Absorption of FITC@APTES Amount of ethanol

15 13 Figure Absorption spectrum of free FITC dye and APTES. The dependence of the absorbance of APTES on ethanol content When the ethanol content increases, PDI index of silica NPs solution increases. When the ethanol content increases at 7, the cloud point phenomenon starts to appear. When the ethanol content increases to 10, clusters are formed instead of individual particles. This phenomenon is explained that when the amount of ethanol increases, the amount of residual water in the solution containing the APTES compound also increases. The rates of hydrolysis and condensation reactions increase that leads to large particle sizes. Simultaneously, the reactions between precursor molecules to form small particles increase. These particles will stick together to form clusters which are not dispersed separately. Therefore, in order to synthesize NPs with desired size and good quality, it is important to control the amount of water in the APTES solution. Therefore, we choose samples of the APTES solution with the lowest amount of ethanol (FITC saturated in ethanol) to synthesize FITCdoped silica NPs Optical properties Standardized absorption and fluorescence spectra of free FITC molecule and the FITC@APTES compound in ethanol and the silica NPs are shown on Figure 3.29 and Absorption normalized FITC_ethanol FITC@APTES SiO2@FITC Fluorescence Normalized FITC-ethanol Wavelength (nm) Wavelength (nm) Figure 3.29 and The normalized absorption and fluorescence spectra of free FITC molecules and the FITC@APTES compound in

16 14 ethanol and the silica NPs Figure 3.29 shows that the absorption spectrum of the FITC molecules in silica particles is different to that of APTES and more different compare to free FITC molecules in ethanol. This difference in absorption spectra is because of the strong interaction of FITC molecules to the silica matrix. Inversely compare to the RB, FITC molecules is introduced to the silica matrix under the form of a precursor involving in the polymerization process. FITC molecules become a part of the network of SiO 2 matrix. Fluorescence spectrum of FITC molecules in silica particle and ethanol is shown in Figure Surprisingly, the fluorescence of doped FITC silica NPs is almost the same to FITC in ethanol. This indicates that the strong interaction between FITC molecules and silica matrix did not affect on the luminescent properties of the molecules. The optimum ethanol amount for the synthesize process of FITC doped NPs was also investigated. The highest luminescent intensity is the sample FITC@APTES7, the lowest one is the sample APTES1. However, as discussed above, the sample of APTES7 are large in sizes and not monodispersed. Thus, the FITC@APTES1 is used to had both optical quality and high morphology Synthesis of silica NPs by the Stöber method Along with the synthesis of FITC-doped silica NPs by the reverse micelle method, we also synthesize and survey the morphology and the optical properties of FITC-doped silica NPs by the Stöber method Optical properties The absorption spectrums and normalized absorption of doped NPs fabricated by Stöber method show the similar absorption spectrum to the spectrum of FITC doped silica participles synthesized by reverse micelle method. This demonstrates the same networks of SiO 2 matrix synthesized by two methods.

17 15 Absorbance (a.u) ST900 ST1000 ST1100 ST Wavelength (nm) Absorption Normalized ST900 ST1000 ST1100 ST Wavelength (nm) Figure The absorption spectrum and normalized absorption of the APTES compound in ethanol and the silica NPs with different sizes Normalized fluorescence spectrums (Figure 3.34) show that the fluorescence spectrum form of FITC dye molecules in NPs is similar to that of free FITC dye molecules in ethanol. However, the spectral peak translates in 6-11 nm range towards shorter wavelength. Fluorescence Intensity (a.u) 1000 ST900 ST1000 ST ST1200 FITC@APTES Wavelength (nm) Fluorescence Normalized ST900 ST1000 ST1100 ST1200 FITC-Ethanol Wavelength (nm) Figure Fluorescence and normalized fluorescence spectrum of FITC dye molecules in ethanol and silica NPs with different sizes As the results, it can be confirmed that the Stober method is also a good method to synthesize FITC- doped silica NPs Functionalization In the framework of the thesis, we functionalized the FITC-doped silica NPs with the amine group (NH 2 ) and the carboxyl group (COOH) that are two very common functional groups in the molecular structure of biology Results of functionalization of NPs with the functional group containing -NH 2

