Langmuir 2007, 23, 1981-1987
1981
Immobilized Horseradish Peroxidase as a Reusable Catalyst for Emulsion Polymerization Alliny F. Naves, Ana M. Carmona-Ribeiro, and Denise F. S. Petri* Instituto de Quı´mica, UniVersidade de Sa˜ o Paulo, P.O. Box 26077, Sa˜ o Paulo SP, 05513-970, Brazil ReceiVed June 29, 2006. In Final Form: NoVember 13, 2006 The study on the adsorption of horseradish peroxidase (HRP) onto silicon wafers was carried out by means of in situ ellipsometry, atomic force microscopy (AFM) and contact angle measurements. A smooth HRP layer adsorbed onto Si wafers. The enzymatic activity of free or adsorbed HRP was determined by the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and by the emulsion polymerization of ethylene glycol dimethacrylate (EGDMA). Upon adsorbing, HRP molecules might have undergone some conformational changes, which caused a small reduction of enzymatic activity in comparison to that observed for HRP solution. However, it was possible to reuse the same HRP-covered Si wafer as catalyst in the polymerization of EGDMA three times.
Introduction In Vitro polymer synthesis catalyzed by enzymes has gained much attention because it offers many advantages in comparison to polymerization catalyzed by traditional catalysts, for instance: (i) high selectivity under mild reaction conditions; (ii) enzymes can be used in water miscible organic solvents, anhydrous organic solvent and micelles; (iii) enzymes can be obtained from renewable sources; (iv) they might substitute potentially toxic catalysts and (v) they have the potential of producing products in high yield at room temperature.1,2 Horseradish peroxidase (HRP) is a glycoprotein containing 18% carbohydrate with heme (iron protoporphyrin) as prosthetic group.3,4 The oxidation states of HRP in the presence of hydrogen peroxide (H2O2) are schematically represented in Figure 1.3,4 Radical intermediates (Compound I) can oxidize a wide variety of organic compounds.3,4 HRP in solution as catalyst for free radical polymerization has proved to be an interesting alternative for the conventional catalysts because the polymerization can be carried out at room temperature, yields reach acceptable levels and the solvent is not restricted to one special class, although organic solvents induce HRP activity loss.5 One of the earliest studies was the polymerization of phenols catalyzed by HRP in nonaqueous media.5 Later the polymerization of acrylamide in the presence of HRP, H2O2 and β-diketones revealed β-diketone as important substrate for HRP.6 Highly syndiotatic poly(methyl methacrylate) was obtained at room temperature in the presence of HRP, H2O2 and 2,4-pentanedione.7 Acetylacetone, abbreviated as acac, seems to act as the free radical initiator for the chain growth of polyacrylamide.8 Polyaniline has been successfully synthesized in the presence of HRP and phosphate buffer9,10 or in micelle solutions.11,12 HRP-mediated emulsion polymerization of polystyrene in the presence 2,4-pentandione led to high yield,
Figure 1. Schematic representation of the HRP catalytic cycle in the presence of H2O2. M, M•, and MM• represent the monomer, monomer radical, and dimer radical, respectively. Nhis refers to the N atom belonging to the histidine 170 residue. Scheme adapted from refs 3 and 4.
* Corresponding author. E-mail:
[email protected]. Tel: 0055 11 3091 3831. Fax: 0055 11 3818 5579.
high conversion and stable nanoparticles (30 to 50 nm).13 Water soluble polyphenols containing sugar moieties obtained in the presence of HRP and organic aqueous solvents presented low conversion rate and low molecular weight. The addition of dioctyl sulfosuccinate to the reaction led to higher conversion rates and longer polymer chains.14 Immobilizing enzymes provides the advantages of reusability, greater convenience in handling, and, in some cases, increases
(1) Kobayashi, S. J. Polym. Sci. Part A: Polym. Chem. 1999, 37, 3041. (2) Gross, R. A.; Kumar, A.; Kalra, B. Chem. ReV. 2001, 101, 2097. (3) Veitch, N. C. Phytochemistry 2004, 65, 249. (4) Berglund, G. I.; Carlsson, G. H.; Smith, A. T.; Szo¨ke, H.; Henriksen, A.; Hajdu, J. Nature 2002, 417, 463. (5) Dordick, J. S.; Marletta, M. A.; Klibanov, A. M. Biotechnol. Bioeng. 1987, 30, 31. (6) Teixeira, D.; Lalot, T.; Brigodiot, M.; Mare´chal, R. Macromolecules 1999, 32, 70. (7) Kalra, B.; Gross, R. A. Biomacromolecules 2000, 1, 501. (8) Kalra, B.; Gross, R. A. Green Chemistry 2002, 4, 174.
