Silicification and Biosilicification - ACS Symposium Series (ACS

Chapter 30

Silicification and Biosilicification Part 2. Silicification at pH 7 in the Presence of a Cationically Charged Polymer in Solution and Immobilized on Substrates 1

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on October 8, 2014 | http://pubs.acs.org Publication Date: March 10, 2003 | doi: 10.1021/bk-2003-0838.ch030

Siddharth V. Patwardhan , Michael F. Durstock , and Stephen J. Clarson 1,*

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Department of Materials Science and Engineering, University of Cincinnati, Cincinnati, O H 45221-0012 Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, O H 45433

Biosilicification is facilitated by proteins and occurs under modest conditions in an aqueous medium (pH 7 and ambient temperature). Silicification at neutral pH in vitro has been shown to occur in the presence of various cationically charged synthetic macromolecules in solution. Here, the synthesis of silica from an aqueous silica precursor in the presence of poly(allylamine hydrochloride) (PAH) and polyacrylic acid (PAA) both in solution and immobilized on substrates is investigated. The results show that the formation of ordered silica structures under these modest conditions was favored for the PAH and the PAH-PAA in solution but neither for PAA in solution nor when the polymers were immobilized as PAH-PAA bilayers on flat substrates. It is possible that the immobilization of the PAH by the electrostatically self-assembly (ESA) technique may allow it to retain its catalytic function, while not allowing it to fulfill its role as a template / structure directing agent. The silica structures were characterized using scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS). For the PAH system in solution silica spheres were seen and for PAH-PAA in solution hexagonal silica structures were observed in co-existence with silica spheres. The results presented herein may be helpful in elucidating biosilicification mechanism(s) and should lead to a better understanding of silicification.

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© 2003 American Chemical Society

In Synthesis and Properties of Silicones and Silicone-Modified Materials; Clarson, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Introduction


Investigations of silicification at neutral pH and under ambient conditions are important due to their close relationship with biosilicification, which also occurs under such mild conditions. The silicified structures that are formed in biological systems are highly sophisticated and are species specific. Examples of organisms, which form ornate silica structures, include diatoms, sponges, grasses and higher plants [1]. Studies of biosilicification have led to the isolation of silaffin proteins from the diatom Cylindrotheca fusiformis that catalyze silica formation and the amino acid primary sequence was determined. Silicatein proteins from sponges have been investigated [2, 3] and related studies on the key amino acids from silica forming proteins in grasses have also been described [4]. To verify the specificity and significance of the aforesaid proteins responsible for silicification in biological systems, a variety of synthetic polymers including polypeptides and diblock copolypeptides have been investigated for their role in silicification [5,6,7]. A synthetic polymer poly(allylamine hydrochloride), PAH, that is cationically charged under the conditions for silicification at neutral pH, has been studied in detail [8, 9]. It was demonstrated that PAH can facilitate the formation of nanometer and micrometer sized spherical silica particles under mild conditions from an aqueous solution of a silica precursor. It was shown by Energy Dispersive Spectroscopy (EDS) and Fourier Transform Infra Red Spectroscopy (FTIR) that the PAH was incorporated into the final silica structures. In the absence of PAH the reaction mixture gelled in one day. These results indicate that PAH acts as a catalyst as well as a template or structure directing agent for silicification [8]. In this context, Tacke has described how macromolecules facilitate silica formation via scaffolding [9]. In further investigations, various parameters that govern the silica synthesis and morphology of the silicified products were studied [10]. Among the important parameters were the reaction time, the precursor concentration and the precursor pre-hydrolysis time. Small Angle Light Scattering (SALS) experiments on the polymeric solutions in the reaction medium (a buffer) revealed the existence of polymer domains that have a periodic correlation distance [11]. When another cationically charged polymer was added to the reaction mixture disc-like silica particles were observed rather than spherical silica particles seen with just PAH. It is postulated that the two polymers may affect the chain conformations of each other, thus altering the product morphology [5]. Experiments in vitro using a mixture of polyamines and silaffins extracted from diatoms resulted in similar disc-like structures [12]. A shear force was externally applied to the reaction mixture and this resulted in the formation offiber-likesilica structures for the PAH system. This might be due


