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Composite nanoparticles: A new way to siliceous materials and a model of biosilica synthesis
Materials Chemistry and Physics xxx (2015) 1e8
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Composite nanoparticles: A new way to siliceous materials and a model of biosilica synthesis Vadim V. Annenkov a, *, Viktor A. Pal'shin a, Olga N. Verkhozina a, Lyudmila I. Larina b, Elena N. Danilovtseva a a b
Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, Russian Federation A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, Russian Federation
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A polyampholyte with pendant polyamine chains is obtained. The polymer catalyses condensation of silicic acid giving stable solutions. Gravity-induced (50,000 g) formation of solid silica was observed in these solutions. The obtained silica is close to biosilica from diatom frustules. A new approach to inorganic and composite materials is proposed.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2015 Received in revised form 7 September 2015 Accepted 14 September 2015 Available online xxx
A new polyampholyte based on poly (acrylic acid) which bears pendant polyamine oligomeric chains (average number of the nitrogen atoms is 11.2) is obtained. This polymer is a model of silaffins e proteins playing important role in formation of siliceous structures in diatom algae and sponges. The polymer catalyses condensation of silicic acid. The obtained solutions contain oligosilicates coordinated with the polymer chains. The action of 50,000 g gravity on this solution results in concentrating-induced condensation of the pre-condensed siliceous oligomers. The obtained solid silica contains 4% admixture of the organic polymer which is close to the silica from diatom frustules. These results confirm the hypothesis about formation of biosilica under the action of desiccation agent, e.g. aquaporins. The formation of solid substances during centrifugation of solutions containing soluble oligomers is a new promising approach to inorganic and composite materials which allows to work in aqueous medium and to reuse the organic polymer. © 2015 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Composite materials Nanostructures Polymers Chemical synthesis Precipitation
1. Introduction Bioinspired approaches to new inorganic and composite materials are intensively studied in the hope of elaboration environmentally friendly procedures which allow to avoid high
* Corresponding author. E-mail addresses: [email protected], [email protected] (V.V. Annenkov).
temperatures and hazardous reactants. Silicon dioxide (silica) is used by some living organisms as the construction material for elements of the skeleton. Diatom algae are the most known and studied silicon-containing organisms because of their high ecological importance (more than 20% of primary carbon assimilation [1]) and beautiful siliceous exoskeletons e frustules [2]. The frustules consist of delicate 2e2000 mm valves having species specific structure of elements (holes, nodes and etc.) ordered on micro- and nanolevel (Fig. 1A). Siliceous sponges are the other
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Fig. 1. (A) SEM of siliceous valves of the diatom Stephanodiscus meyeri Genkal et Popovsk; (B) confocal images of siliceous spicules of the sponge Lubomirskia baicalensis Pallas stained in vivo with a fluorescent dye. Scale bar represents 1 (A, insertion), 5 (A) and 20 mm (B).
organism which applies silica for the skeleton formation. Sponges build their bodies from needle-like spicules (Fig. 1B) which are organo-silica composites [3,4]. Diatom frustules and sponge siliceous spicules are attractive for biologists and chemists as the example of silicon dioxide and composite materials which are synthesized at moderated conditions, without high temperature, pressure and toxic reagents usually applied in the silicon industry. The study of biosilicification during the last decades have resulted in some achievements including finding of silica-associated proteins (frustulins, pleuralins, silaffins, silacidins) [5e7], identification of the silicon transporter gene [8] and understanding the cytoskeleton role in the formation of siliceous elements in the silicon deposition vesicles (SDVs) [9-11,12and references in this publication]. The using of pH-sensitive dye allowed to measure pH in the SDV which was equal to 5.5 [13]. In spite of the evident progress, there are several key stages in the biosilicification mechanism which remain unclear. The living cells capture silicon from the environment in the form of silicic acid and transport it to SDV in the slightly condensed form [14e16]. But how the cell synthesizes the highly ordered silica in SDV? There are two principal possibilities to influence the silica formation in SDV: internal and external control [17]. The internal control is considered in the frames of matrix hypothesis proposed by Sumper and Kroger [18]. According to this hypothesis, solid silica is formed around an organic matrix which consists of selforganized biopolymeric chains. Peptides bearing posttranslational polyamine and phosphate modifications (silaffins) are considered as the basis of the matrix [19]. These compounds have been found in minor amounts under analysis of diatom frustules [20]. The main doubt about this hypothesis arises from the absence of the silaffin matrix in the diatom silica. The siliceous frustules contain <5% of organic compounds [21,22]. The finding of silaffins has promoted study of the silicic acid condensation in the presence of various amine-containing polymers [23e28]. These works were aimed on simulating silica formation in SDV and on the elaboration of new siliceous materials. A lot of interesting composite materials have been obtained, including monodisperse spheres, porous and other ordered structures. These materials contain more than 30% of the organic matrix which is tightly mixed with the siliceous phase [23,24,29e31]. Thus, the precipitation of silica in the presence of polymers capable of the interaction with growing siliceous nanoparticles gives rise to a material considerably different from the diatom silica. The other way to control silica formation in the SDV is external
control from the cell. The cell cytoskeleton influences the SDV shape, and consequently the shape of growing silica valve [11,32]. The external control consists also in SDV feeding with silica precursors which have been demonstrated in the first computer simulation of the diatom valve growth [33]. The other way to influence silica formation in the SDV was supposed in the “aquaporine hypothesis” which assumes local desiccation by the action of aquaporines as the driving force of the silica formation in the required point of the SDV [34]. One of the reasons to involve aquaporines was the high amount of water which must be removed from the SDV during silica solidification. According to various estimations [35], concentration of the silica precursors in SDV is below 340 mM which corresponds to 2% calculating on SiO2. Thus, condensation of these precursors to relatively dry silicon dioxide must be accompanied by removing of a large amount of water from the SDV. Aquaporines are the proteins which are used by various living cells to remove water from vesicles [36,37]. On the other hand, desiccation can induce fast condensation of silicic acid up to instantaneous formation of the solid phase (syneresis) [38]. In this study we tried to prepare a solution of silica precursors close to hypothesized SDV content and to study behavior of these precursors under increase of the concentration. Silicic acid was precondensed in the presence of polyamine-containing polyampholyte (poly (acrylic acid) with grafted polyamine chains, PAAPA, Scheme 1). The resulting solution was undergone an action of enhanced gravity by means of centrifugation with the aim to initiate concentrating-induced condensation. We have chosen the polymer-containing system as a model of SDV content from the following reasons: i. free silicic acid was not found in diatoms in a reliable amount according to NMR data [39,40]; ii. the other possible precursor is oligosilicates (oligomers of the pre-condensed silicic acid) which can be formed in the process of silicon assimilation from the environment [15,16,24]; iii. stabilization of the oligosilicates in solution and prevention of the further condensation can be achieved by complexing with organic polymers [41]; iv. directed cytoplasmic transport of the oligosilicates from the outer cell membrane to the SDV implies interaction with some biopolymer. Silaffins are polymers associated with siliceous frustules, their structure allows to suppose their affinity to oligosilicates and the in vitro experiments have proven formation of silaffin e silica complexes [6,16]. The polymer obtained in this work is the first synthetic polyampholyte which bears long polyamine side chains, so this polymer is the closest analogue of the natural silaffins. 2. Materials and methods 2.1. Materials 1,4-Dioxane, ethanol, acetic acid, sodium hydroxide, sodium silicate, hexafluorobutanoic acid, reagents for the molybdenum blue assay [ammonium molybdate, oxalic acid, 4methylaminophenol sulfate, sodium sulphite, standard silicate solution, hydrochloric acid (35%), and sulphuric acid (98%)] and 0.1 M and 1 M solutions of NaOH and HCl were purchased from Sigma Aldrich, Fisher, and Acros Chemicals and used without further treatment. Acryloyl chloride (AC, Sigma Aldrich) was distilled before polymerization. 2,20 -Azobis(isobutyronitrile) (AIBN, Sigma Aldrich) was recrystallized from ethanol. The mixture of oligomeric amines (OA, H3CeNH-
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Scheme 1. Synthesis of poly (acrylic acid) with grafted polyamine chains (PAA-PA).
