Tổng hợp và khảo sát các tính chất của vật liệu nano phát quang nền NaYF4 chứa ion đất hiếm er3+ và yb3+ định hướng ứng dụng trong y sinh tt tiếng anh
MINISTRY OF EDUCATION AND TRAINING
VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY -----------------------
Ha Thi Phuong
SYNTHESIS AND LUMINESCENT PROPERTIES CHARACTERIZATION OF NANOMATERIALS BASED ON NaYF4 MATRICES CONTAINING Er3+ AND Yb3+ FOR BIOMEDICAL APPLICATION Major: Optical, optoelectronic and photonic materials Code: 9 44 01 27
SUMMARY OF DOCTORAL THESIS IN MATERIAL SCIENCE
Hanoi – 2019
The thesis has been completed at: Institute of Materials Science - Vietnam Academy of Science and Technology
Science supervisors: 1. Dr. Tran Thu Huong 2. Prof. Dr. Le Quoc Minh
Reviewer 1: Assoc. Prof. Dr. ….. Reviewer 2: Prof. Dr. ………… Reviewer 3: Assoc. ……………
The thesis was defended at Evaluation Council held at Graduate University of Science and Technology - Vietnam Academy of Science and Technology on, 2019.
Thesis can be further referred at: - The Library of Graduate University of Science and Technology. - National Library of Vietnam.
Nowadays, there are many applications of nanomaterials and products made of nanomaterials such as material science, energy, environment, electronics and especially in biomedical sciences. In biomedical science, luminescent nanomaterials could make flouorescent labelling possible and effective. Notably, in the last few years, many types of nanomaterials have become the current subjects of basic and applied research, such as nanomaterials containing rare-earth upconversion nanophosphors (UCNP). When stimulating this materialby infrared light, radiant emission will be aborted in the visible region. Therefore, they have become one of the new and recognized research objects in many sectors such as health care, security, energy .... When applying in the health care sector, the UCNP has two specific advantages compared to other luminescent materials. First of all, using infrared stimulation sources minimizes the object's self-emitting ability and enhances the contrast of micro-images. In addition, infrared light is safer for human body, which does not cause cell changes, and it can penetrate a few millimeters into human tissue, which will make a deeper intervention into the affected area.
There are many published works on UCNP types, in which oxide, fluoride base materials of ytri and gadoli rare-earth ion-doped Er3+, Yb3+, Tm3+, Ho3+ are more prominent. Studies show that nanometer-sized NaYF4 network will create upconversion luminescent effect with high, durable and luminescent performance in different conditions. This material promises many potential applications bio security, optoelectronic and especially in biomedical as image recognition (bioimaging), biological sensors (biosensing), therapy Cancer (cancer therapy). Biological identification applications in vitro and in vivo of rare earth ionic doped UCNP have high contrast ratio. Therefore, UCNP materials have great potential in designing and manufacturing biological nano complexes. Very accurately identify some types of cancer cells. In the world, some researchers’ groups collected UCNP materials in sizes ranging from a few tens to several hundred nm, luminescent green areas, fingerprint recognition applications, drug guides, nanoscale oil or they could be used in association with biomarkers that could test certain types of cells such as lung cancer cells, Hela cells. In particular, many UCNP materials of several hundred nm can mark nm-sized cells by extracellular method. Because the structure, luminescent properties and biological conjugation are some of the determinants of biomedical application, the study of these factors has always played an important role in material production. Studies in Vietnam on luminescent nanomaterials containing rare soil. Although those research have been just able to access nanotechnology, they have made important changes, creating new attraction for scientists. For upconversion nanophosphors luminescent nanomaterials, several research groups have investigated UCNPs containing Er, Yb, Tm ions, with oxides of ytri, sodiumytrifloride substrate. These works mainly on the synthesis method, structure, size and shape as well as the characteristics and mechanisms of fluorescence upconversion in a multi-photon form with the orientation of the application in imaging, optoelectronics, security printing and some research results applied in the energy industry. However, the problem of applying UCNP in cancer detection and treatment is not much. The question is how to use upconversion nanophosphors into biomedical and choose which solution is suitable for the process of functionalization, conjugation with biological activity objects. How does the combination of nanotechnology and biology allow the application of nano-sized fluorescent materials for the purpose of detecting and detecting biological molecules used in biomedical sensors and images?
2 For all the above mentioned points, I selected the topic of research on nanomaterials containing rare earth upconverted luminescence application in medical biology as the content for the thesis with the title: "Synthesis and luminescent properties characterization of nanomaterials based on NaYF4 matrices containing Er3+ and Yb3+ for biomedical application". The goal of the thesis: 1. Successfully synthesized upconversion nanophosphors NaYF4: Yb3+, Er3+, hexagonal crystal structure (β), rod shape, red light emission areas. 2. The morphology, structure and optical properties of NaYF4: Yb3+, Er3+ materials had investigated. The process of covering, functionalizing, conjugating NaYF4 materials: Yb3+, Er3+ had constructing for biomedical applications. From there, select the most appropriate material to fabricate the micro-image fluorescent marker complex. 3. The experimental research and application of biomedical nano complexes of NaYF4: Yb3+, Er3+ @ silica-N = FA to identify breast cancer cells MCF7 through fluorescence immunity technique with reverse fluorescence microscope Research methods: The thesis was conducted by experimental method. 1. The upconversion nanophosphors were synthesized by wet chemical methods (hydrothermal and hydrothermal assisted soft template PEG) to make. 2. The structure and morphology of the sample was analyzed by modern measurements such as X-ray diffraction pattern, infrared spectrometry, Field Emission Scanning Electron Microscopy. The luminescent properties of studied samples were measured on high-resolution steady-state photoluminescent setups. 3. The application of biomedical nano complexes of NaYF4: Yb3+, Er3+ @ silica-N = FA were investigated by fluorescence immunity technique. i.
