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The amorphous frustule with a mesopore pattern of the peanut-shaped marine diatom Psammodictyon panduriforme (Gregory) Mann comb. nov. (Bacillariophyta) was analyzed. Two dominating photoluminescence emission peaks in P. panduriforme were observed at 417 nm and 534 nm, and are attributed to radiative luminescence caused by oxygen-vacancy defects on the diatom frustules. Under the 355 nm pulse laser illumination, a narrow PL spectrum of frustules from the diatom P. panduriforme was observed at 475 nm with a 9.3 nm linewidth that may be caused by the resonance cavity effect on the quasi-regular pore structure on the frustules. Diatom frustules from Psammodictyon panduriforme (Gregory) Mann comb. nov. (Bacillariophyta) have quasi-regular pore patterns on the biosilica valves that can be utilized in optoelectronic devices with mesoporous structures.
© 2016 Optical Society of America
1. Introduction
Diatoms are unicellular algae with a cell wall (also named as frustules) composed primarily of glass-like SiOx [1]. The diatomic frustules are distinct, highly ornamented because they are perforated with unique pores that vary greatly depending on the species. As a result of their unique morphology and porous biosilica walls, diatoms have been applied in nanotechnology as gratings [2] and in photolithography as masks [3]. The surface of the diatom frustules is covered with arrays of nano-pores and micro-pores with pore diameters ranging from several nm up to micron scale. The light weight and strong mechanical strength properties of diatom frustules are believed to be derived from the tiny pore structures. Sub-micro regular arrays of holes have been characterized in frustules [4,5]. Gale et al. [6] reported that the antibody-functionalized diatom biosilica frustules serve as a microscale biosensor platform for selective and label-free photoluminescence (PL) based detection of immunocomplex formations. The photonic properties [7] and the light focusing ability [8] of diatoms had also been reported.
In this study, the peanut-shaped marine diatom, Psammodictyon panduriforme (Gregory) Mann comb. nov. (Bacillariophyta), was analyzed as an amorphous SiO2 structure of the diatom frustules with mesoporous patterns in the valves [9]. Optical and material properties of diatom frustules from P. panduriforme are analyzed in detail. Characterization of chemical and optical properties of diatom frustules is important and interesting because its diverse shapes, porous/patterned micro-structures, various optical properties such as fluorescence emission and light focusing ability, and biocompatibility allow these to be utilized in the field of biophotonics, sensors, microfluidics, and especially optoelectronic devices.
2. Experiments
The photosynthetic peanut-shaped marine diatom Psammodictyon panduriforme (Gregory) Mann comb. nov. (Bacillariophyta), was grown by controlling at a temperature of 24°C for 14hrs illumination/10hrs darkness cycles in a 100rpm shaker (orbital shaker). Intact biosilica of P. panduriforme cells were isolated by treating them with a dilute H2SO4 solution. 50ml aliquots of culture suspension were collected and centrifuged at 2000rpm for 10minutes. The pellet was re-suspended in 50ml of de-ionized water, centrifuged in the same conditions, and washed again for five times. The washed diatom cell pellet from P. panduriforme was resuspended in dilute H2SO4 solution, and reacted in a 60°C water bath for 10minutes. The liquid in the suspension turned dark green. The treated cell mass from P. panduriforme was washed five times in de-ionized water, and then stored at 4°C. Unstained P. panduriforme cells and clean diatom frustules (also from P. panduriforme) were placed on a 0.17mm cover slip and observed using confocal microscopy (Olympus FV1000, Japan). The objective used was 40 × from Olympus (0.9 N.A.,Olympus, Japan). The samples were excited by using a 405 nm laser diode as an excitation source. Field emission scanning electron microscopy (cold cathode type, JEOL Ltd., JSM-6700F, Tokyo, Japan) was conducted on an Energy Dispersion X-ray Spectroscopy (EDS) system (Noran Phage-ID). The SEM micrographs were used to observe the morphology and microstructure of the diatom Psammodictyon panduriforme (Gregory) Mann comb. nov. (Bacillariophyta). The surface morphology, micro-structure, and crystal structures of the diatom P. panduriforme were examined by high resolution transmission electron microscopy (HRTEM/JEM-2100, Tokyo, Japan) and operated at an accelerating voltage of 160 kV. A piece of glass with diatoms from P. panduriforme was scraped and dispersed on a carbon coated copper mesh (200mesh, Agar Scientific Ltd., U.K.). The crystal structures and the crystal lattices were determined by using selected area electron diffraction patterns. Elemental analysis was performed by energy dispersion spectroscopy (EDS, OXFORDEXL 10/T). It analyzed the elements in diatom frustules from P. panduriforme. The functional groups and chemical bonding of the diatom frustules valve biosilica from P. panduriforme were directly analyzed by transmission Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra were collected by a Spectrum One (Perkin Elmer, Norwalk, CT, USA). The scan number was eight with a range from 400 to 4000cm−1 with a resolution of 2 cm−1. A HRXRD diffractometry pattern was another type of data used to characterize the micro-structures and crystal structures of the diatom P. panduriforme. HRXRD patterns were detected by a Bruker D8 Advance diffractometer (Germany). The source of HRXRD was Cu Kα (1.542 angstrom) and the diffractometer was operated at 40kV and 41mA. The scanning rate was 0.05° at 2θ/sec and the range of 2θ was from 5° to 80°. The micro-photoluminescence (µ-PL) spectra were measured by using a 325nm He–Cd laser (Kimmon IK3452R-F) and a 355nm Nd-YVO4 pulse laser as the excited sources. Laser beam was focused to a 10 µm diameter laser spot size with a convex lens. The PL spectra were collected by using a Jobin Yvon Triax 550 monochromator with a 600lines/mm grating and a matched liquid nitrogen cooled CCD detector.