(9) Liu, W.; Kumar, J.; Tripathy, S.; Senecal, K. J.; Samuelson, L. J. Am. Chem. Soc. 1999, 121, 71. (10) Wang, X.; Schreuder-Gibson, H.; Downey, M.; Tripathy, S.; Samuelson, L. Synth. Met. 1999, 107, 117-121. (11) Liu, W.; Kumar, J.; Tripathy, S. Langmuir 2002, 18, 9696. (12) Namani, T.; Walde, P. Langmuir 2005, 21, 6210. (13) Shan, J.; Kitamura, Y.; Yoshizawa, H. Colloid Polym. Sci. 2005, 284, 108. (14) Tawaki, S.; Uchida, Y.; Maeda, Y.; Ikeda, I. Carbohydrate Polym 2005, 59, 71.
10.1021/la061884o CCC: $37.00 © 2007 American Chemical Society Published on Web 12/23/2006
1982 Langmuir, Vol. 23, No. 4, 2007
their stability.15 For instance, HRP immobilized onto cinnamic carbohydrate esters was more resistant than the soluble form against inactivation by excess of H2O2 and by heat.16 The activity and stability of HRP immobilized onto mesoporous silica with variable pore size were investigated.17 The highest enzymatic activity and best thermal stability were observed for HRP adsorbed onto silica with mean pore diameter of 5.0 nm. This pore size matches the HRP molecular dimensions, preventing enzymatic conformational changes, and therefore, keeping high activity and stability.17 Monolayers of HRP adsorbed either onto Si wafers or onto succinylated modified Si wafers presented half-life time longer than 40 days at 6 °C.18 Assembling HRP and bipolar quaternary ammonium salt on gold electrode led to devices with fast response and good chemical and mechanical stability.19 Alternated layer deposition of HRP and surfactant preserved HRP activity for prolonged period of time.20 So far the potential of using immobilized HRP as catalyst for emulsion polymerization has been seldom explored. In this work, HRP has been adsorbed onto Si wafers in order to produce a catalyst, which can be inserted in a polymerization and after reaction it can be removed for reuse. The adsorption process was monitored by means of in situ ellipsometry. HRP adsorbed layer was characterized by means of contact angle measurements and atomic force microscopy (AFM). The catalytic activity and reusability of HRP-covered wafers were determined in the emulsion polymerization of ethylene glycol dimethacrylate (EGDMA). Materials Silicon (100) wafers purchased from Crystec (Berlin, Germany) were used as substrates. Si wafers with dimensions of (0.8 × 0.8) cm2 were previously rinse d in a standard manner,21 dried under a stream of N2 and characterized prior to use. Horseradish peroxidase, HRP, type VI-A, from Amoracia rusticana, (molecular weight ∼ 44,000 g mol-1, P-6782, EC 1.11.1.7, 1380 units mg-1) and 2,2′azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma, USA) were purchased from Sigma, USA, and used without further purification. HRP purity, also called Reinheitzahl (Rz), was estimated from the ratio of absorbance at 403 nm and at 280 nm (Rz ) A403/ A280).22 The Rz of HRP solution at 0.25 g.L-1 amounted to 2.9 ( 0.1, indicating low contamination by other proteins.22 Emulsion polymerization of ethylene glycol dimethacrylate (EGDMA, Sigma, USA) was studied in the presence of cetyltrimethylammonium bromide (CTAB, Aldrich, USA) and immobilized HRP or free HRP. H2O2 30% (w/v) solution (Nuclear, Brazil) was added in order to enable HRP catalytic action. The exact amount of each reactant is given in Table 1. All reactants were used without prior purification, except for the monomer EGDMA, which was purified by distillation prior to polymerization.
Methods Ellipsometry. Ellipsometric measurements were performed in situ and ex situ using a vertical computer-controlled DRE-EL02 ellipsometer (Ratzeburg, Germany). The angle of incidence was set at 70.0° and the wavelength, λ, of the He-Ne laser was 632.8 nm. (15) Chibata, I.; Tosa, T.; Sato, T.; Mori, T.; In: Immobilized Enzymes. Wiley & Sons, New York, 1978. (16) Rojas-Melgarejo, F.; Rodrı´gues-Lo´pez, J. N.; Garcı´a-Ca´novas, F.; Ga´rciaRuiz, P. J. Chem. Technol. Biotechnol. 2004, 79, 1148. (17) Takahashi, H.; Li, B.; Sasaki, T.; Miyazaki, C.; Kajino, T.; Inagaki, S. Chem. Mater. 2000, 12, 3301. (18) Vianello, F.; Zennaro, L.; Di Paolo, M. L.; Rigo, A.; Malacarne, C.; Scarpa, M. Biotechnol. Bioeng. 2000, 68, 488 (19) Cui, A.; Tang, J.; Li, W.; Wang, Z.; Sun, C.; Zhao, M. Mater. Chem. Phys. 2001, 71, 23. (20) Berzina, T. S.; Piras, L.; Trotsky, V. I. Thin Solid Films 1998, 327, 621. (21) Petri, D. F. S.; Wenz, G.; Schunk, P.; Schimmel, T. Langmuir 1999, 15, 4520. (22) Chattopadhyay, K.; Mazumdar, S. Biochemistry 2000, 39, 263.
