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Noncovalent Imprinting in the Shell of Core-Shell Nanoparticles Natalia Pe´rez-Moral and Andrew G. Mayes* School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, United Kingdom Received September 8, 2003. In Final Form: February 6, 2004 Propranolol was imprinted using noncovalent interactions in the shell of core-shell nanoparticles prepared by aqueous emulsion polymerization in the presence and absence of toluene. The imprinted particles were characterized, and their capacity to rebind propranolol from both organic and aqueous media was analyzed. Results showed that the amount of template incorporated into the polymer and the presence of toluene as a “porogenic” agent influenced the ability of the nanoparticles obtained to rebind propranolol. The presence of toluene during imprinting increased rebinding by about 2-fold in buffer and by 3-fold in toluene, compared with similar materials made in the absence of toluene during imprinting. It also influenced the final surface area of the particles. Binding site affinity, assessed by radioligand displacement, was measured as IC50 values of about 1-10 µM. This compares with about 3 µM for bulk polymer made with a similar composition. Finally, to demonstrate the advantages of structured particles for analytical applications a new property, fluorescence, was incorporated into the core of the particles without interfering with the imprinted shell and its ability to rebind propranolol.
Introduction Molecular imprinting is by now established as one of the most promising methods to construct artificial receptors that can achieve specific recognition at a molecular level under practically relevant conditions. The field has been extensively reviewed over the past few years.1-6 Traditionally, due to the simplicity and convenience of the method, molecularly imprinted polymers (MIPS) have been synthesized in bulk, and their rebinding capacity has been analyzed in the same organic solvent in which the synthesis was carried out. In recent years, however, the need to extend their uses and applications has led to a number of groups reporting new methods for the synthesis of imprinted polymers, where the morphology of the polymers can be controlled and particles can be produced in different formats and environments. The interest in controlling the morphology of the polymers resides in the importance of controlling the surface area and the improvement in the diffusion of template and analyte molecules from and into the polymer. Some of the new methodologies developed in recent years to achieve imprinting of organic molecules in polymeric particles include the synthesis of particles in a fluorocarbon solvent by suspension polymerization7 or in acetonitrile by precipitation polymerization.8 In aqueous solvents, imprinted particles have been described using a two-step swelling method,9 emulsion seeded polymerization10-12 and microemulsion polymerization.13 Along with control of particle size and a reduction in size polydispersity, when compared with ground and sieved bulk polymer, these (1) Andersson, L. I. J. Chromatogr., B 2000, 745, 3-13. (2) Alexander, C.; Davidson, L.; Hayes, W. Tetrahedron 2003, 59, 2025-2057. (3) Hentze, H. P.; Antonietti, M. Curr. Opin. Solid State Mater. Sci. 2001, 5, 343-353. (4) Sellergren, E. B. Molecularly Imprinted Polymers: Man-Made Mimics of Antibodies and their Applications in Analytical Chemistry; Elsevier Science B.V.: Amsterdam, 2001; Vol. 23. (5) Haupt, K. Analyst 2001, 126, 747-756. (6) Haupt, K. Chem. Commun. 2003, 2, 171-178. (7) Mayes, A.; Mosbach, K. Anal. Chem. 1996, 68, 3769-3774. (8) Ye, L.; Cormack, P. A. G.; Mosbach, K. Anal. Commun. 1999, 36, 35-38.
