NJC View Article Online Published on 09 January 2014 Downloaded by University of Massachusetts - Amherst on 26/10/2014 06:27:40 PAPER Cite this: New J Chem., 2014, 38, 2114 View Journal | View Issue Fabrication and optical characterization of multimorphological nanostructured materials containing Eu(III) in phosphate matrices for biomedical applications T T Huong,*a L.T Vinh,ab T K Anh,a H T Khuyen,a H T Phuongac and L Q Minhad EuPO4ÁH2O nanorods/nanoparticles with a rhabdophane-type hexagonal form have been successfully synthesized using microwave assisted co-precipitation The effects of chemical composition and pH on the size, shape, morphology and luminescence properties have been investigated by powder X-ray diffraction, Field Emission Scanning Electron Microscopy (FESEM), and photoluminescence spectroscopy The mean size of the nanorods is about 15–30 nm in diameter and 200–400 nm in length and the size Received (in Victoria, Australia) 7th October 2013, Accepted 9th January 2014 DOI: 10.1039/c3nj01206a of nanoparticles is 10–20 nm A powdered sample of these EuPO4ÁH2O nanorods/nanoparticles emitted yellow-green light with narrow bands at 594, 619, 652, and 697 nm under UV-vis excitation The surface effects of the built core–shell structure on the fluorescent properties, and the compatibility of the EuPO4ÁH2O nanomaterials with a biological system, have been studied to develop a new fluorescent label for biomedical imaging The primary test results in using a EuPO4ÁH2O–Immunoglobulin G conjugate for recognizing the measles virus in a vaccine is presented www.rsc.org/njc Introduction In the field of in vivo biomedical imaging, recently, there are three main types of luminescent materials used in labelling and imaging:1–3 organic dyes, semiconductor quantum dots, and rare-earth fluorescent materials Traditional organic dye materials (e.g rhodamine), are still being used in fluorescence imaging, however, they show a disadvantage because of their instability in biological environments, consequently limiting the sensitivity and selectivity of the investigation Nano-crystals based on semiconductor quantum dots such as CdS or CdSe, which exhibit excellent fluorescent properties, durability and good solubility in water, are well developed in the field of biological fluorescent labelling However, these materials in practice exhibit high toxicity, which is a barrier for their use in potential applications Therefore, in recent years, fluorescent nano-materials containing rare-earth ions have become promising candidates as labelling materials in biomedical imaging techniques because of their non-toxicity and strong luminescence properties.4–7 a Institute of Materials Science, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay Distr., Hanoi, Vietnam E-mail: huongtt@ims.vast.ac.vn, tthuongims@gmail.com b Department of Chemistry, Hanoi University of Mining and Geology, Vietnam c Department of Chemistry, Hanoi Medical University, Vietnam d University of Engineering and Technology, National University Hanoi, Vietnam 2114 | New J Chem., 2014, 38, 2114 2119 There are several kinds of nano-materials containing rare earth ions with high luminescent efficiencies of up to and above ten percent Such as YVO4:Eu3+ nanoparticles,8,9 LnPO4ÁH2O:Eu, Tb nanomaterials10–14 and ZrO2:Yb3+, Er3+ nanoparticles,15 which have been developed for agrobiological and medical applications.4,5,16 In previous studies, we have been successful in synthesizing nanorods/nanowires of EuPO4, and TbPO4.17,18 It was found that the size and shape of the products have important effects on their luminescent intensity However, nano-sized materials with high luminescent yields are still required for medical and biological applications Therefore, we are continuously trying to achieve higher luminescent intensities from these nanomaterials containing the rare earth ion Eu3+ in these phosphate matrices In this report, we focus on the fabrication and luminescent characterization of EuPO4ÁH2O, a multimorphological nanostructured material (e.g particles, rods) The EuPO4ÁH2O nanoparticles and nanorods were