Biocatalysis in the development of functional polymer–ceramic nanocomposites

Colloids and Surfaces B: Biointerfaces 39 (2004) 143–150

Biocatalysis in the development of functional polymer–ceramic nanocomposites Christy Ford a , Mohit Singh a , Louise Lawson a , Jibao He b , Vijay John a,∗ , Yunfeng Lu a , Kyriakos Papadopoulos a , Gary McPherson c , Arijit Bose d a

Department of Chemical Engineering, Tulane University, New Orleans, LA 70118, USA Coordinated Instrumentations Facility, Tulane University, New Orleans, LA 70118, USA c Department of Chemistry, Tulane University, New Orleans, LA 70118, USA Department of Chemical Engineering, University of Rhode Island, Kingston, RI 02881, USA b

d

Available online 28 January 2004

Abstract Fluorescent silica/polymer nanocomposites have been synthesized by condensing tetramethyl orthosilicate (TMOS) around fluorescent polymer strands of poly(2-naphthol). The polymer is biocatalytically synthesized via peroxidase catalyzed polymerization in micelles of the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Silica condensation at the micelle–water interface results in encapsulation of the polymer. Fluorescence spectroscopy and fluorescent light microscopy provide critical evidence that the polymer luminescence properties are conferred to the composite material. The fabrication of polymer entrapped in ordered, mesoporous materials represents a viable step toward the development of functional polymer–ceramic nanocomposites. © 2003 Elsevier B.V. All rights reserved. Keywords: Horseradish peroxidase; Polymer–ceramic nanocomposites; Fluorescence; Mesoporous silica

1. Introduction In recent years, there has been significant interest in the use of enzymes as catalysts for polymer synthesis. Enzymebased reactions are environmentally benign and enzyme catalysis is highly specific resulting in polymers that have unique functionalities reflecting the biomimetic aspect of such natural processes [1]. Some examples include the use of lipases for polyester synthesis [2], cellulases for polysaccharide synthesis [3], and peroxidases for polyphenol and polyaromatic amine synthesis [4]. The use of oxidative enzymes such as peroxidase are especially relevant here as they catalyze the polymerization of substituted phenols following pathways identical to the biological synthesis of lignin [5,6]. Such polyphenolics have applications in traditional coatings technologies as environmentally benign synthetic replacements for phenol–formaldehyde based polymers. Additionally, they can be developed into a variety ∗ Corresponding author. Tel.: +1-504-865-5883; fax: +1-504-865-6744. E-mail address: [email protected] (V. John).

0927-7765/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2003.12.010

of functional conjugated materials that have the potential to exhibit novel electro-optical properties including electrical conductivity [7] and luminescence [8]. In conducting enzymatic polymerization of substituted phenols, the sparingly soluble nature of the monomers in water must be considered. Chain growth in an entirely aqueous medium is not feasible because the even lower solubility of dimmers and trimers cause phase separation and precipitation. Enzymatic polymerization in organic solvents can be carried out through retention of enzyme activity in solvents such as 1,4-dioxane [4,9]. From the perspective of interfacial phenomena, conducting the enzymatic polymerization reaction in micellar systems is a feasible alternative approach. The monomers can be solubilized in the micelles, while the enzyme is able to retain catalytic activity in aqueous media. In this report, we employ nanometer scale confined geometries of micelles of the cationic surfactant, cetyltrimethylammonium bromide (CTAB), as an environment for the polymerization of 2-naphthol. We have previously reported the enzymatic synthesis of fluorescent poly(2-naphthol) in reversed micelles [8] of the double-tailed anionic surfactant AOT (bis e-ethylhexyl sodium sulfosuccinate), and enzy-

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Synthesis Scheme Monomer

Horseradish Peroxidase

Surfactant (CTAB)

Silica HRP, H 2 02

CTAB Micelles Swollen with monomer

TMOS

CTAB Micelles Swollen with polymer

Mesoporous silica/polymer composite

Fig. 1. Proposed polymer/mesoporous silica composite synthesis scheme.