NaVes et al. Table 1. Amounts of EGDMA and H2O2 Used for Emulsion Polymerization in 10 mL of 2 × 10-3 mol L-1 CTAB at 25 °Ca adsorbed HRP formu EGDMA lation (µL) 1 2 3 4 5
200 200 200 900 900
H2O2 (0.003%)
dHRP (nm)
mHRP (µg)
8 × 5 µL 1.59 ( 0.04 0.14 ( 0.05 8 × 50 µL 1.71 ( 0.01 0.15 ( 0.01 8 × 50 µL 8 × 50 µL 1.9 ( 0.1 0.17 ( 0.01 8 × 50 µL
free HRP mHRP (µg)
0.175 0.175
a
Aliquots of H2O2 were added to the reaction eight times at intervals of 5 min. dHRP and mHRP correspond to the thickness of the adsorbed HRP layer and the mass of HRP either adsorbed or free in solution, respectively. mHRP was calculated by multiplying Γ by the Si wafer area of 0.64 cm2. dHRP values were determined by means of ellipsometry.
For the data interpretation, a multilayer model composed by the substrate, the unknown layer and the surrounding medium should be used. Then the thickness (dk) and refractive index (nk) of the unknown layer can be calculated from the ellipsometric angles, ∆ and Ψ, using the fundamental ellipsometric equation and iterative calculations with Jones matrices23 ei∆ tan Ψ )
Rp ) f(nk, dk, λ, φ) Rs
(1)
where Rp and Rs are the overall reflection coefficients for the parallel and perpendicular waves. They are a function of the angle of incidence φ, the wavelength λ of the radiation and of the refractive index and the thickness of each layer of the model, nk, dk, respectively. From the ellipsometric angles ∆ and Ψ and a multilayer model composed by silicon, silicon dioxide, enzyme layer and HRP solution (in situ) or air (ex situ) it is possible to determine only the thickness of the HRP layer, dHRP. The thickness of the SiO2 layers was determined in air, considering the refractive index for Si as n ) 3.88 - i0.01824 and its thickness as an infinite one, for the surrounding medium (air) the refractive index was considered as 1.00. Because the native SiO2 layer is very thin, its refractive index was set as 1.46224 and just the thickness was calculated. The mean thickness of the native SiO2 layer amounted to (1.9 ( 0.2) nm. Adsorption kinetics of HRP onto Si substrates was studied by means of in situ ellipsometric experiments filling a poly(tetrafluoroethylene) cell with 30 mL of HRP solution at the concentration of 0.001 mg mL-1 and pH 6.5, at (24 ( 1) °C. The thickness of HRP adsorbed layer onto Si wafers was monitored as a function of time, considering the nominal index of refraction of HRP as 1.515. Ex situ adsorption isotherms of HRP on silicon wafers were obtained by immersing the substrates into HRP solutions prepared in the concentration range of 0.005 mg mL-1 to 0.100 mg mL-1 at pH 6.5, during 9 h. After that period of time the samples were washed with abundant distilled water and dried under a stream of N2. Then the mean thickness of adsorbed HRP layers dHRP was determined in the air, as presented in Table 1. The adsorbed amount of HRP, Γ (mg m-2), can be estimated by multiplying the enzyme layer thickness, dHRP, by the density F of a dry enzyme layer (F ∼ 1.37 g cm-3):25 Γ ) FdHRP
(2)
In situ desorption experiments were performed for HRP covered substrates by exchanging the HRP solution either by distilled water or by monomer dispersion or by CTAB solution during 9 h. The temperature was kept at (24 ( 1) °C. (23) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light, North Holland Publication, York, 1982. (24) Edwards, D. P. Ed. Handbook of Optical Constants of Solids, Academic Press: London 1985. (25) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundstro¨m, I. Thin Solid Films 1998, 324, 257.