synthesized using microwave assisted co-precipitation The effects of chemical composition and pH on the size, shape, morphology and luminescence properties of the prepared materials were also investigated Then, we investigated the surface effects of the built core–shell structure on the fluorescent properties, and the compatibility of the EuPO4ÁH2O nanomaterials with a biological system The first test results using a linked product of EuPO4ÁH2O This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 View Article Online Paper nanoparticles and immunoglobulin G (IgG) as a fluorescent immuno-label for recognizing the measles virus is also briefly demonstrated Published on 09 January 2014 Downloaded by University of Massachusetts - Amherst on 26/10/2014 06:27:40 Results and discussion Morphological and structural properties Morphology Field Emission Scanning Electron Microscopy (FESEM) images of the EuPO4ÁH2O nanomaterials with changes in the mole ratio of Eu3+ and PO43À are shown in Fig 1(a–f) From these images, we can conclude that the synthesized material with a 1/1 mole ratio of Eu3+ and PO43À (Fig 1a) consists of nano-rod shapes The mean sizes of the EuPO4ÁH2O nanorods are about 15–30 nm in diameter and 200–400 nm in length NJC The other mole ratios of Eu3+ and PO43À, 1/3, 1/5, 1/10, 1/15, and 1/30 are related to the FESEM images (b) to (f), respectively We observed that the EuPO4ÁH2O nanorods were in fact rods of connected particles, which then broke up into nanoparticles with diameters of about 15–20 nm Based on the FESEM results in Fig 1, we can primarily summarize that the shape (from rod to particle) of the synthesized samples can be controlled by adjusting the mole ratio of Eu3+ and PO43À The results show that at a mole ratio of Eu3+/PO43À = 1/15, a breaking up process has started, and we start to see the formation of nanoparticles in place of nanorods So we chose this ratio to study the effects of other factors such as pH, etc The investigation of the influence of a pH range between and 12 on the morphology of the EuPO4ÁH2O nanomaterial was Fig FESEM images of the EuPO4ÁH2O multimorphological nanostructured material with changes in the mole ratio of Eu3+ and PO43À, synthesized at pH = 6: (a) Eu3+/PO43À = 1/1; (b) Eu3+/PO43À = 1/3; (c) Eu3+/PO43À = 1/5; (d) Eu3+/PO43À = 1/10; (e) Eu3+/PO43À = 1/15; (f) Eu3+/PO43À = 1/30 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J Chem., 2014, 38, 2114 2119 | 2115 View Article Online Paper Published on 09 January 2014 Downloaded by University of Massachusetts - Amherst on 26/10/2014 06:27:40 NJC Fig FESEM images of the EuPO4ÁH2O multimorphological nanostructured materials with changing pH values: pH = (a), pH = (b), pH = (c), pH = (d), pH = 10 (e), pH = 12 (f) performed using a molar ratio of Eu3+/PO43À 1/15 The results indicate that the pH value of the reaction solution plays an important role in controlling the morphology of the prepared products Fig 2(a–f) show the typical FESEM images of the samples The FESEM image in Fig 2(a) indicates that the product prepared at pH = (without the addition of NaOH) consists of nanowires 15–30 nm in diameter and 200–400 nm in length When the pH was increased to 4, 6, 8, 10 and 12 (Fig 2b–f) the wires started to break up to form fine particles However, with pH values below 10, the nanoparticles mostly stuck together to build a chain Therefore we suggest a possible formation mechanism of the EuPO4ÁH2O nanoparticles from the nanorods/ nanowires and their internal breaking and reconstruction 2116 | New J Chem., 2014, 38, 2114 2119 When the pH value of the reaction solution (without NaOH) is (Fig 2a), the oxonium cations (H3O+) cannot perturb the EuPO4 growth along a single direction, and the rate of growth is faster than in other directions, which limits the growth anisotropy of EuPO4 and leads to the formation of a EuPO4ÁH2O nanorod/ nanowire, with a 1-dimensional morphology Gedanken et al reported that EuPO4ÁH2O nanorods/nanowires were obtained using microwave assisted hydrothermal synthesis only between a pH range of 1.8 to 2.2.11 In the case of the pH of the reaction