Nano-gold biosynthesis by silica-encapsulated ...

View Article Online / Journal Homepage / Table of Contents for this issue

PAPER

www.rsc.org/materials | Journal of Materials Chemistry

Nano-gold biosynthesis by silica-encapsulated micro-algae: a ‘‘living’’ bio-hybrid material† ‡ Clemence Sicard,ab Roberta Brayner,*a Jeremie Margueritat,a Miryana Hemadi,a Alain Coute,c Claude Yepremian,c Chakib Djediat,c Jean Aubard,a Fernand Fievet,a Jacques Livageb and Thibaud Coradin*b

Downloaded by Université Claude Bernard Lyon on 25 January 2013 Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0JM01735C

Received 3rd June 2010, Accepted 23rd July 2010 DOI: 10.1039/c0jm01735c Klebsormidium flaccidum algal cells exhibiting the ability to form gold nanoparticles intra-cellularly in suspension were encapsulated within silica gels. Optical and electronic microscopy indicate that entrapped cells maintain their ability to reduce gold salts. A difference in the kinetics of gold colloid formation within silica in the absence or presence of cells could be followed by UV-visible absorption spectroscopy, confirming the bio-mediated nature of the reduction process. Study of the photosynthetic activity of the algae showed that the encapsulation process protects the cells from lethal effects arising from gold toxicity. Moreover, the first in situ imaging of entrapped cells using Raman spectroscopy allowed the investigation of the influence of the gold colloids on the photosynthetic system of the algae, in particular through modification of chlorophyll fluorescence and carotenoid signals. Such a coupling of sol–gel encapsulation and Raman imaging should allow the future development of novel photosynthesis-based cellular biosensors.

Introduction The term ‘‘bio-hybrid’’ can be associated with materials that have in common to associate a biological component and an inorganic component.1 However, they can exhibit very different structures and properties mainly depending on the nature of the biological system. In a first case, this system consists of a biological molecule or macromolecule, which is used for its physico-chemical properties (self-organization, mechanical properties,.) (structural bio-hybrids).2 In a second case, natural systems exhibit a specific biological activity (enzymatic or metabolic activity) that is preserved within the inorganic material (functional biohybrids).3 Finally, these biological activities can be used to elaborate materials or to modify their properties (‘‘living’’ materials).4 These three kinds of materials may find a wide range of applications, in particular in catalysis, in drug delivery and for the design of biosensors and bioreactors.

a Universit e Paris Diderot (Paris 7), CNRS, UMR 7086, Interfaces, Traitements, Organisation et Dynamique des Syst emes (ITODYS), 2 place Jussieu, F-75251 Paris Cedex 05, France. E-mail: roberta. [email protected]; Fax: +33 157277263; Tel: +33 157278764 b UPMC Univ Paris 06, CNRS, Chimie de la Mati ere Condens ee de Paris, Coll ege de France, 11 place Marcelin Berthelot, F-75005 Paris, France. E-mail: [email protected]; Fax: +33 1 44274769; Tel: +33 1 44275517 c Mus eum National d’Histoire Naturelle, D epartement RDDM, USM 505, 57 rue Cuvier, F-75005 Paris, France † This paper is part of a Journal of Materials Chemistry themed issue on Advanced Hybrid Materials, inspired by the symposium on Advanced Hybrid Materials: Stakes and Concepts, E-MRS 2010 meeting in Strasbourg. Guest editors: Pierre Rabu and Andreas Taubert. ‡ Electronic Supplementary Information (ESI) available: SEM images of encapsulated cells before gold production; SEM image and EDX analysis of encapsulated cells after gold production; TEM image of Au colloids in cell-free silica gel. XRD and XPS data for silica-encapsulated cells after gold production. See DOI: 10.1039/c0jm01735c/