3. Results and discussion
The fluorescence emission spectra of live P. panduriforme diatoms and diatom frustules with the organic matter removed from them were observed with a 405nm diode laser illumination as shown in Fig. 1. A distinct emission peak wavelength at 670nm (red fluorescence) was measured that indicated an emission from the chlorophyll[R] in live diatom P. panduriforme cells. After removing the organic material from the diatom P. panduriforme, the peak wavelengths of the diatom frustules were observed at 470nm and 520nm.
Fig. 1 The PL spectra of live diatoms and diatom frustules P. panduriforme diatoms were measured. The PL intensity of diatom frustules was amplified about 3-fold to show the peaks.
The SEM micrographs and the EDS characterization of frustules of the diatom P. panduriforme were measured as seen in Fig. 2. In Figs. 2(a) and 2(b), the full view of the diatom is observed showing that P. panduriforme has peanut-like shaped frustules. The silica frustule consists of two overlapping valves that are joined by girdle bands. The length of the frustules ranges from 8 to 15μm and the width from 5 to 8μm. Krögerl et al. [10] reported that a diatom cell wall consists of two thecae: an epitheca (ET) and a hypotheca (HT) as the valve structures, with the girdle bands (GB) at the sidewall surface. The isoalted diatom cell wall in P. panduriforme was observed to have a species-specific pore structure on the valve surface and on the girdle band sidewall as shown in Fig. 2(a). In Fig. 2(b), the complete diatom frustules from P. panduriforme with the ET and the HT structures were observed in detail for structural analysis. In Fig. 2(c), the mesoporous frustule structure of the diatom P. panduriforme is observed in side view of the girdle band structure. Figures 2(d),(e) show the magnified surface of the frustules biosilica valve in the diatom P. panduriforme with the porous structures. The frustules exhibit regular shapes, ranging between 350 and 400nm in size and are occluded by internal cribra with circular pores of 40–60 nm in diameter. Figure 2(f) is the EDS spectrum of the frustule biosilica from the diatom P. panduriforme. The peaks of sodium, calcium and magnesium are attributed to tiny amounts of residual sea water salts. The aluminum signal is correlated to the Al-coated glass substrate. There are two main peaks, silicon (1.72keV) and oxygen (0.52keV). The molar ratio of Si/O is 1/3.6 which is close to the 1/4 and corresponding to chemical stoichiometric composition of SiO2.
Fig. 2 SEM micrographs and EDS characterization of frustules biosilica from the diatom Psammodictyon panduriforme: (a)(b) Full views of single diatom frustules. (c) The porous structure of the sidewall region of P. panduriforme. (d)(e) Magnified micrographs of the valve structures of P. panduriforme. The mesoporous structure can be observed in the valve structure. (f) The EDS spectrum of frustules from the diatom P. panduriforme showing the biosilica composition.
The X-ray diffraction curves of the frustules from the diatom P. panduriforme and the glass substrate serving as a template were measured and are shown in Fig. 3(a). The material properties of frustules from the diatom P. panduriforme are identified by a broad peak indicating that diatom frustules are composed of an amorphous biosilica similar to glass substrate. Diatom frustules consist of porous and amorphous bio-mineralized architectures. From the EDS and the x-ray diffraction results, the frustules from the diatom P. panduriforme were analyzed as amorphous silicic acid, SiO2, with a porous wall structure.