solution being between and 12, our experimental results indicate that the direction of growth and the anisotropy is inhibited Therefore, the formed nanorods/nanowires started to break up, and started to form nanoparticles This reconstruction maybe due to a preferential ionic interaction, in which the This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 View Article Online Published on 09 January 2014 Downloaded by University of Massachusetts - Amherst on 26/10/2014 06:27:40 Paper hydroxide anions (OHÀ) compete with the phosphate anions In addition, the rapidly changing electric field of the microwave reactor may result in a nanoscale sized slit in the microdomain of the as formed nanorods/nanowires In short it can be summarized that with pH values between and 12 the breaking up process of the EuPO4ÁH2O nanorods/nanowires became obvious When the pH = 12 the formation of the nanoparticles was nearly complete and the FESEM image clearly shows individual EuPO4ÁH2O nanoparticles Nevertheless we found that when the pH was increased, the size of the EuPO4 particles decreased and that the particle size was more uniform Phase and structure XRD patterns of the prepared EuPO4Á H2O nanomaterials show that a rhabdophane-type hexagonal form (PDF card No 20-1044) is the dominant phase (Fig 3) Qualitatively, as shown in Fig (lines 1–6), the changing of the mole ratio of Eu3+ and PO43À causes no change in the crystalline phase composition or crystallinity of the prepared samples The X-ray diffraction analysis results show that the phase of the obtained EuPO4ÁH2O nanophosphors is almost a hexagonal form in the pH range of to 12 Photoluminescence Photoluminescence excitation (PLE) spectra of the EuPO4ÁH2O nanomaterials have been reported in a previous study.8 The excitation wavelengths were in the UV region at 317, 361, 375, 393, and the visible region at 414 and 464 nm Excitation at any of these wavelengths resulted in similar luminescence spectra of the EuPO4ÁH2O products It is noted that under all of the chosen excitation wavelengths, the EuPO4ÁH2O products emit yellow-green light, which can be observed by the naked eye The photoluminescence (PL) spectra of the EuPO4ÁH2O nanomaterials under an excitation wavelength of 393 nm at room temperature are presented in Fig The main emission peaks of the EuPO4ÁH2O product were observed at 594, 619, 652, and 697 nm, which relate to 5D0–7F1, 5D0–7F2, 5D0–7F3, and D0–7F4 optical transitions of Eu3+, respectively The EuPO4ÁH2O product yielded the emission characteristics of Eu3+, in which the 5D0–7F4 transition at 594 nm is the most Fig XRD patterns of EuPO4ÁH2O with Eu3+/PO43À = 1/1–1/30 at pH = (line 1: Eu3+/PO43À = 1/1; line 2: Eu3+/PO43À = 1/3; line 3: Eu3+/PO43À = 1/5; line 4: Eu3+/PO43À = 1/10; line 5: Eu3+/PO43À = 1/15; line 6: Eu3+/ PO43À = 1/30) NJC Fig PL spectra of the EuPO4ÁH2O nanomaterials synthesized at a mole ratio of Eu3+/PO43À = 1/15, with changing pH values: pH = 2–12 at lexc = 393 nm prominent emission line for the EuPO4ÁH2O nanomaterials at pH = 2, 4, 6, 8, 9, 10, 11, 12 These fluorescence properties of EuPO4ÁH2O have attracted a great deal of attention in biology and medicine To use EuPO4ÁH2O products in the study of biomedical processes, the first step is the need to functionalize the nanomaterials, i.e to connect its surface to a number of functional groups (organic), such as OH, NH2, SH 1,7 We have studied the appropriate chemical reactions for functionalizing luminescent nanomaterials such as EuPO4ÁH2O using sol–gel technology.18 We present here the effect of shells and organic functionalization on the photoluminescent characterization of EuPO4Á H2O nanomaterials Due to the large surface-to-volume ratio of nanophosphores their surface plays an important role in their optical properties Therefore the modification of their surface by sol–gel coating technology can bring about a large change in emission intensity of these nanophosphores The conditions used in the sol–gel deposition were chosen to optimize the fabrication of an outer layer that contained the chosen functional group so as to maintain