matic polynaphthol synthesis has also been reported to be feasible in organic solvents [9]. In addition to the initial objective of enzymatic polymer synthesis, we report a method to encapsulate the polymer into mesoporous ceramics to fabricate novel polymer– ceramic nanocomposites. CTAB micelles are extremely effective in the synthesis of mesoporous silicas [10,11] and have been widely studied in this regard [12–16]. Hydrolysis of silica precursors occurs at the micelle–water interface and surfactant charge stabilization of the hydroxylated intermediates causes condensation to occur in the water phase at the micelle–water interface. Such interactions lead to ordered silicas of the M41S family [10,11] that exhibit a range of hexagonal, cubic, and lamellar mesopore morphologies. Our objectives are illustrated by the proposed synthesis scheme shown in Fig. 1. Initially, CTAB micelles form with the monomer (represented by a peg) solubilized in the micelle. The enlarged micelle depiction demonstrates the hydroxyl groups (represented by the peg head) are oriented toward the water phase, while the hydrophobic portion of the monomer is oriented toward the interior of the micelle. The enzyme is shown to be located in the bulk water, but interactions with the surfactant may result in the enzyme being resident at the micelle–water interface. Polymerization is then conducted (with the addition of H2 O2 ) and we hypothesize that the polymer will remain solubilized in the micelles. Subsequently, the silica precursor (tetramethyl orthosilicate—TMOS) is added to the micellar solution with the non-polar precursor also being solubilized in the micelles. It was hypothesized that interfacial silica condensation would result in the formation of mesoporous silica channels with the encapsulation of the polymer. We report an approach to encapsulate enzymatically synthesized poly(naphthol) in mesoporous silicas, and thereby

develop fluorescent polymer–ceramic nanocomposites. Such composites may exhibit the dual functionalities of both the polymer and the ceramic component, exhibiting the structural characteristics of the ceramic and the functional luminescence of the polymer. Such nanocomposites may have widespread applications in the development of highly robust chemical and biochemical sensors.

2. Materials and methods 2.1. Materials Horseradish peroxidase type II (HRP), 2-naphthol, hydrogen peroxide, cetyltrimethylammonium bromide, HEPES, tetramethyl orthosilicate (TMOS) were purchased from Sigma–Aldrich and used without further purification. 2.2. Enzymatic polymerization 2-Naphthol was dissolved in a micellar solution containing 56 mM CTAB and 0.1 M HEPES buffer, pH 5.3. The monomer-to-surfactant ratio ranged from 0.056 to 6 (or 3.1 mM to 0.34 M). Horseradish peroxidase (0.5 mg/ml) was added to the above micellar solution. Polymerization was initiated by adding 30 wt.% hydrogen peroxide drop-wise, with the hydrogen peroxide to monomer molar ratio equal to 1.3. The reaction mixture was stirred for 1 h at room temperature to complete the polymerization before introducing the silica precursor. 2.3. Poly(2-naphthol)/silica composite synthesis After polymerization, 0.625 ml (0.43 M) of tetramethyl orthosilicate was added to the micellar solution containing

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poly(2-naphthol). The solution was stirred vigorously to allow the micelles to accommodate TMOS. An amount of 0.65 ␮l of 50 wt.% NaOH was added to the solution, increasing the pH to 11. A slurry formed upon TMOS hydrolysis and condensation. The hydrolysis and condensation were continued for 2 h before removing excess surfactant and unreacted monomer. The slurry was washed with distilled water and filtered using a 0.22 ␮m Millipore filter membrane several times. All samples were air-dried after filtration. 2.4. Polymer/silica composite characterization FT-IR spectra were performed using a Perkin-Elmer Spectrum GX by casting the sample on ZnSe windows. Molecular weight distributions were measured using gel permeation chromatography (Perkin-Elmer 200 Series UVVis Detector and Perkin-Elmer Series 200 Pump) using a Jordi-Gel DVB Mixed Bed (length 250 mm and inner diameter 10 mm). The UV detector was set at 270 nm. THF was used as the mobile phase at a flow rate of 0.8 ml/min. An amount of 5–10 mg of dry synthesized poly(2-naphthol) was dissolved in 1 ml of THF and 5 ␮l of this solution was used for molecular weight analysis. Polystyrene samples with molecular weights ranging from 687 to 4000 were used as calibration standards. Luminescence measurements were carried out using a Perkin-Elmer Luminescence Spectrometer LS50B. An inverted Olympus light microscope with a fluorescent attachment and an Optronics CCD camera was