18 16 Results of DLS measurement and Zeta potential showed that particles before and after functionalization both have low PdI. It means that the particles are well monodispersed. The increment in size of silica NPs after functionalization proves that the NPs is coated by APTES. At the same time, Zeta potential increases from mv to -0.8 mv. It indicates the replacement of a proportion of OH groups functional group containing - NH 2 (existing as the NH 3+ ions) which tents to a positive value of Zeta potential. Table 3.10: Results of DLS measurement and Zeta potential of silica NPs with the functional group containing -NH 2 synthesized by the Stöber method Samples TEM sizes (nm) Parameters of DLS Hydrodynamic diameter PdI (nm) Zeta potential (mv) ST ST1000NH In order to be able to used synthesized particles, one of criteria characteristic is the stability of NPs. Therefore, three months after synthesis process, we do the investigation of all synthesized NPs. All samples preserved in water has been agglomerated to large clusters which can be observed by naked eyes. Table 3.11: Results of DLS measurement and Zeta potential of samples of silica NPs with the functional group containing -NH 2 Samples Parameters of DLS Hydrodynamic diameter (nm) PdI Notes ST1000NH 2 (Tris) After 03

19 17 months Samples preserved in Tris buffer showed that the PdI index of samples after 3 months still remains at low value (0.056). Which corresponds to a well monodispersed sample (Table 3.11). We conclude that particles are well preserved in Tris medium. This results has been predicted since Tris is an amine consisting of one amine group and three hydroxyl groups. This structure can compensate and naturalized the statistic charge on the surface of silica NPs. In this condition, the NPs are tent to stay separate in the solution Functionalization of silica NPs with the functional group containing -COOH The thesis also presents the results of functionalization of silica NPs with the functional group containing - COOH on the particle surface. When amount of precursor C 3 H 6 Na 2 O 5 Si (DDOS) used for consecutively polymerize on the particle surface increases, Zeta potential also increases. The functionalization occurs until the Zeta potential reaches the value leading to stable participle samples and monodispersed participles in the medium (ς > 30 mv). CHAPTER 4. SILICA NPs USED AS A BIOLOGICAL MARKER 4.1. Recognition E. coli O157: H7 bacteria by immunofluorescence method Imaging by fluorescence microscopy Photos taken by fluorescence microscopy shows that E. coli O157: H7 bacteria after binding with the SiO 2 KT complex has high luminescence (gold red - light spots, Figure 4.1b). Figure 4.1.c shows that the silica NPs cling on the bacterium surface. The tomography image taken by using confocal technique (Figure 4.1.d) shows that silica particles only cling on the target bacterium surface but inside bacteria. It was confirmed that the SiO 2 KT complex only clings in a high rate on the surface

20 18 under the immune principle (antigen - antibody). a b c d Figure 4.1. Images of cells of E. coli O157:H7 bacteria Development of a fluorescent spectral method for quantitatively determining of E. coli O157:H7 bacteria To determine the quantity of E. coli O157:H7 bacteria, the bacteria surface were fully cover by antibody-silica NPs complex under the immunofluorescence principle. The fluorescence intensity is expected that depends linearly to the number of bacteria in the sample. In figure 4.3b, the experiment data, red square line, shows a good agreement to the assumption. Using this results as a calibration data, we can accurately determine the number of bacteria in solution.

