Summary of doctoral thesis in Material science: Research on the synthesis, properties of GdPO4:Tb3+ and Gd2O3:Eu3+ nanomaterials oriented for biomedical applications
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Successful synthesis of GdPO4:Tb3+ nanorods and Gd2O3:Eu3+ nanospheres; coating GdPO4:Tb3+ nanorods and Gd2O3:Eu3+ nanospheres with a silica shell, functionalizing with NH2 functional group, conjugating with anti-venom and anti-CEA antibodies for further biomedical applications.
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Nội dung Text: Summary of doctoral thesis in Material science: Research on the synthesis, properties of GdPO4:Tb3+ and Gd2O3:Eu3+ nanomaterials oriented for biomedical applications
- MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY ----------------------------- GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY PHAM THI LIEN RESEARCH ON THE SYNTHESIS, PROPERTIES OF GdPO4:Tb3+ AND Gd2O3:Eu3+ NANOMATERIALS ORIENTED FOR BIOMEDICAL APPLICATIONS Major: Optical, optoelectronic and photonic materials Code: 62 44 01 27 SUMMARY OF DOCTORAL THESIS IN MATERIAL SCIENCE Ha Noi - 2020
- The thesis has been completed at: Graduate University of Science and Technology - Vietnam Academy of Science and Technology Science supervisors: 1. Dr. Nguyen Thanh Huong 2. Prof. Dr. Le Quoc Minh Reviewer 1: Reviewer 2: Reviewer 3: The thesis was defended at Evaluation Council held at Graduate University of Science and Technology - Vietnam Academy of Science and Technology on , 2020. Thesis can be further referred at: - The Library of Graduate University of Science and Technology. - National Library of Vietnam.
- 1 INTRODUCTION 1. 1. The necessity of the thesis Luminescent nano-materials containing Rare Earth (RE-NP) have received much attention in recent years. Notably, the materials GdPO4: Tb3+ and Gd2O3:Eu3+ become the critical research tool for the wide- ranging biomedical applications in the diagnosis and treatment of human disease. Magnetic resonance imaging (MRI) examination and especially live-cell fluorescence microscopy are widely utilized in hospitals and clinics. In Vietnam, many research groups have focused on the synthesis of luminescent materials as photonic biomarkers, creating the analytical tool for assessing the quality of biologicals, foods and vaccines. However, most of the biomedical applications are primarily based on a single functional tool, e.g. either luminescent material as a biomarker or magnetic material as a hyperthermia agent for enhancing the contrast of image. Therefore, it is urgent to develop a multifunctional tool, especially simultaneously possessing fluorescence and magnetic properties in a single system to improve the efficiency of diagnostic and treatment processes. According to the latest reports in the field of rare-earth luminescent materials, we decide to chose Terbium (Tb3+) and Europium (Eu3+)-activated Gadolinium (Gd3+) compounds as research objects. To our knowledge, these kinds of materials can meet the requirement of multifunctional tool because Gd3+ ions simultaneously exhibit fluorescence and magnetic properties, thus making them well- suited to serve as nanoprobes in the diagnostic and treatment of human disease.
