Enzyme Multilayers on Colloid Particles: Assembly, Stability, and

Colloidal biocatalysts, comprising polystyrene (PS) carrier particles coated with ...... Cooper, T. M.; Campbell, A. L.; Crane, R. L. Langmuir 1995, 1...
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Enzyme Multilayers on Colloid Particles: Assembly, Stability, and Enzymatic Activity Frank Caruso* and Corinna Schu¨ler Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany Received July 5, 2000. In Final Form: August 14, 2000 Colloidal biocatalysts, comprising polystyrene (PS) carrier particles coated with enzyme multilayers, were fabricated via the layer-by-layer self-assembly method. Glucose oxidase (GOD), horseradish peroxidase (POD), or preformed enzyme-polyelectrolyte complexes were assembled in alternation with oppositely charged polyelectrolytes onto PS particles. Microelectrophoresis, single-particle light scattering, and transmission electron microscopy confirmed stepwise growth of the multilayer films on the colloid particles. The high surface area enzyme multilayer-coated particles were successfully employed as specific enzyme reactors (i.e., as catalysts). Whereas no loss in activity was observed for the enzymes immobilized directly onto particle surfaces, precomplexing the enzymes with polymer in solution drastically reduced their activity (by up to 70%). The enzymatic activity (per particle) was found to increase with the number of enzyme layers immobilized, irrespective of whether the enzyme was precomplexed. However, particles coated with preformed enzyme-polyelectrolyte complexes displayed a significantly lower enzymatic activity than those fabricated by the direct adsorption of free enzyme. Multicomponent films of GOD and POD on colloid particles were also prepared, and sequential enzymatic catalysis was demonstrated. Furthermore, experiments were conducted with particles exhibiting both magnetic and catalytic functions. These particles, premodified with a layer of magnetic nanoparticles to impart a magnetic property and subsequently coated with enzyme multilayers, were repeatedly used as catalysts following their rapid and easy separation with a magnet. Such biocolloids are expected to find applications in biotechnology.

Introduction Organized ultrathin protein architectures have attracted great interest because of their broad application in biotechnology. Protein films are routinely employed in bioseparations, immunoassays, diagnostics, localization, and catalysis.1-5 Proteins have traditionally been immobilized onto solid surfaces by a variety of techniques, including physical adsorption, solvent casting, covalent binding, and electropolymerization.1 However, these methods often produce irregular films with a low density of protein. Ordered protein multilayer films with a high protein density have been constructed by using Langmuir-Blodgett deposition methods1,6 or by exploiting biospecific interactions.7-10 Denaturation of the immobilized proteins, restricted film permeability to substrates, and the highly specific nature of the assembly limit the wide applicability of these methods. The process of * To whom correspondence should be addressed. Fax: +49 331 567 9202. E-mail: [email protected]. (1) In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000. (2) In Proteins at Interfaces: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (3) In Antibodies: A Practical Approach; Catty, D.; Raykundalla, C., Eds.; IRL Press: Oxford, 1989; Vol. II, p 97. (4) In Biosensors: Fundamentals and Applications; Turner, A. P. F., Karube, I., Wilson, G. S., Eds.; Oxford University Press: Oxford, 1987. (5) Guesdon, J. L.; Avrameas, S. In Applied Biochemistry and Bioengineering; Wingard, L. B., Katchalski-Katzir, E., Goldstein, L., Eds.; Academic Press: New York, 1981; Vol. 3, p 207. (6) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1992, 210/211, 710. (7) Ahlers, M.; Mu¨ller, W.; Reichert, A.; Ringsdorf, H.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1990, 29, 1269. (8) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706. (9) Cassier, T.; Lowack, K.; Decher, G. Supramol. Sci. 1998, 5, 309. (10) Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1998, 120, 1665.