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the orientation of the polymer in the solution under externally applied shear [13]. Apart from fiber-like structures, various other silica structures were also synthesized and they were found to be amorphous [5]. Further investigations of these structures and the conditions under which they formed are in progress. With this background in mind, some questions still remain unanswered such as: can silicification under such modest conditions be carried out heterogeneously? What silica morphologies may result upon the use of a mixture of a positively and a negatively charged polymer? Here we have made use of PAH and polyacrylic acid (PAA) in an attempt to answer these questions. In one case they were coated by electrostatically selfassembled (ESA) onto a flat substrate, which was then dipped into a silicic acid solution. In another case, a mixture of PAH and PAA in solution was exposed to a silica precursor solution. The results were compared to the PAH system at neutral pH and under ambient conditions.

Experimental

1.

Chemical Reagents:

Tetramethoxysilane (TMOS), 99+%, was used as the silica precursor. Hydrochloric acid (HC1) was used for the TMOS pre-hydrolysis. The polymers used for silicification in solution were poly(allylamine hydrochloride), PAH, (Molecular Weight = 15,000 g mole" ) and polyacrylic acid, PAA, (Molecular Weight = 50,000 g mole" , 25 % solution in water). The reaction medium used was a potassium phosphate buffer (pH 7.0). Deionized ultra filtered (DIUF) water was used for washing the samples. All reagents were used as received without any further purification. The details of the reagents used for the ESA experiments are described in the respective section. 1

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Silica Synthesis in Solution using PAH or PAA only:

Tetramethoxysilane (TMOS) was chosen as the silica precursor and potassium phosphate buffer as a solvent. A stock solution of ImM HC1 in DIUF water was prepared and was used for all the reactions. The TMOS solution in 1 mM HC1 and the polymer solution in buffer were always freshly prepared for each experiment, as the TMOS solution was found to gel within 24 hours. A typical reaction mixture contained 80 uL of the buffer, 20 pL polymer solution (50 mg/ml in the buffer for PAH and as received for PAA) and 10 uL TMOS


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solution, which was pre-hydrolyzed in HC1 solution. All the reactants were measured and added to micro sample polypropylene test tubes (1.5 mL). The tubes were then closed and shaken well to thoroughly mix the reactants in each case. All the reactions were carried out at 20° C, atmospheric pressure and neutral pH. After the desired reaction time, the samples were centrifuged at 14,000 χ G force for 5 minutes. It was observed that a white solid precipitated in the tubes in the case of PAH only. The liquid was removed and DIUF water was then added to the tubes. The precipitate was then re-dispersed in the DIUF water. This washing of samples was repeated three times to remove any free polymer, which ensures that the reaction has been terminated. This dispersion was diluted further and 2-4 drops of this solution were placed on an aluminum SEM sample holder in each case. The solution was then left to dry under ambient conditions overnight.

3.

Silica Synthesis in Solution using P A H + PAA:

The reaction mixture contained 80 uL of the buffer, 10 pL PAH solution in the buffer (50 mg/ml), 10 uL of PAA (used as received) and 10 uL TMOS solution, which was pre-hydrolyzed in HC1 solution. All the reactions were carried out at 20° C, atmospheric pressure and neutral pH. Rest of the procedure was as described in the previous section.