Horseradish Peroxidase as a Reusable Catalyst Contact angle measurements were performed at (24 ( 1) °C in a home-built26 apparatus equipped with a digital camera, which is connected to a computer. Sessile water drops of 8 µL were used for advancing contact angle (θA). All the surfaces were measured after long periods (9 h) of adsorption. Atomic force microscopy (AFM) topographic images were obtained using a PicoSPM-LE Molecular Imaging system with cantilevers operating in the intermittent-contact mode (AAC mode), slightly below their resonance frequency of approximately 290 kHz in the air. All topographic images represent unfiltered original data and refer to scan areas of 1 µm × 1 µm with a resolution of (512 × 512) pixels. At least two samples of the same material were analyzed at different areas of the surface. Image processing and the determination of the root-mean-square (rms.) roughness were performed by using the PicoScan 5.3.2. software. Catalytic Activity of Adsorbed and Free HRP. The catalytic activity of adsorbed HRP and free HRP was tested in two different ways. The first one involved the oxidation of ABTS catalyzed by HRP in the presence of H2O2, into the radical cation ABTS•+,27 as schematically shown in Supporting Information (see supp-mater-0). The reaction was started by the addition of HRP-covered Si wafer to a solution containing 0.14 µmol L-1 ABTS and 50 µL H2O2 0.003% (w/v). The oxidation of ABTS (λmax ) 340 nm) into ABTS•+ (λmax ) 414 nm) was monitored by UV-vis spectrophotometry (Beckmann-Coulter DU 600) as a function of time. The concentration of ABTS•+ was calculated using Beer-Lambert equation and considering the optical pathway as 1 cm and extinction coefficient of 3.6 × 104 mol L-1 cm-1.28 A typical ABTS•+ spectrum is presented as Supporting Information (see suppl-mater-1). The formation of ABTS•+ was evidenced by the appearance of green color solution. The mass of adsorbed HRP was calculated by multiplying Γ by Si wafer area (see Table 1). Control experiments were done using free HRP in solution under the same conditions and reactants amounts. The catalytic activity of adsorbed or free HRP was also tested in a typical emulsion polymerization recipe,29 where the medium was 10 mL of aqueous solution of CTAB at the concentration of 2.0 mmol L-1. The polymerization of EGDMA was carried out in the presence of the HRP-covered Si wafers or free HRP under additions of aliquots of diluted hydrogen peroxide, H2O2, solution (0,003%), in intervals of 5 min each (see Table 1). The medium was purged with N2 during H2O2 addition, at (24 ( 1) °C. The polymerization was carried out at (24 ( 1) °C, under mechanical stirring (500 rpm) in the pH range of 5 to 6 during 1 h. After that the system was dialyzed (dialysis tubing cellulose 12,400 MW, Sigma-Aldrich, USA) against water with daily changes until the conductivity of dialysis water reached 5 µScm-1. In this process no buffer was used. The dialyzed dispersions presented pH in the range of 5.5 to 6.0. Syntheses were performed for each system in triplicate with the same HRP covered substrate without desorption of HRP from the substrate. After polymerization HRP covered Si wafers were rinsed with abundant distilled water in order to remove impurities, dried under a stream of N2 and characterized. The stability of adsorbed HRP layer was observed by means of ellipsometry and AFM. Control experiments were performed using free HRP in solution under the same conditions and reactants amounts. Particle Characterization. Solid content and the conversion of monomer into polymer were determined by gravimetry measurements. Fourier Transform infrared vibrational spectra (FTIR) were obtained in a Bomen MB100 equipment for the dried latex PEGDMA particles, pure CTAB and pure HRP. SEM images of dried dispersions were obtained in a Jeol JSM-6460LV equipment. (26) Adamson A.; “Physical Chemistry of Surfaces”. John Wiley & Sons, New York, 1982. (27) Kadnikova, E. N.; Kostic, N. M. J. Molecul. Catalysis B: Enzymatic 2002, 18, 39. (28) Childs, R. E. Bardsley, W. G. Biochem. J. 1975, 145, 93. (29) Gilbert, R. Emulsion Polymerization: A Mechanistic Approach; Academic Press: London, 1995. (30) Almeida, A. T.; Salvadori, M. C.; Petri, D. F. S. Langmuir 2002, 18, 6914. (31) Pancera, S. M.; Gliemann, H.; Schimmel, Th.; Petri, D. F. S. Langmuir 2002, 18, 3517.
Langmuir, Vol. 23, No. 4, 2007 1983
Figure 2. Adsorption kinetics of HRP (c ) 0.001 mg mL-1) onto a Si wafer at (24 ( 1) °C.