Fig. 3. Fourier transform infrared spectra of (a) poly(2-naphthol) synthesized in CTAB micelles and (b) 2-naphthol. The polymer has significant naphthol character as noted by retention of the hydroxyl group. The loss of the 844 cm−1 C–H out of plane bending vibration for the polymer provides evidence of polymer formation.

used to image fluorescent samples. Image-Pro Plus from Media Cybernetics Inc. was the software used to image the samples. X-ray diffraction measurements were done using a Scintag, XDS 2000 instrument equipped with a Peltier cooled Si(Li) detector. The diffractometer possessed Cu K␣ radiation of 1.54059 Å. The diffraction data were recorded for 2θ angles between 1◦ and 10◦ , with a step size of 0.030◦

Fig. 2. Simplified schematic of the polymerization reaction. The polymerization of 2-naphthol is catalyzed by horseradish peroxidase in the presence of an initiator, hydrogen peroxide. Various radical resonance structures are formed and some of the coupling products are shown. Because of the wide variety of resonance structures only the coupling products are shown. The polymer chain grows from 2-naphthol binding to the dimmers. The coupling between dimmers and 2-naphthol units may occur through a variety of resonance structures of both the monomer and the dimmer.

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and step rate of 1.00◦ /min. The divergence slit was 0.5 mm (1 mm scatter) and the receiving slit was 0.2 mm (0.3 mm scatter). Polymer morphology was examined using a JEOL 5410 scanning electron microscope with an acceleration voltage of 20 kV. The samples were coated with platinum before imaging. 3. Results and discussion Horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide can be written in the simplified form [5] HRP + H2 O2 → compound I

compound I + R–H → R∗ + compound II compound II + R–H → R∗ + HRP Compounds I and II are intermediate states—compound I results from a two-electron oxidation of the heme group and compound II results from the one-electron reduction of compound I. R–H is the substrate and R∗ is the phenoxy radical species. The overall coupling reaction can be expressed as 2R–H + H2 O2 → R–R + 2H2 O Fig. 2 illustrates the mechanistic aspects of poly(naphthol) synthesis and indicates resonance structures that lead to

Fig. 4. (a) Photograph of the solution containing poly(2-naphthol) synthesized in CTAB micelles before the introduction of silica precursor. The polymer is sustained by the micelles and does not precipitate. (b) Photograph of the system after silica formation, illustrating the precipitation of polymer/silica leaving a transparent supernatant. (c) FT-IR spectra of poly(2-naphthol) synthesized in CTAB micelles (solid line) and the supernatant from the poly(2-naphthol)/silica composite (dashed line). (d) Dried mesoporous silica not containing polymer (the control) is white in color while the (e) dried poly(2-naphthol)/silica composite is light green in color. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