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Preparation of PAH-PAA Bflayers on Substrates:

A set of experiments was carried out to study silicification in the presence of PAH when it was immobilized on flat silicon and silica glass substrates. This was achieved by sequentially electrostatically self adsorbing alternate layers of polyallylamine hydrochloride (PAH) and poly(acrylic acid) (PAA) onto either a glass or silicon wafer substrate, as described briefly below and in full detail elsewhere [14,15]. For the ESA bilayers the PAH used had a molecular weight of 60,000 g/mole and was obtained from Polysciences. The PAA had a molecular weight of 240,000 g/mole and was obtained from Scientific Polymer Products. Solutions were made of each polymer with deionized water with a resistivity of at least 18 Mohm»cm obtained from a Milli-Q filtration setup. The pH of both solutions was adjusted to 3.5 by the addition of a small amount of HC1. 2

First, a substrate was dipped into a ΙΟ" M aqueous solution of PAH for 15 minutes. The substrate was then rinsed in three separate water baths for 1-2 minutes each with the result being that the surface was now positively charged due to the adsorbed layer of PAH. It was then dipped into a ΙΟ" M aqueous 2


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solution of PAA for 15 minutes and againrinsedin the three separate water rinse baths. The adsorption of a thin layer of the PAA renders the surface negatively charged. This process of alternating between polycation (PAH) and polyanion (PAA) solutions, with arinsingstep in between, was then consecutively repeated to build up a film of the desired thickness. The films used comprised of 10.5 or 11.5 bilayers and had PAH as the outermost layer.

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Silica Synthesis on PAH-PAA Coated Substrates:

The PAH-PAA coated substrates were dipped into either a 1 M or 0.1 M prehydrolyzed TMOS solution, removed after 5 minutes, washed with DIUF water and then dried. The samples were then examined by SEM for silica formation on the substrates.

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Product Characterization:

The products obtained in each case were characterized by Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS). A palladiumgold alloy was vacuum evaporated onto the dried samples. They were then investigated using a Hitachi S-4000 Field Emission Scanning Electron Microscope at the Advanced Materials Characterization Center (AMCC), Department of Materials Science and Engineering (MSE), University of Cincinnati (UC). EDS analysis was performed using an OXFORD ISIS system attached to the SEM.

Results and Discussion

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Silica Synthesis in Solution using PAH:

PAH is able to facilitate the formation of ordered silica structures from a silica precursor in aqueous solution at neutral pH and the observed structures include spheres when the system is unperturbed and fibers when the system is under flow /shear [6, 8,11,13]. Representative silica structures from an unperturbed reaction solution at pH 7 and under ambient conditions are shown in Figure 1. The silica spheres were in the size range of 800 nm - 2 pm.


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2.

Silica Synthesis in Solution using PAA:


Further, to investigate the activity of PAA for facilitating the silica formation, we carried out similar experiments with PAA as described above for PAH. The PAA did not facilitate the precipitation of ordered silica structures at neutral pH and formed gel in a day [6].

3.

Synthesis in Solution using PAH+PAA:

When the PAH and PAA solutions were mixed they formed a turbid white solution, which did not precipitate. Upon addition of the silica precursor solution to this mixture silica was seen to precipitate after 5 minutes reaction time. A representative SEM micrograph of the resulting silica is shown in Figure 2. Two kinds of morphologies were observed to coextist: spheres (~ 100-200 nm diameter) and hexagons (~ 350-700 nm length of sides). Energy dispersive spectra (EDS) revealed that both types of structures were composed of silica. Similar co-existence of different silica structures was reported in an investigation of the poly-L-lysine system [5]. Detailed studies on the nature of the hexagons and the mechanism of their formation are in progress.

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Synthesis on Substrates (ESA bilayers of PAH-PAA):

There was no evidence for the controlled formation of silica on the PAH-PAA coated substrates except for a few irregular shaped silica regions. This silica found on the substrates is most likely gel that had simply adhered to the surface and not formed due to the PAH immobilized on the substrate. If this hypothesis is wrong, then the silica should have been found uniformly covering the substrates as the PAH was coated uniformly. Furthermore, it should have been well structured as seen when PAH was used in solution [8-10].

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Homogeneous versus Heterogeneous Silicification:

It was observed that PAH was incorporated into the silica structures during silicification in solution and hence the cationically charged polymer not only acts as a catalyst for the silica formation but also has the role of template / structure directing agent. In Figure 3 it is shown how silica may grow over the charged polymer (PAH) in the silica precursor solution / reaction medium thus illustrating its scaffolding role.