21 19 Fluorescence Intensity (a.u) x10 4 CFU 7.5x10 3 CFU 5.0x10 3 CFU 2.5x10 3 CFU 1.0x10 3 CFU 7.5x10 2 CFU 5.0x10 2 CFU 2.5x10 2 CFU Fluorescence Intensity (a.u) Wavelength (nm) x x x x10 4 Number bacteria(cfu) Figure 4.3. a) The fluorescence spectrum of bacterium samples with different concentrations marked with the SiO 2 RB@KT complex b) Fluorescence intensity according to the number of bacteria (red line) and line of best fit (green, Y = 0,008.X) These results were compared with colony counting methods on agar plates. The results show that there is a high correlation between the two methods (Figure 4.4). Thus, fluorescent spectral method can use to detect and quantify bacteria. 9.0x10 4 Number of bacteria (CFU) 6.0x x Plate counting Spectrofluorometer Figure 4.4. Comparison of two methods of detection of bacteria: colony counting method (left) and fluorescent spectral method (right)

22 Identifying breast cancer cells Detection of breast cancer cells by images of fluorescence microscopy Fluorescent images of KPL-4 cells freshly incubated with the SiO 2 RB@HER2 complex (Figure 4.5.A) and with the SiO 2 RB@BSA complex (Figure 4.5.B). On the image of fluorescence microscopy (Figure 4.5.A), we can see the silica particles (red) on membrane and surround the nucleus of KPL-4 cells. In order to verify nonspecific, control samples of the SiO 2 RB@BSA particles (without antibodies) were incubated with KPL- 4 cells. The results showed that (Figure 4.5.B) almost no silica cling on KPL-4 cells. It confirms the binding of SiO 2 RB@HER2 in figure 4.5A is specific. Figure 4.5. Fluorescent images of KPL-4 cells freshly incubated with the SiO 2 RB@HER2 complex (A) and with the SiO 2 RB@BSA complex (B) Detection of quantity of breast cancer cells by cytometry device After staining, we count cells by a FACS Canto flow cytometer (BD). The control samples contains two types of He La and KPL4 cells with a ratio of 1 to 1 with or without being incubated with SiO 2 RB@BSA complex. The results of two control samples show identical distribution

23 21 which have maximum intensity at about 2, This histogram is taken as the background. The samples containing two kinds of HeLa and KPL-4 cells with a ratio of 1:1 and 5:1 is used to verify the technique. At the ratio 1:1, another distribution is found at the fluorescence intensity 2, This distribution can be firmly assigned to be the contribution of KPL-4. At lower portion of KPL-4, ratio 5 : 1, only one distribution is found at the fluorescence intensity equals to 6, The counted number is larger than that when the two cells haves the same concentration. However, a narrower distribution, lower fluorescence proposes a same number of KPL-4 cell. This result confirms that the SiO 2 HER2 complex has the ability to specifically recognize KPL-4 cells in the mixture with HeLa cells even at low ration. Figure 4.11 and Count of KPL-4 cells in the mixture of Hela and KPL-4 cells with a ratio of 1 to 1 and 5 to 1 CONCLUSIONS The dissertation revealed the methods of synthesis and functionalization of organic dye-doped silica NPs. The optical properties of synthesized NPs are investigated. Several attempt to apply the material in identifying biological elements has been done. The research objectives have

24 22 been achieved and can be summarized as follows: A. Synthesis 1. Silica NPs are successful synthesized with diameter in range of nm well dispersed in water by the normal micelle method. The NPs has been functionalized with biocompatible group such as the amino group (NH 2 ), the sulfhydryl group (SH) and the carboxyl group (COOH) and coatings of bovine serum albumin (BSA) and streptavidin protein. 2. FITC - doped monodispersed silica NPs are synthesized with a nm range in diameter by the reverse micelle method and the Stöber method. These NPs were functionalized with the amino group (NH 2 ) and the carboxyl group (COOH) by the polymerization method with the appropriate precursors. The NPs functionalized with the amino group (NH 2 ) are very stable in Tris buffer (PDI <0.1) for a storage period of 3 months. B. Optical properties 1. Interaction between RB molecules and silica matrix is weak. Interactions between FITC molecules and silica matrix is a strong interaction leading to which has effects on absorption spectrum of this molecule. However, the survey the form of fluorescence spectra showed that both those interactions do not affect optical properties of the dye molecules studied. The functional groups, coatings of BSA and SA protein do not affect the optical properties of the RB and FITC molecules in particles. 2. The study of optical and physical properties of the RB molecules in silica matrix showed that the quantum efficiency Q and luminescence lifetimes τ of the RB molecules in NPs is greater and longer than that of free RB in water due to minimization of fluorescence quenching. The increase of QE is primarily due to the increase of the speed of radiative relaxation rate Γ r and the increase of luminescence lifetime τ (LF) is mainly due to the reduction of the speed of non-radiative relaxation rate Γ nr. C. Applications