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multiple coupling modes to form dimmers. Subsequent step condensations to larger structures are not shown for simplicity, since there are numerous possible structures that result from such coupling. 3.1. Enzymatic polymer synthesis in CTAB micelles We have found that polymerization in CTAB micelles results in the polymer being sustained in the micellar system. Even with monomer-to-surfactant ratios of 6:1, both the monomer and the polymer remain solubilized in the micelles. After initiating the polymer reaction with hydrogen peroxide, the solution undergoes a color change from cloudy white to green. Gel permeation chromatography indicates that poly(2-naphthol) synthesized in CTAB micelles has a number average molecular weight (Mn ) of 630, weight average molecular weight (Mw ) of 940, and a polydispersity of 1.5. This reflects the formation of oligomers with 4–7 units and is in agreement with results obtained in AOT inverse micellar systems [8]. Fig. 3 illustrates the FT-IR spectra of (a) poly(2-naphthol) synthesized in CTAB micelles and (b) 2-naphthol. The polymer has significant naphthol character as evidenced by strong retention of the OH stretch. Information about the substitution patterns in an aromatic ring can be deduced from the C–H out of plane bending vibrations in the 900– 650 cm−1 region. The 2-naphthol FT-IR spectrum shows C–H out of plane bending vibrations at 844, 810, and 742 cm−1 representative of an isolated hydrogen, two adjacent hydrogen atoms, and four adjacent hydrogen atoms, respectively. The C–H bending vibration at 844 cm−1 (C–H bend for an isolated hydrogen atom) is significantly reduced after polymerization, suggesting that the carbon at the C1 position is involved in polymerization. The other two C–H out of plane bending vibrations at 810 and 742 cm−1 are preserved after polymerization. A small shoulder is observed on the left of the 810 cm−1 vibration, which may result from unreacted monomer. The remaining vibrations in the poly (2-naphthol) spectra can be assigned to the C–H aromatic stretch (3060 cm−1 ), aliphatic CH2 and CH3 stretch from residual CTAB (2960, 2830 cm−1 ), C=O stretch resulting from quinonoid groups (1670 cm−1 ), C=C aromatic stretch (1640, 1599, 1506, 1470 cm−1 ), in-plane OH bend (1380 cm−1 ), and C–OH stretch (1210 cm−1 ). 3.2. Polymer encapsulation and synthesis of polymer–ceramic nanocomposite The observation that poly(2-naphthol) remains solubilized in CTAB micelles indicates the possible feasibility of subsequent encapsulation in mesoporous silicas. Accordingly, the composite was synthesized by adding TMOS to CTAB micelles containing poly(2-naphthol) and allowing hydrolysis and condensation of the silica precursor to proceed. As mentioned earlier, upon enzymatic polymerization, the clear colorless micellar solution becomes green with no

Fig. 5. (a) XRD pattern of mesoporous silica not containing polymer exhibits hexagonal structure. (b) A typical XRD pattern is presented for poly (2-naphthol)/silica composites indicates lamellar structure. The composite has a surfactant-to-monomer molar ratio = 1.

observable precipitation (Fig. 4a). Upon condensation of silica, the system becomes a slurry with the solid component precipitating out and leaving a clear supernatant (Fig. 4b). FT-IR analysis indicates that the supernatant from the polymer/silica composite does not contain an appreciable amount of poly(2-naphthol) as evidenced by the essentially baseline spectrum of the supernatant (Fig. 4c). Mesoporous silica not containing polymer (Fig. 4d) is white in color while the silica/poly(2-naphthol) composite is a light green color (Fig. 4e). This is a visual indication that the polymer may be trapped in the pores of silica, but is not complete evidence. X-ray diffraction patterns for the control mesoporous silica without any polymer incorporated, and for the poly(2naphthol)/silica composite are shown in Fig. 5a and b, respectively. The diffraction pattern for the control sample fully indexes to the hexagonal structure of MCM-41 [10,11]. On the other hand, the diffraction pattern of the composite exhibits evenly spaced peaks with d-spacing of 4.2 nm (d1 0 0 ) and 2.1 nm (d2 0 0 ), indexing as lamellar structures. Such lamellar structures are observed for poly(2naphthol)/silica composites with monomer-to-surfactant molar ratios ranging from 0.056 to 6. There is an accompanying loss of crystallinity in the composite upon

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Fig. 6. Scanning electron micrograph of (a) silica not containing polymer and (b) poly(4-ethylphenol)/silica with monomer-to-surfactant ratio = 1. Transmission electron micrograph illustrating (c) hexagonal arrangement of pores for mesoporous silica without polymer and (d) lamellar arrangement for poly(4-ethylphenol)/silica composite with monomer-to-surfactant ratio = 3. Note the SEM and TEM image of (b) and (d) here are of poly(4-ethylphenol)/silica composite. Poly(2-naphthol)/silica is expected to be similar.