the emission properties as much as possible PL spectra of EuPO4ÁH2O synthesized at pH = and the core–shell nanophosphores of EuPO4Á H2O@silica–NH2 under excitation at lex = 393 nm are presented in Fig The results reveal that the luminescent intensity was substantially changed after the EuPO4ÁH2O nanoparticles had been coated with a shell layer, which was linked with an amine group (NH2) (Fig 5) These luminescent experimental results indicate that the NH2 group in the shell effectively quenches the luminescent intensity of EuPO4ÁH2O However, the influence of the sol–gel shell layer on the structure of the fluorescent spectra is negligible, and it also improves the stability of the emission intensity of EuPO4ÁH2O in different biomedical solutions This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J Chem., 2014, 38, 2114 2119 | 2117 View Article Online Published on 09 January 2014 Downloaded by University of Massachusetts - Amherst on 26/10/2014 06:27:40 NJC Paper Fig PL spectra of EuPO4ÁH2O (1); EuPO4ÁH2O@silica–NH2 (2); pH = and lexc = 393 nm To develop a new conjugate which is suitable for labelling we focused on some molecules with strong bioaffinities such as biotin, protein IgG and bovine serum albumin (BSA) Based on the immune-reactions between an antibody of a conjugate and an antigen of a virus/vaccine, they can be detected using a fluorescence microscope and imaged with a digital camera In this study, we have used a chemical coupling reaction to connect functionalized EuPO4Á H2O nanoparticles with the protein IgG via a direct reaction between functional groups, using an intermediate linker.19 Using the obtained conjugates containing IgG as the targeting biomolecule, we have demonstrated an analysis of the measles vaccine, which is one of the key products of the Industrial Centre for Investigation and Production of Vaccine and Biologicals (POLYVAC-centre) Application as a fluorescent immunoassay (FIA) of viruses/vaccines We applied a comparative analysis method and used the conjugated IgG-linked nanomaterials, as well as a commercial product as the reference, in the cell incubation procedure of the POLYVAC-centre.18 We have experimentally researched the conjugated product IgG– EuPO4ÁH2O in an incubation process with vaccine fabricates The images obtained from fluorescent microscopic measurements are shown in Fig In Fig 6(a), one can see the image of a commercial conjugate incubated with standard Vero cells from the vaccine production processing line of the POLYVAC-centre Fig 6(b), shows the image of Vero cells infected with the measles virus, and the commercial conjugate Fig 6(c) shows the image of measles virus infected Vero cells and EuPO4ÁH2O@silica–NH2–IgG Incubation processing with an exposure procedure of the conjugate in the POLYVAC-centre was used for the preparation of the tested specimens The obtained fluorescent images in the Fig 6(b) and (c) show clearly the locations of the products of the immune reactions between the antibody of the MP commercial conjugate and the EuPO4ÁH2O@silica–NH2–IgG conjugate and the antigen of the measles virus in the vaccine The results indicate that the fabricated conjugates could be used for the detection and recognition of the measles virus, and for controlling the quality of the fabrication process The performance of EuPO4ÁH2O@silica–NH2 linked with IgG for fluorescence immunoassay (FIA) analysis using a fluorescent optical microscope may be comparable with the commercial conjugate (label) for reference Nevertheless, the prepared EuPO4Á H2O@silica–NH2–IgG conjugates have shown a strong, stable, yellow green fluorescence emission and a reproducible intensity in a broad range of pH values, and in the biological microenvironments of vaccine fabrication The quality of the fluorescence images of the assayed specimens mostly remained stable for several months Conclusions In summary, EuPO4ÁH2O multimorphological nanomaterials with a rhabdophane-type hexagonal form were successfully synthesized using microwave assisted co-precipitation The mean size of the EuPO4ÁH2O nanorods is about 15–30 nm in diameter and 