Results and Discussion Adsorption Behavior of HRP on Si Wafers. The adsorption kinetics of HRP on silicon wafers in the dilute regime (cHRP ) 0.001 mg mL-1) is presented in Figure 2. The thickness of adsorbed HRP, dHRP, increased linearly but very slowly compared to that for other enzymes with similar molecular weights. For instance, considering a comparable enzyme concentration, the adsorption equilibrium for enolase,30 creatine phosphokinase,31,32 or hexokinase33 onto Si wafers is achieved after 3 h of adsorption. After approximately 6 h of adsorption, dHRP reached a constant mean thickness of (1.1 ( 0.1) nm, which corresponded to the equilibrium condition. At this plateau, the adsorbed amount Γ was 1.5 mg m-2. Topographic images were obtained by AFM at different adsorption times. After 1 h of adsorption, the mean dHRP value amounted to (0.35 ( 0.05) nm. The corresponding topographic image (Figure 3a) shows that the substrate is partially covered with small entities. Upon increasing the adsorption time to 3 h, the dHRP value increased to (0.63 ( 0.05) nm, and the entities seem to be more densely packed on the surface (Figure 3b). After 9 h of adsorption, the thickness at limiting adsorption amounted to (1.1 ( 0.1) nm and the substrate is almost completely covered by HRP entities, but one can still observe some tiny spots of free substrate (Figure 3c). Interestingly, the mean roughness values (rms) did not vary significantly as a function of adsorption time. Considering the kinetic data in Figure 2, the adsorption time has been set to 9 h for all adsorption experiments in order to ensure adsorption equilibrium. Figure 4 shows the adsorption isotherm (solid diamonds) obtained for HRP on silicon wafers. It presents a continuous increase in the adsorbed amount Γ as a function of bulk concentration until a plateau value of (2.5 ( 0.2) mg m-2 is reached, which could be observed for HRP concentrations greater than 0.04 mg mL-1. Considering the Γ value of (2.5 ( 0.2) mg m-2, an HRP molecular weight of 44 000 g mol-1, Avogadro’s number (6.02 × 1023 molecules mol-1), the surface density of the HRP molecules deposited on silicon wafers can be estimated to be 3.4 × 1012 molecules cm-2, yielding an average area per HRP molecule of 2933 Å.2 This figure is in excellent agreement with 2800 Å2 determined for HRP on Si wafers by means of fluorescence measurements.18 The radius of gyration of the HRP monomer was calculated by dynamics simulations34 to be 2.65 nm, considering the (32) Pancera, S. M.; Gliemann, H.; Schimmel, H.; Petri, D. F. S. J. Phys. Chem. B 2006, 110, 2674. (33) Pancera, S. M.; Glemman, H.; Schimmel, Th.; Petri, D. F. S. Journal of Coloid and Interface Science 2006, 302, 417. (34) Laberge, M.; Huang, Q.; Schweitzer-Stenner, R.; Fidy, J. Biophys. J. 2003, 84, 2542.
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Figure 3. AFM images of adsorbed HRP (c ) 0.001 mg mL-1) on Si wafers after (a) 1, (b) 3, and (c) 9 h of adsorption, with the corresponding HRP roughness (rms) and layer thickness (dHRP) values. dHRP was determined by means of ellipsometry. Scan area size is 1 µm × 1 µm.
Figure 4. Adsorption isotherm obtained for HRP on Si wafers after a period of 9 h of adsorption at (24 ( 1)° C ((). Desorption isotherm obtained for HRP adsorbed on Si wafers after a period of 9 h of immersion in pure water at (24 ( 1)° C (4).
crystallographic dimensions of 4.0 nm × 6.7 nm × 11.7 nm (pdb code 1H58, www.rcsb.org/pdb).35 The values of dHRP determined by means of ellipsometry in the plateau region after 16 h of adsorption amounted to (1.8 ( 0.1) nm, indicating that the adsorbed layer of HRP does not correspond to a monolayer, so tiny spots on the Si wafers remain free, as observed in Figure 3c. Desorption experiments were performed by immersing HRPcovered Si wafers in the ellipsometric cell full of distilled water for 9 h. Changes in the thickness of the adsorbed HRP layer were monitored by means of ellipsometry. From the thickness of the remaining HRP film and eq 2, the remaining adsorbed amount was calculated as a function of HRP concentration, as presented (35) www.rcsb.org/pdb ID: 1H58, accessed in November 10, 2006.
Figure 5. Contact angle as a function of the adsorbed amount of HRP on a Si wafer.