processes also offer other benefits. These include the reduction of wasted polymer (important if expensive templates or monomers are used) and a much better control of the process during polymerization with respect to temperature control and reproducibility. These factors become highly significant as processes are scaled up. The controlled synthesis of imprinted polymers in the form of spherical particles shares all these advantages. The synthesis of polymeric structured nanoparticles in aqueous environments was first combined with imprinting by investigating the imprinting of cholesterol in the shell of two-stage core-shell particles. Interactions based on a combination of covalent-noncovalent bonds or hydrophobic forces for surface imprinting were employed.10,11 In our group, we also proved that the classical noncovalent approach is also successful in producing molecularly imprinted core-shell particles.14 Recently, this has been confirmed by another group, using the same core-shell approach but utilizing different templates and assay methods.15 In this paper, we present a more detailed study of imprinting in core-shell particles. We have imprinted propranolol in the shell of nanoparticles produced by aqueous emulsion polymerization using noncovalent interactions. The particles were subsequently characterized, and the effects of some parameters such as the presence of solvent during shell synthesis and the amount of template were assessed by measuring template reuptake by the polymers. The specific rebinding of ligands from aqueous and organic solutions was analyzed by (9) Hosoya, K.; Yoshikazo, K.; Tanaka, N.; Kimata, K.; Araki, T.; Haginaka, J. Chem. Lett. 1994, 1437-1438. (10) Perez, N.; Whitcombe, M. J.; Vulfson, E. N. J. Appl. Polym. Sci. 2000, 77, 1851-1859. (11) Pe´rez, N.; Whitcombe, M. J.; Vulfson, E. N. Macromolecules 2001, 34, 830-836. (12) Carter, S. R.; Rimmer, S. Adv. Mater. 2002, 14, 6687-6670. (13) Vaihinger, D.; Landfester, K.; Krauter, I.; Brunner, H.; Tovar, G. E. M. Macromol. Chem. Phys. 2002, 203, 1965-1973. (14) Pe´rez-Moral, N.; Mayes, A. G. In Molecularly Imprinted Materials: Sensors and other Devices; Shea, K. Y., Roberts, M. J., Eds.; MRS Symposium Proceedings, Vol. 723; Materials Research Society: Warrendale, PA, 2002; pp 61-66. (15) Carter, S.; Lu, S. Y.; Rimmer, S. Supramol. Chem. 2003, 15, 213-220.
10.1021/la0356755 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/24/2004
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radioligand binding assay, and the results obtained were related to the morphology of the polymers. Also, a new property, fluorescence, has been incorporated into the core of the imprinted particles without interfering with the imprinted shell and its capacity to recognize and rebind the targeted ligand. Such particles have considerable potential in a variety of assay technologies. Experimental Section General. To remove the inhibitor prior to polymerization, methacrylic acid (MAA) was distilled under a vacuum, while methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EDMA) were washed with 1 M aqueous sodium hydroxide, dried over MgSO4, and stored with 3 Å molecular sieves at 4 °C until required. D,L-Propranolol hydrochloride was transformed to the free base by dissolution in water, addition of sodium hydroxide, and extraction into dichloromethane. The solvent was removed, and the propranolol was dried to constant weight under a high vacuum before use. Sodium dodecyl sulfate (SDS), ammonium peroxodisulfate (APS), and other solvents and reagents were used as received. D,L-3H-propranolol (specific activity of 29 Ci/mmol, 1 µCi/µL in ethanol) was obtained from Sigma. Scintillation counting was performed in a Wallac 1409 DSA β-radiation counter. Scintillation cocktail Ecoscint A (National Diagnostics) was used for aqueous samples. Organic samples were counted in toluene containing 2,5 diphenyloxazole (3 g/L) and 1,4-bis(5-phenyloxazol-2-yl)-benzene (0.2 g/L), both obtained from Aldrich. Particles were characterized by transmission electron microscopy (TEM) using a JEOL JEM 200E X transmission electron microscope. Samples were prepared by drying a drop of a solution of particles in water with SDS over a grid coated by a carbon film. Surface area was measured by the BrunauerEmmett-Teller (BET) method. Fluorescence spectra were measured on a JobinYvon-Spex FluoroMax-2 spectrofluorometer. Zeta potential measurements were obtained in a Malvern Zetasizer NanoZS. Synthesis of Particles. In the first stage, seed particles were synthesized to create the core of the final particles. The seed was prepared using a standard batch emulsion polymerization in a 1 L three-necked