Article pubs.acs.org/crystal
Transparent Aggregates of Nanocrystalline Hydroxyapatite Anders C. S. Jensen, Casper J. S. Ibsen, Duncan Sutherland, and Henrik Birkedal* Department of Chemistry and iNANO, Aarhus University, DK-8000 Aarhus C, Denmark S Supporting Information *
ABSTRACT: Assemblies of nanoparticles into transparent aggregates have solicited strong research interest in the form of both crystalline or amorphous aggregates of nanoparticles. In the present work, we make short-range ordered several millimeter-sized transparent aggregates of citrate modified calcium phosphate nanoparticles and discuss the mechanism of their formation. Microparticles of hydroxyapatite (HAP) nanocrystals and amorphous calcium phosphate (ACP) were synthesized with citrate as a growth and assembly modifier. Millimeter-sized transparent aggregates of these microparticles were made with 0 to 7.5% citrate/Ca2+. The degree of crystallinity, i.e., the ratio between nanocrystalline HAP and ACP in the microparticles, was determined by Rietveld refinement of powder X-ray diffraction data with an internal standard. It was found to decrease with increasing citrate concentration. Citrate also reduced the nanocrystallite size at low citrate concentrations. Above ∼3% added citrate, the crystallite size did not reduce further. Transparent aggregates were obtained by drying a suspension of particles. The aggregates lacked long-range order and in many cases featured spiral fractures partially propagating through the aggregates. The assembly mechanisms were studied by in situ video imaging, polarized optical microscopy, transmission electron microscopy, and confocal microscopy. The transparent aggregates consisted of polydisperse microparticles. The transparent aggregates form due to evaporation, but sedimentation leads to vertical size segregation with larger microparticles preferentially located at the bottom of the sample.
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INTRODUCTION In nature, highly ordered and homogeneous assemblies of nanoparticles are widely used to make versatile materials.1−5 These find use in structural support,5 sensing,6 and even in reproductive organs.7 One biomineral that is famous for its optical properties is mother of pearl or nacre. It shows remarkable iridescent colors as a result of diffraction from layers of aragonite platelets with a periodicity of ∼500 nm, thereby reflecting visible light. In order to create this mesostructure, the platelets must be homogeneous and ordered.2,4,8 By the example of nacre, it is clear that any transparent aggregates of nanoparticles must avoid any ordered structure in the range of visible light. Several examples of these materials have already been made. However, many of these materials are thin films made from inorganic/organic composites.9−13 They can also be made from Ag nanoribbons14 or by denaturized cellulose.15 Although there are numerous examples of transparent thin films, there are only a handful of transparent bulk materials 16−19 made from either TiO 2 or CaCO 3 in combination with polymers. They are typically densely packed disordered, homogeneous structures. In the thin films, high aspect ratio building blocks are common with a tendency for the materials to display local ordering of particles. Transparent bulk materials have thus far only been reported for aggregates of isotropic particles of 99% Sigma-Aldrich) that was temperated under stirring at 25 °C for 5 min. A mixture of 10 mL of 0.36 M NaH2PO4· H2O (>99% Sigma-Aldrich) and 0.84 M NaOH (>98% SigmaAldrich) and 10 mL 0−0.045 M Na3(citrate)·2H2O (>99% SigmaAldrich) was added. The reaction was kept at 25 °C for 24 h using a water bath controlled by a Julabo ED/F 12 (Julabo GmbH, Seelbach, Germany) and a MIXdrive 15 2Mag magnetic stirrer (2mag AG, München, Germany). The pH started at a value of about 12.5 and fell during the reaction as phosphate and hydroxy ions were consumed during formation of calcium phosphates. All samples were purified by Received: July 18, 2014 Revised: October 10, 2014
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dx.doi.org/10.1021/cg501080c | Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