Fig. 3 (a) The X-ray diffraction pattern of the biosilica in the diatom Psammodictyon panduriforme compared to commercial glass. (b) The FT-IR spectrum of the diatom biosilica in P. panduriforme.
The FT-IR spectrum of the frustules in the diatom P. panduriforme is shown in Fig. 3(b). The most common peaks of the diatom frustules consisted of the Si-O-Si stretching at 1040-1095cm−1, the Si-O-Si bending at 797-800cm−1, the Si-O stretching of Si-OH groups at 797cm−1, and O-H stretching of hydroxyl groups at 3435cm−1. The weak signals of 664 and 2200cm−1 represents very tiny amounts of Si-H bands. The peaks of 3740-3780 and 1630-1640cm−1 were identified as Si-OH bonds. There are also peaks of carboxyl groups, including C = O stretching at 1735cm−1, C-H stretching at 2850 (CH2) and 2925 (CH3) cm−1. A small C-H peak at 2917cm−1 and a possible peak at 800 cm−1 covered by the Si-O-Si at the same location. The source of the carboxyl groups are owed to the residual organic materials in the diatom P. panduriforme. They correspond to the primary amide of proteins bounded on the surface of the diatom biosilica.
The μ-PL spectra of frustules of the diatom P. panduriforme were measured as shown in Fig. 4(a) excited by a continuous-wavelength 325nm HeCd laser. The two dominant peak wavelengths and the line-widths of the PL spectra were measured at 417 nm (39nm) and 534nm (121nm). The peak wavelength of the frustules from the diatom P. panduriforme was measured at 534nm (2.32 eV) similar to what Stefano et al.’s reports [11]. Zhu et al. [12] reported that three PL bands centered around 1.7, 2.1, and 2.9 eV have been detected from a-SiOx:H material prepared by dual-plasma chemical vapor deposition. The radiative defect luminescence mechanisms are attributed to Si–OH groups (2.9 eV) or to oxygen-vacancy defects (2.1 eV) [13–15]. The biosilica composition and the radiative recombination from oxygen vacancy of the frustules of the diatom P. panduriforme were analyzed through FTIR analysis and the PL spectra, respectively. By reducing the measurement temperature from 300K to 10K in the vacuum chamber, the PL emission intensities of the diatom frustules was slightly increased at 10K indicating that the thermal quench phenomenon was not clearly observed in the diatom frustules. The radiative defect luminescence was confined by the mesoporous [16] structure in the frustules of P. panduriforme. In Fig. 4(b), the PL spectrum of the frustules of the diatom P. panduriforme was measured when excited by a 355nm Nd-YVO4 pulse laser at 300K. The peak wavelength and the line width of the diatom frustules were measured at 475.2 nm and 9.3nm, respectively. The broadened PL emission linewidth was measured through a 325nm CW HeCd laser, and a quasi-regular pore structure was observed in the diatom frustules. Under a pulsing 355nm laser excitation, the PL emission linewidth was reduced which could have been caused by the resonance cavity effect in the quasi-regular pore structure. This linewidth reducing phenomenon is similar to the photonic crystal structure in Si- and GaAs-based optoelectronic devices with a regular sub-micro air holes pattern [17,18].
Fig. 4 The micro-photoluminescence spectra of diatom frustule were measured by (a) 325 CW laser and (b) 355nm pulse laser acted the excitation laser sources.
High resolution TEM micrographs of frustule microstructure from the diatom Psammodictyon panduriforme are shown in Figs. 5. Full view of the single peanut-shaped diatom frustules measured 11μm in length and 5μm in width, respectively (Fig. 5a). The surface morphology of frustules from the diatom P. panduriforme consisted of the biosilica valve and the pore pattern structures shown in Figs. 5(b),(c). The pore array is periodic with regular nanostructures on the frustules biosilica valve of P. panduriforme. In Fig. 5(d), the edge of the girdle band of the diatom frustules is filled with mesoporous structures. There are two to four mesoporous in a micro-grain structure. The fine structure of the pore array on frustules valves from the diatom P. panduriforme was observed in Fig. 5(e). The diameter of the pore size measured in average, 50nm. In Fig. 5(f), the ring diffraction pattern of the frustules from the diatom P. panduriforme was observed demonstrating its amorphous biosilica structure.
Fig. 5 HR-TEM images and diffraction ring of frustule biosilica in the diatom Psammodictyon panduriforme. (a) The HRTEM micrographs of full view of single diatom frustules from P. panduriforme, (b) and (c) the surface of diatom frustule biosilica valve from P. panduriforme with the pore structures, (d) The edge of the girdle band of frustules from the diatom P. panduriforme with plenty of mesoporous, (e) The fine structure of pore array on the valve frustules from the diatom P. panduriforme. (f) The ring diffraction pattern of diatom frustules biosilica in P. panduriforme.