jacketed reactor connected to a water bath to control the temperature. The system was equipped with a condenser, a mechanical overhead stirrer with a glass four-blade impeller, and a gas inlet to maintain an inert argon atmosphere. A solution of NaHCO3 (0.95 g, 11.3 mmol) in distilled water (520 g) with SDS (0.913 g, 3.16 mmol) was added into the reactor and purged with argon to remove oxygen under gentle stirring, while increasing the temperature to 90 °C. Once the temperature was reached, the monomer mixture (MMA, 35 g, 0.35 mol; EDMA, 3.64 g, 0.018 mol; and for the fluorescent seed, fluorescent monomer 9-anthrylmethyl methacrylate, 2.41 g, 8.72 mmol) was introduced and the stirring speed was increased to 600 rpm. After 1 min, the initiator, APS (0.18 g, 0.0036 mol, dissolved in 1 mL of water), was added to initiate the polymerization. The temperature was maintained at 90 °C for 24 h to ensure total decomposition of the initiator. The final latex was filtered through a fine nylon mesh and its solid content (7%) was calculated before being used in the next step. Core-shell particles were synthesized using a 500 mL reactor similar to that described above. First, a solution containing water (88.5 g) and SDS (0.5 g, 1.73 mmol) was added into the reactor and purged with argon under gentle stirring while the temperature was raised to 70 °C. A solution of monomers (EDMA, 10.1 g, 50.9 mmol; MAA, 1.1 g, 12.7 mmol), template, and seed (51.4 g) that had been previously mixed for 20 min was also charged into the reactor followed by an aqueous solution of 0.112 g (0.84 mmol) of APS. The stirring speed was increased to 200 rpm, and the reaction was allowed to proceed for 6 h before cooling to room temperature. The yield in the preparation of the nanoparticles was calculated from the measurement of the final solid content, and it was found to be around 98-100% in all the cases. The resultant polymer particles were washed in an ultrafiltration cell with a regenerated cellulose membrane (Amicon YM10) to remove any surfactant or electrolyte adsorbed on the particles by passing ultrapure water through the cell until the conductivity of the effluent equaled that of the pure water. The
Pe´ rez-Moral and Mayes Table 1. Composition of Polymeric Shell (Second Stage of Polymerization) in Core-Shell Particlesa % prop/M tol/M (mol) (vol) Dp (nm) C1 I1.1 I1.2 I1.3 I1.4 I1.5 C2 I2.1 I2.2 I2.3 I2.4
0.5 1 2 4 6 0.5 1 2 4
1/1 1/1 1/1 1/1 1/1 1/1
70 37 55 70 66 72 60 60 59 52 60
surface expected surface area (m2/g) area (m2/g) Z (mV) 103 232 218 96 66 68 111 196 155 93 44
86 162 109 86 92 83 100 100 102 115 100
-19.69 -16.13 -14.15 -9.21 -11.34 -7.23 -20.42 -17.92 -17.69 -17.92 -18.5
a M ) EDMA + MAA; prop ) propranolol; tol ) toluene; Dp ) diameter of the particle; Z) zeta potential. The seed diameter was 53 nm.
template was then removed by washes with 1 M ammonium acetate dissolved in a mixture of ethanol/acetic acid/water (40/ 25/35 v/v), followed by washes in acetic acid/ethanol (1/3 v/v) and methanol.16 Radioligand Binding Assay. Particles (0.25 mg) were mixed with 1 mL of a solution of 3H-propranolol in the solvent mixture (0.07 µL 3H-propranolol mL-1) and incubated overnight at room temperature. The suspension was then centrifuged at 12 000 rpm for 5 min, and 0.5 mL of the supernatant was mixed with 3 mL of scintillation liquid. The radioactivity was measured by liquid scintillation counting. The displacement of radiolabeled propranolol with unlabeled template was performed in Eppendorf tubes. To each tube was added 200 µL of a solution containing 0.25 mg of polymer, 300 µL of solvent containing the required amount of unlabeled propranolol, 200 µL of solvent, and 200 µL of solvent containing 0.07 µL 3H-propranolol mL-1. After incubating the tubes overnight at room temperature, the tubes were centrifuged at 3500 rpm for 5 min and 0.5 mL of the resulting supernatants were mixed with 3 mL of the scintillation liquid. The unbound radioactivity was quantitated by scintillation counting. Fluorescence Measurements. The fluorescent monomer anthrylmethyl methacrylate was synthesized according to the published procedure by Holden et al.17 Fluorescence spectra were measured in a quartz cuvette with emission slits set at 5 nm. An excitation wavelength of 340 nm was used to obtain the fluorescence emission spectra of all polymer particles, which were resuspended in THF. Zeta Potential Measurements. The particles were resuspended in an aqueous buffer (25 mM sodium citrate with 0.5% acetic acid and 2% ethanol, pH 4.2) and transferred to a disposable folded capillary cell (Malvern Instruments). The zeta potential was measured using the supplied software.