9342 | J. Mater. Chem., 2010, 20, 9342–9347

What is the difference between a bio-hybrid and a traditional organic-inorganic hybrid material? In terms of properties, the chemical and structural complexity of biopolymers, the specificity and efficiency of enzymatic reactions as well as the metabolic diversity of cells overpass by far the current performance of synthetic organic molecules and materials. In terms of synthesis, the reaction conditions are more and more drastic (reagents, solvents, pH, temperature, etc.) as one wishes to preserve (and use) the biological function of the system in an optimal manner. In this context, it is not surprising that cyanobacteria and micro-algae have been extensively used for the design of cellbased bio-hybrid materials.5 In terms of the encapsulation process, these organisms may survive in a wide range of pH conditions and can produce an external network of polysaccharide (EPS) that can protect them against detrimental interactions with the host internal surface. In terms of applications, they can be used for the design of biosensors due to their environment-sensitive photosynthetic activity as well as bioreactors for the production of hydrogen and molecular metabolites of interest.6,7 In the present work, we took advantage of another property of photosynthetic organisms related to their ability to form metal and metal oxide nanoparticles extra- or intra-cellularly.8 This pathway constitutes one of the most promising route to ‘‘green’’ nanomaterials. However, up to now, this process was only studied in solution. Here we wished to perform the nanoparticle production reaction within an inorganic solid in order to stabilize the cells. Moreover, encapsulation was expected to confine the metal nanoparticles within the algae or at their close vicinity, enhancing their influence on cellular activity. Here we show that entrapped algae maintain their ability to form gold colloids that are localized intra-cellularly and on the cell outer-coating. The encapsulation significantly decreases the impact of gold formation on the photosynthetic activity of the cells. In addition, for the first time to our knowledge, it was possible to use Raman This journal is ª The Royal Society of Chemistry 2010

View Article Online

spectroscopy imaging for the in situ study of encapsulated cells, opening the route to the design of novel cell-based biosensors.9

Experimental section


Cell description and culture Klebsormidium flaccidum cells were selected on the basis of previous reports showing that they are able to synthesize Au nanoparticles via an enzymatic route.8c K. flaccidum, benthic green-algae, strain ALCP 749 (MNHN culture collection), was isolated from a sample of a black soiling developing on buildings near Paris (France). Morphologically, K. flaccidum green-algae possess cells, cylindrical to barrel-shaped, forming long filaments slightly constricted at cross-walls, one parietal and cup-shaped chloroplast, covering approximately 2/3 of the cells. K. flaccidum was grown in 250 mL Erlenmeyer flasks, in sterile Bold’s basal medium (BB medium), under controlled temperature of 20 1 C and luminosity of 50–80 mmol m2 s1 photosynthetic photon flux (PPF) for 16 h (8 h dark period) under ambient CO2 conditions. The cell suspension was transferred (10% (v/v) of inoculum) into 25 mL of culture medium, grown for 4 weeks and centrifuged to recover 5 mL of cell suspension. Encapsulation procedure Silica encapsulation was performed as previously described in the literature using sodium silicate and colloidal silica (Ludox) as precursors.10 However, compared to previous reports on bacteria encapsulation, the addition of glycerol was found unnecessary. A second layer of gel (1 mL), resulting from the acidification in the same ratio of a mixture of sodium silicate and Ludox, was added on top of the gel to limit leaching of algae from the gels. For colloidal gold formation, an aqueous solution of HAuCl4 at 103M was added above the gels.

otherwise reduce the maximum fluorescence yield. A ratio of variable-over-maximal fluorescence (Fv/Fm) can then be calculated which approximates the potential quantum yield of PS II. Raman spectra were obtained with a LABRAM Jobin-Yvon microspectrometer. The microscope was equipped with a long working distance X 100 objective (NA: 0.60). The spectra have been recorded using two different excitation wavelength: 514 nm (Ar laser) and 633 nm (He–Ne laser), and a XY micrometre displacement stage was used to performed Raman mapping.

Results and discussion When HAuCl4 was added to Kf cells encapsulated in a silica gel, the inorganic matrix slowly turned from green to pink, suggesting the formation of colloidal metallic gold (Fig.1a). The process starts at the gel/supernatant interface and then proceeds to the bottom of the gel. Optical microscopy of cells shows that the green chloroplasts inside algal cells turn purple after gold addition and reduction (Fig. 1b,c). SEM allows the observation of algal filaments trapped within the silica network (ESI†). Secondary electron imaging turns the cell wall into a transparent membrane allowing the visualization of the algae plasts (Fig. 2a). After gold reduction, metallic nanoparticles become apparent as bright dots on the cell surface (Fig. 2b, c), whose chemical nature could be confirmed by EDX (ESI†). TEM images before gold addition shows a well-preserved cell, with intact wall and intra-cellular structure, including thylakoids (Fig. 3a). The colloidal silica network is surrounding the algal cell but does not appear to be in direct contact with the cell wall but rather to stick on the surface of the cell gelatineous sheath. Noticeably, the proximity of the silica network with the