alternating adsorption of charged macromolecules (also known as the layer-by-layer, LbL, technique) has recently emerged as a promising approach to fabricate controlled and highly ordered molecular assemblies according to a specifically designed architecture.11,12 The strategy entails the stepwise adsorption of charged species onto a charged substrate, utilizing primarily electrostatic interactions for multilayer film growth. Charge overcompensation occurs with deposition of each layer, thereby facilitating adsorption of the next layer. The LbL strategy was first introduced almost a decade ago for the creation of pure polyelectrolyte multilayer films on macroscopically flat surfaces.11 Multicomponent films of inorganic particles13-16 or dye molecules17-21 alternating with polyelectrolyte were later prepared by the LbL technique. Similarly, multilayer films of a wide array of water-soluble proteins, alternately assembled with oppositely charged polyelectrolytes,9,22-26 (11) Decher, G.; Hong, J. D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (12) For a recent review, see Decher, G. Science 1997, 277, 1232. (13) Schmitt, J.; Decher, G.; Dressick, W. J.; Brandow, S. L.; Geer, R. E.; Shashidhar, R.; Calvert, J. M. Adv. Mater. 1997, 9, 61. (14) Feldheim, D. L.; Grabar, K. C.; Natan, M. J.; Mallouk, T. E. J. Am. Chem. Soc. 1996, 118, 7640. (15) Kotov, N. A.; Dekany, I.; Fendler, J. H. J. Phys. Chem. 1995, 99, 13065. (16) Musick, M. D.; Keating, C. D.; Keefe, M. H.; Natan, M. J. Chem. Mater. 1997, 9, 1499. (17) Cooper, T. M.; Campbell, A. L.; Crane, R. L. Langmuir 1995, 11, 2713. (18) Araki, K.; Wagner, M. J.; Wrighton, M. S. Langmuir 1996, 12, 5393. (19) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224. (20) Yoo, D.; Wu, A. P.; Lee, J.; Rubner, M. F. Synth. Met. 1997, 85, 1425. (21) Tedeschi, C.; Caruso, F.; Mo¨hwald, H.; Kirstein, S. J. Am. Chem. Soc. 2000, 122, 5841. (22) Onda, M.; Ariga, K.; Kunitake, T. J. Biosci. Bioeng. 1999, 87, 69. (23) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427.

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were constructed, and their successful application in catalysis22,25,26 and immunosensing23 was demonstrated. Recently, we extended the LbL method to colloid particles to produce a new class of nanocomposite coreshell particles.27 Pure polyelectrolyte multilayers28-33 and multicomponent multilayers of nanoparticles34-38 or proteins39-41 with polyelectrolyte were constructed on particle surfaces. These multilayered colloids are of particular interest because of their inherently high surface area. For example, enzyme multilayers on particle surfaces are attractive enzyme reactors for catalysis reactions, with the potential to yield higher enzymatic reaction efficiencies than their planar film counterparts.41 In this paper, we provide a detailed investigation of the formation of glucose oxidase (GOD) and horseradish peroxidase (POD) enzyme multilayers on polystyrene (PS) particles, their stability, and their subsequent use as catalysts (see Scheme 1). We examined the assembly of the enzymes under conditions where the GOD and POD exhibit an overall net negative and positive charge, respectively. Microelectrophoresis, single-particle light scattering (SPLS), and transmission electron microscopy (TEM) were used to characterize the formation of the enzyme multilayers on the colloids. The thermostability of the immobilized enzymes as well as their activity as a function of pH were studied. Active catalysts, consisting of enzyme multilayers on particles, with different architectures and quantities of enzyme were produced for catalysis investigations. The effect of premixing enzyme with polyelectrolyte prior to assembly was also investigated with respect to layer formation on the colloids and catalysis efficiency. Sequential reactions catalyzed by multienzyme films were successfully demonstrated. The introduction of a magnetic function onto the PS particles prior to enzyme layer construction allowed the efficient and repeatable separation of the particles before and after catalysis reactions. Experimental Section Materials. Poly(allylamine hydrochloride) (PAH), MW 70 000; poly(sodium 4-styrenesulfonate) (PSS), Mw 70 000; poly(ethyleneimine) (PEI), Mw 25 000; sodium chloride (NaCl); and (24) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (25) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163. (26) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. J. Ferment. Bioeng. 1996, 82, 502. (27) For a review, see Caruso, F. Chem.sEur. J. 2000, 6, 413. (28) Caruso, F.; Schu¨ler, C.; Kurth, D. G. Chem. Mater. 1999, 11, 3394. (29) Kurth, D. G.; Caruso, F.; Schu¨ler, C. Chem. Commun. 1999, 1579. (30) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo¨hwald, H. Macromolecules 1999, 32, 2317. (31) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2201. (32) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Popov, V. I.; Mo¨hwald, H. Polym. Adv. Technol. 1998, 9, 759. (33) Caruso, F.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 1998, 102, 2011. (34) Rogach, A.; Susha, A.; Caruso, F.; Sukhorukov, G.; Kornowski, A.; Kershaw, S.; Mo¨hwald, H.; Eychmu¨ller, A.; Weller, H. Adv. Mater. 2000, 12, 333. (35) Susha. A.; Caruso, F.; Rogach, A. L.; Sukhorukov, G. B.; Kornowski, A.; Mo¨hwald, H.; Giersig, M.; Eychmu¨ller, A.; Weller, H. Colloids Surf., A: Physicochem. Eng. Aspects 2000, 163, 39. (36) Caruso, F.; Mo¨hwald, H. Langmuir 1999, 15, 8276. (37) Caruso, F.; Susha, A. S.; Giersig, M.; Mo¨hwald, H. Adv. Mater. 1999, 11, 950. (38) Caruso, F.; Lichtenfeld, H.; Mo¨hwald, H.; Giersig, M. J. Am. Chem. Soc. 1998, 120, 8523. (39) Caruso, F.; Fiedler, H.; Haage, K. Colloids Surf., A: Physicochem. Eng. Aspects 2000, 169, 287. (40) Caruso, F.; Mo¨hwald, H. J. Am. Chem. Soc. 1999, 121, 6039. (41) Schu¨ler, C.; Caruso, F., Macromol. Rapid Commun. 2000, 21, 750.