200–400 nm in length, the size of the EuPO4ÁH2O nanoparticles is about 10–20 nm The structure, morphology and luminescence of the EuPO4ÁH2O nanorods/nanoparticles can be controlled by pH and chemical composition The luminescence spectra of EuPO4ÁH2O contain four main bands at 594, 619, 652, and 697 nm assigned to the 5D0–7F1, 5D0–7F2, D0–7F3, and 5D0–7F4 transitions of Eu3+, respectively EuPO4ÁH2O@silica–NH2–IgG conjugates have been successfully fabricated by coating EuPO4ÁH2O with a thin silica layer containing NH2 and then linking to IgG via a coupling reaction The primary test results showed that the EuPO4ÁH2O@silica– NH2–IgG conjugate could be used for the detection and recognition of the measles virus This is a promising result for using rare earth luminescent nanomaterials for the development of fluorescent labelling and imaging tools in biomedicine Experimental Synthesis of multimorphological nanostructured materials Fig (a) Micro-image of specimens of standard Vero cells using the MP Biomedicals commercial conjugate, (b) measles virus infected Vero cells using the MP Biomedicals commercial conjugate and (c) measles virus infected Vero cells using EuPO4ÁH2O@silica–NH2–IgG 2118 | New J Chem., 2014, 38, 2114 2119 EuPO4ÁH2O nanostructured materials were prepared using a microwave assisted method combined with co-precipitation using Eu(NO3)3Á5H2O (Sigma-Aldrich, 99.9%), and NH4H2PO4 (Merck, 99%) as starting materials The reaction solution was magnetically stirred for 120 and the pH of this solution was adjusted in the range of 2–12 by adding 10 mol LÀ1 NaOH After that, at each selected pH value, this reacting solution was irradiated using a MAS-II microwave synthesis extraction workstation (Sineo Co.) for 15 minutes with the microwave power adjusted from 300 to 900 W The mole ratio of Eu3+ and PO43À This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 View Article Online Published on 09 January 2014 Downloaded by University of Massachusetts - Amherst on 26/10/2014 06:27:40 Paper was changed as follows: Eu3+/PO43À = 1/1; 1/3; 1/5; 1/10; 1/15; 1/30 The final products were collected, centrifuged at 5900 rpm, and cleaned several times using ethanol and distilled water The primary silicate shell as a protecting layer and functionalization with NH2 was fabricated as following: 10 ml of tetraethylorthosilicated (TEOS) (1/2) in absolute ethanol and 10 ml of as-synthesized EuPO4ÁH2O solution was mixed with a magnetic stirrer at room temperature (24 hours) The pH of this solution was adjusted to the range of 11–12 by adding NH4OH 10 M The resulting products were collected, centrifuged and cleaned several times with ethanol and distilled water The final products were dried at 60 1C for 24 h in air The results, which were repeated several times, showed good reproducibility We used 3-aminopropyltrimetoxysilane (APTMS) which contains an NH2 functional group In these typical syntheses, 22.5 ml of absolute ethanol and ml of APTMS were put into a 100 ml three-necked flask under magnetic stirring at room temperature for 30 The solution was heated up to 60 1C under reflux Then, ml of the EuPO4ÁH2O@silica nanomaterial solution at pH was added drop wise The reaction time was about h The solution was next gently stirred for 20 h The resulting products were collected via three centrifugation/dispersion steps in a water–ethanol mixture (2 : 5, v/v) The final products were again washed with deionized water and then dried at 60 1C for 24 h in air Protein binding with sol–gel functionalized nanophosphors: coupling the protein immunoglobulin G to the APTMS functionalized nanomaterial, was achieved using glutaraldehyde as a reactive amine linker The APTMS treated EuPO4ÁH2O nanomaterial solution and glutaraldehyde were dispersed in phosphate buffered saline (PBS, 0.1 M, pH 5) with a concentration of g lÀ1 The above solution was added to different concentrations of immunoglobulin G (IgG) (Aldrich) These reaction mixtures were incubated at 30 1C for h The resulting products were collected, centrifuged at 5900 rpm, and washed several times using ethanol–water and distilled