Novel contributions of thesis: NaYF4: Yb3+, Er3+ upconversion nanophosphors with a hexagonal crystal structure (β) form were successfully synthesized by hydrothermal method. The mean size of the NaYF4: Yb3+, Er3+ upconversion nanophosphors is about 100 nm ÷ 200 nm in diameter and 300 nm ÷ 800 nm in length. Under an excitation wavelength of 980 nm, luminescence spectra consists of two characteristic emission bands of Er3+ ion from 510 nm ÷ 570 nm and 630 nm ÷ 700 nm. The biomedical nano complexes of NaYF4: Yb3+, Er3+ @ silica / TPGS and NaYF4: Yb3+, Er3+ @ silica – N = FA were successfully synthesized. They have luminescent properties upconversion against dominant red light emission areas. The results of experimental research and application of biomedical nano complexes NaYF4: Yb3+, Er3+ @ silica-N = FA to identify breast cancer cells MCF7 through fluorescence immunity technique with
reverse fluorescence microscope shows that the pairing of nano complexes was observed with the cells. Layout of thesis: The layout of the thesis: In addition to the introduction, conclusion, list of symbols and abbreviations, list of tables, list of images and drawings, list of published works related to the thesis. Project, appendices and references. Thesis content is presented in 4 chapters: Chapter 1: Overview of upconversion nanophosphors containing rare earth ions in NaYF4 matricies Chapter 2: Experimental techniques Chapter 3: Presents the results of synthesis and investigate characterization of NaYF4: Yb3+, Er3+ upconversion nanophosphors. Chapter 4: Presents the results of synthesis and Application of upconversion nanophosphors for marking, identification of breast cancer cells MCF7
3 CHAPTER 1. OVERVIEW OF UPCONVERSION NANPPHOSPHORS CONTAINING RARE EARTH IONS IN NAYF4 MATRICIES 1.1. Luminescent nanomaterials containing rare earth ions 1.1.1. General characteristics of rare earth elements Common electron configurations of rare earth elements: 1s22s22p63s23p63d104s24p64d104fn5s25p65dm6s2 (n = 0 ÷ 14; m = 0 or 1). The emission characteristic of rare earth ions is due to the presence of electrons inside the 4fn shell (containing up to 14 electrons) that are not filled, as they are excited to high energy levels, lowering the energy level down or down to the basics will result in luminescence. 1.1.2. Luminescent nanomaterials containing rare earth ions Luminescent materials are coming from two main parts: substrate and doped material or luminescence centers. Substrates are materials that have mechanical, structural stability and optical inertia. 188.8.131.52. Mechanism of luminescence containing rare earth ions For rare-ion-doped luminescent materials, the luminescence mechanism of rare earth ions doped basically is to shift the level of electrons in the atom. The luminescent mechanism of the material depends on the electronic configuration of the rare-earth elements. 184.108.40.206. The separation of energy levels in 4f class of rare earth elements Consider the influence of the Crystal field of the background. The 4f (not filled) electronic layer of rare earth ions is covered by 2 layers of 5s25p6, so the effect of the surrounding crystal field is weak, so it is possible to see the crystal field as a distortion. The key to this phenomenon is less dependent on the RE background, but different backgrounds will have different levels of energy depending on the different symmetries of the backgrounds. 220.127.116.11. The luminescence process of Lanthanide For a fluorescence system based on Lanthanide, two major fluorescent processes occur: excitation radiation is absorbed directly by the activator and absorbed radiation is absorbed by other ions or groups of ions. 1.2. Upconversion luminescence process 1.2.1. Upconversion Mechanism of Lanthanide Upconversion Nanophosphors (UCNPs) Most of these conventional materials exhibit luminescent emission with a Stokes shift (Scheme 1). That is they emit lower-energy photons under excitation with higher-energy photons. A few processes have been found to possess the ability to generate anti-Stokes photoluminescence. In these cases the emitted photons have higher energy than those used for the excitation. Two-photon absorption-based luminescence and secondharmonic generation are two kinds of anti-Stokes processes, which require coherent lasers as the excitation sources and which have been well-investigated. Upconversion luminescence is a distinct antiStokes process. This process can be performed by low-power and incoherent excitation sources, such as continuous-wave (CW) lasers, standard xenon or halogen lamps, or even focused sunlight. The general principle of the upconversion luminescence process is illustrated in Scheme 1.4 which demonstrates the diﬀerence to the conventional photoluminescence process.
lanthanide upconversion processes can be divided into three main classes: excited-state absorption, energy-transfer upconversion, and photon avalanche. Because photon avalanche is seldom found in nanoscale lanthanide materials, we will mainly discuss the excited-state absorption and energy-transfer upconversion processes.