- 2 To meet the requirement for diverse applications, the aim of our thesis is to synthesize the luminescent nanomaterials with good repeatability, well-controlled morphology, high solubility in water and highly efficient luminescence. My doctoral thesis is entitled: “Research on the synthesis, properties of GdPO4:Tb3+ and Gd2O3:Eu3+ nanomaterials oriented for biomedical applications”. 2. Research objectives of the thesis - Successful synthesis of GdPO4:Tb3+ nanorods and Gd2O3:Eu3+ nanospheres. - Coating GdPO4:Tb3+ nanorods and Gd2O3:Eu3+ nanospheres with a silica shell, functionalizing with NH2 functional group, conjugating with anti-venom and anti-CEA antibodies for further biomedical applications. - Initial experimental application of the nanocomplexs based GdPO4:Tb3+ nanorods and Gd2O3:Eu3+ nanospheres using immunofluorescence analysis method for labelling cells and detecting the antigen of cobra venom and CEA antigen of colorectal cancer cells. 3. Research contents of the thesis In the following chapter 1, we present a brief overview of rare- earth doped luminescent material; GdPO4:Tb3+/Gd2O3:Eu3+ based luminescent materials and objects for biomedical application. Next, we describe experimental methods and material characterizations used in the thesis. We then present the material characterization results of GdPO4:Tb3+ nanorods and Gd2O3:Eu3+ nanospheres. We also examine and determine the magnetic hyperthermia property of GdPO4 and GdPO4:Tb3+ nanorods. These nanomaterials were then conjugated
- 3 with anti-venom and anti-CEA antibodies for further biomedical applications. Finally, we demonstrate the great potential of these multifunctional nanocomplexs for detecting cobra venom antigen and the CEA antigen of colorectal cancer cells. We conclude in the last chapter. In this thesis, the luminescent nanomaterials have been synthesized by using hydrothermal process and multistep synthesis approach. Surface morphology and structural characteristics of as- synthesized nanomaterials were examined with field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDX). The optical properties of as-prepared samples were measured with the spectrometers (iHR550, Acton SP2300i and Fluorolof FL3- 2-2, Horiba (USA)). The magnetic properties were investigated with magnetic field-dependent magnetization measurement using vibrating sample magnetometer (VSM) system. The measurement of magnetic induction heating is performed with the commercial RDO-HFI (IMS). The biological experiments were performed at the Laboratory of Molecular Biology (Vietnam Military Medical Academy). Novel attribution of the thesis: - Successful synthesis of GdPO4:Tb3+ nanorods with a length of 300– 500 nm and width of 10–30 nm through hydrothermal process and Gd2O3:Eu3+ nanosphere with a diameter of 145–155 nm via multistep synthesis approach. - Successfully coating GdPO4:Tb3+ nanorods and Gd2O3:Eu3+ nanospheres with a silica shell, functionalizing with NH2 functional group, conjugating with anti-venom antibodies and anti-CEA