[(CH2)3eN(CH3)-]n-H) was obtained as previously described [42], the average chain length (n) is 10.2 according to 1H NMR data [CDCl3, 400.13 MHz, ppm: 2.36 (terminal CH3), 2.10 (median CH3)]. OA contains chains of 4e30 nitrogen atoms according to MS analysis [Fig. 1 in Supplementary data (SD)]. The sample of diatom algae Stephanodiscus meyeri was obtained from the Lake Baikal and the siliceous valves were prepared according to [43]. Fluorescent staining of the sponge spicules was performed as described in [44].
2.2. Polymer synthesis PAA-PA was synthesized according to Scheme 1. Firstly, poly (acryloyl chloride) (PAC) was prepared via radical polymerization in dioxane (3 g of acryloyl chloride and 12 mL of dioxane) using AIBN as an initiator (2% of the monomer mass) at 60 C in argon atmosphere in a hermetically sealed 50 mL vial during 72 h [45]. The polymer yield was estimated by dissolution of the reaction mixture in water following with dialysis against water through a cellophane membrane (4 kDa cutoff) and freeze-drying of the resulted poly (acrylic acid). The yield was 90%. Polymerization degree of PAC is 280 [determined from viscometry data for poly (acrylic acid)] [46]. Taking into account the high yield of PAC and its inclination to hydrolysis, we used the obtained dioxane solutions of PAC in the reaction with oligomeric amine without PAC isolation. 4.2 g of the PAC solution was diluted with 10 mL of dioxane and mixed with OA solution (0.257 g of OA in 5 mL of dioxane) at 0 C. A yellow precipitate was observed after the solutions mixing. The reaction mixture was stirred 5 min at 0 C and 20 min at room temperature. After that the reaction mixture was diluted with 100 mL of water, stirred overnight, a precipitate was removed by centrifugation (3500 g for 5 min) and washed with 10 mL of 0.1 M NaOH. The obtained solution of PAA-PA was purified by dialysis against water through a cellophane membrane (4 kDa cutoff), and freeze-dried. PAA-PA was obtained in 68% yield. 1 H NMR (Scheme 2, D2O, 400.13 MHz, ppm): 1.2e1.8 (Hb), 2.07 (Ha), 2.15 (Hc), 2.67 (He), 2.8 (Hf), 3.09 (Hd). 13C NMR (Scheme 2, D2O, 100.61 MHz, ppm): 37 (Cc), 38 (Ca), 44.7 (Cf), 45.5e46.5 (Cb), 53
Scheme 2. Structure of the PAA-PA copolymer and labels of atoms.
(Cd), 184.5 (Cg, h). FTIR spectrum of the polymer (Fig. 2, film) contains bands of amine (1120, 1050 cm1, nCeN; broad absorbance band at 2200e3000 cm1, nNeH in protonated groups), carboxylic group (1320 cm1, nCeO; 1558 cm1, ~COO, naC]O; 1406 cm1, ~COO, nsC]O), methyl and methylene groups (1458 cm1, da; 2946, 2866 cm1, n). The IR bands were assigned according to [47,48] taking into account IR spectrum of OA [42]. The composition of the PAA-PA copolymer was estimated from the FTIR data by means of calibration with poly (acrylic acid) e OA mixtures which were prepared at pH 7.5 and freeze-dried. PAA-PA copolymer was also dissolved in water, pH was adjusted to 7.5 and the solution was freeze-dried. The bands at 1558 (~COO) and 1050 cm1 (OA) were used for the analysis. PAA-PA contains 18.0% of OA which corresponds to 2.22% grafting degree.
Fig. 2. FTIR spectra of PAA-PA copolymer, silica obtained from 100 mM sodium silicate and HCl at pH 5.5 and the precipitate obtained by centrifugation of the PAA-PA e silicic acid solutions (1 mg/mL of PAA-PA, 50 mM of silicic acid). The insertion represents ATR-FTIR spectra of solutions containing 50 mM silicic acid and 50 mM silicic acid with 1 mg/mL PAA-PA at pH 5.5. The solution spectra were recorded relative to water after 1 h after pH adjusting to 5.5.
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2.3. Study of silicic acid condensation and gravity-induced synthesis of the siliceous materials The silicic acid condensation was studied at pH 5.5 in the presence of 30 mM acetate buffer. Stock solutions of 90 mM acetic acid, 150 mM sodium silicate, 5 mg/mL PAA-PA and water were mixed in the desired proportions and 1 M HCl was added in the amount corresponded to pH adjusting to 5.5 (this amount was estimated in a separate experiment). Dynamic light scattering (DLS) and molybdenum blue measurements of non-condensed silicic acid were started immediately after mixing of the components. The gravity-induced synthesis of the siliceous materials consisted in centrifugation of the aged solutions at 50,000 g during 1 h. The aging time was 30 min for a solution of 1 mg/mL PAA-PA and 50 mM of silicic acid, and 20 h for a blank 50 mM solution of silicic acid and for a solution of 1 mg/mL PAA-PA and 10 mM of silicic acid. This aging times correspond to 70e80% condensation of silicic acid. The precipitates obtained after centrifugation were washed with water (4 C) and freeze-dried. Composition of the precipitates was determined by dissolving of 3e5 mg in 1 mL of 0.3 M NaOH at 60 C during 3 h. Silicon concentration in the resulted solutions was measured by the molybdate method [49]. Silica content in the precipitates was calculated based on silicon content in silica obtained from 0.1 M Na2SiO3 by precipitation with 1 M HCl at pH 5.5. 2.4. Instrumentation Mass spectrometric analysis was performed on an Agilent 6210 TOF LC/MS System. Samples were dissolved in water containing 0.1% formic acid at concentrations of 17 mg/l. Water and acetonitrile with 0.05% (v/v) hexafluorobutanoic acid were used as eluting solvents A and B, 40% and 60% respectively. The flow rate of the mobile phase was set at 0.1 mL/min, the injection volume of sample solution was 10 ml, and the stop time was set at 5 min. The usual conditions for TOF MS were as follows: mass range was m/z 300e2500 and scan time was 1 s with interscan delay of 0.1 s; mass spectra were recorded under ESI þ V mode, centroid, normal dynamic range, and cone voltage using tune page; capillary voltage 3500 V, desolvation temp 325 C, nitrogen flow 50 L/min. Under these conditions, peaks of the amines appeared as protonated ions. Potentiometry measurements were performed on a “Multitest” ionometer using a combined pH-electrode in a temperaturecontrolled cell at 20 ± 0.02 C. 0.1 M NaOH was used for adjusting pH of the solutions up to 11, and 0.1 M HCl was employed as a titrant. FTIR spectra were recorded with an Infralum FT-801 spectrometer using KBr pellets, film of ZnSe surface (PAA-PA polymer) or attenuated total reflection (ATR) module (water solutions). 