Fig 1.4. Scheme 1. (a) Schematic Principle of Conventional Photoluminescence and (b) the Upconversion Luminescence Processes 1.2.2. UCNP composition Host, Activator, and Sensitizer for Lanthanide UCNPs 18.104.22.168. Host lattice considerations Selection of appropriate host materials is essential for high eﬃciency UC emissions. Basically, an ideal host material should be transparent in the spectral range of interest, have high optical damage threshold, and be chemically stable. Moreover the host lattice can aﬀect the UC eﬃciency in two ways: (i) by the phonon dynamics, and (ii) by the local crystal ﬁeld. Lanthanide UCNPs are generally comprised of an inorganic host and lanthanide dopant ions, although some complexes showed upconversion luminescence in the literature. To date, the upconversion process has been widely studied in various nanoscale host matrices, such as ﬂuorides and other halides (chlorides, bromides, and iodides), oxides, oxysulﬁdes, phosphates, vanadates, among others. Ideal host materials should have low lattice phonon energies so as to minimize nonradiative loss and maximize the radiative emission. This is because nonradiative energy loss requires the assistance of phonons present in the host lattice. Heavy halides, such as chlorides, bromides, and iodides generally exhibit low phonon energies of less than 300 cm−1. However, they are hygroscopic and are of limited use. Oxides show high chemical stability, but their phonon energies are relatively high, generally larger than 500 cm−1. In comparison, ﬂuorides usually exhibit low phonon energies (∼500 cm−1) and high chemical stability and thus are often used as host materials for upconversion processes. To date, NaYF4 has been the most popular host for lanthanide UCNPs. 22.214.171.124. Activators Since inorganic crystalline host materials do not partake in UC processes luminescent centers, referred to as activators, are required. Most Ln3+ species can theoretically be used to produce UC emissions as they have more than one excited 4f energy level (exceptions are La3+, Ce3+, Yb3+, and Lu3+). These Ln3+ species offer long-lived metastable excited states (up to 0.1s, due to low f-f transition probabilities), as well as multiple and equally spaced intermediate metastable energy levels in a ladder-like arrangement. This is exempliﬁed, using the activators of Er3+. 126.96.36.199. Sensitizers Yb3+ has a larger absorption cross-section than those of the lanthanide activators. The 2F7/2 → 2F5/2 transition of Yb3+ is conveniently resonant with many f−f transitions of Er3+, Tm3+, and Ho3+, thus facilitating eﬃcient energy transfer from Yb3+ to these ions. Thus, Yb3+ is often codoped with Er3+, Tm3+, or Ho3+ as a sensitizer to enhance upconversion emission. A CW 980 nm laser is applied as the excitation source to match the 2F7/2 → 2F5/2 transition of Yb3+ (Scheme 3). Additionally, in this review, we concentrate on summarizing
5 the advances in Yb3+ sensitized UCNPs, although some transition-metal ions have also been reported to serve as sensitizers or emitters to achieve upconversion emissions. 1.3. Several methods of synthesis luminescent nanomaterials contain rare earth ions for biomedical applications The chemical synthesis method controls the uniform size of the nanoparticle but usually produces only very small amounts, suitable for applications in sophisticated technology, such as in nano electronics, nano optics, and high-definition television, more recently in biomedical medicine. From different reaction conditions, it is possible to synthesize nanomaterials with diverse shapes such as particles, rods, fibers, disks, etc… One of those products is light-emitting nano materials containing rare earth ions such as Y2O3: Eu3+; YVO4: Eu3+; NaYF4: Yb3+, Er3+ etc. In this thesis, we present three main chemical methods for synthesizing luminescent nanomaterials containing rare earth ions for biomedical application: hydrothermal, sol-gel and microwave. 1.4. Application of upconversion nanophosphors (UCNP) in biomedical engineering. 1.4.1. UCNP for Bioimaging Luminescent imaging is very useful for early diagnosis and treatment of some incurable diseases. Over the years, much research has been focused on developing new ﬂuorescence imaging techniques and luminescent labels in order to improve the signal-to-noise ratio (SNR). Due to the special CW-excited upconversion process, upconversion emissive materials exhibit unique large anti-Stokes shifts. Thus, upconversion luminescence imaging with UCNPs as labels could be expected to completely eliminate autoﬂuorescence from biotissues in bioimaging. To date, UCNPs have been successfully applied to the bioimaging of various biological samples, including living cells and small animals. Because of the high image contrast, in vitro and in vivo biological format applications of UCNP can be determined with high accuracy, especially in in vitro conditions. 1.4.2. Lanthanide UCNP for biosensing Optical sensing and assays play vital roles in theranostics due to the capability to detect hint biochemical entities or molecular targets as well as to precisely monitor speciﬁc fundamental physiological processes. UCNPs are promising for these endeavors due to the unique frequency converting capability of biocompatible NIR light that is silent to tissues. They have the potential to reach a high detection sensitivity deeply located in the living body systems. However, the PL of UCNPs is not directly related to any biochemical property of a system except for temperature. Therefore, to be useful in a biochemical recognition process (the fundamental process in chemical sensing), UCNPs have to be used in combination with suitable recognition elements such as indicator dyes. The recognition element of a biosensor may consist of an enzyme, an antibody, a polynucleotide, or even living cells. Next, the process of biochemical recognition has to be transduced into an optical signal given by the UCNPs. The transduction was generally implemented by a FRET and/or LRET mechanism. In the following, we summarized UCNP-based in vitro temperature sensing, detection of ions (cyanide, mercury, etc.), sensing of small gas molecules (oxygen, carbon dioxide, ammonia, etc.), as well as UCNP-based bioassays for biomolecules (avidin, ATP, DNA, RNA, etc.). 1.4.3. Photothermal therapy (PTT) Photothermal therapy (PTT) employs photoabsorbers to generate heat from light absorption, leading to thermal ablation of cancer cells. In recent years, PTT has emerged as an increasingly recognized alternative to classical cancer therapies such as surgery, radiotherapy, and chemotherapy. Various nanomaterials with high optical absorbance have been highly successful in this application.
6 1.4.4. Photodynamic therapy (PDT) Photodynamic therapy (PDT) is a clinical treatment that utilizes phototriggered chemical drugs (photosensitizers) to produce singlet oxygen (1O2) to kill tumors. Typical PDT treatments involve three components: the photosensitizer, the light source, and the oxygen within the tissue at the disease site. Under appropriate light excitation (generally in the visible range), the photosensitizer can be excited from a ground singlet state to an excited singlet state, which undergoes intersystem crossing to a longer-lived triplet state and then reacts with a nearby oxygen molecule to produce highly cytotoxic 1O2. PDT has been used for therapy in prostate, lung, head and neck, or skin cancers. However, conventional PDT is limited by the penetration depth of visible light needed for its activation. NIR light in the “window of optical transparency” (750-1100 nm) of tissue can penetrate signiﬁcantly deeper into tissues than the visible light, because absorbance and light scattering for most body constituents are minimal in this range. Importantly, UCNPs can eﬃciently convert the deeply penetrating near-infrared light to visible wavelengths that can excite photosensitizer to produce cytotoxic 1O, promising their use in PDT treatment of pertinent located deeply tumors.