- 4 antibodies to detect the cobra venom antigen and CEA antigen of colorectal cancer cells. - The magnetic inducting heater effect of GdPO4 and GdPO4:Tb3+ have been investigated, at 300 Oe, 390 kHz, the temperature reach to 44 - 49 oC in 1500 s. The results indicating the synthesized materials are well-suited for application in magnetic hyperthermia therapy. The thesis is divided into 4 chapters: Chapter 1: Rare-earthe doped luminescent nanomaterials. Chapter 2: Experimental methods. Chapter 3: Synthesis of GdPO4:Tb3+ nanorods and the application of GdPO4:Tb3+ nanocomplexs for the detection of cobra venom antigen. Chapter 4: Synthesis of Gd2O3:Eu3+ nanospheres and the application of Gd2O3:Eu3+ nanocomplexs for the detection of the CEA antigen of colorectal cancer cells. The main results of the thesis were published in 7 scientific papers. CHAPTER 1: RARE EARTH ION-BASED LUMINESCENT NANO MATERIALS 1.1. Introduction of luminescent nano materials 1.2. Rare earth ion-based fluorescente materials 1.2.1. Luminescent properties of rare earth compounds 1.2.1.1. Characteristics of Gd3+ ion 1.2.1.2. Characteristics of Tb3+ ion 1.2.1.3. Characteristics of Eu2+ and Eu3+ ions 1.2.2. Allowed electronic transition in RE3+ ion 1.2.3. Energy transfer process
- 5 (a) (b) Figure 1. 7. Energy level diagram and energy transfer mechanism of Gd3+ ion to Eu3+ ion (a), Gd3+ ion to Tb3+ ion (b) 1.3 Features and applications of Gadolini 1.3.1. Gadolini material 1.3.2. The luminescent host material of Gd2O3 1.3.3. The luminescent host nanomaterial of GdPO4. 1.4. Characteristics of cobra venom and CEA antigens 1.4.1. Characteristics of Naja atra cobra venom 1.4.2. Characteristics of CEA colorectal cancer antigen CHAPTER 2. EXPERIMENTAL METHOD 2.1. Chemical methods 2.1.1. Hydrothermal method 2.1.1.1. Introduction of hydrothermal method Characteristics of material can tune by adjusting the following parametrs: pH, temperature, speed of reaction, time of hydrolysis, and crystallographic influence on morphology, magnitude and properties of final products. Synthesis process of GdPO4:Tb3+
- 6 Figure 2.4: Schematic representation for synthesis process of GdPO4:Tb3+ 2.1.2 Multi-step chemical synthesis method 2.1.2.1 Introduction of a multi-step chemical synthesis method 2.1.2.2. Synthesis process of Gd2O3:Eu3+ Figure 2.5. Experimental diagram for preparation of Gd2O3: Eu3+ 2.1.3. Synthetic method of biomedical nano compounds GdPO4:Tb3+; Gd2O3:Eu3+. 2.1.3.1. Surface treatment method 2.1.3.2. Methods of surface functionalization and conjugation between luminescent nanomaterials and biologically active elements 2.1.3.3. Coating GdPO4: Tb3+ nanorods by Silica 2.1.3.4. Functionalizing GdPO4:Tb3+@Silica nanorods by -NH2
- 7 Figure 2. 7. Experimental diagram for wrapping and surface functionalization of GdPO4:Tb3+ nanorods 2.1.3.5. Preparation of GdPO4:Tb3+@Silica-NH2 nano compound with the snake venom antibody (IgG) Figure 2. 8. Diagram of synthesizing nano compound by integrating GdPO4:Tb3+@silica-NH2 with the snake venom antibody (IgG) 2.1.3.6. Coating Gd2O3:Eu3+ by silica 2.1.3.7. Functionalization of Gd2O3:Eu3+ @Silica by -NH2 Figure 2.9. Diagram of wrapping Gd2O3:Eu3+ by Silica and integrating NH2 group
- 8 2.1.3.8. Synthesis of Gd2O3:Eu3+@Silica-NH2 nano compound with the CEA antibody. Figure 2.10. Schematic illustration for the synthesis of nano compound by integrating Gd2O3:Eu3+@silica-NH2 with the CEA antibody (IgG) 2.2. Methods of material analysis CHAPTER 3: RESEARCHING RESULTS OF GdPO4:Tb3+ AND APPLICATION OF GdPO4:Tb3+ NANO COMPOUND TO DETECT THE VENOM ANTIGEN 3.1. Crystal structure, morphology, optical and magnetic properties of synthesized materials 3.1.1. FESEM analysis 3.1.1.1 FESEM images of GdPO4. 3.1.1.2. FESEM images of GdPO4: Tb3+ Figure 3.2 exhibits as-prepared materials have nanorod morphology with the length of 300-500nm and the width of 10-30nm. It can be seen the nanorods in the sample with [Tb3+]/[Gd3+] = 7% are rather uniform and not clustered in bundles compared to others.