1H and 13C NMR spectra were obtained on a BrukereDPX 400 spectrometer (400.13 and 100.61 MHz, correspondingly) in D2О and CDCl3. Turbidimetry titration was conducted using a UNICO 2100 spectrophotometer at 420 nm. 0.6 mg/mL solutions of the copolymer and poly(acrylic acid) samples were prepared in 0.1 M NaOH and titrated with propan-2-ol. The results are presented (Fig. 2 in SD) as corrected absorption [A2 ¼ A/(1-g)] vs volume fraction of the precipitant (g). Scanning electron microscopy (SEM) was performed using FEI Quanta 200 and Philips SEM 525 M instruments. Transmission electron microscopy (TEM) was performed using a LEO 906E instrument on freeze-dried solutions. The solid products were dispersed in hexane and drops of the dispersion were placed on formvar film coated copper grids. Energy dispersive X-ray analysis (EDAX) was performed with FEI Quanta 200. The samples were placed on aluminum sample holders and then sputter coated with
gold using SDC 004 (BALZERS) coater. Atomic force microscopy (AFM) was conducted using Scanning Probe Microscope CMM2000 (PROTON-MIET, ZAVOD, JSC, Russia) operated in contact mode in air at room temperature using silicon probes (nominal probe curvature radius of 10 nm). Height mode images (512 512 pixels) were collected with a scan speed between 1 and 2 Hz. The samples were dispersed in hexane using ultrasonic bath (30 min) and placed on glass coverslips. The software package Gwyddion was used for AFM image processing. A Zeiss LSM710 microscope was used to obtain confocal images with the following parameters: excitation 488 nm, detector slit e 501e572 nm. Dynamic light scattering experiments were performed using a LAD-079 instrument built in The Institute of Thermophysics (Novosibirsk, Russia). All solutions were purified from dust using filter units with 0.45 mm pore size (Sartorius 16555-Q Minisart syringe filters). The experiments were performed at 20 C ± 0.02 C. Measurements were done with 650 nm solid-state laser at 90 scattering angle. Correlation functions were analyzed with a polymodal model using random-centroid optimization method [50]. 3. Results and discussion We have obtained poly (acrylic acid) with grafted polyamine chains (PAA-PA) by the reaction of poly (acryloyl chloride) with oligomeric polyamine which contains chains of 6e30 nitrogen atoms (average chain length e 11.2 propylamine units). Polymerization degree of the resulting polymer is 280 and the grafting degree is 2.22% which corresponds to ~ COOH: amine ratio of z4: 1. Turbidimetry titration curve for the PAA-PA polymer differs from the poly (acrylic acid) curve (Fig. 2 in SD) and does not contain flat segments which points on the absence of non-modified poly (acrylic acid) chains. Potentiometry titration of the PAA-PA solution proceeds with precipitation of the polymer at pH below 5.3 (Table 1) due to polyampholyte character of PAA-PA. An addition of sodium silicate to the PAA-PA solution decrease pH of the precipitation (Table 1) or prevent the precipitation at all. The homogeneity of the PAA-PA e sodium silicate mixtures at pH 5.5 (pH in SDVs) makes it possible to study this interesting point by DLS and molybdenum blue method which allows to measure concentration of non-condensed silicic acid (in the form of monomer and dimer) [49,15]. PAA-PA drastically accelerates condensation of silicic acid at the first minutes of the reaction (Fig. 3): the concentration of free Si(OH)4 drops below 20 mM in 2 min and after that gradually decreases to the equilibrium level (2e3 mM, [49]). The light scattering data (Fig. 4) show the existence of PAA-PA in solution in the form of 180 nm particles. These data are obtained at pH 5.5, near precipitation point of the polymer, so we suppose these relatively large particles are multi-molecular aggregates formed from PAA-PA macromolecules. The condensation of silicic acid gives rise to bimodal distributed particles: 60e150 and 650e900 nm. The condensation in the presence of PAA-PA results in solutions which show the same size distribution as the organic polymer. TEM analysis of the freeze-dried solutions after silicic acid condensation in the presence of PAA-PA (Fig. 5A) confirms DLS data. The particles look like organic formations with inclusions of more electron-dense material which are probably oligosilicate chains similar to observed in poly(vinyl amine) e silica system [24]. Silica and oligosilicates are able to interact with aminecontaining polymers [23e28] by means of hydrogen and ionic bonds between silanol and amine moieties. This interaction starts when primary oligosilicate particles are formed in the solution and results in increase of the fraction of silanol anions which are the most active groups in condensation with monomeric silicic acid [49]. Silicic acid condensation accelerates and gives rise to soluble or insoluble aggregates between silica and organic polymer. IR
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Table 1 Precipitation intervals for PAA-PA and PAA-PA e sodium silicate solutions (titration from alkali area). System
PAA-PA, PAA-PA, PAA-PA, PAA-PA, PAA-PA,
Heterogeneity interval, pH
1 mg/L 1 mg/L; Na2SiO3, 10 mM 1 mg/L; Na2SiO3, 20 mM 0.25 mg/L; Na2SiO3, 20 mM 1 mg/L; Na2SiO3, 50 mM
Titration end point
Precipitation
Dissolution
5.3 3.8 4.4 no precipitation no precipitation
no dissolution no dissolution 2.4
Fig. 3. Effect of PAA-PA on condensation of silicic acid at pH 5.5. Initial concentration of Si(OH)4 is 50 mM, PAA-PA e 1 mg/mL.