7 CHAPTER 2. EXPERIMENTAL TECHNIQUES 2.1. Synthesis of NaYF4: Yb3+, Er3+ nanophosphors by hydrothermal method NaYF4:Yb3+, Er3+ nanophosphors were prepared by hydrothermal method 2.2. The ways of the synthesis of NaYF4: Yb3+, Er3+ biomedical nanocomposite complexes To make NaYF4: Yb3+, Er3+ biomedical nanocomposite complexes, it is necessary to first treat the surface of the material, then the functionalization and conjugation of materials with biological agents. 2.2.1. Functionalization Bio-compatible property of the UCNPs requires a core shell structure to protect the luminescent materials and the cells involved. Besides that, the biolabels need to have specific property to target a specific tumor such as its surface have to have proper ligands that bind to the cells. 2.2.2. Method of surface functionalization and conjugation between upconversion luminescent nanomaterials and biological agents. Surface silanization (or silica coating) is an inorganic surface treatment strategy to make nanoparticles water-dispersible and biocompatible. Silica is known to be highly stable, biocompatible, and optically transparent. When utilized as a coating material, surface silanization methods can ﬂexibly oﬀer abundant functional groups (e.g., -COOH, -NH, -SH, etc.) and thus satisfy various needs of conjugation with biological molecules (e.g., folic acid, peptides, proteins, DNA, succinimid, biotin etc.). There are two types of related chemistry to coat silica onto the nanoparticles, depending on the polar nature of the capping ligands on the particle surface. One is the Stober method, which can be utilized to coat silica on hydrophilic UCNPs. Tetraethyl silicate (TEOS) is added to an excess of water containing a low molar-mass ethanol and ammonia, together with the hydrophilic UCNPs. A precise control of the amounts of involved reagents as well the pH value can lead to a uniform growth of silica onto the UCNPs. 2.3. The structure, morphology and luminescent properties of materials. The morphological observation and crystalline phase identification of all prepared samples were carried out by the way of using Field Emission Scanning Electron Microscopy (FESEM, Hitachi, S-4800), (TEM, S-4800-HITACHI và JEOL-1010) and X-ray diffraction (XRD, Siemens. The luminescent properties of studied samples were measured on high-resolution steady-state photoluminescent setup based on luminescence spectrum photometer system, MicroSPEC-2356 với Laser He-Cd. FTIR were measured on IMPACT-410, NICOLET. The microsized images of the specimens which from the virus infected cells exposure with the conjugates from nanomaterials have been viewed by a fluorescent microscopic equipment Olympus BX-40 (Japan).
CHAPTER 3. THE RESULTS OF SYNTHESIS AND INVESTIGATE CHARACTERIZATION OF NAYF4: Yb3+, Er3+ UPCONVERSION NANPPHOSPHORS 3.1. The synthesis of NaYF4: Yb3+, Er3+ upconversion nanophosphors 3.1.1. Synthesis process of NaYF4: Yb3+, Er3+ upconversion nanophosphors (Process 1) Synthesis process of nano materials containing rare earth ions NaYF4: Yb3+, Er3+ (process 1) by hydrothermal method is shown in Figure 3.1.
Solution NaOH (200; 4000; 6000; 20000) Solution A
C2H5OH + PEG
Stir / 30 minutes
Y3+ : Yb3+: Er3+ (79,5: 20,0: 0,5)
Solution B Stir / 120 minutes
190 oC/ 24 hours
Solution D Centrifuge, wash, dry
Powder NaYF4: Yb3+, Er3+ Fig 3.1. Synthesis process of nano materials containing rare earth ions NaYF4:Yb3+, Er3 (process 1) NaYF4: Yb3+, Er3+ samples were synthesized according to process 1 and listed in Table 3.1. Table 3.1. The list of NaYF4: Yb3+, Er3+samples were synthesized according to process 1. No. 1 2 3 4
Samples M1 M2 M3 M4
Y3+ (% mol) 79,00 79,25 79,50 79,75
Yb3+ (% mol) 20,50 20,25 20,00 19,75
Er3+ (% mol) 0,5 0,5 0,5 0,5
3.1.2. The results of Investigate of the structure and morphology of NaYF4: Yb3+, Er3+ upconversion nanophosphors according to process 1. 188.8.131.52. XRD pattern of the nanophosphors of NaYF4: Yb3+, Er3+ were synthesized according to process1. XRD pattern of the nanophosphors of NaYF4: Yb3+, Er3+ at 190 oC, 24 hours with different Yb3+/Y3+ ratios : M1 (20,5/79) - line 1; M2 (20,25/79,25) - line 2; M3 (20/79,5) - line 3 and M4 (19,75/79,75) - line 4 were synthesized according to procedure 1 presented in Figure 3.2 .
9 The phase structures of NaYF4:Yb3+, (1) M1 (2) M2 (3) M3 (4) M4 Cubic NaYF4
Er3+ nanophosphors were investigated by X-ray diffraction (XRD) and the results are showed in Fig. 3.2. In the XRD pattern of the sample, there are diffraction peaks at 2: 29.5o; 30.8o; 34.7o; 39.5o; 43.5o; 53.2o; 59.8o; 61.2o; 62.2o; 68.3o; 71o; 78.95o equal to hexagonal phase of NaYF4
Hex NaYF4 (4) (3) (2) (1)
(JCPDS card No. 28-1192); at 2: 28,3 o; 32,8 o; 46,9 o; 55,7 o; 58,4 ; 75,7o, 78,06 o 20
and 87,50 o. equal to hexagonal phase of 2- Theta (degree) NaYF4 (JCPDS card No. 77-2042 of α – Fig 3.2. XRD pattern of the nanophosphors of NaYF4: Yb3+, Er3+ at NaYF4 (cubic). We found that all measured 190 oC, 24 hours with different Yb3+/Y3+ ratios: M1 (20,5/79); M2 peaks are belonging to this standard (20,25/79,25); M3 (20/79,5) và M4 (19,75/79,75) pattern. 184.108.40.206. The morphology of NaYF4: Yb3+, Er3+ were synthesized according to process 1 FESEM images of the nanophosphors of NaYF4: Yb3+, Er3+ with different Yb3+/Y3+ ratios 20,5/79 (M1); 20,25/ 79,25 (M2); 20,0/79,5 (M3); 19,75/ 79,75 (M4) were presented in Figure 3. 3. The results showed that all samples were in the form of square blocks with dimensions of about 100 nm 300 nm.