Gold nanoparticles formation and characterization TEM observations were performed in bright field mode with two different electron microscopes - a Philips CM12 and a JEOL 1011 equipped with a numeric camera - operating both at low accelerating voltage (60 kV) to avoid organic phase damage under the beam. Both imaging and diffraction modes were employed for characterization. Specimens were dispersed on 3 mm carbon-coated copper grids by deposition of a drop of a diluted suspension of particles in ethanol, after sonication. SEM observations were performed with a JEOL JSM 6100 (tungsten filament) operating at 20 kV, with a working distance of 10 mm. Prior to observation the samples were fixed with glutaraldehyde, dehydrated in acetone and dried with a critical point dryer BAL-TEC CPD 030 with liquid CO2 (critical point 31 C, 73.8 bar). Imaging was also performed with a high voltage Secondary Electron Detector. The photosynthetic activity of microalgae was measured using the pulsed amplitude modulation (PAM) method with a Handy PEA (Hansatech instruments) fluorometer. This method uses the saturation pulse method, in which a phytoplankton sample is subjected to a short beam of light that saturates the photosystem II (PS II) reaction centers of the active chlorophyll molecules.11 This process suppresses photochemical quenching, which might This journal is ª The Royal Society of Chemistry 2010

Fig. 1 (a) Evolution with time of silica gels encapsulating Kf cells after HAuCl4 addition. (b,c) Optical microscopy images of encapsulated cells before and after gold reduction (x 2000).

J. Mater. Chem., 2010, 20, 9342–9347 | 9343


View Article Online

Fig. 2 SEM secondary electron imaging of Kf cells within silica gels before (a) and after (b) gold reduction. (scale bar ¼ 1 mm). At higher magnification (c), white dots correspond to Au nanoparticles (scale bar ¼ 250 nm).

cell membrane was observed to vary strongly from sample to sample, suggesting that it may depend not only on the preparation procedure but also on the physiological state of the algae. One week after gold salt addition, cell integrity appears preserved within the silica network (Fig. 3b). Dark spots 10–15 nm in diameter are clearly visible in the thylakoid membranes (Fig. 3c). At higher magnification, particles are also observed within the cell EPS network (Fig. 3d). These observations are in agreement with our previous studies on non-encapsulated cells suggesting that gold reduction occurs in thylakoids where reducing enzymes are present.8c Resulting colloids were observed to diffuse out of the cells and become trapped in the surrounding EPS. It was also possible to detect particles within the silica gel although they were only sparingly present and of similar size as SiO2 colloids (Fig. 3e). Electron diffraction pattern confirms the presence of Au in the cfc crystalline phase (Fig. 3f). Attempts to characterize further gold colloids by XRD and XPS were unsucessful, probably due to (i) the low concentration of metallic nanoparticles within the gel and (ii) the fact that most particles are buried inside the cells which are trapped inside the silica gel (ESI†). The kinetics of gold reduction was followed by UV-visible spectroscopy. Initially, a broad band in the 450–500 nm and an absorption near 680 nm can be distinguished (see arrows on Fig. 4a), corresponding to carotenoids and similar pigments and to chlorphyll a and b, respectively. After 100 h, an intense band at 560 nm has grown, corresponding to colloidal gold surface plasmon resonance absorption (Fig. 4a). The intensity of this band continuously increase up to ca. 80 h and then reach a plateau. (Fig. 4b). It is worth noticing that even when cells were 9344 | J. Mater. Chem., 2010, 20, 9342–9347

Fig. 3 TEM images of Kf cells within silica before (a) and after (b) gold addition. Gold colloids could be found within the cells in the thylakoids (c), in the cell EPS (d) and in the surrounding silica gel (e). ED pattern of dark dots with examples of attribution to the cfc Au structure (f).