spectroscopy of the silicic acid e PAA-PA solutions (Fig. 2, insertion) confirms participation of silanol groups in interaction with organic polymer. The region of SieOeSi (1100e1150 cm1) and SieOH (960 cm1) vibrations [51] is free from the water vibrations which allows to study these groups with ATR technique. In the case of Si(OH)4 solution without organic polymer, SieOH band is rather weaker than SieOeSi band similar to the spectrum of solid silica (Fig. 2). Condensation in the presence of PAA-PA polymer drastically increases SieOH absorption which is the evidence of interaction between these groups and electron-donating groups of organic polymer. Thus, the condensation of silicic acid in the presence of PAA-PA gives rise to solutions stable during several days after mixing of the components. The obtained solutions of composite nanoparticles were subjected to enhanced gravitation force by centrifugation at 50,000 g during 1 h. We have expected sedimentation of the composite particles following with increase of their concentration
2.6 2.3 2.0 2.1 2.3
and concentrating-induced condensation to a solid phase. As expected, precipitates were found at the bottom of centrifuge tube after centrifugation when initial silicic acid concentration was 10 and 50 mM and PAA-PA concentration was 1 mg/mL. The precipitate obtained at 10 mM Si(OH)4 was gel-like substance and in the case of 50 mM silicic acid we have obtained a hard precipitate which contained 4% of the organic polymer only. No precipitates were observed under centrifugation of the solutions prepared without PAA-PA polymer. The precipitates formed under centrifugation contain structures of three types: the main part in the form of 100e200 nm “sintered” particles (Fig. 5B, C and Fig. 3A, B in SD), a minor part in the form of fibrous material (Fig. 5F and 3C in SD) and combination of the particles and fibers (Fig. 5E). The presence of silicon in these precipitates is confirmed with EDAX analysis (Fig. 5D, G). FTIR spectrum of the precipitate (Fig. 2) contains bands of SieOeSi (1075 cm1) and SieOH (960 cm1). Bands of the organic polymer (1380e1730 cm1) are weak due to its low content in the precipitate. The silanol band is more intensive comparing with silica obtained without organic polymer which is the result of the interactions between amine groups of PAA-PA and silanol groups. We propose the following scheme of the reaction (Fig. 6): The fast condensation of silicic acid occurs at the very beginning of the process due to the catalytic action of polyamine chains similar to the other amine-containing polymers [24,27]. The primary siliceous nanoparticles interact with polyamine chains and the PAA-PA 180 nm aggregates are being filled with oligosilicate chains (Fig. 6B and C). The condensed silicic acid is a weak polymeric acid (pKa ¼ 6e7, [49]) and the PAA-PA e oligosilicate composite particles can be considered as polyampholytes with increased amount of acidic groups comparing with initial PAA-PA polymer. The change of acidic/basic group ratio decreases isoelectric point of the aggregates and increases their solubility in acidic medium (Table 1). The centrifugation of these solutions results in concentrating of
Fig. 4. Size distribution of 1 mg/mL PAA-PA solution (a and b), of the particles formed on condensation of silicic acid (c, 10 mM and d, 20 mM) and of the mixed PAA-PA e Si(OH)4 solutions (e, f e 10, g, h e 20 and i e 50 mM Si(OH)4) at pH 5.5. The data for a, e and g curves were obtained at 15 min after pH adjusting to 5.5, i e after 2 h and the other curves e after 4 days.
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Fig. 5. TEM (A) images of PAA-PA e silicic acid freeze-dried solutions and SEM (BeG) images of the precipitates obtained by centrifugation of the PAA-PA e silicic acid solutions: 1 mg/mL of PAA-PA, 20 (A) 10 (B), and 50 (CeG) mM of silicic acid. (D) and (G) represent SEM image and EDAX silicon mapping of the precipitate. Scale bar: 200 nm (A), 500 nm (B, C, E and F) and 5 mm (D and G).
the composite particles near bottom (Fig. 6D). The oligosilicate e PAA-PA interactions are an equilibrium process, so we have also local increase of the oligosilicate concentration in the solution. Thus, fast formation of solid silica occurs and the polymeric chains are released from the coordination with siliceous phase (Fig. 6E). This process considerably differs from the known precipitation of polymer-silica composites from the relatively diluted solutions. Silicic acid condensation in the presence of polymeric base is similar to formation of polymerepolymer complexes: precipitation starts at some stoichiometric ratio between the interacting groups. This reaction does not depend on total concentration of the components (in the reasonable boundaries) and the product consists of the two components mixed on the level of individual polymeric chains. The obtained polymerepolymer complex is soluble or
insoluble depending on the components nature, pH, ionic strength, temperature, and etc. The peculiarity of silica-containing systems consists in the ability of siliceous component to further condensation which gives more bulky siliceous phase and decrease number of silanol groups capable of interaction with the organic polymer. Centrifugation of the composite particles provides the conditions for this condensation. The organic polymer is needed for creation of dense composite nanoparticles in the solution but during the final condensation of the siliceous phase the polymer e silica contacts are decreased and PAA-PA is involved into final precipitate in a minor amount. The centrifugation-induced transformation of siliconcontaining nanoparticles is a model of hypothesized processes in living cells when silica nanoparticles stabilized with an organic
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Fig. 6. Scheme of the silicic acid condensation in the presence of PAA-PA (AeC) following with centrifugation of the soluble composite nanoparticles (D and E).
polymer undergo desiccation under the action of aquaporines [34]. Our experiments show that concentrating (desiccation) of the solution containing stabilized silica can result in formation of solid siliceous material close to biogenic silica. PAA-PA used as stabilizing polymer contains pendant polyamine chains and acidic groups in the main chain similar to silaffins found in diatoms [18]. On the basis of our results, we hypothesize that biological role of the silaffins consists in stabilization of oligosilicates before synthesis of siliceous constructions in the diatom cell. The absence of considerable amount of organic matrix in the bio-silica is explainable with transformation of oligosilicate-silaffin aggregates under some desiccation process which can be guided by the cell with the use of aquaporines [35]. 4. Conclusions We have obtained a new polyampholyte PAA-PA which bears pendant polyamine oligomeric chains. This polymer is a model of silaffins e proteins playing important role in formation of siliceous structures in diatom algae and sponges. The study of silicic acid condensation in the presence of PAA-PA showed catalytic effect of the polymer on the condensation. The obtained solutions contain oligosilicates coordinated with PAA-PA chains. Sedimentation of the composite nanoparticles under increased gravity resulted in concentrating induced condensation of the pre-condensed siliceous oligomers giving rise to solid silica containing admixture of the organic polymer. This product is rather closer to the silica from diatom frustules than composite precipitates fallen from solutions of silicic acid and polymeric amines. We consider our results confirm the hypothesis about formation of biosilica under the action of desiccation agent, e.g. aquaporins [34].
The formation of solid substances during centrifugation of solutions containing soluble oligomers is a new promising approach to inorganic and composite materials. This bioinspired approach allows to obtain a stable aqueous solution of the composite precursor and to carry out the final condensation without evaporation of the solvent or precipitation with the addition of non-solvent. The remaining solution contains the main part of the initial organic polymer and can be reused. Acknowledgments V. Annenkov and E. Danilovtseva thanks the Russian Academy of Sciences (project #0345e2014e0001) for the financial support; V. Pal'shin and O. Verkhozina were supported by Project # 14-0331644 of the Russian Foundation for Basic Research. The authors wish to thank the United Instrumental Center (Irkutsk Scientific Center) and the Center of Ultramicroanalysis (Limnological Institute) for providing equipment. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.matchemphys.2015.09.022. References [1] P. Treguer, D.M. Nelson, A.J. Van Bennekom, D.J. Demaster, A. Leynaert, B. Queguiner, Science 268 (1995) 375e379. [2] R. Gordon, D. Losic, M.A. Tiffany, S.S. Nagy, F.A.S. Sterrenburg, Trends Biotechnol. 27 (2009) 116e127. [3] M.J. Uriz, X. Turon, M.A. Becerro, G. Agell, Microsc. Res. Tech. 62 (2003) 279e299. [4] M.J. Uriz, X. Turon, M.A. Becerro, Prog. Mol. Subcell. Biol. 33 (2003) 163e193.