Fig. 3.3. FESEM images of the nanophosphors of NaYF4: Yb3+, Er3+ with different Yb3+/Y3+ ratios 20,5/79 (M1); 20,25/ 79,25 (M2); 20,0/79,5 (M3); 19,75/ 79,75 (M4) 220.127.116.11. The upconversion luminescence (UCL) spectra of NaYF4: Yb3+, Er3+ were synthesized according to process 1. Combining morphological and structural studies, we continue to investigate the luminescent properties of NaYF4: Yb3+, Er3+ samples through upconversion luminescence (UCL) spectra.
4 - I
4 - I S 15/2 3/2
4 4 - I F 15/2 9/2
M1 upconversion luminescence M2 (UCL) spectra analysis in Figure M3 M4 3.4 show that when excited at 980 nm, all samples have a M3 fluorescence effect that converts M4 in reverse with blue at wavelengths (510 nm ÷ 570 nm) M2 and red at wavelengths (630 nm ÷ 700 nm) corresponding to 2 H11/2 → 4I15/2; 4S3/2 → 4I15/2 and M1 4 F9/2 → 4I15/2 of Er3+ ions. Thus, from the results of the 500 550 600 650 700 750 upconversion luminescence Wavelength (nm) (UCL) spectra, the red zone Fig 3.4. The upconversion luminescence (UCL) spectra of the emission of the M3 sample 3+ 3+ nanophosphors of NaYF4: Yb3+, Er3+ with different Yb3+/Y3+ ratios: (NaYF4: Yb , Er with molar ratio Yb3+ / Y3+ = 20.0 / 79.5 is 20,5/79 (M1); 20,25/ 79,25 (M2); 20,0/79,5 (M3) and 19,75/ 79,75 (M4) under NIR laser excitation at 980 nm dominant). Schematic diagram of energy, radiation and non-radiation processes of Yb3+/ Er3+codoped materials were shown in Figure 3.5.
Fig. 3.5. Schematic diagram of energy, radiation and non-radiation processes of Yb3+/Er3+codoped materials 3.2. The synthesis of NaYF4: Yb3+, Er3+ upconversion nanophosphors assisted soft template PEG 3.2.1. Synthesis process of NaYF4: Yb3+, Er3+ upconversion nanophosphors assisted soft template PEG Synthesis process of NaYF4: Yb3+, Er3+ upconversion nanophosphors assisted soft template PEG is shown in Figure 3.7.
Solution NaOH (200; 4000; 6000; 20000) Solution A
C2H5OH + PEG
Stir / 30 minutes
(79,5: 20,0: 0,5)
Stir / 120 minutes
190 oC/ 24 hours
Solution D Centrifuge, wash, dry
Powder NaYF4: Yb3+, Er3+ Fig 3.7. Synthesis process of NaYF4: Yb3+, Er3+nanomaterials assisted soft template PEG NaYF4: Yb3+, Er3+ - PEG samples were synthesized according to process 1 and listed in Table 3.4. Table 3.4. The list of NaYF4: Yb3+, Er3+ (Y3+/ Yb3+/ Er3+ = 79,5/ 20/ 0,5) with PEG =200; 4000; 6000; 20000 were synthesized according to procedure 1. No.
0 2 MP2
3.2.2. The results of Investigate of the structure and morphology of NaYF4: Yb , Er - PEG 18.104.22.168. XRD pattern of the nanophosphors of NaYF4: Yb3+, Er3+ - PEG
Fig 3.8. XRD pattern of the nanophosphors of NaYF4: Yb3+, Er3+ at 190 oC, 24 hours (M1 - line 1) , NaYF4:Yb3+, Er3+ - PEG 200 (MP2 - line 2); PEG 4000 (MP4 - line 3); PEG 6000 (MP6 - line 4); PEG 20000 (MP20 - line 5) 3+ 3+ NaYF4: Yb , Er assisted soft template PEG with NaYF4: Yb3+, Er3+ at 190 oC, 24 hours (M1 - line 1), NaYF4:Yb3+, Er3+ - PEG 200 (MP2 - line 2); PEG 4000 (MP4 - line 3); PEG 6000 (MP6 - line 4); PEG 20000 (MP20 - line 5) are showed in Fig. 3.8. The analysis results on the X-ray diffraction diagram in Figure 3.10 show the phase structure of M3 (line 1), MP2 (line 2), MP4 (line 3), MP6 (line 4) and MP20 (line 5) models. ) still has a two-phase mixed structure α, -NaYF4. The diffraction peaks on the diagram are all sharp to show that the samples are crystallized. This proves that the presence of PEG in the sample does not change the phase structure of the material. 22.214.171.124. FESEM images of the nanophosphors of NaYF4: Yb3+, Er3+ - PEG After investigating the structure of the material, we continue to investigate the effect of PEG on the morphology of NaYF4 materials: Yb3+, Er3+ (Figures 3.11 and 3.12).
Fig 3.9. FESEM images of the nanophosphors of NaYF4: Yb3+, Er3+ - PEG 200 (a) and PEG 4000 (b) FESEM image in Figure 3.9 of the MP2, MP4 samples and Figure 3.10 of the MP6 and MP20 samples show that the samples are still in the square shape with the size of about 100 nm ÷ 300 nm. This proves that the presence of soft-forming agent PEG does not change the morphology of the material.