Caruso and Schu¨ ler Scheme 1. Schematic Illustration of the Layer-by-Layer Assembly of Polyelectrolyte and Enzyme Multilayers on Colloid Particles

morpholinoethane sulfonic acid (MES) were all obtained from Aldrich. Glucose oxidase (GOD), horseradish peroxidase (POD), glucose, N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) and o-dianisidine were purchased from Sigma. Tris(hydroxymethyl) aminomethane (Tris), sodium acetate (NaAc), and hydrogen peroxide (H2O2) were obtained from Fluka. Buffers for the pH-dependent experiments (pH 2-6, citrate-hydrochloric acid; pH 7, phosphate; pH 8-11, boric acid-potassium chloridesodium hydroxide) were from Merck. All chemicals were used as received except for PSS, which was dialyzed against Milli-Q water (Mw cutoff 14 kDa) and lyophilized before use. The sulfatestabilized polystyrene (PS) particles (diameter 470 or 640 nm) were prepared as described previously.42 Details of the preparation of the magnetic PS particles used can be found in an earlier publication.37 The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18.2 MΩ cm. Enzyme Multilayer Formation on Colloids. The general procedure used to assemble the enzyme multilayers on colloids was the same as that described previously.39-41 Briefly, 1 mL of a 1 mg mL-1 polyelectrolyte solution (containing no additional salt for PEI, 0.5 M NaCl for PAH and PSS solutions) was added to approximately 1010 PS particles in 200 µL of water. The polyelectrolyte was allowed to assemble onto the colloids for 20 min, during which time the mixture was occasionally stirred. Following this, the dispersion was centrifuged for 6 min at 13 000 g, the particles were washed with water, and the centrifugation/ wash cycle was repeated a further two times. The particles were then redispersed in ca. 100 µL of water. Enzyme solution (2 mg mL-1, 10 or 50 mM buffer) was then added to the polyelectrolytecoated particles, and 1 h was allowed for enzyme adsorption onto the particles. All enzyme layers were assembled at 4 °C and maintained at this temperature throughout. For GOD, Tris (pH 8.0-8.2) and MES (pH 6.0) buffers were used. Under these conditions, GOD has a net negative charge (isoelectric point, pI, GOD ) 4.2). NaAc (pH 5.2) and HEPES (pH 6.8) buffers were used for POD assembly, giving POD an overall positive charge (pI POD ) 8.8). Excess enzyme was removed by three wash/ centrifugation cycles. Subsequent enzyme/polyelectrolyte layers were deposited using the same procedure and conditions described above. (42) Furusawa, K.; Norde, W.; Lyklema, J. Kolloid Z. Z. Polym. 1972, 250, 908.