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Composite nanoparticles: A new way to siliceous materials and a model of biosilica synthesis Vadim V. Annenkov a, *, Viktor A. Pal'shin a, Olga N. Verkhozina a, Lyudmila I. Larina b, Elena N. Danilovtseva a a b
Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, Russian Federation A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, Irkutsk 664033, Russian Federation
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A polyampholyte with pendant polyamine chains is obtained. The polymer catalyses condensation of silicic acid giving stable solutions. Gravity-induced (50,000 g) formation of solid silica was observed in these solutions. The obtained silica is close to biosilica from diatom frustules. A new approach to inorganic and composite materials is proposed.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2015 Received in revised form 7 September 2015 Accepted 14 September 2015 Available online xxx
A new polyampholyte based on poly (acrylic acid) which bears pendant polyamine oligomeric chains (average number of the nitrogen atoms is 11.2) is obtained. This polymer is a model of silaffins e proteins playing important role in formation of siliceous structures in diatom algae and sponges. The polymer catalyses condensation of silicic acid. The obtained solutions contain oligosilicates coordinated with the polymer chains. The action of 50,000 g gravity on this solution results in concentrating-induced condensation of the pre-condensed siliceous oligomers. The obtained solid silica contains 4% admixture of the organic polymer which is close to the silica from diatom frustules. These results confirm the hypothesis about formation of biosilica under the action of desiccation agent, e.g. aquaporins. The formation of solid substances during centrifugation of solutions containing soluble oligomers is a new promising approach to inorganic and composite materials which allows to work in aqueous medium and to reuse the organic polymer. © 2015 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Composite materials Nanostructures Polymers Chemical synthesis Precipitation
1. Introduction Bioinspired approaches to new inorganic and composite materials are intensively studied in the hope of elaboration environmentally friendly procedures which allow to avoid high
* Corresponding author. E-mail addresses: [email protected], [email protected] (V.V. Annenkov).
temperatures and hazardous reactants. Silicon dioxide (silica) is used by some living organisms as the construction material for elements of the skeleton. Diatom algae are the most known and studied silicon-containing organisms because of their high ecological importance (more than 20% of primary carbon assimilation [1]) and beautiful siliceous exoskeletons e frustules [2]. The frustules consist of delicate 2e2000 mm valves having species specific structure of elements (holes, nodes and etc.) ordered on micro- and nanolevel (Fig. 1A). Siliceous sponges are the other
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Fig. 1. (A) SEM of siliceous valves of the diatom Stephanodiscus meyeri Genkal et Popovsk; (B) confocal images of siliceous spicules of the sponge Lubomirskia baicalensis Pallas stained in vivo with a fluorescent dye. Scale bar represents 1 (A, insertion), 5 (A) and 20 mm (B).
organism which applies silica for the skeleton formation. Sponges build their bodies from needle-like spicules (Fig. 1B) which are organo-silica composites [3,4]. Diatom frustules and sponge siliceous spicules are attractive for biologists and chemists as the example of silicon dioxide and composite materials which are synthesized at moderated conditions, without high temperature, pressure and toxic reagents usually applied in the silicon industry. The study of biosilicification during the last decades have resulted in some achievements including finding of silica-associated proteins (frustulins, pleuralins, silaffins, silacidins) [5e7], identification of the silicon transporter gene [8] and understanding the cytoskeleton role in the formation of siliceous elements in the silicon deposition vesicles (SDVs) [9-11,12and references in this publication]. The using of pH-sensitive dye allowed to measure pH in the SDV which was equal to 5.5 [13]. In spite of the evident progress, there are several key stages in the biosilicification mechanism which remain unclear. The living cells capture silicon from the environment in the form of silicic acid and transport it to SDV in the slightly condensed form [14e16]. But how the cell synthesizes the highly ordered silica in SDV? There are two principal possibilities to influence the silica formation in SDV: internal and external control [17]. The internal control is considered in the frames of matrix hypothesis proposed by Sumper and Kroger [18]. According to this hypothesis, solid silica is formed around an organic matrix which consists of selforganized biopolymeric chains. Peptides bearing posttranslational polyamine and phosphate modifications (silaffins) are considered as the basis of the matrix [19]. These compounds have been found in minor amounts under analysis of diatom frustules [20]. The main doubt about this hypothesis arises from the absence of the silaffin matrix in the diatom silica. The siliceous frustules contain <5% of organic compounds [21,22]. The finding of silaffins has promoted study of the silicic acid condensation in the presence of various amine-containing polymers [23e28]. These works were aimed on simulating silica formation in SDV and on the elaboration of new siliceous materials. A lot of interesting composite materials have been obtained, including monodisperse spheres, porous and other ordered structures. These materials contain more than 30% of the organic matrix which is tightly mixed with the siliceous phase [23,24,29e31]. Thus, the precipitation of silica in the presence of polymers capable of the interaction with growing siliceous nanoparticles gives rise to a material considerably different from the diatom silica. The other way to control silica formation in the SDV is external
control from the cell. The cell cytoskeleton influences the SDV shape, and consequently the shape of growing silica valve [11,32]. The external control consists also in SDV feeding with silica precursors which have been demonstrated in the first computer simulation of the diatom valve growth [33]. The other way to influence silica formation in the SDV was supposed in the “aquaporine hypothesis” which assumes local desiccation by the action of aquaporines as the driving force of the silica formation in the required point of the SDV [34]. One of the reasons to involve aquaporines was the high amount of water which must be removed from the SDV during silica solidification. According to various estimations [35], concentration of the silica precursors in SDV is below 340 mM which corresponds to 2% calculating on SiO2. Thus, condensation of these precursors to relatively dry silicon dioxide must be accompanied by removing of a large amount of water from the SDV. Aquaporines are the proteins which are used by various living cells to remove water from vesicles [36,37]. On the other hand, desiccation can induce fast condensation of silicic acid up to instantaneous formation of the solid phase (syneresis) [38]. In this study we tried to prepare a solution of silica precursors close to hypothesized SDV content and to study behavior of these precursors under increase of the concentration. Silicic acid was precondensed in the presence of polyamine-containing polyampholyte (poly (acrylic acid) with grafted polyamine chains, PAAPA, Scheme 1). The resulting solution was undergone an action of enhanced gravity by means of centrifugation with the aim to initiate concentrating-induced condensation. We have chosen the polymer-containing system as a model of SDV content from the following reasons: i. free silicic acid was not found in diatoms in a reliable amount according to NMR data [39,40]; ii. the other possible precursor is oligosilicates (oligomers of the pre-condensed silicic acid) which can be formed in the process of silicon assimilation from the environment [15,16,24]; iii. stabilization of the oligosilicates in solution and prevention of the further condensation can be achieved by complexing with organic polymers [41]; iv. directed cytoplasmic transport of the oligosilicates from the outer cell membrane to the SDV implies interaction with some biopolymer. Silaffins are polymers associated with siliceous frustules, their structure allows to suppose their affinity to oligosilicates and the in vitro experiments have proven formation of silaffin e silica complexes [6,16]. The polymer obtained in this work is the first synthetic polyampholyte which bears long polyamine side chains, so this polymer is the closest analogue of the natural silaffins. 2. Materials and methods 2.1. Materials 1,4-Dioxane, ethanol, acetic acid, sodium hydroxide, sodium silicate, hexafluorobutanoic acid, reagents for the molybdenum blue assay [ammonium molybdate, oxalic acid, 4methylaminophenol sulfate, sodium sulphite, standard silicate solution, hydrochloric acid (35%), and sulphuric acid (98%)] and 0.1 M and 1 M solutions of NaOH and HCl were purchased from Sigma Aldrich, Fisher, and Acros Chemicals and used without further treatment. Acryloyl chloride (AC, Sigma Aldrich) was distilled before polymerization. 2,20 -Azobis(isobutyronitrile) (AIBN, Sigma Aldrich) was recrystallized from ethanol. The mixture of oligomeric amines (OA, H3CeNH-
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Scheme 1. Synthesis of poly (acrylic acid) with grafted polyamine chains (PAA-PA).