Fig. 3.10. FESEM images of the nanophosphors of NaYF4: Yb3+, Er3+ - PEG 6000 (a) and PEG 20000 (b) 3.2.3. The luminescent properties of the nanophosphors of NaYF4: Yb3+, Er3+ - PEG Under NIR laser excitation at 980 nm, the upconversion luminescence (UCL) spectra of NaYF4: Yb3+, Er3+ - PEG were shown in Figure 3.11. Observing the spectra on the samples MP2, MP4, MP6, MP20 all appear emission peaks in the region with wavelength from 400 700 nm. The emission peaks wavelength range from 510 570 nm and from 630 700 nm, corresponding to 2H11/2 → 4I15/2; (520nm), 4S3/2 → 4I15/2
4 - I 15/2
(540nm) and 4F9/2 → 4I15/2 (650nm) of Er3+ ions. λexc = 980 nm
4 - I 15/2 4 S
H11/2 - 4I15/2
Fig 3.11. The upconversion luminescence (UCL) spectra of the nanophosphors of NaYF4: Yb3+, Er3+- PEG 200 (MP2 - line 1); PEG 4000 (MP4 - line 2); PEG 6000 (MP6 - line 3) and PEG 20000 (MP20 - line 4) under NIR laser excitation at 980 nm. Observing the emission peaks between the red and blue regions shows that the red zone emission is more dominant, especially the emission intensity of NaYF4:Yb3+, Er3+ - PEG 20000 emitting red is much higher than intensity NaYF4: Yb3+, Er3+ - PEG nanomaterial emissions have low molecular weight under NIR laser excitation at 980 nm. The β-NaYF4 hexagonal structure material has the ability to luminesce reverse conversion about 10 times larger than the -NaYF4 form. In order to synthesize materials with hexagonal structure β-NaYF4, a number of factors can be changed such as reaction time, annealing temperature, concentration of sensitizers, luminescent center concentration, pH, etc. In the thesis, in order to synthesize materials with the desired β-
14 NaYF4 hexagonal structure, we have changed the order of creating NaYF4 matrices in the process of material synthesis. 3.3. The synthesis of NaYF4: Yb3+, Er3+ upconversion nanophosphors with NaYF4 matrices order change. 3.3.1. Synthesis process of NaYF4: Yb3+, Er3+ upconversion nanophosphors with NaYF4 matrices order change (process 2) Table 3.5. The list of NaYF4: Yb3+, Er3+samples were synthesized according to process 2 No.
NaYF4: Yb3+, Er3+ - PEG 20000 samples were synthesized according to process 2 (Fig 3.13) was listed in Table 3.5 SolutionNaOH
C2H5OH + PEG Y3+ + NaF
Stir / 30 minutes
Solution (Yb3+ + Er3+)
Solution B Stir / 120 minutes
Solution C 190 oC/ 24 hours
SolutionD Centrifuge, wash, dry
Powder NaYF4: Yb3+, Er3+
Fig 3.13. Synthesis process of nanomaterials containing rare earth ions NaYF4: Yb3+, Er3+ with NaYF4 matrices order change (Process 2) 3.3.2. The result of Investigate of the structure and morphology of NaYF4: Yb3+, Er3+ upconversion luminescent nanomaterials according to process 2 126.96.36.199. XRD pattern of the nanophosphors of NaYF4: Yb3+, Er3+ were synthesized according to process 2.
15 The results of XRD diagram EY2 Hexagonal NaYF4
analysis of EY2 sample presented in Figure 3.14 show at the 2 angles: 17,1 53,2 ; 55,3 ; 62,3 ; 71,03 ; 86,7 . There are diffraction peaks equivalent to the -NaYF4 hexagonal. The XRD schema results of the EY2 model match the results on the JCPDS standard card number 00-028-1192. In addition to the
; 29,9 ; 30,8 ; 34,7 ; 43,5 ; 46,5 ;
diffraction peaks of the -NaYF4 phase,
on the XDR diagram of the sample, no strange peaks were observed. This shows that NaYF4:Yb3+, Er3+ samples with PEG 20000 were synthesized according to the structured procedure 2 with the
2 - Theta (degree)
-NaYF4 hexagonal phase structure, indicating that the molar ratio of Er3+/ Y3+ did not affect the structure crystal of material.
Fig 3.14. XRD pattern of the nanophosphors of NaYF4:Yb3+, Er3+ PEG20000 (EY2) at 190 oC, 24 hours synthesized according to desired -NaYF4 phase structure. process 2 3+ We continue to investigate the effect of the molar ratio of Er / Y3+ on the crystal structure of the synthesized samples according to process 2 (Fig 3.15). The results showed that, with (1) EY1 (2) EY2 the change of the molar ratio of (3) EY3 (4) EY4 Er3+/ Y3+, the samples still showed Hex NaYF4 diffraction peaks equivalent to the
(4) (3) (2) (1)
2 - Theta (degree)
Fig . 3.15: XRD pattern of the nanophosphors of NaYF4:Yb3+, Er3+ PEG20000 (EY2) at 190 oC, 24 hours with change of Er3+/ Y3+ ratio synthesized according to process 2 ( EY1 = 0,25/ 79,75; EY2 = 0,5/ 79,5; EY3 = 1,0/ 79,0; EY4 =2,0/ 78,0) Thus, changing the matricies order in the synthesis process obtained NaYF4: Yb3+, Er3+ materials with desired hexagonal structure -NaYF4. 188.8.131.52. The morphology of NaYF4: Yb3+, Er3+ has the structure of β-NaYF4 FE-SEM images of the nanophosphors were presented in Figure 3.17. The FE-SEM image of the NaYF4:Yb3+, Er3+ indicates that the nanorods have bundles shape with the lengths of rod about 300 800 nm and diameter of rod about 100 200 nm.