Figure 1. (A) Optical microscopy images showing the transparency of all samples with the corresponding citrate content (scale bar 2 mm). (B) Cross-polarized microscopy images of the same samples showing the Maltese cross indicating local order. Note that the sample orientation is not the same in panels A and B. dialysis for 24 h in DI water using dialysis membranes with a 3500 g/ mol cut off to remove NaCl. The samples were centrifuged and then resuspended in DI water using a magnetic stirrer. The slurries were dried at 60 °C overnight. Characterization. All samples where characterized using powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). For the PXRD, a Rigaku Smartlab diffractometer (Rigaku Corporation, Tokyo, Japan) with a Cu Kα rotating anode was used in Bragg-Brantano mode with a Ni filter. The samples were mounted on a flat sample spinner at 30 rpm. Synthetic monocrystalline powder Diamond (∼1 μm SigmaAldrich) was used as an internal standard. Rietveld refinement was done in Fullprof38 using spherical harmonics to model the size anisotropy.39 The instrumental line broadening was determined using LaB6 (NIST). Aggregates were investigated for possible preferred orientation by mounting aggregates on glass needles and analyzing diffraction patterns using 2D detectors. This was done at the Swiss-Norwegian Beamlines station BM-01A of the European Synchrotron Radiation Facility using X-rays of a wavelength of 0.6829 Å. The diffraction patterns were measured using a Pilatus 2 M detector (Dectris Ltd., Baden, Switzerland). The samples investigated were 0.17−0.2 mm in size. No significant preferred orientation of crystallites was observed. TGA was performed on a Netzsch STA 449C (NETZSCHGeratebau GmbH, Selb, Germany). Approximately 10 mg of sample was heated to 1200 °C in an Al2O3 crucible using a heating rate of 10 K/min in an atmosphere of Ar and O2. FTIR analysis was carried out on a Nicolet 380 FTIR (Thermo Electron Corporation), with an ATR sample mount and samples were measured from 400 to 4000 cm−1 averaging 32 spectra and employing 4 cm−1 resolution. Optical microscopy and cross polarized microscopy was done on an Olympus szx16 microscope with an Olympus sc30 camera using the program Cellsens entry (Olympus America Inc., PA, USA). SEM micrographs were taken on a novo nanoSEM 600 (FEI) with an ETD detector. μCT was carried out at the TOMCAT beamline at SLS at 25 keV using 1601 projections with 500 ms exposure resulting in a voxel size of 0.325 μm. 3D rendering and adjustments of contrast and threshold was done in ImageJ.40 Time lapse imaging was done in an aluminum block at 60 °C, with a SONY video camera at 1.55 mega pixel and a 30 s time resolution. UV/vis spectroscopy was measured on a Shimadzu UV-3600 UV− vis-NIR spectrometer (Shimadzu Scientific Instruments Kyoto, Japan). A dual beam setup was used for absorption measurements, and an integrating sphere was used to determine the percentage of scattered light from the forward transmitted light. The samples were mounted on black cardboard with a 2 mm pin aperture behind the sample. A 1 mm aperture was set in front of the sample to ensure an equal area of sample was measured on all samples. The setup is shown in Figure S1, Supporting Information. To elucidate the role of nanocrystal containing microparticles in the assembly and structure of the transparent aggregates, we performed
confocal laser scanning microscopy (CLSM) by adding a small amount of fluorescently labeled microparticles to the microparticle mixture used for transparent aggregate formation. The fluorescently labeled transparent aggregate samples were made by mixing microparticles from a normal synthesis with 1 wt % of microparticles synthesized with 0.1% (in relation to calcium ions) calcein disodium salt (SigmaAldrich). The experiment was conducted with particles synthesized with 7.5% citrate in relation to calcium ions. CLSM was performed on a Zeiss LSM 700 confocal microscope (Zeiss, Jena, Germany) using an excitation laser with a wavelength of 488 nm and detecting fluorescence above 500 nm. Imaging was done with a 63× plan apochromat NA 1.4 oil immersion lens. TEM was done on a Philips CM 20 HRTEM (Philips, Eindhoven, The Netherlands) at 200 keV using a LaB6 source. The sample was prepared by focused ion beam (FIB) cutting of a transparent aggregate made with 7.5% citrate. For sample preparation, the sample mounted on a aluminum stub and a 25 × 10 × 1 μm sample was cut free using a FIB VERSA 3D (FEI, Eindhoven, The Netherlands) equipped with a gallium ion gun and transferred to a PELCO lift out 4 narrow post copper TEM grid. On the holder, the sample was further thinned down in the FIB to