not present, gold reduction also occurred but with a different reduction rate, with a threshold value being reached after ca. 6 h (Fig. 4b). The plasmon resonance absorption band maximum shifted from 540 nm to 650 nm during the 100 h reaction time (Fig. 4a), and the corresponding nanoparticles were larger (> 50 nm) (ESI†). The PAM method was previously shown to be well-adapted to follow the photosynthetic activity of micro-algae entrapped in silica gels.4d Here, measurements were performed over two weeks for cell suspensions and encapsulated cells without or with gold salt addition (Fig. 5). Over this period, Kf in suspension maintains a constant high activity whereas entrapped cells also show a constant PSII efficiency but a much lower Fv/Fm value that appears to result from their immobilisation in silica. After gold addition, free cells are not significantly perturbed after one week but their photosynthetic activity drops down to a negligible value after two weeks. In the same conditions, entrapped cells maintain a photosynthetic activity similar to that observed without gold. We then turned our attention on the possibility to use gold colloids for Surface Enhanced Raman Scattering (SERS) measurements, with the aim of designing a cell-based biosensor.9 Raman spectra and images of free and encapsulated Klebsormidium flaccidum, excited at 514 nm and 633 nm are presented in Fig. 6 and 7. Before gold synthesis, the Raman spectra excited at 633 nm (Fig. 6b) show a broad band centred at 1152 cm1 (683 nm), corresponding to the fluorescence of chlorophyll localised inside the chloroplast, as observed in Fig. 7e and 7g. The signal intensity is weaker for encapsulated cells than for free This journal is ª The Royal Society of Chemistry 2010


View Article Online

Fig. 4 (a) Evolution with time of the UV-visible absorption spectra of silica gels after gold salt addition in the absence (dashed lines) or presence (plain lines) of Kf cells. (b) Evolution with time of the intensity of the plasmon resonance band (corrected with the optical density at 450 nm) within the silica gels after gold salt addition in the absence (open circle) or presence (dark circle) of Kf cells.

Fig. 5 Evolution of the photosynthetic activity of Kf, as given by the Fv/Fm ratio by PAM fluorimetry for cell suspension (circles) and encapsulated cells (squares) without (dark symbols) and with (open symbols) added HAuCl4.

ones, due to their lower photosynthetic activity as revealed by the PAM measurements (vide infra). When excited at 514 nm, Raman spectra (Fig. 6a) show intense lines at 994, 1174 and 1506 cm1. These lines are readily attributed to the presence of carotenoids. Indeed, at that excitation wavelength, carotenoids are specifically excited in resonance, leading to a huge enhancement of Raman lines of the chromophore (i.e. the polyenic chain in carotenoids).12 Therefore, the three main lines correspond to CH3 in plane rocking vibrations, C–C stretching coupled with C– C–H bands and C]C stretching mode, respectively. Once gold nanoparticles were produced by cells, the chlorophyll fluorescence is strongly attenuated (Fig. 6b, 7f and 7h). This quenching may arise from two phenomena: i) the inhibition of the photosynthetic process during particle formation; ii) the quenching of the chlorophyll fluorescence by the gold nanoparticles that are located at their vicinity, as observed in Fig. 3c.13 In parallel, the high density of gold nanoparticles produced in situ should also enhance the Raman signal of the carotenoids via the SERS effect.14 On the contrary, as shown on Fig. 6a, the Raman spectrum of carotenoids, excited at 514 nm, disappears while an unstructured broad emission spectrum, red-shifted with respect to chlorophyll fluorescence, appears. This observation could be explained by a strong modification of carotenoids during gold nanoparticles formation, leading to a drastic change in the electronic levels of these molecules and thus the loss of the resonance excitation of the Raman spectra. Noticeably, This journal is ª The Royal Society of Chemistry 2010

Fig. 6 Raman spectra excited at (a) 514 nm and (b) 633 nm on free (grey curves) and encapsulated (green curves) Kf before (lines) and after (dots) gold production.

a modification of carotenoid production was previously reported as an effect of heavy metals interaction with algae.15 The disappearance of the carotenoid Raman spectrum could also result from a strong activation of the charge transfer between J. Mater. Chem., 2010, 20, 9342–9347 | 9345


View Article Online

Fig. 7 Optical images (right column) and reconstructed images from the intensity of Raman spectra with excitation at 514 nm (left column) and 633nm (middle column), for encapsulated cell before (top line) and after (second line) gold production and for free cell before (third line) and after (bottom line) gold production.