(c) (d) Fig. 3.17. FESEM images of the nanophosphors of EY1 (a), EY2 (b) , EY3 (c) và EY4 (d) with β-NaYF4 (synthesized according to process 2) 3.3.3. Luminescent properties of NaYF4:Yb3+, Er3+ has the structure of β-NaYF4 Luminescent properties of (1) EY1- 0,25% Er NaYF4:Yb3+, Er3+-PEG has the (2) EY2 - 0,5% Er (3) EY3 - 1,0% Er structure of β-NaYF4 with change (4) EY4 - 2,0% Er of Er3+/ Y3+ ratio were investigated. Fig.3.18 shows the (4) upconversion luminescence (UCL) spectra of the samples (3) EY1, EY2, EY3 and EY4. The results showed that the samples (2) emitted blue and red areas corresponding to the transitions of (1) Er3+, the red emission rate of EY2 450 500 550 600 650 700 750 was strongest (EY2 compared to Wavelength (nm) EY1 is 1.53 times; compared to Fig 3.18. The upconversion luminescence (UCL) spectra of the EY3 is 4.58 times). nanophosphors of NaYF4:Yb3+, Er3+ with change of Er3+/ Y3+ ratio synthesized according to process 2, under NIR laser excitation at 980 nm F9/2- 4I15/2
S3/2- 4I15/2 4
H11/2- 4I15/2 2
CHAPTER 4. THE RESULT OF SYNTHESIS AND APPLICATION OF UPCONVERSION NANPPHOSPHORS CONTAINING RARE EARTH IONS FOR MARKING, IDENTIFICATION BREAST CANCER CELLS MCF7 4.1. Surface treatment, functionalization and conjugation of NaYF4 materials containing Yb3+ and Er3+ ions 4.1.1. Surface treatment of NaYF4 material containing Yb3+ and Er3+ ions with silica TEOS + C2H5OH
Stir / 15 minutes
CH3COOH + H2O
Stir /30 minutes
NaYF4: Yb3+, Er3+@NaYF4 + C2H5OH
Stir / 6 hours
Solution NaYF4:Yb3+, Er3+
Centrifuge, wash, dry
Fig.4.1. Surface treatment process of NaYF4:Yb3+, Er3+ with silica 4.1.2. Functionalization of NaYF4: Yb3+, Er3+@silica with APTMS APTMS + C2H5OH
Fig. 4.7: Conjugation process of NaYF4:Yb3+, Er3+@silica-NH2 material with folic acid
Fig. 4.5. Functionalization process of NaYF4:Yb3+, Er3+@silica with TPGS
4.1.4. Conjugation of NaYF4: Yb3+, Er3+@silica-NH2 materials with folic acid Fig. 4.7 discribed conjugation process of NaYF4:Yb3+, Er3+@silica-NH2 material with folic acid. Complex reaction NaYF4:Yb3+, Er3+@ silica-N = FAbiomedical nanoparticles reaction is shown in Figure 4.8.
R – NH2
NHS + DCC
(NaYF4:Yb3+, Er3+ @silica-N=FA)
NHS: N-Hydroxysuccinimide (C4H5NO3)
DCC: N, N’-Dicyclohexylcarbodiimide (C13H22N2)
Figure 4.8. Reaction to form NaYF4:Yb3+, Er3+@ silica-N=FA biomedical nanoparticles The teste to investigate the ability to pair of NaYF4:Yb3+, Er3+@silica-N=FA biomedical nanoparticles with mark MCF7 breast cancer cell (Fig. 4.9).
Fig. 4.9. Biological conjugation process of NaYF4:Yb3+, Er3+ @ silica-N = FA with MCF7 cancer cells 4.2. The results of morphology, structure and luminescent properties of functionalized and conjugated materials 4.2.1. The morphology, structure and luminescent properties of functionalized and conjugated of NaYF4:Yb3+, Er3+@silica
Fig 4.10. FESEM images of the nanophosphors of NaYF4:Yb3+, Er3+(a) và NaYF4:Yb3+, Er3+@ silica (b) at 190 oC, 24 hours FESEM image results show that, with samples NaYF4:Yb3+, Er3+ uncoated silica (Figure 4.10a) the rod-shaped material, the length is about 300 nm ÷ 800nm and the diameter of 100 nm ÷ 200nm and is not sticky. With the presence of silica, samples NaYF4:Yb3+, Er3+ @ silica (Figure 4.10b), the morphology of the material is still in the form of rods with increasing but not significant size. Morphology of NaYF4:Yb3+, Er3+@ silica after functionalization, conjugation by attaching -NH2 group with APTMS, TPGS and FA attachment were observed on field electron microscopes and were shown in Figure 4.11. Looking at FESEM image shows the presence of TPGS in Figure 4.11a (NaYF4:Yb3+, Er3+@ silica / TPGS), of -NH2 in Figure 4.11b (NaYF4:Yb3+, Er3+@ silica-NH2) and FA in Figure 4.11c (NaYF4:Yb3+, Er3+@ silica-N = FA) shows that the material is still rod-shaped with a length of about 300nm
20 ÷ 800nm and a diameter of about 200 ÷ 300 nm. Thus, the materials NaYF4:Yb3+, Er3+ after being covered with silica and attached with the TPGS or -NH2 or FA groups are not morphologically changed.
Fig 4.11. FESEM images of the nanophosphors of NaYF4: Yb3+, Er3+@ silica-TPGS (a), NaYF4: Yb3+, Er3+@silica-NH2 (b) and NaYF4: Yb3+, Er3+@silica-N=FA (c) at 190 oC, 24 hours 4.2.2. Fourier transform infrared spectrum of NaYF4:Yb3+, Er3+@ silica were functionalized, conjugated.
3+ 3+ (2) NaYF4:Yb , Er @silica
3+ 3+ (3) NaYF4:Yb , Er @silica/TPGS
3+ 3+ (1) NaYF4:Yb , Er
Wavenumber (cm )
Fig 4.12. FTIR spectra of the nanophosphors of NaYF4: Yb3+, Er3+ (1), NaYF4: Yb3+, Er3+@silica (2) and NaYF4: Yb3+, Er3+@silica/TPGS (3) Fig 4.12. presented FTIR spectra of the nanophosphors of NaYF4: Yb3+, Er3+ (line 1), NaYF4: Yb3+, Er3+ @ silica (line 2) and NaYF4: Yb3+, Er3+ @ silica / TPGS (line 3) recorded in the region of 4000 cm-1 ÷ 400 cm-1. Infrared spectroscopy survey of samples showed that TPGS has improved the dispersion ability of domestic materials as well as in some other biological buffer solutions. This is also consistent with a number of published studies. From the above results, we believe that TPGS has been successfully attached to NaYF4:Yb3+, Er3+ silica. Similarly, for materials NaYF4: Yb3+, Er3+ conjugated by FA, infrared spectrum of NaYF4:Yb3+, Er3+ (line 1); NaYF4:Yb3+, Er3+ @ silica (line 2); NaYF4:Yb3+, Er3+@ silica-NH2 (line 3) and NaYF4:Yb3+, Er3+@ silica-N = FA (line 4) were also surveyed and shown in Figure 4.13.