- 9 Figure 3.2. FESEM images of GdPO4:Tb3+ with the ratio of [Tb3+]/[Gd3+] = 0, 1, 3, 5, 7, 9%. 3.1.2 TEM images of GdPO4: Tb3+ Figure 3.3. Images of GdPO4: Tb3+ coated by silica 3.1.2. Powder XRD patterns of synthesized samples As shown in figure 3.4, the X-ray diffraction peaks show that synthesized materials have single-phase tetragonal structure with the tetragonal shape and match with the peaks of standard 98-004-3396 for Gadolinium phosphate. Figure 3. 4. The X-ray diffraction patterns of GdPO4, GdPO4:Tb3+ 3.1.3. Optical properties 3.1.3.1. Luminescent spectra of GdPO4 3.1.3.2. Luminescent spectra of GdPO4:Tb3+
- 10 Figure 3.6 indicates the absorption peaks at 272 and 275nm, which are strong absorption bands of Gd3+, corresponding to 8S – 6I shift. The ones at 305 and 310nm corresponding to 8S – 6P shift are main absorption peaks of Gd3+. The excitation bands at the wavelengths of 350, 367 and 377 nm derive from Tb3+ ion which correspond to f-f electronic transitions between energy levels belonging to 4f configuration. Figure 3. 6. PLE spectra of Figure 3.7. PL spectra of GdPO4:Tb3+ (0- 9%). GdPO4:Tb3+ with the ratio of 0, 1, 3, 5, 7, 9% excited at 272nm. As shown in figure 3.7, GdPO4:Tb3+ (0- 9%) samples emit the blue light due to the 5D4→7FJ (J = 6, 5, 4, 3) transition of Tb3+. The 5 D4→7F6, 5D4→7F5, 5D4→7F4 and 5D4→7F3 transtions of Tb3+ correspond to the wavelengths of 488nm, 543, 586 and 620 nm, respectively. The strongest emission band occurs at 543 nm. 3.1.3.4. Luminescent spectra of GdPO4:Tb3+ @ silica integrated amin functional group and IgG antibody The luminescent intensity of the samples decreases in turn after functionalization, binding to the amine and IgG groups. However, such luminescent intensity slightly declines at the last step of binding to IgG, which is a favorable condition for the application of this material in the rapid detection of the cobra venom antigen.
- 11 Figure 3. 8. PL spectra of GdPO4:Tb3+(1), GdPO4:Tb3+@silica (2), GdPO4:Tb3+@silica –NH2 (3), GdPO4:Tb3+@silica –NH2-IgG (4). 3.1.4. Infrared (IR) spectra Figure 3.9. IR spectra of GdPO4:Tb3+(a), GdPO4:Tb3+@silica (b) GdPO4:Tb3+@silica-NH2 (c) và GdPO4:Tb3+@silica-NH2-IgG (d). The absorption bands of C-H (1400 cm-1), O-H of H2O (3500cm-1 and 1600 cm-1) and PO43- (1043, 622 and 545 cm-1) are observed in the IR spectrum of the samples, see figure 3.9. The presence of O-Si-O (1100-900 cm-1) and N-H (2200-2000 cm-1 and 3450 cm-1) bands proves the silica coating and surface functionalization by NH2 group were successful.
- 12 3.1.5. Magnetism of GdPO4 and GdPO4:Tb3+ Figure 3.10. Magnetic curves of Figure 3.11. The magnetic GdPO4:Tb3+ with the ratio inducting heater effect of [Tb3+]/[Gd3+] of 0, 1, 3, 5, 7, 9%. GdPO4:Tb3+ with [Tb3+]/[Gd3+]: 0, 1, 3, 5, 7, 9% It can be seen from figure 3.10 that the magnetization of GdPO4 reaches the highest value of 1,7 emu/g which is twice larger than that of Zhang’s work and as much as that of Xu’s work. The magnetization of Tb3+ doped samples decrease, nevertheless the lowest value of GdPO4:9%Tb3+ also achieves 0,8 emu/g equivalent to the value of Zhang’s group. Therefore, GdPO4:Tb3+ can be used to increase the contrast level of the magnetic resonance image MRI. The magnetic inducting heater effect of GdPO4 and GdPO4:Tb3+ have been investigated, at 300 Oe, 390 kHz (figure 3.11), the temperature reach to 44 - 49 oC in 1500 s. The results indicating the synthesized materials are well-suited for application in magnetic hyperthermia therapy. 3.2. Application of GdPO4:Tb3+ in the detection of Naja atra venom antigen The experiment of detecting the venom antigen of GdPO4:Tb @silica-NH2-IgG nano compound is shown in figure 3.12. 3+
- 13 Figure 3. 12. Diagrammatic illustration for detection of the venom antigen by Gd2O3:Tb3+@silica-NH2-IgG fluorescent nano compund 1. Attaching simultaneously the snake venom antigen and nano compound on the 6-disc wells. 2. Incubating the nano compound integrated the antivenom antibody with the venom antigen. 3. After washing, only the nano compound integrated the antibody and antigen of the venom. 4. The nano compound emits blue luminescence with excitation wavelength at 405nm 3.2.1. Evaluation of conjugating of nano material – IgG antigen based on FTIR spectra Figure 3. 13. FTIR spectra of IgG antibody samples before and after conjugating luminescent material.