25 23 1. It has successfully synthesized the complexes of RB - doped silica NPs and FITC-doped NPs - specificity monoclonal antibodies E. coli O157:H7 bacteria and HER2 beast cancer cells (SiO 2 RB@KT, SiO 2 RB@HER2, SiO 2 FITC@HER2). 2. The complexes of SiO 2 RB@KT, SiO 2 RB@HER2, SiO 2 FITC@HER2 are used to identify specificity bacterial cells of E. coli O157: H7 and BT-474 and KPL-4 breast cancer cells by the immunofluorescence method. 3. It is to use fluorescence spectral method to construct determining methods of the amount of target bacteria E. coli O157: H7. 4. The correlation between the number of cells with fluorescence intensity was studied to find out capacity of detection breast cancer target cells of the SiO 2 HER2 complex by the flow cytometric method. LIST OF RESEARCH WORKS USED IN THE THESIS 1. Tong Kim Thuan, Tran Hong Nhung, Tran Thanh Thuy, Pham Cong Hoat, Pham Minh Tan, Jean Claude Brochon, Patrick Tauc, Nguyen Thi Thanh Ngan, Utilization of fluorescent nanoparticles for multiplex detecting and monitoring microorganisms, Journal of Biotechnology (2009), 7(4): Pham Minh Tan, Tran Thu Trang, Tran Thanh Thuy, Nghiem Thi Ha Lien, Vu Thi Thuy Duong, Tong Kim Thuan và Tran Hong Nhung. Synthesis, optical properties of dye doped ormosil nanoparticles for biolabeling application, Journal of Science and Technology of Thai Nguyen University (2012), 96(08), Minh Tan Pham, Thi Van Nguyen, Thuy Duong Vu Thi, Ha Lien Nghiem Thi, Kim Thuan Tong, Thanh Thuy Tran, Viet Ha Chu, Jean- Claude Brochon and Hong Nhung Tran, Synthesis, photophysical properties and application of dye doped water soluble silica-based nanoparticles to label bacteria E. coli O157:H7, Advances in Natural

26 24 Sciences: Nanoscience and Nanotechnology, IOP Publishing, 3 (2012) Hong Nhung Tran, Thi Ha Lien Nghiem, Thi Thuy Duong Vu, Minh Tan Pham, Thi Van Nguyen, Thu Trang Tran, Viet Ha Chu, Kim Thuan Tong, Thanh Thuy Tran, Thi Thanh Xuan Le, Jean-Claude Brochon, Thi Quy Nguyen, My Nhung Hoang, Cao Nguyen Duong, Thi Thuy Nguyen, Anh Tuan Hoang and Phuong Hoa Nguyen, Dye-doped silicabased nanoparticles forbioapplications, Advances in Natural Sciences: Nanoscience and Nanotechnology, IOP Publishing, 4 (2013) Hong Nhung Tran, Thi Ha Lien Nghiem, Thi Thuy Duong Vu, Viet Ha Chu, Quang Huan Le, Thi My Nhung Hoang, Lai Thanh Nguyen, Duc Minh Pham, Kim Thuan Tong, Quang Hoa Do, Duong Vu, Trong Nghia Nguyen, Minh Tan Pham, Cao Nguyen Duong, Thanh Thuy Tran, Van Son Vu, Thi Thuy Nguyen, Thi Bich Ngoc Nguyen, Anh Duc Tran, Thi Thuong Trinh and Thi Thai An Nguyen, Optical nanoparticles: synthesis and biomedical application, Advances in Natural Sciences: Nanoscience and Nanotechnology, IOP Publishing, 6 (2015)