3+ 3+ (1) NaYF4:Yb , Er
3+ 3+ (2) NaYF4:Yb , Er @silica 3+ 3+ (3) NaYF4:Yb , Er @silica-NH
3+ 3+ (4) NaYF4:Yb , Er @silica-N=FA
Wavenumber (cm )
Fig 4.13. FTIR spectra of the nanophosphors of NaYF4: Yb3+, Er3+ (1); NaYF4: Yb3+, Er3+@silica (2); NaYF4: Yb3+, Er3+@silica-NH2 (3) and NaYF4: Yb3+, Er3+@silica-N=FA (4) Observing the infrared spectrum of the samples showed that chemical bonds between FA molecules and inorganic components were formed. These results demonstrate that FA ligands have been successfully paired to NaYF4: Yb3+, Er3+@slica-NH2. 4.2.3. Luminescent properties of NaYF4:Yb3+, Er3+@silica material: Yb3+, Er3+ @ silica have functionalized and conjugated.
4 4 F9/2 - I15/2
(1) NaYF4:Yb3+, Er3+
(2) NaYF4:Yb3+, Er3+@Silica
4 4S 3/2 - I15/2
4 - I
Fig 4. 14. The upconversion luminescence (UCL) spectra of the nanophosphors of NaYF4: Yb3+, Er3+(1); NaYF4: Yb3+, Er3+@silica (2) under NIR laser excitation at 980 nm Figure 4. 14 shows the upconversion luminescence (UCL) spectra of the nanophosphors of NaYF4: Yb3+, Er3+(1); NaYF4: Yb3+, Er3+@silica (2) under NIR laser excitation at 980 nm. The results showed that when coved in silica, the intensity of fluorescence increased 2.5 times. After covering the silica, we functionalized them with TPGS and APTMS.
H11/2 - I15/2
4 4 S3/2 - I15/2
4 4 F11/2 - I15/2
NaYF4: Yb , Er @silica/TPGS
Fig 4.15. The upconversion luminescence (UCL) spectra of the nanophosphors of NaYF4: Yb3+, Er3+@ silica/TPGS under NIR laser excitation at exc = 980nm Fluorescence results of the materials Fig, 4.15 and Fig.4.17 show that all materials emitted blue at wavelengths (510 nm ÷ 570 nm) and red at wavelengths (630 nm ÷ 700 nm) corresponding to 2H11/2 → 4I15/2; 4 S3/2 → 4I15/2 and 4F9/2 → 4I15/2 of Er3+ ions. However, when fitted with FA, NaYF4: Yb3+, Er3+ @ silica-N = FA materials still glow UCL although the luminescence intensity of this sample is weaker than that of nonFA models. 3+ 3+ (1) NaYF4:Yb , Er
4 F9/2- I15/2
3+ 3+ (2) NaYF4:Yb , Er @silica 3+ 3+ (3) NaYF4:Yb , Er @silica-NH2
3+ 3+ (4) NaYF4:Yb , Er @silica-N=FA
4 3/2- I15/2
Hình 4.17. The upconversion luminescence (UCL) spectra of the nanophosphors of NaYF4:Yb3+,Er3+(1); NaYF4:Yb3+,Er3+@silica (2); NaYF4: Yb3+, Er3+@silica-NH2 (3) and NaYF4: Yb3+, Er3+@silica-N=FA (4) at exc = 980nm 4.3. Result of application upconversion luminescent nanomaterials to identify MCF7 cancer cells In vitro cellular imaging was performed to prove the localization of the functionalized UCNPs within the cytoplasm of breast cancer cells. The UCNPs were incubated the with MCF-7 cells for 24 hours. We observed the UCNPs have been clearly localized within the cell cytoplasm by microscope with no significant signs of cytotoxicity. The evidence conﬁrms that the UCNPs can be potentially used as bio-labels for MCF-7
23 breast cancer cells. This is possible because the high afﬁnity via FA–FR. After the binding of the ligands, the UCNPs is internalized into the cell via invagination process. Fig. 4.19 demonstrates imaging results of biological test for UCNPs. In Fig. 4.19 (a), we show images of MCF7 cells only in three cases: upper one is a bright field image, middle one is a dark field image, lower one is merged image of two above pictures. Similarly, Fig. 6 (b) presents images for the case of MCF7 cells after incubated with NaYF4:Yb3+, Er3+@silica-N=FA. When we use right field regime of microscope, we only see a typical MCF7 cell type, in both cases (a) and (b). For the dark field images, we do not see luminescence from bare MCF7 cells but we observed the brightness emitting dots in the MCF7 incubated with UCNPs. The merge pictures show clearly that the brightness emitting dots (represent for existance of UCNPs) were attached around MCF7 cells. This indicated that the rich folate receptor was captured to the MCF7 cells. Bright field
NaYF4: Yb , 3+
Er @silicaNH2 MCF7
NaYF4: Yb , 3+
Er @ silicaN=FA Fig. 4.19. Upconversion fluorescent imaging of MCF7 cells and MCF7 cells after incubated with NaYF4:Yb3+, Er3+@Silica-N=FA was observed by reverse fluorescence microscope. Thus, through rigorous samples showed that folate groups were attached to MCF7 cells or in other words, biomedical nanoparticles: NaYF4: Yb3+, Er3+ @ silica – N = FA were paired on the surface of MCF7 breast cancer cells. The pairing position of this nanoscale with cancer cells was observed by reverse microscopy Zeiss axio vert A1. The results showed that for the first time using biomedical nanoparticles: NaYF4: Yb3+, Er3+ @ silica – N = FA attached to MCF7 breast cancer cells and surveyed on the backilluminated fluorescence microscope Zeiss axio vert A1 has shown that the biomedical nanoparticles: NaYF4: Yb3+, Er3+ @ silica – N = FA can be used as a marker for identifying breast cancer cells MCF7 in vitro.