in Figure 4 (open triangles). On average, 7% of immobilized HRP desorbed from silicon substrates when immersed in water, indicating the strong affinity of HRP for SiO2 surfaces. Similar behavior was observed when HRP-covered Si wafers were immersed in a monomer dispersion or in CTAB solution. Figure 5 shows the dependence of the advancing contact angle (θ) on the amount of adsorbed HRP (Γ). Upon increasing the mean Γ value from (0.7 ( 0.2) mg m-2 to (2.5 ( 0.3) mg m-2 (plateau value), the mean θ value increased from 42 to 52°. At very low Γ values, the uncovered substrate area is larger than at the Γ value corresponding to the plateau. Because Si wafers are very hydrophilic substrates (θ ) 5°), the increase in θ values with increasing Γ value is expected. The mean θ value of 52° found for Γ ) (2.5 ( 0.3) mg m-2 (plateau value) indicates that both HRP hydrophilic and hydrophobic residues are oriented with respect to the air, as well as the absence of any preferential
Horseradish Peroxidase as a Reusable Catalyst
Langmuir, Vol. 23, No. 4, 2007 1985 Table 2. Solid Content and Conversion of Monomer into Polymer Determined after 160 min of Emulsion Polymerization of EGDMA Catalyzed Either by Free HRP (Formulations 3 and 5) or Immobilized HRP (Formulations 1, 2, and 4) formulation
solid content (mg mL-1)
conversion (%)
1 2 3 4 5
0.55 ( 0.05 0.94 ( 0.06 1.1 ( 0.4 1.7 ( 0.8 0.93 ( 0.03
2.6 ( 0.2 4.6 ( 0.2 5(2 1.8 ( 0.9 0.98 ( 0.03
orientation. According to pdb data,35 HRP is a glycoprotein composed of 308 amino acid residues (60% hydrophilic residues and 40% hydrophobic residues). Most hydrophobic amino acids surround the active site, where the heme prosthetic group is located. The correlation between enzymatic activity and the presence of many hydrophilic residues on the surface of HRPcovered surfaces is discussed below. Activity of Adsorbed HRP on Silicon Wafers. When immobilized enzymes retain their activity, they can be very useful because they can be transported and immersed in the desired reaction, they can be reused, and sometimes they bring about a shift in the optimal pH.15,32,36,37 The enzymatic activity of immobilized HRP on Si wafers (Γ ) 2.5 ( 0.2 mg m-2) was investigated in the presence of H2O2 by means of (i) oxidation of ABTS into the radical cation ABTS•+ 27 and (ii) emulsion polymerization of ethylene glycol dimethacrylate (EGDMA) in CTAB solution. Figure 6 shows the formation of radical cation ABTS•+ in the presence of H2O2 as a function of time catalyzed either by free HRP (solid square) or immobilized HRP (open triangles). The green color characteristic of this reaction was observed under both conditions (Supporting Information, suppl-mater-2). The initial stage, from 0 to 200 s, shows that the formation of ABTS•+ is much faster when HRP molecules are free in solution than when HRP molecules are immobilized, as evidenced by the initial slopes. The access to the substrate is more difficult when HRP molecules are immobilized than when they are free in solution; in other words, the difference in the initial rate might be based on substrate diffusion within the heterogeneous catalyst (immobilized HRP). After 200 s, the slopes of both curves are very similar, suggesting that the initial stage is the rate-determining step. After 10 min of reaction, one notices that when HRP molecules are free in solution the oxidation is faster and the amount of product formed (19 µmmol L-1 ABTS•+) is about 50% higher than that observed when HRP molecules are immobilized (12 µmmol L-1 ABTS•+). The enzymatic activity of immobilized HRP molecules is lower than that observed for free HRP molecules because access to the substrate is more difficult when they are immobilized and HRP might undergo conformational changes. Emulsion polymerization of ethylene glycol dimethacrylate (EGDMA) was carried out in CTAB solution in the presence of H2O2 and catalyzed either by free HRP (mHRP ) 0.175 µg) or immobilized HRP (mHRP ≈ 0.160 µg). (See Table 1 for formulation details.) Table 2 shows the conversion of monomer
into polymer, which corresponds to the polymer yield, and the solid content determined for the different formulations. HRPcovered Si wafers have been applied for formulations 1,2, and 4. Comparing formulations 1 and 2, one notices that increasing the H2O2 concentration 10 times favored the polymerization yield considerably. The increase in the amount of H2O2 added to the reaction increased the formation of compound I (Figure 1), which is the species responsible for the radical polymerization. However, the increase in monomer volume from 200 µL (formulation 2) to 900 µL (formulation 4) led to low conversion. The polymerization of EGDMA in the presence of free HRP (formulations 3 and 5) presented a similar dependence of conversion on monomer volume. Moreover, for the same amounts of H2O2 and EGDMA the polymerization yields obtained in the presence of free HRP or immobilized HRP were comparable. Considering the general mechanism for emulsion polymerization,29 radical intermediates (compound I) react with EGMDA monomer in the aqueous phase, bringing about the