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Figure 1. A representative scanning electron micrograph of silica structures formed upon the use of PAH in solution at neutral pH. Bar = 1 pm.

Figure 2. Representative scanning electron micrograph of silica structures formed upon the use of PAH and PAA in solution at neutral pH. In Synthesis and Properties of Silicones and Silicone-Modified Materials; Clarson, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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Silica

Figure 3. The scaffolding role of PAH in solution in facilitating silicification at neutral pH The failure of the formation of silica on PAH coated substrates may be due to the non-availability of the charges on the protonated amines of the PAH as the PAA may be tying up the PAH by ionic and hydrogen bonds [13, 15]. It is possible that the immobilization of the PAH by the ESA technique may allow it to retain its catalytic function, while not allowing it to fulfill its role as a template / structure directing agent. It might also suggest that the silica formation is favorable in homogeneous systenis and not in the heterogeneous system investigated here. It was hypothesized that the PAH + PAA reaction mixture may not have facilitated the formation of ordered structures silica but would gel due to the non-availability of charged PAH which might be tied by PAA as discussed for the PAH+PAA bilayers. This experiment indicates the importance of the availability of charge(s) present on macromolecules in the silicification and the templating / structure directing role of the polymer in solution.

Conclusions Silicification at neutral pH in the presence of a cationically charged polymer in solution and immobilized on substrates is described here. It is proposed that the silica formation may not be favorable in the heterogeneous systems considered above due to immobilization of the cationic polymer and the elimination of its role as a structure directing agent. The results discussed herein indicate the


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importance of availability of active sites (charges in this case) present on macromolecules in silicification. A novel hexagonal morphology was seen when a mixture of PAH and PAA was used in solution while ordered silica spheres were seen in the case of unperturbed PAH solutions. These results may help elucidate the mechanism(s) of biosilicification and lead to a better understanding of silicification, in general. The use of synthetic polymers in synthesizing novel morphologies has many potential applications in the field of bioinspired materials science.

Acknowledgements We thank DAGSI for providing the financial support for this research. We appreciate the help and expertise in the SEM analysis provided by Niloy Mukherjee. We also thank Dr. Jeff Baur for several helpful discussions.

References 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Simpson, T. L.; Volcani, Β. E. Eds., Silicon and Siliceous Structures in Biological Systems, New York: Springer-Verlag, 1981. Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129. Shimizu, K.; Morse, D. E . In: Biomineralization: From Biology to Biotechnology and Medical Application, E. J. Baeuerlein, ed., Wiley -VCH: New York, 2000, pp 207. Harrison (formerly Perry), C. C., Phytochemistry 1996, 41(1), 37. Patwardhan, S. V.; Mukherjee, N; Clarson, S. J. J. Inorg. Organomet. Polym. 2001, 11(3), 193. Patwardhan, S. V.; Clarson, S. J. Silicon Chemistry 2002, in press. Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature 2000, 403, 289. Patwardhan, S.V.; Clarson,S.J.Polym. Bull. 2002, 48(4-5), 367. Tacke, R. Angew. Chem. Int. Ed. 1999, 38(20), 3015. Patwardhan, S. V.; Mukherjee, N; Clarson, S. J. Silicon Chemistry 2002, 1(1), 47. Patwardhan, S. V., Masters Thesis, Department of Materials Science and Engineering, University of Cincinnati, OH, USA, 2002. Kroger, N.; Deutzmann, R.; Bergsdorf, C.; Sumper, M . PNAS 2000, 97(26), 14133. Patwardhan, S. V.; Mukherjee, N; Clarson, S. J. J. Inorg. Organomet. Polym. 2001, 11(2), 117. Shiratori, S. S.; Rubner, M . F. Macromolecules 2000, 33, 4213. Decher, G. Science 1997, 277, 1232. Durstock, M. F.; Rubner, M. F. Proceedings of SPIE 1997, 3148, 126.


Silicification and Biosilicification - ACS Symposium Series (ACS

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