carotenoids and chlorophyll due to the presence of gold nanoparticles within the photosynthetic complex, as previously described.16

Conclusion The design of cell-based biosensors is one of the most challenging area in biotechnology because it requires (i) the preservation of the cell viability and activity and (ii) a suitable signal/transduction system.17 In many cases where a 3D immobilization of the active cells is required, it is necessary to develop a cytocompatible chemical route for encapsulation and identify in situ techniques for the detection of biological activities. In this context, the present work confirms that the sol–gel process is suitable for the encapsulation of algal species within silica gels.5 In particular, it indicates that immobilized cells 9346 | J. Mater. Chem., 2010, 20, 9342–9347

maintain their ability to synthesize gold nanoparticles intracellularly,8 while limiting detrimental side-effects of this process. Moreover, our results demonstrate that Raman imaging is a suitable tool to image silica-encapsulated green algae, allowing the monitoring of the influence of gold nanoparticles on their photosynthetic system. Evaluation of these novel ‘‘living’’ bio-hybrid systems coupled to in situ Raman detection as photosynthesis-based environmental biosensors can now be foreseen.18

Acknowledgements C.S. thanks the Ile-de-France Region and CNRS for providing her PhD grant in the frame of the CNano IdF program. The help of A. Anglo (LCMCP), F. Herbst and D. Montero (ITODYS) for electronic microscopy experiments is also acknowledged. This journal is ª The Royal Society of Chemistry 2010

View Article Online


Notes and references 1 (a) Bio-inorganic Hybrid Nanomaterials, E. Ruiz-Hitzky, K. Ariga and Y. Lvov (ed.), Wiley-VCH, Weinheim, 2008; (b) M. Darder, P. Aranda and E. Ruiz-Hitzky, Adv. Mater., 2007, 19, 1309; (c) T. Coradin, J. Allouche, M. Boissiere and J. Livage, Curr. Nanosci., 2006, 2, 219. 2 (a) M. Sarakaya, C. Tamerler, A. K. Y. Schulten and F. B. Baneyx, Nat. Mater., 2003, 2, 577; (b) C. B. Mao, D. J. Solis, B. D. Reiss, S. T. Kottmann, R. Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson and A. M. Belcher, Science, 2004, 303, 213; (c) M. B. Dickerson, K. H. Sandhage and R. R. Naik, Chem. Rev., 2008, 108, 4935; (d) S. Mann, Nat. Mater., 2009, 8, 781. 3 (a) I. Gill and A. Ballesteros, Trends Biotechnol., 2000, 18, 282; (b) W. Jin and J. D. Brennan, Anal. Chim. Acta, 2002, 461, 1; (c) H. B€ ottcher, U. Soltmann, U. M. Mertig and W. Pompe, J. Mater. Chem., 2004, 14, 2176; (d) G. Carturan, R. Dal Toso, S. Boninsegna and R. Dal Monte, J. Mater. Chem., 2004, 14, 2087; (e) D. Avnir, T. Coradin, O. Lev and J. Livage, J. Mater. Chem., 2006, 16, 1013; (f) J. Livage and T. Coradin, Rev. Mineral. Geochem., 2006, 64, 315. 4 (a) P. Mukherjee, A. Ahmad, D. Mandal, S. Senapati, S. R. Saikar, M. I. Khan, R. Ramani, R. Parischa, P. V. Ajaykumar, M. Alam, M. Sastry and R. Kumar, Angew. Chem., Int. Ed., 2001, 40, 3585; (b) S. S. Shankar, A. Ahmad, R. Pasricha and M. Sastry, J. Mater. Chem., 2003, 13, 1822; (c) H. K. Baca, C. Ashley, E. Carnes, D. Lopez, J. Flemming, D. Dunphy, S. Singh, Z. Chen, N. G. Liu, H. Y. Fan, G. P. Lopez, S. M. Brozik, M. Werner-Washburne and C. J. Brinker, Science, 2006, 313, 337; (d) C. Gautier, J. Livage, T. Coradin and P. J. Lopez, Chem. Commun., 2006, 4611; (e) F. Wang and C. Mao, Chem. Commun., 2009, 1222. 5 (a) J. C. Rooke, C. F. Meunier, A. Leonard and B.-L. Su, Pure Appl. Chem., 2008, 80, 2345; (b) C. F. Meunier, P. Dandoy and B.-L. Su, J. Colloid Interface Sci., 2010, 342, 211. 6 (a) J. C. Rooke, A. Leonard, H. Sarmento, J.-P. Descy and B.-L. Su, J. Mater. Chem., 2008, 18, 2833; (b) E. Pena-Vasquez, E. Maneiro, C. Perez-Conde, M. C. Moreno-Bondi and E. Costas, Biosens. Bioelectron., 2009, 24, 3538; (c) H. Nguyen-Noc and C. Tran-Minh, Anal. Chim. Acta, 2007, 583, 161. 7 (a) D. Fiedler, U. Hager, H. Franke, U. Soltmann and H. B€ ottcher, J. Mater. Chem., 2007, 17, 261; (b) J. C. Rooke, A. Leonard and