[(CH2)3eN(CH3)-]n-H) was obtained as previously described [42], the average chain length (n) is 10.2 according to 1H NMR data [CDCl3, 400.13 MHz, ppm: 2.36 (terminal CH3), 2.10 (median CH3)]. OA contains chains of 4e30 nitrogen atoms according to MS analysis [Fig. 1 in Supplementary data (SD)]. The sample of diatom algae Stephanodiscus meyeri was obtained from the Lake Baikal and the siliceous valves were prepared according to [43]. Fluorescent staining of the sponge spicules was performed as described in [44].
2.2. Polymer synthesis PAA-PA was synthesized according to Scheme 1. Firstly, poly (acryloyl chloride) (PAC) was prepared via radical polymerization in dioxane (3 g of acryloyl chloride and 12 mL of dioxane) using AIBN as an initiator (2% of the monomer mass) at 60 C in argon atmosphere in a hermetically sealed 50 mL vial during 72 h [45]. The polymer yield was estimated by dissolution of the reaction mixture in water following with dialysis against water through a cellophane membrane (4 kDa cutoff) and freeze-drying of the resulted poly (acrylic acid). The yield was 90%. Polymerization degree of PAC is 280 [determined from viscometry data for poly (acrylic acid)] [46]. Taking into account the high yield of PAC and its inclination to hydrolysis, we used the obtained dioxane solutions of PAC in the reaction with oligomeric amine without PAC isolation. 4.2 g of the PAC solution was diluted with 10 mL of dioxane and mixed with OA solution (0.257 g of OA in 5 mL of dioxane) at 0 C. A yellow precipitate was observed after the solutions mixing. The reaction mixture was stirred 5 min at 0 C and 20 min at room temperature. After that the reaction mixture was diluted with 100 mL of water, stirred overnight, a precipitate was removed by centrifugation (3500 g for 5 min) and washed with 10 mL of 0.1 M NaOH. The obtained solution of PAA-PA was purified by dialysis against water through a cellophane membrane (4 kDa cutoff), and freeze-dried. PAA-PA was obtained in 68% yield. 1 H NMR (Scheme 2, D2O, 400.13 MHz, ppm): 1.2e1.8 (Hb), 2.07 (Ha), 2.15 (Hc), 2.67 (He), 2.8 (Hf), 3.09 (Hd). 13C NMR (Scheme 2, D2O, 100.61 MHz, ppm): 37 (Cc), 38 (Ca), 44.7 (Cf), 45.5e46.5 (Cb), 53
Scheme 2. Structure of the PAA-PA copolymer and labels of atoms.
(Cd), 184.5 (Cg, h). FTIR spectrum of the polymer (Fig. 2, film) contains bands of amine (1120, 1050 cm1, nCeN; broad absorbance band at 2200e3000 cm1, nNeH in protonated groups), carboxylic group (1320 cm1, nCeO; 1558 cm1, ~COO, naC]O; 1406 cm1, ~COO, nsC]O), methyl and methylene groups (1458 cm1, da; 2946, 2866 cm1, n). The IR bands were assigned according to [47,48] taking into account IR spectrum of OA [42]. The composition of the PAA-PA copolymer was estimated from the FTIR data by means of calibration with poly (acrylic acid) e OA mixtures which were prepared at pH 7.5 and freeze-dried. PAA-PA copolymer was also dissolved in water, pH was adjusted to 7.5 and the solution was freeze-dried. The bands at 1558 (~COO) and 1050 cm1 (OA) were used for the analysis. PAA-PA contains 18.0% of OA which corresponds to 2.22% grafting degree.
Fig. 2. FTIR spectra of PAA-PA copolymer, silica obtained from 100 mM sodium silicate and HCl at pH 5.5 and the precipitate obtained by centrifugation of the PAA-PA e silicic acid solutions (1 mg/mL of PAA-PA, 50 mM of silicic acid). The insertion represents ATR-FTIR spectra of solutions containing 50 mM silicic acid and 50 mM silicic acid with 1 mg/mL PAA-PA at pH 5.5. The solution spectra were recorded relative to water after 1 h after pH adjusting to 5.5.
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2.3. Study of silicic acid condensation and gravity-induced synthesis of the siliceous materials The silicic acid condensation was studied at pH 5.5 in the presence of 30 mM acetate buffer. Stock solutions of 90 mM acetic acid, 150 mM sodium silicate, 5 mg/mL PAA-PA and water were mixed in the desired proportions and 1 M HCl was added in the amount corresponded to pH adjusting to 5.5 (this amount was estimated in a separate experiment). Dynamic light scattering (DLS) and molybdenum blue measurements of non-condensed silicic acid were started immediately after mixing of the components. The gravity-induced synthesis of the siliceous materials consisted in centrifugation of the aged solutions at 50,000 g during 1 h. The aging time was 30 min for a solution of 1 mg/mL PAA-PA and 50 mM of silicic acid, and 20 h for a blank 50 mM solution of silicic acid and for a solution of 1 mg/mL PAA-PA and 10 mM of silicic acid. This aging times correspond to 70e80% condensation of silicic acid. The precipitates obtained after centrifugation were washed with water (4 C) and freeze-dried. Composition of the precipitates was determined by dissolving of 3e5 mg in 1 mL of 0.3 M NaOH at 60 C during 3 h. Silicon concentration in the resulted solutions was measured by the molybdate method [49]. Silica content in the precipitates was calculated based on silicon content in silica obtained from 0.1 M Na2SiO3 by precipitation with 1 M HCl at pH 5.5. 2.4. Instrumentation Mass spectrometric analysis was performed on an Agilent 6210 TOF LC/MS System. Samples were dissolved in water containing 0.1% formic acid at concentrations of 17 mg/l. Water and acetonitrile with 0.05% (v/v) hexafluorobutanoic acid were used as eluting solvents A and B, 40% and 60% respectively. The flow rate of the mobile phase was set at 0.1 mL/min, the injection volume of sample solution was 10 ml, and the stop time was set at 5 min. The usual conditions for TOF MS were as follows: mass range was m/z 300e2500 and scan time was 1 s with interscan delay of 0.1 s; mass spectra were recorded under ESI þ V mode, centroid, normal dynamic range, and cone voltage using tune page; capillary voltage 3500 V, desolvation temp 325 C, nitrogen flow 50 L/min. Under these conditions, peaks of the amines appeared as protonated ions. Potentiometry measurements were performed on a “Multitest” ionometer using a combined pH-electrode in a temperaturecontrolled cell at 20 ± 0.02 C. 0.1 M NaOH was used for adjusting pH of the solutions up to 11, and 0.1 M HCl was employed as a titrant. FTIR spectra were recorded with an Infralum FT-801 spectrometer using KBr pellets, film of ZnSe surface (PAA-PA polymer) or attenuated total reflection (ATR) module (water solutions). 