Results and Discussion A structured core-shell arrangement is usually produced by a two-stage emulsion polymerization. In this procedure, a second layer of imprinted polymer is polymerized around the initial seed or core of the particle. Conditions for the second step in the polymerization procedure were carefully chosen in order to avoid undesired structures or secondary nucleation of new particles when the mixture containing the cross-linker monomer and the template are added. Although initial reports indicated a possible unexpected effect of the core in the performance of the core-shell particles,11 recent results have shown that the composition of the core does not influence the performance of the imprinted particles.14 The formation of a core-shell structure is locked and kinetically favored when a small amount of cross-linker (16) Andersson, L. Anal. Chem. 1996, 68, 111-117. (17) Holden, D. A.; Guillet, J. E. Macromolecules 1980, 13, 9-295.
Noncovalent Imprinting in Core-Shell Nanoparticles
Figure 1. TEM micrographs of control and imprinted coreshell monodisperse particles: (a) nonimprinted polymer C1 and (b) imprinted polymer I1.4. The scale bar indicates 200 nm.
is incorporated in the composition of the seed. Therefore, a monodisperse seed containing 5% w/w EDMA and 95% w/w MMA was synthesized and characterized by TEM. The particles produced were 53 nm in diameter and monodisperse. In the second stage of the polymerization, some of the main factors affecting the morphology of the particles were assessed. The influence of porogen (toluene) and amount of template (propranolol) was studied by preparing coreshell particles imprinted with an increased amount of template from 0.5 to 6% with and without porogen. Control particles were also prepared in the absence of template in order to analyze the nonspecific rebinding. Table 1 summarizes the composition of the shells and the size and surface areas of the resulting core-shell nanoparticles. In most of the cases, the diameters of the core-shell particles obtained by measurement of TEM images were similar to the values expected taking into account the size of the particles used for the core and the amount of monomer added in the second stage. In a few cases, however, secondary nucleation occurred with new particles being nucleated during the polymerization of the shell, producing smaller particles with an average diameter below the expected value. This may also account, at least in part, for the increase in the final surface area (see Table 1 and Figure 1). Attempts to synthesize polymers containing more than 6% (mol) of template to the total amount of monomer under the conditions set for the polymerizations resulted in latices that were nonstable. Presumably an adequate adjustment of some of the parameters such as feeding method, percentage of surfactant, or final solid content should allow an increase in the amount of template incorporated to the system. Such factors are known to affect latex stability in other core-shell systems.18 In previous sections, the solvent included during the imprinting stage has been termed “porogenic”, since this is its role in conventional imprinting polymerizations. It creates a pore structure due to the insolubility of the growing polymer chains and networks in the solvent, leading to precipitation of polymer “nanonodules” (typically some tens of nanometers in size). Physical aggregation and chemical linkage of these nodules leads to the final material with a network of permanent pores. The role of the solvent porogen within the imprinted shells of these core-shell nanoparticles is clearly somewhat different. Since the entire imprinted shell is only about 10 nm thick, there should be little chance for a permanent pore structure to form by precipitation in the growing (18) Lee, S.; Rudin, A. In Polymer Latexes; ACS Symposium Series, Vol. 492; American Chemical Society: Washington, DC, 1992; p 234.