incorporation of polymer. We are carrying out further studies to understand these transitions from hexagonal to lamellar structures through cryo-transmission electron microscopy of the micellar structure upon doping with monomer and polymer, to attempt to correlate micellar structure with the final microstructure of the composite. Fig. 6 illustrates electron micrographs of the control and the polymer–ceramic composites. The control MCM-41 (Fig. 6c) exhibits an hexagonal structure while the polymer/silica composite (Fig. 6d) exhibits a lamellar structure. These observations are consistent with the structural information obtained from XRD. In these figures, the sample is a poly(4-ethylphenol)/silica composite, to emphasize the observation that enzymatically synthesized polyphenols all induce the transition from hexagonal to lamellar microstructures in silicas when encapsulated at sufficient levels (a full description of doping level and corresponding microstructures will be reported separately). Other

researchers have also found interesting structural changes in silicas upon incorporating additives. Zink and coworkers also observed the transition from hexagonal to lamellar upon doping silicate thin films with molecules such as carbazole and fluorine [17]. Anderson et al. found that increasing concentrations of cosolvents in silica synthesis cause a transition from ordered hexagonal to disordered hexagonal in the case of methanol and a transition from ordered hexagonal to lamellar in the case of THF as a cosolvent [18]. 3.3. Fluorescence properties of poly(2-naphthol)/silica composites The functional characteristics of these composites have been evaluated through their luminescence properties. The dried polymer/silica composite was cast on a quartz slide to obtain films whose fluorescence characteristics were

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Fig. 7. (a) Fluorescence spectra of dried poly(2-naphthol)/silica composite and mesoporous silica without polymer or monomer. All samples were cast on a quartz slide. (b) Fluorescent light microscope image of poly(2-naphthol)/silica composite with monomer-to-surfactant ratio = 1. The control sample of MCM-41 does not exhibit fluorescence (data not shown).

measured (Fig. 7a). The poly(2-naphthol)/silica composite exhibits intense peaks at 370 and 470 nm, while the control sample does not exhibit any fluorescence. Powders of the composite were slurried in water and dilute suspensions were dropped on to a glass slide and dried. Fluorescence light microscopy shows distinct particles of poly(2-naphthol)/silica composites producing an intense fluorescence (Fig. 7b). In contrast, the control sample of mesoporous silica does not exhibit any fluorescence. We have also noted that continued stirring of the slurry in water does not result in any significant degradation of the fluorescence characteristics indicative of a robust encapsulation of the polymer and the potential to use such composites in sensor applications.

4. Conclusions A novel two-step approach to encapsulate polymer in mesoporous materials is reported. We have demonstrated

the feasibility of carrying out enzymatic polymerization in CTAB micelles. Importantly, the polymer is solubilized in the micelles even at high monomer-to-surfactant ratios. Enzymatic polymerization is followed by the synthesis of mesoporous silicas to generate poly(2-naphthol)/silica composites. Poly(2-naphthol) appears to be incorporated in the mesoporous silica, and the composite materials exhibit luminescent properties. Fluorescent polymers and polymer composites have important applications in plastic scintillators [19,20], luminescent solar concentrators [21], laser materials [22,23], paint and varnishes [24], and fiber optic sensors [25]. Many fluorescent dyes contain segments obtained from naphthol units, an inexpensive raw material [26]. This work demonstrates a reasonably facile method of coupling biocatalysis and ceramic synthesis to encapsulate fluorescent polymers in nanoscale confined geometries. The resulting composite may indeed, find technological applications, especially as robust sensor materials.

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Acknowledgements Financial support from the National Science Foundation (Grants 0329311 and 9909912) and from NASA (NCC3-946 and NAG-1-02070) is gratefully acknowledged.

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