1H and 13C NMR spectra were obtained on a BrukereDPX 400 spectrometer (400.13 and 100.61 MHz, correspondingly) in D2О and CDCl3. Turbidimetry titration was conducted using a UNICO 2100 spectrophotometer at 420 nm. 0.6 mg/mL solutions of the copolymer and poly(acrylic acid) samples were prepared in 0.1 M NaOH and titrated with propan-2-ol. The results are presented (Fig. 2 in SD) as corrected absorption [A2 ¼ A/(1-g)] vs volume fraction of the precipitant (g). Scanning electron microscopy (SEM) was performed using FEI Quanta 200 and Philips SEM 525 M instruments. Transmission electron microscopy (TEM) was performed using a LEO 906E instrument on freeze-dried solutions. The solid products were dispersed in hexane and drops of the dispersion were placed on formvar film coated copper grids. Energy dispersive X-ray analysis (EDAX) was performed with FEI Quanta 200. The samples were placed on aluminum sample holders and then sputter coated with
gold using SDC 004 (BALZERS) coater. Atomic force microscopy (AFM) was conducted using Scanning Probe Microscope CMM2000 (PROTON-MIET, ZAVOD, JSC, Russia) operated in contact mode in air at room temperature using silicon probes (nominal probe curvature radius of 10 nm). Height mode images (512 512 pixels) were collected with a scan speed between 1 and 2 Hz. The samples were dispersed in hexane using ultrasonic bath (30 min) and placed on glass coverslips. The software package Gwyddion was used for AFM image processing. A Zeiss LSM710 microscope was used to obtain confocal images with the following parameters: excitation 488 nm, detector slit e 501e572 nm. Dynamic light scattering experiments were performed using a LAD-079 instrument built in The Institute of Thermophysics (Novosibirsk, Russia). All solutions were purified from dust using filter units with 0.45 mm pore size (Sartorius 16555-Q Minisart syringe filters). The experiments were performed at 20 C ± 0.02 C. Measurements were done with 650 nm solid-state laser at 90 scattering angle. Correlation functions were analyzed with a polymodal model using random-centroid optimization method [50]. 3. Results and discussion We have obtained poly (acrylic acid) with grafted polyamine chains (PAA-PA) by the reaction of poly (acryloyl chloride) with oligomeric polyamine which contains chains of 6e30 nitrogen atoms (average chain length e 11.2 propylamine units). Polymerization degree of the resulting polymer is 280 and the grafting degree is 2.22% which corresponds to ~ COOH: amine ratio of z4: 1. Turbidimetry titration curve for the PAA-PA polymer differs from the poly (acrylic acid) curve (Fig. 2 in SD) and does not contain flat segments which points on the absence of non-modified poly (acrylic acid) chains. Potentiometry titration of the PAA-PA solution proceeds with precipitation of the polymer at pH below 5.3 (Table 1) due to polyampholyte character of PAA-PA. An addition of sodium silicate to the PAA-PA solution decrease pH of the precipitation (Table 1) or prevent the precipitation at all. The homogeneity of the PAA-PA e sodium silicate mixtures at pH 5.5 (pH in SDVs) makes it possible to study this interesting point by DLS and molybdenum blue method which allows to measure concentration of non-condensed silicic acid (in the form of monomer and dimer) [49,15]. PAA-PA drastically accelerates condensation of silicic acid at the first minutes of the reaction (Fig. 3): the concentration of free Si(OH)4 drops below 20 mM in 2 min and after that gradually decreases to the equilibrium level (2e3 mM, [49]). The light scattering data (Fig. 4) show the existence of PAA-PA in solution in the form of 180 nm particles. These data are obtained at pH 5.5, near precipitation point of the polymer, so we suppose these relatively large particles are multi-molecular aggregates formed from PAA-PA macromolecules. The condensation of silicic acid gives rise to bimodal distributed particles: 60e150 and 650e900 nm. The condensation in the presence of PAA-PA results in solutions which show the same size distribution as the organic polymer. TEM analysis of the freeze-dried solutions after silicic acid condensation in the presence of PAA-PA (Fig. 5A) confirms DLS data. The particles look like organic formations with inclusions of more electron-dense material which are probably oligosilicate chains similar to observed in poly(vinyl amine) e silica system [24]. Silica and oligosilicates are able to interact with aminecontaining polymers [23e28] by means of hydrogen and ionic bonds between silanol and amine moieties. This interaction starts when primary oligosilicate particles are formed in the solution and results in increase of the fraction of silanol anions which are the most active groups in condensation with monomeric silicic acid [49]. Silicic acid condensation accelerates and gives rise to soluble or insoluble aggregates between silica and organic polymer. IR
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Table 1 Precipitation intervals for PAA-PA and PAA-PA e sodium silicate solutions (titration from alkali area). System
PAA-PA, PAA-PA, PAA-PA, PAA-PA, PAA-PA,
Heterogeneity interval, pH
1 mg/L 1 mg/L; Na2SiO3, 10 mM 1 mg/L; Na2SiO3, 20 mM 0.25 mg/L; Na2SiO3, 20 mM 1 mg/L; Na2SiO3, 50 mM
Titration end point
Precipitation
Dissolution
5.3 3.8 4.4 no precipitation no precipitation
no dissolution no dissolution 2.4
Fig. 3. Effect of PAA-PA on condensation of silicic acid at pH 5.5. Initial concentration of Si(OH)4 is 50 mM, PAA-PA e 1 mg/mL.