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shell since the critical size would probably not be reached. The solvent could, however, have two other roles. First, it could plasticize the surface of the polymer seed. Since the seed is cross-linked in this case, it will not dissolve or swell substantially, but the surface structure could begin to interact with the forming shell. The fact that particle formation and performance of the imprinted particles do not appear to be influenced by the nature of the core14 suggests that this is probably a minor effect. A much more significant effect is probably produced by the solvation of the monomers and templates during imprinting. The solvent will affect the dielectric of the monomer mixture and hence the relative strengths of the various different intermolecular interactions taking place. It will also ensure that the cross-linked imprinted network is formed in a solvent-swollen state, making the structure more extended and flexible and hence facilitating diffusion of template through the polymer structure. This would give better access to binding sites deeper in the shell. Particle measurements made in the dry state by TEM indicate that the shells made in the presence of toluene are somewhat thicker than those made with no solvent present, even though the amount of monomer supplied during the shell synthesis was the same in either case. This indicates that the polymer is thus more extended in structure even in the dry state. This type of measurement gives no indication as to the relative swellability of the shell, however, and this will be investigated further in future work. There also appears to be a correlation between the amount of template used during shell imprinting and the surface area of the particles. Use of more template appears to lead to particles with lower final surface areas, both in the presence and absence of solvent. At this stage, we can only speculate on the reason for this. It is possible that the presence of the template affects the distribution of the carboxylic acid groups in the growing polymer network (this, after all, is how the imprinted receptor sites should be created). This, in turn, would affect the solubility of the growing polymer network in the monomer or monomer/ solvent mixture and might lead to different degrees of polymer precipitation. As stated previously, this is unlikely to produce much permanent pore structure but could affect factors such as the surface roughness of the final shell, giving surface areas greater than those expected from geometric calculations on smooth spheres of equivalent size. As the amount of template increases, roughness is reduced and the particle area tends toward that expected from geometric calculations and, at the highest template loadings, even falls below the expected geometric area. This may be due to a degree of size heterogeneity. The zeta potential measurements give an indication of the surface charge on the particles. Under the conditions of measurement, this is related to the number of surface accessible carboxylic acid groups. It might be expected that this would mirror the surface area measurements, since the amount of MAA in the system was the same in all cases. In reality, the behavior was more complex than this. In the presence of toluene, the zeta potential dropped steadily as the amount of template increased, whereas the surface area first increased and then decreased. This implies that the carboxylic acid distribution is indeed affected by the presence of the template, as discussed above. This is clearly not the case for the particles imprinted in the absence of toluene. In this series, the zeta potential remains remarkably constant as the amount of template increases, even though the surface area first increases and then decreases. In this case, the increasing amount of template appears to be localizing relatively
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Table 2. Specific Rebinding of Propranolol to 0.25 mg of Polymera
ID code (C1) I1.1 I1.2 I1.3 I1.4 I1.5 (C2) I2.1 I2.2 I2.3 I2.4
toluene
% prop/M
1/1 1/1 1/1 1/1 1/1 1/1
0.5 1 2 4 6 0.5 1 2 4
% specific rebinding in buffer
% specific rebinding in toluene*
(16) 2 15 18 19 24 (11) 3 6 10 14
(2) 2 9 15 27 31 (16) 3 9 8 8
a Buffer ) Na citrate 25 mM + 0.5% acetic acid + 2% ethanol, pH ) 4.2; toluene* ) toluene + 0.5% acetic acid. Values are means of 4 replicates; the standard deviation was typically about 3.5% in buffer and 4.5% in toluene.
more carboxy groups at the accessible surface. This is consistent with the increasing number of accessible binding sites (see rebinding data) but does not explain the surface area behavior. Perhaps, in the absence of solvent, the balance of MAA and EDMA incorporation into growing polymer chains has a different effect on the solubility of the growing polymer chains and hence precipitation and generation of surface roughness. The capacity to rebind propranolol was assessed from organic and aqueous systems by radioligand binding assay (see Table 2). As expected, particles with the shell imprinted with a higher amount of template showed a higher capacity of uptake of propranolol, both in organic and aqueous environments. This effect was more significant when the accessibility to the sites was facilitated by the effect of the solvent present during polymerization. Rebinding was calculated by monitoring the amount of radioactive ligand (3H-propranolol) left in the supernatant after uptake by the imprinted polymer occurred. Specific rebinding was defined as the percentage of propranolol rebound to the imprinted polymer after subtraction of the amount rebound nonspecifically to the nonimprinted control particles (shown in parentheses in Table 2). Rebinding experiments were performed both in organic and aqueous solutions. Uptake from a solution of propranolol in toluene containing 0.5% acetic acid was higher than when the analysis was performed in an aqueous buffer (sodium citrate 25 mM with 0.5% acetic acid and 2% ethanol, pH 4.2). The imprinting effect was significant in both cases, although it is lower than that observed for imprinting of propranolol with the same monomers and very similar monomer ratios using other established polymerization methods.19 This is likely as a result of the imprinting polymerization taking place very close to an aqueous interface. Under these conditions, it might be expected that hydration of both template and functional monomers would disturb the template-monomer interaction equilibrium and hence reduce the efficiency of imprinting. In both aqueous and organic rebinding media, particles synthesized with a toluene-solvated shell exhibited a higher uptake of propranolol than when the shell was made without added solvent. It is assumed that in the latter case the only imprinted sites accessible are located at or very near the surface of the particle, hence the reduced level of template rebinding. Variable levels of nonspecific binding were observed in the control samples, with and without solvated shells, (19) Pe´rez-Moral, N.; Mayes, A. G. Anal. Chim. Acta 2004, 504, 1521.