water The IgG linked EuPO4ÁH2O@silica–NH2 products were stored in closed box at 1C in a refrigerator Characterization methods The morphological observations and crystalline phase identifications of all of the prepared samples were carried out using Field Emission Scanning Electron Microscopy (FESEM, Hitachi, S-4800), and X-ray diffraction (XRD, Siemens D5000 with l = 1.5406 Å in the range of 101 r 2y r 801) The luminescent properties of the studied samples were measured on a highresolution steady-state photoluminescent setup based on a luminescence spectrum photometer system, Horiba Jobin Yvon IHR 320 (USA) The excitation wavelength was 393 nm The microsized images of the virus infected cells exposed to the nanomaterial conjugates were viewed using fluorescent microscopic equipment, an Olympus BX-40 (Japan), and pictured using a digital camera, Nikon D5000, with a resolution of 12.30, f/3.5–5.6 G VR NJC Acknowledgements This work was supported by Vietnam’s National Foundation for Science and Technology Development (NAFOSTED), project code: 103.06-2012.72 and implemented in the framework of long-term Research Topics of Rare Earth Nanoluminophores and Application, and partly supported by the National Key Lab of Electronic Materials and Devices in Institute of Materials Science, Vietnam Academy of Science and Technology References F Wang, W B Tan, Y Zhang, X Fan and M Wang, Nanotechnology, 2006, 17, R1 O V Salata, J Nanobiotechnol., 2004, 2, 3–6 S N Misra, M A Gagnani, I M Devi and R S Shukla, Bioinorg Chem Appl., 2004, 2, 155 J Feng, G M Shan, A Maquieira, M E Koivunen, B Guo, B D Hammock and I M Kennedy, Anal Chem., 2003, 75, 5282 C R Patra, R Bhattacharya, S Patra, S Basu, P Mukherjee and D Mukhopadhyay, J Nanobiotechnol., 2006, 4(11), K L Wong, G L Law, M B Murphy, P A Tanner, W T Wong, P K S Lam and M H W Lam, Inorg Chem., 2008, 47(12), 5190 D Giaume, M Poggi, D Casanova, G Mialon, K Lahlil, A Alexandrou, T Gacoin and J.-P Boilot, Langmuir, 2008, 24, 11018 E Beaurepaire, V Brissette, M.-P Sauviat, D Giaume, K Lahlil, A Mercuri, D Casanova, A Huignard, J.-L Martin, T Gacoin, J.P Boilot and A Alexandrou, Nano Lett., 2004, 4(11), 2079 ărkcan, G Mialon, M Poggi, D Casanova, T L Nguyen, S Tu A Alexandrou, T Gacoin and J P Boilot, J Lumin., 2009, 129(12), 706–1710 10 S Cho, G K Choi, J S An, J Kim and K S Hong, Mater Res Bull., 2009, 44, 173 11 C R Patra, G Alexandra, S Patra, D S Jacob, A Gedanken, A Landau and Y Gofer, New J Chem., 2005, 29, 733 12 J Yang, G Li, C Peng, C Li, C Zhang, Y Fan, Z Xu, Z Cheng and J Lin, J Solid State Chem., 2010, 183(2), 451 13 Z G Yan, Y W Zhang, L P Youb, R Si and C H Yan, J Cryst Growth, 2004, 262, 408–414 14 C Yu, M Yu, C Li, X Liu, J Yang, P Yang and J Lin, J Solid State Chem., 2009, 182, 339347 ănen, J H Kankare, M Lastusaari and L Pihlgren, 15 I Hyppa J Nanomater., 2007, 2007, 16391 16 J Kang, X Y Zhang, L D Sun and X X Zhang, Talanta, 2007, 71, 1186–1191 17 T T Huong, T K Anh, L T Vinh, W Strek, H T Khuyen and L Q Minh, J Rare Earths, 2011, 29(12), 1174 18 L Q Minh, T T Huong, N T Huong, H T Khuyen, N T Binh, D K Tung, T K Anh, N D Hien, L T Luan, N T Quy, D M Dung, N N Anh Thu and N V Man, Adv Nat Sci.: Nanosci Nanotechnol., 2012, 3, 035003, DOI: 10.1088/2043-6262/ 3/3/035003 19 G T Hermanson, Bioconjugate Techniques, Academic Press (Elsevier), 2nd edn, 2008, [Paperback] ISBN-10: 0123705010, ISBN-13: 978-0123705013 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J Chem., 2014, 38, 2114 2119 | 2119 ... for using rare earth luminescent nanomaterials for the development of fluorescent labelling and imaging tools in biomedicine Experimental Synthesis of multimorphological nanostructured materials. .. indicate that the fabricated conjugates could be used for the detection and recognition of the measles virus, and for controlling the quality of the fabrication process The performance of EuPO4ÁH2O@silica–NH2... demonstrated an analysis of the measles vaccine, which is one of the key products of the Industrial Centre for Investigation and Production of Vaccine and Biologicals (POLYVAC-centre) Application as a fluorescent