To analyze the light extraction properties, P. panduriforme frustules were spreaded on an InGaN-based light emitting diodes epitaxial wafer. Wang et al. [19] reported a self-assembly of nanostructured diatom microshells into patterned arrays. During the coating process, the diatom frustules were dispersed in an IPA solvent and spin coated onto the ITO layer which acted as a transparent conductive layer for the fabricated InGaN LED wafer. The thickness was from a 1 to a 2-layer diatom layer. The LED structure consisted of a low-temperature grown 30nm-thick GaN buffer layer, a 4μm-thick n-type GaN:Si layer, 0.2μm-thick InGaN/GaN multiple quantum wells active layers, and a 0.2μm-thick magnesium-doped p-type GaN:Mg layer that grown on the sapphire substrate through a metalorganic chemical vapor deposition system [20,21]. The PL spectra were analyzed through an angle-resolved photoluminescence measurement [22] with a 405nm excitation laser illuminated from the backside sapphire substrate to excite the InGaN active layer. The PL emission light from the InGaN active layer transmitted through the diatom frustules layer and detected at the normal direction by varying the detected angles. The PL emission intensity of the InGaN active layer was larger than the PL emission intensity of P. panduriforme frustules. The PL emission spectra were measured by a multi-channel CCD detector with a 550mm focal length monochromator. The PL spectra was detected at the front-side of the flat LED wafer without and with the P. panduriforme frustules structure shown in Figs. 6(a),(b). The wavelength-resolved angular far-field patterns of the P. panduriforme PL spectra were measured. In Fig. 6(a), the Fabry–Pérot (FP) interference line-patterns were observed in the LED wafer that indicated the smooth InGaN epitaxial layer at top air/GaN surface. In Fig. 6(b), the FP interference line-patterns were not observed in the LED wafer with the P. panduriforme frustules layer. This is the results of the PL emission light scattered by P. panduriforme frustules with micro- and meso-pore structure on the LED wafer.
Fig. 6 The wavelength-resolved angular far-field patterns of the PL spectra of the LED wafer (a) without Psammodictyon panduriforme frustule layer and (b) with Psammodictyon panduriforme frustule layer. (c) The far-field radiation patterns of the LED wafer without and with the frustule layer from the diatom Psammodictyon panduriforme were measured through angle-resolved PL measurements.
The far-field radiation patterns of both LED samples were measured through angle-resolved PL measurements shown in Fig. 6(c). The divergent angles of the LEDs are identified as the angle of the half-maximum PL emission intensity. The divergent angles of the LED wafer with and without diatom frustule layer were measured. The divergent angles of the LED wafer without and with P. panduriforme frustule layer were calculated at 147° and 136°, respectively. The PL emission light can be extracted through the ITO layer and coupled with the diatom frustules. For the far-field radiation pattern, the interference lines pattern were observed in the ST-LED due to the light reflectance at the top of the air/GaN and at the bottom of the GaN/sapphire flat interfaces. No interference and narrowing divergent angles were observed in the InGaN LED with the top diatom frustule layer. The light extraction efficiency in the InGaN LED can be increased through a low light refractive index diatom frustule layer above the ITO conductive layer. The divergent angle is slightly reduced due to the light coupling into the diatom frustules and the light has been redirected in the normal direction of the LED chips. The slightly narrow divergent angle of LED wafer with P. panduriforme frustule layer was caused by the PL emission light coupled with the pore structure of the diatom frustule to improve the light extraction efficiency.
4. Conclusion
The frustules of the peanut-shaped marine diatom Psammodictyon panduriforme (Gregory) Mann comb. nov. (Bacillariophyta) were analyzed as the amorphous silica structure with mesoporous structure. The two dominant PL emission peaks were observed at 417nm and 534nm and were attributed to the radiative-defect luminescence from the oxygen-vacancy defects on the diatom frustules from P. panduriforme. Under pulse laser illumination, the narrow PL spectrum of the diatom frustules from P. Panduriforme was observed to be caused by the quantum confinement effect on the mesoporous structure in the diatom frustules. The diatom frustules in P. panduriforme consisted of periodic nano-scale biosilica valves that can be utilized in optoelectronic devices with mesoporous structures.
Acknowledgment
The authors gratefully acknowledge the financial support for this research by the Ministry of Science and Technology of Taiwan under grant No. 102-2218-E-005-010-MY3 and 104-2221-E-005-014-MY2. Chia-Feng Lin, Su-Yuan Lai, and Min-Ying Wang contributed equally to this work.
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