- 14 The line (a) in figure 3.13 indicates the presence of the O-H (3459 cm ), N-H (2078 cm−1), C=O (1639 cm−1 to1442 cm−1) and the bond −1 of N-H in the first amine group of carbonyl (C-N) group. The peak at 1389cm-1 in the line (b) is due to the formation of C - N bond by means of the aldehyd group (O = C - H) reaction of GDA bond with NH2 of IgG. As a consequence, the combination of luminescent nanorods and anti-venom antibodies were formed. 3.2.2. Evaluating the ability of GdPO4:Tb3+@silica-NH2-IgG nano compound in the detection Naja atra venom antigen. (a) (b) (c) Figure 3. 14. Image of Naja atra venom antigen under the visible light (a); Naja atra antigen combined with nano material under the visible light (b); Naja atra antigen combined with nano material under the visible light observed under fluorescence microscope of 405nm, the magnification of 40x and eyepiece of 10x (c). The results showed that the wells incubated with nanorods-antibodies provided a green image and nanoparticles distributed uniformly throughout the field green light under a 40x fluorescence microscope (Figure 3.14 (c)). This result shows that these wells have a specific association between the nanorods-antibodies and the Naja atra cobra antigen so that when it was washed, it does not wash away. Meanwhile, other emissions were not green due to the absence of a combination of antibody-coated nanoparticles and Naja atra cobra venom antigens so they were washed away during washing. Based on
- 15 these results, it could be concluded that this Naja atra venom detection procedure can shorten the time needed for early diagnosis and improve the efficacy of treatment and thus could facilitate early treatment of snake bite and save lives. Thus, with respect to other green-emitting luminescent materials like Yb, Er-codoped NaYF4 or TbPO4·H2O- based nanomaterials, our synthesized immunoglobulin G-conjugated GdPO4·nH2O:Tb3+ nanorods are compatible for detection of antigen- antibody reaction. CHAPTER 4: RESEARCHING RESULTS OF Gd2O3:Eu3+ AND APPLICATION OF Gd2O3:Eu3+ NANO COMPOUND TO DETECT THE CEA ANTIGEN OF COLORECTAL CANCER CELLS 4.1. The analyses of structure, morphology, optical and magnetic properties of samples 4.1.1. FESEM images • FESEM images of Gd(OH)CO3.H2O:Eu3+ with the different ratio of [Urê]/ [Gd3++ Eu3+] Figure 4.1. FESEM images of Gd(OH)CO3.H2O:Eu3+ with the ratio [Urê]/[Gd3+ + Eu3+] of 20 (a), 25 (b), 30 (c), 35 (d), 40 (e) All samples obtained have nanospherical shape as seen in Figure 4.1. The diameter of such nanospheres increases with the increase of molar ratio [Urê]/ [Gd3+ + Eu3+]. Typically, the average diameter of