formation of the EGDMA monomer radical, which can react with neighbor monomers, forming oligoradicals. Oligoradicals formed in the aqueous phase migrate to the CTAB micelle core, which is swollen by monomers, characterizing particle nucleation, because the monomer concentration in the particles is continuously replenished by monomer diffusion from the monomer droplets to the particles. Diffusion and subsequent polymerization increase the diameter of particles. Finally, all monomer droplets have vanished, and only monomer in the particles can still polymerize. EGDMA is a bifunctional monomer often used as a cross-linking agent. Upon increasing the monomer volume (formulations 4 and 5), the probability of a termination reaction among oligoradicals still in the aqueous phase increases, reducing the conversion. The low yield values observed in Table 2 might be explained by the low HRP concentration used in the present study (0.0175 µg mL-1). Polymerization reactions catalyzed by free HRP often apply large HRP concentrations. Polymerizations of sugar-based phenols used 0.16 mg mL-1 38 or 0.40 mg mL-1 14 HRP in the reactant mixture, obtaining conversions between 10 and 20%. A yield of ∼60% was observed for the emulsion polymerization of aniline in the presence of HRP at a concentration of 0.06 mgmL-1.9,11 HRP-catalyzed polymerization (HRP at 70-90 mg mL-1) of methyl methacrylate (MMA) in a water-mixed organic solvent led to highly stereoregular chains and product yields ranging from 2 to 85%.7 The polymerization of acrylamide and sodium acrylate in the presence of CTAB or bis(2-ethylhexyl)sodium sulfosuccinate (AOT) using HRP at a concentration of 40 mg mL-1 led to 94% polymer yield.8 Nanoparticles (30-50 nm in diameter) of polystyrene were synthesized using 20 mg mL-1 HRP as a catalyst, reaching a mean conversion of ∼90%.13 In that case, 2,4-pentanedione was added to the reaction to act as the substrate for HRP, generating primary radicals in the presence of hydrogen peroxide. Although acetylacetone, a
(36) Quimqampoix, H. Biochemie 1987, 69, 753. (37) Frenkel-Mullerad, H.; Avnir, D. J. Amer. Chem. Soc. 2005, 127, 8077.
(38) Akita, M.; Tsutsumi, D.; Kobayashi, M.; Kise, H. Biosci. Biotechnol. Biochem. 2001, 65, 1581.
Figure 6. Radical cation ABTS•+ formation in the presence of H2O2 as a function of time. The reaction is catalyzed by free HRP (9) or immobilized HRP (4).
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Figure 7. AFM images and rms and mean thickness (dHRP) values obtained for the same HRP-covered Si wafer (a) before and (b) after being used in three different polymerizations of EGDMA. dHRP was determined by means of ellipsometry. Scan areas size is 1 µm × 1 µm.
β-diketone compound, has been reported to be a key compound in free HRP-mediated polymerization,7,8,13 in this study the addition of acetylacetone did not improve the conversion observed in Table 2. It is possible that when HRP is immobilized the access for 2,4-pentanedione is difficult so that the conversion of monomer into polymer is not improved. PEGDMA polymerized in the presence of free HRP and HRPcovered Si wafers was characterized by means of FTIR. PEGDMA was polymerized in bulk using potassium persulfate as an initiator and pure HRP as a control. All spectra are available as Supporting Information (suppl-mater-3 and suppl-mater-4). The characteristic PEGDMA bands are present in the spectra obtained for PEGDMA polymerized in the presence of free HRP and HRP-covered Si wafers. Because the characteristic PEGDMA bands superimpose the characteristic HRP bands, FTIR spectra do not allow us to infer about PEGDMA contamination by HRP. SEM images of dried dispersions evidenced that PEGDMA formed irregular particles with size varying from 300 to 15 000 nm. The images are available as Supporting Information (suppl-mater-5 and supplmater-6). As control experiments, the mean thickness values of adsorbed HRP layer were determined by ellipsometry in the air before and after three polymerization reactions performed with the same HRP-covered wafer. A decrease of ∼15% in the dHRP values was observed (Figure 7). Comparing AFM images obtained for HRPcovered Si wafers before (Figure 7a) and after three polymerizations (Figure 7b), one notices in the latter a larger area of the bare Si wafer (small holes) exposed, corroborating with the thickness decrease of 15%. Nevertheless, the mean roughness values remained unchanged. Surface-enhanced laser desorption ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) data indicated that upon oxidation (formation of compound I in Figure 1) HRP desorbs from the silica surface and readsorbs upon returning to the native state.39 The stability, activity, and reusability of HRP-covered Si wafers used in the polymerization of EGDMA were investigated. One of the HRP-covered wafers used in formulation 2 was chosen because it led to the highest product yield. The same HRPcovered Si wafer has been used consecutively in three polymerization reactions. After each reaction, the HRP-covered Si (39) Kirkor, E. S.; Scheeline, A. J. Phys. Chem. B 2001, 105, 6278.