spectroscopy of the silicic acid e PAA-PA solutions (Fig. 2, insertion) confirms participation of silanol groups in interaction with organic polymer. The region of SieOeSi (1100e1150 cm1) and SieOH (960 cm1) vibrations [51] is free from the water vibrations which allows to study these groups with ATR technique. In the case of Si(OH)4 solution without organic polymer, SieOH band is rather weaker than SieOeSi band similar to the spectrum of solid silica (Fig. 2). Condensation in the presence of PAA-PA polymer drastically increases SieOH absorption which is the evidence of interaction between these groups and electron-donating groups of organic polymer. Thus, the condensation of silicic acid in the presence of PAA-PA gives rise to solutions stable during several days after mixing of the components. The obtained solutions of composite nanoparticles were subjected to enhanced gravitation force by centrifugation at 50,000 g during 1 h. We have expected sedimentation of the composite particles following with increase of their concentration
2.6 2.3 2.0 2.1 2.3
and concentrating-induced condensation to a solid phase. As expected, precipitates were found at the bottom of centrifuge tube after centrifugation when initial silicic acid concentration was 10 and 50 mM and PAA-PA concentration was 1 mg/mL. The precipitate obtained at 10 mM Si(OH)4 was gel-like substance and in the case of 50 mM silicic acid we have obtained a hard precipitate which contained 4% of the organic polymer only. No precipitates were observed under centrifugation of the solutions prepared without PAA-PA polymer. The precipitates formed under centrifugation contain structures of three types: the main part in the form of 100e200 nm “sintered” particles (Fig. 5B, C and Fig. 3A, B in SD), a minor part in the form of fibrous material (Fig. 5F and 3C in SD) and combination of the particles and fibers (Fig. 5E). The presence of silicon in these precipitates is confirmed with EDAX analysis (Fig. 5D, G). FTIR spectrum of the precipitate (Fig. 2) contains bands of SieOeSi (1075 cm1) and SieOH (960 cm1). Bands of the organic polymer (1380e1730 cm1) are weak due to its low content in the precipitate. The silanol band is more intensive comparing with silica obtained without organic polymer which is the result of the interactions between amine groups of PAA-PA and silanol groups. We propose the following scheme of the reaction (Fig. 6): The fast condensation of silicic acid occurs at the very beginning of the process due to the catalytic action of polyamine chains similar to the other amine-containing polymers [24,27]. The primary siliceous nanoparticles interact with polyamine chains and the PAA-PA 180 nm aggregates are being filled with oligosilicate chains (Fig. 6B and C). The condensed silicic acid is a weak polymeric acid (pKa ¼ 6e7, [49]) and the PAA-PA e oligosilicate composite particles can be considered as polyampholytes with increased amount of acidic groups comparing with initial PAA-PA polymer. The change of acidic/basic group ratio decreases isoelectric point of the aggregates and increases their solubility in acidic medium (Table 1). The centrifugation of these solutions results in concentrating of
Fig. 4. Size distribution of 1 mg/mL PAA-PA solution (a and b), of the particles formed on condensation of silicic acid (c, 10 mM and d, 20 mM) and of the mixed PAA-PA e Si(OH)4 solutions (e, f e 10, g, h e 20 and i e 50 mM Si(OH)4) at pH 5.5. The data for a, e and g curves were obtained at 15 min after pH adjusting to 5.5, i e after 2 h and the other curves e after 4 days.
Please cite this article in press as: V.V. Annenkov, et al., Composite nanoparticles: A new way to siliceous materials and a model of biosilica synthesis, Materials Chemistry and Physics (2015), http://dx.doi.org/10.1016/j.matchemphys.2015.09.022
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Fig. 5. TEM (A) images of PAA-PA e silicic acid freeze-dried solutions and SEM (BeG) images of the precipitates obtained by centrifugation of the PAA-PA e silicic acid solutions: 1 mg/mL of PAA-PA, 20 (A) 10 (B), and 50 (CeG) mM of silicic acid. (D) and (G) represent SEM image and EDAX silicon mapping of the precipitate. Scale bar: 200 nm (A), 500 nm (B, C, E and F) and 5 mm (D and G).
the composite particles near bottom (Fig. 6D). The oligosilicate e PAA-PA interactions are an equilibrium process, so we have also local increase of the oligosilicate concentration in the solution. Thus, fast formation of solid silica occurs and the polymeric chains are released from the coordination with siliceous phase (Fig. 6E). This process considerably differs from the known precipitation of polymer-silica composites from the relatively diluted solutions. Silicic acid condensation in the presence of polymeric base is similar to formation of polymerepolymer complexes: precipitation starts at some stoichiometric ratio between the interacting groups. This reaction does not depend on total concentration of the components (in the reasonable boundaries) and the product consists of the two components mixed on the level of individual polymeric chains. The obtained polymerepolymer complex is soluble or
insoluble depending on the components nature, pH, ionic strength, temperature, and etc. The peculiarity of silica-containing systems consists in the ability of siliceous component to further condensation which gives more bulky siliceous phase and decrease number of silanol groups capable of interaction with the organic polymer. Centrifugation of the composite particles provides the conditions for this condensation. The organic polymer is needed for creation of dense composite nanoparticles in the solution but during the final condensation of the siliceous phase the polymer e silica contacts are decreased and PAA-PA is involved into final precipitate in a minor amount. The centrifugation-induced transformation of siliconcontaining nanoparticles is a model of hypothesized processes in living cells when silica nanoparticles stabilized with an organic
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Fig. 6. Scheme of the silicic acid condensation in the presence of PAA-PA (AeC) following with centrifugation of the soluble composite nanoparticles (D and E).
polymer undergo desiccation under the action of aquaporines [34]. Our experiments show that concentrating (desiccation) of the solution containing stabilized silica can result in formation of solid siliceous material close to biogenic silica. PAA-PA used as stabilizing polymer contains pendant polyamine chains and acidic groups in the main chain similar to silaffins found in diatoms [18]. On the basis of our results, we hypothesize that biological role of the silaffins consists in stabilization of oligosilicates before synthesis of siliceous constructions in the diatom cell. The absence of considerable amount of organic matrix in the bio-silica is explainable with transformation of oligosilicate-silaffin aggregates under some desiccation process which can be guided by the cell with the use of aquaporines [35]. 4. Conclusions We have obtained a new polyampholyte PAA-PA which bears pendant polyamine oligomeric chains. This polymer is a model of silaffins e proteins playing important role in formation of siliceous structures in diatom algae and sponges. The study of silicic acid condensation in the presence of PAA-PA showed catalytic effect of the polymer on the condensation. The obtained solutions contain oligosilicates coordinated with PAA-PA chains. Sedimentation of the composite nanoparticles under increased gravity resulted in concentrating induced condensation of the pre-condensed siliceous oligomers giving rise to solid silica containing admixture of the organic polymer. This product is rather closer to the silica from diatom frustules than composite precipitates fallen from solutions of silicic acid and polymeric amines. We consider our results confirm the hypothesis about formation of biosilica under the action of desiccation agent, e.g. aquaporins [34].
The formation of solid substances during centrifugation of solutions containing soluble oligomers is a new promising approach to inorganic and composite materials. This bioinspired approach allows to obtain a stable aqueous solution of the composite precursor and to carry out the final condensation without evaporation of the solvent or precipitation with the addition of non-solvent. The remaining solution contains the main part of the initial organic polymer and can be reused. Acknowledgments V. Annenkov and E. Danilovtseva thanks the Russian Academy of Sciences (project #0345e2014e0001) for the financial support; V. Pal'shin and O. Verkhozina were supported by Project # 14-0331644 of the Russian Foundation for Basic Research. The authors wish to thank the United Instrumental Center (Irkutsk Scientific Center) and the Center of Ultramicroanalysis (Limnological Institute) for providing equipment. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.matchemphys.2015.09.022. References [1] P. Treguer, D.M. Nelson, A.J. Van Bennekom, D.J. Demaster, A. Leynaert, B. Queguiner, Science 268 (1995) 375e379. [2] R. Gordon, D. Losic, M.A. Tiffany, S.S. Nagy, F.A.S. Sterrenburg, Trends Biotechnol. 27 (2009) 116e127. [3] M.J. Uriz, X. Turon, M.A. Becerro, G. Agell, Microsc. Res. Tech. 62 (2003) 279e299. [4] M.J. Uriz, X. Turon, M.A. Becerro, Prog. Mol. Subcell. Biol. 33 (2003) 163e193.
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Please cite this article in press as: V.V. Annenkov, et al., Composite nanoparticles: A new way to siliceous materials and a model of biosilica synthesis, Materials Chemistry and Physics (2015), http://dx.doi.org/10.1016/j.matchemphys.2015.09.022