- 16 nanospheres is 140, 190, 210, 240 and 270nm corresponding to the molar ratio [Urê]/[Gd3+ + Eu3+] of 20, 25, 30, 35 and 40, respectively. With the ratio [Urê]/[Gd3+ + Eu3+] of 25, the nanospheres are generally the most uniform. As a consequence, this ratio is used to synthesize Eu3+-doped Gd2O3 with the molar ratio Eu3+/ Gd3+ of 3,5; 5; 6; 7; 7,5 and 8%. • FESEM images of Gd(OH)CO3.H2O: Eu3+ dried at 70oC Figure 4.3. FESEM images of Gd(OH)CO3.H2O:Eu3+ dried at 70oC with the ratio [Urê]/[Gd3++Eu3+] of 0 (a); 3,5(b); 5(c); 6(d); 7(e); 7.5(f) and 8%(g) As shown in Figure 4.3, when Eu3+ is not doped, the nanospheres are quite uniform with the diameter of about 157-173nm (Figure 4.3a). When doping Eu3+ with different molar ratios Eu3+/Gd3 + of 3.5; 5; 6; 7; 7.5 and 8%, the nanospheres have the diameter in the range of 100- 230nm. From FESEM images, for Eu3+-doped sample with the concentration of 6%, the nanospheres are rather uniform with the diameter of 185-195 nm compared to others. As a result, this sample is selected for next experiments. 4.1.2. Thermogravimetric analysis (TGA) From Figure 4.4, one can observe 4 phases including: Phase 1 (25- 200oC): the weight of sample decreases 5.79% due to the evaporation
- 17 of water molecules; Phase 2 (200-380oC): the 8.13 wt % loss is observed because of the decomposition of OH group. Phase 3: the decomposition of cacbonate group in the range of 380-550oC results in 15.21wt% loss. Phase 4: a further 28.49 wt% loss between 550 and 1000oC is caused by the disintegration of cacbonate group. In all 4 phases, there are strong points of heat absorption at 170, 319, 427 and 609 oC. These can be considered as the phase transition points, yielding to can be selected suitable processing temperature to obtain the material with a stable phase structure. Therefore, we choose the sample processing temperature of 650 oC, at this temperature the product obtained will be Gd2O3:Eu3+. Figure 4. 4. TG-DTG curves of the as-prepared Gd(OH)CO3.H2O:Eu3+ Figure 4.5 shows the diameter of Gd2O3 nanospheres (a) after annealing at 650oC with the diameter of about 104-130nm. The diameter of nanospheres in the sample containing 3.5% Eu3+ (b), 5% Eu3+ (c), 6% Eu3+ (d), 7% Eu 3+ (e), 7.5% Eu3+ (f ) and 8% Eu 3+ (g) is about 105-143nm, 117-130nm, 145 -155nm, 136-150nm, 99-130nm and 176-200nm, respectively.
- 18 Figure 4.5. FESEM của vật liệu Gd2O3:Eu3+ khi ủ ở nhiệt độ 650oC với tỷ lệ [Eu3+]/[Gd3+] là 0 (a); 3,5(b); 5(c); 6(d); 7(e); 7.5(f);8%(g). 4.1.3. TEM images of Gd2O3: 6%Eu3+ Figure 4.6. TEM images of Gd2O3: 6%Eu3+ (a) and Gd2O3: 6%Eu3+@silica (b). Figure 4.6 exhibits that the nanospheres are uniformly synthesized with the diameter of about 145-155 nm which is similar to the FESEM results (Figure 4.6a). The diameter of such nanospheres increase significantly after coating silica, around 155-165nm. The silica shell can be easily observed by naked eyes (Figure 4.6b). 4.1.4. Powder XRD patterns of as-synthesized samples The sample of Gd(OH)CO3.H2O:6%Eu3+ is almost amorphous annealed at 200oC. However, X-ray diffraction peaks are observed at 2θ: 28,70; 33,10; 47,60; 56,40; and 59.10º, in agreement with the standard of JCPDS:110604, when annealing at 650oC. This can be confirmed that the material obtained is cubic crystal.
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