Figure 8. Mean conversion determined after 160 min of polymerization of EGDMA using formulations 2 (green column) and 3 (red column). HRP-covered Si wafers (green column) were used just after preparation and after storing for 3 or 6 days.
wafer was rinsed, dried, characterized, and stored for 3 days inside a snap glass at room temperature before use in the next polymerization. The mean conversion obtained with freshly prepared HRP-covered Si wafers (4.7 ( 0.4%) was comparable to that observed for freshly prepared free HRP in solution (4.9 ( 0.5%), as shown in Figure 8. After storing HRP-covered Si wafers for 3 or 6 days, the conversions amounted to 4.1 ( 0.3 and 3.8 ( 0.4%, respectively. These findings show that the loss of activity of immobilized HRP after 3 and 6 days of storage was small, 12 and 19%, respectively. From a practical and economical point of view, immobilizing HRP molecules onto Si wafers seems to provide the unique possibility of storing and reusing HRP-covered Si wafers. The adsorption of HRP onto polystyrene (PS), poly(styrene/ acrolein) (P(SA)), and polyacrolein (PA) lattices, which are hydrophobic, has been previously studied .40 HRP was not suitable for the covalent immobilization on P(SA) latex, and lost its activity after adsorption onto PS latex. The present study shows that immobilized HRP molecules on Si wafers, which are very hydrophilic surfaces, retained their enzymatic activity. Moreover, (40) Basinska, E.; Slomkowski, S. Colloid & Polym. Sci. 1995, 273, 431.
Horseradish Peroxidase as a Reusable Catalyst
contact angle measurements performed for HRP-covered Si wafers (Figure 5) indicated the orientation of both hydrophilic and hydrophobic residues with respect to the air. Concerning the forces that keep the conformation of immobilized HRP on Si wafers so that the enzymatic activity is retained long term and after continuous use, one should consider the hydration forces.41,42 There are many reports that correlate the hydration layers around the enzyme upon adsorbing with its enzymatic activity. Kim and Cremer43 showed by means of sum frequency generation that the water structure at the silica/water interface changes upon adsorbing bovine serum albumin (BSA) as a function of pH. At pH 5.6, the “icelike” structure was slightly decreased, and the waterlike structure showed a substantial reduction in intensity.43 By lowering the pH to 3.8, the intensity of the ice-like mode increased, and that of the water-like mode decreased. The activity of phosphatases was preserved under extreme pH when they were entrapped in sol-gel matrices. The hydration level inside the pores seems to cause the enzyme preservation.44 Similarly, the activity behavior of creatine phosphokinase (CPK) immobilized onto Si wafers under different pH conditions showed that under alkaline conditions the contribution of strong hydration forces might weaken the CPK unfolding upon electrostatically driven adsorption.32 Hydration forces are due to not only the structure of the water layers but also the osmotic effect, which can be expressed as the changes in the number of water molecules associated with the single molecule undergoing conformational changes.45 The preservation of enzymatic activity of HRP (41) Leikin, S.; Parsegian, V. A.; Rau, D. C.; Rand, R. P. Annu. ReV. Phys. Chem. 1993, 44, 369. (42) Marcelja, S.; Israelachvili, J. N.; Wennerstro¨nm, H. Nature 1997, 385, 689. (43) Kim, J.; Cremer, P. S. Chem. Phys. Chem. 2001, 8-9, 543. (44) Frenkel-Mullerad, H.; Avnir, D. J. Am. Chem. Soc. 2005, 127, 8077. (45) Rand, R. P. Phil. Trans. R. Soc. Lond. B 2004, 359, 1277.
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adsorbed onto Si wafers might be associated with the interface hydration, which avoids HRP denaturation after use or upon storing.
Conclusions Ellipsometry and AFM evidenced the formation of a smooth HRP layer on Si wafers. Contact angle measurements on HRPcovered Si wafers indicated no preferential orientation of HRP hydrophilic or hydrophobic residues with respect to the air. The enzymatic activity of free HRP in solution was higher than that determined for immobilized HRP. Upon adsorbing, HRP might undergo conformational changes, which bring about a reduction in catalytic activity. The reusability of HRP-covered Si wafers as catalysts in the polymerization of EGDMA was verified. From a practical point of view, HRP-covered Si wafers are advantageous catalysts for polymerization because they can be easily prepared, stored, transported, reused, and immersed in the reactant mixture without losing catalytic properties. Acknowledgment. We acknowledge FAPESP, CNPq, and Laborato´rio de Filmes Finos do IFUSP (Proc. FAPESP no. 00/ 08231-1) for use of the SEM facility. Supporting Information Available: Typical spectrum obtained for ABTS•+. Setup used to verify qualitatively the formation of radical cation ABTS•+ in the presence of H2O2 catalyzed either by free or immobilized HRP. Typical FTIR spectrum obtained for HRP. FTIR transmittance spectra obtained for PEGDMA polymerized in the presence of HRP-covered Si wafers in the presence of free HRP and in the bulk. SEM images of dried dispersions. Schematic representation of the oxidation reaction of ABTS to radical cation ABTS•+. This material is available free of charge via the Internet at http://pubs.acs.org. LA061884O