Figure 2. Displacement of propranolol from the shell of coreshell particles imprinted with 0 (reference polymer), 2, and 4% of template in the presence of toluene. Displacement was measured in toluene containing 0.5% acetic acid: (9) C1 (reference polymer); (2) I1.3 (imprinted with 2% propranolol); (b) I1.4 (imprinted with 4% propranolol). The continuous line represents the fit of each data set to a four-parameter logistic function (ref 20). Table 3. Specific Rebinding of Propranolol to Particles Prepared with Fluorescent Cores and Imprinted with 4% Propranolol/M % specific rebinding in buffer
(control) imprinted
% specific rebinding in toluene*
with porogen
without porogen
with porogen
without porogen
(10) 22
(15) 17
(3) 22
(15) 6
a Buffer ) Na citrate 25 mM + 0.5% acetic acid + 2% ethanol, pH 4.2; toluene* ) toluene + 0.5% acetic acid. Values are means of 4 replicates; the standard deviation was typically about 3.5% in buffer and 4.5% in toluene.
under both the rebinding conditions tested. This again suggests that the conditions of synthesis and the presence of solvent may have a significant effect on the surface properties of the particles and the distribution of functional monomers in the imprinted polymer structure. This is currently being evaluated further using laser light scattering and zeta potential measurements. Figure 2 shows the results of a typical displacement experiment where 3H propranolol was displaced by unlabeled propranolol in polymers imprinted with 2% and 4% of template to monomer. IC50 values, calculated by fitting the obtained experimental data to a four-parameter logistic function,20 were 9.6 µM for the polymer imprinted with 2% template and 0.9 µM for the polymer imprinted with 4% template. These values compare extremely well with values of 2.6 measured by us for a bulk polymer prepared with a similar recipe under equivalent polymerization conditions. This value is identical to that reported in the literature16 for a similar propranolol-imprinted polymer, suggesting that MIP synthesis and performance are highly reproducible. One of the main advantages that the core-shell structure offers is the possibility to add specific properties exclusively into the core of the particles without interfering with the imprinted polymer situated in the shell of the particle. As an example, we have synthesized a seed that contains a fluorescent monomer (9-anthrylmethyl methacrylate) covalently incorporated into its composition and polymerized an imprinted shell around it. The excitation and emission spectra of the core-shell polymeric particles showed that the fluorescent monomer is incorporated into the polymer (Figure 3). The peak ratios in the emission spectra of the fluor alter slightly once it is incorporated (20) Christopoulos, T. K.; Diamandis, E. P. In Immunoassay; Diamandis, E. P., Christopoulos, T. K., Eds.; Academic Press: San Diego, 1996; pp 44-46.
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Figure 3. Emission spectra of (a) fluorescent monomer, (b) fluorescent seed, (c) core-shell particles with fluorescent monomer in the core, and (d) core-shell particles without fluorescent monomer in the core.
into the polymer structure of the seed particle. This is probably as a result of it experiencing a slightly different microenvironment with altered polarity compared with when it is in free solution (in THF). The spectra from the seed (core) and the core-shell particles are almost identical as expected. The uptake experiments showed that the polymer behaves in a similar way to nonfluorescent particles (Table 3). Conclusions Successful noncovalent imprinting of propranolol in the shell of core-shell nanoparticles of about 60-70 nm in diameter has been demonstrated. The presence of solvent during polymerization of the imprinted shell significally increases the binding capacity of the polymers. The affinity of the binding sites in toluene with 0.5% of acetic acid,
estimated from radioligand displacement curves, was very similar to that of bulk imprinted material, despite the presence of water at the interface of the particles during imprinting. A fluorescent monomer was successfully incorporated into the nanoparticle core. The presence of an imprinted shell around the core had no effect on the fluorescence of the core; neither did the fluorescent monomer in the core affect the imprinting in the shell. It is expected that this type of particle will find applications in assay technology. Acknowledgment. The authors thank Dr. A. I. Cooper from the University of Liverpool for the surface area measurements and BBSRC, Grant Number 83/E 13283, for funding this work. LA0356755