This journal is ª The Royal Society of Chemistry 2010

8

9 10

11 12 13 14 15 16 17 18

B.-L. Su, J. Mater. Chem., 2008, 18, 1333; (c) D. J. Dickson, C. J. Page and R. L. Ely, Int. J. Hydrogen Energy, 2009, 34, 204; (d) S. Ramachandran, T. Coradin, P. K. Jain and S. K. Verma, Silicon, 2009, 1, 215; (e) A. Leonard, J. C. Rooke, C. F. Meunier, H. Sarmento, J.-P. Descy and B.-L. Su, Energy Environ. Sci., 2010, 3, 370. (a) K. B. Narayanan and N. Sakthivel, Adv. Colloid Interface Sci., 2010, 156, 1, and references therein; (b) M. F. Lengke, M. E. Fleet and G. Southam, Langmuir, 2006, 22, 2780; (c) R. Brayner, H. Barberousse, M. Hemadi, C. Djediat, C. Yepremian, T. Coradin, J. Livage, F. Fievet and A. Coute, J. Nanosci. Nanotechnol., 2007, 7, 2696; (d) M. F. Lengke, M. E. Fleet and G. Southam, Langmuir, 2007, 23, 8982; (e) R. Brayner, C. Yepremian, C. Djediat, T. Coradin, F. Herbst, J. Livage, F. Fievet and A. Coute, Langmuir, 2009, 25, 10062. (a) T. Vo-Dinh, Sens. Actuators, B, 1995, 29, 183; (b) I. Notingher, Sensors, 2007, 7, 1343. (a) N. Nassif, O. Bouvet, M. N. Rager, C. Roux, T. Coradin and J. Livage, Nat. Mater., 2002, 1, 42; (b) N. Nassif, C. Roux, T. Coradin, M. N. Rager, O. M. M. Bouvet and J. Livage, J. Mater. Chem., 2003, 13, 203; (c) N. Nassif, C. Roux, T. Coradin, O. M. M. Bouvet and J. Livage, J. Mater. Chem., 2004, 14, 2264. (a) K. Rohacek and M. Bartak, Photosynthetica, 1999, 37, 339; (b) T. K. Antal, T. E. Krendeleva and A. B. Rubin, Photosynth. Res., 2007, 94, 13; (c) R. J. Ritchie, J. Photosynth. Res., 2008, 96, 20. A. T. Tu, Raman Spectroscopy in biology: principles and applications, John Wiley & Sons, 1982. J. R. Lakowicz, Principles of fluorescence spectroscopy, 3rd Edition, Springer, 2006. Q. Liao, C. Mu, D.-S. Xu, X.-C. Ai, J.-N. Yao and J.-P. Zhang, Langmuir, 2009, 25, 4708. E. Pinto, T. C. S. Sigaud-Kutner, M. A. S. Laitao, O. K. Okamoto, D. Morse and P. Colepicolo, J. Phycol., 2003, 39, 1008. S. Mackowski, S. W€ ormke, A. J. Maier, T. H. P. Brotosudarmo, H. Harutyunyan, A. Hartschuh, A. O. Gorovov, H. Scheer and C. Br€auchle, Nano Lett., 2008, 8, 558, and references therein. Y. Lei, W. Chen and A. Mulchandani, Anal. Chim. Acta, 2006, 568, 200. M. Campas, R. Carpentier and R. Rouillon, Biotechnol. Adv., 2008, 26, 370.

J. Mater. Chem., 2010, 20, 9342–9347 | 9347