Enzyme Multilayers on Colloids For the premixing experiments, polyelectrolyte and enzyme were added to a vial and then diluted with the appropriate buffer so that the final concentrations of polymer and enzyme in the solution were 4 and 2 mg mL-1, respectively. The polyelectrolyteenzyme complexes in solution exhibited a zeta-potential similar to that of the same polyelectrolyte adsorbed onto particles. Alternate adsorption of the charged complex and oppositely charged polyelectrolyte was conducted by the standard procedure outlined above. Microelectrophoresis. Enzyme multilayer growth was followed qualitatively by measuring the microelectrophoretic mobility of coated particles using a Malvern Zetasizer 4 by taking the average of 5 measurements at the stationary level. The mobilities (u) were converted to the zeta (ζ) potential using the Smoluchowski relation ζ ) uη/, where η and  are the viscosity and permittivity of the solution, respectively. All measurements were performed in air-equilibrated pure water (pH ∼ 5.6) without added electrolyte. Single-Particle Light Scattering (SPLS). Enzyme multilayer growth was measured quantitatively by SPLS using a homebuilt instrument. The SPLS equipment and measurement principle are described in detail elsewhere.43,44 Briefly, the dispersion of uncoated or coated particles was passed through a capillary with a 0.1-mm diameter orifice at the end and directed through an argon laser (Innova 305) beam, focused to allow only a single particle in focus at a particular time. Forward-scattered light pulses recorded from particles flowing through the scattered volume were detected in the angular region of 5-10°. The intensity distributions were collected by a multichannel analyzer and then stored on a PC. Further details can be found in earlier publications.30-33 Temperature-Dependent Measurements. The samples were incubated at a certain temperature for 10 min in a water bath. 100 µL of the samples was then pipetted into a cuvette, and the activity was measured as described below. Transmission Electron Microscopy (TEM). TEM measurements were performed on a Philips CM12 microscope operated at 120 kV. TEM samples were prepared by depositing a diluted particle suspension onto a carbon-coated copper grid. The mixture was allowed to air-dry for one min, and the extra solution was then blotted-off. Enzymatic Activity Assays. To measure the activity of the GOD multilayers, 2.4 mL of a 0.21 mM o-dianisidine solution in 50 mM sodium acetate buffer (pH 5.1), 0.5 mL of a 10% (w/v) β-D-glucose solution, and 0.1 mL of a POD solution (containing approximately 60 units mL-1) were mixed in a cuvette and airequilibrated until the absorbance at 500 nm was constant. GODcoated PS particles were then added to the mixture, and the increase in absorbance at 500 nm for 4 min was recorded immediately after mixing. For each experiment, the same number of enzyme-coated particles was added (approximately 108 particles in 0.1 mL) to this mixture (total volume of 3 mL). The activity of immobilized POD was measured in the same way as GOD, although the solution components were different. For POD, 2.4 mL of a 0.21 mM o-dianisidine solution in 50 mM sodium acetate buffer (pH 5.1) and 0.1 mL of a H2O2 solution (30%) were used. The activity of the GOD/POD multilayers was measured in the same way as for the single-component GOD multilayers, the only exception being that immobilized POD was used for the sequential reaction rather than POD in solution.

Results and Discussion Enzyme Multilayer Assembly and Characterization. Proteins are known to adsorb through a variety of interactions, including hydrophobic, van der Waals, electrostatics, and hydrogen bonding. Discernment of the main factor driving protein adsorption is often not straightforward since there commonly exists a number of interactions operating simultaneously.1 For example, although it can be expected that at several pH units away (43) Lichtenfeld, H.; Knapschinsky, L.; Sonntag, H.; Shilov, V. Colloids Surf., A: Physicochem. Eng. Aspects 1995, 104, 313. (44) Lichtenfeld, H.; Knapschinsky, L.; Du¨rr, C.; Zastrow, H. Prog. Colloid Polym. Sci. 1997, 104, 148.

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Figure 1. ζ-Potential of (a) PAH/GOD, (b) PSS/POD, and (c) PSS/POD + PEI/GOD multilayers on either 470- or 640-nm PS particles as a function of layer number. For single-component enzyme layers, GOD was assembled from a 50 mM MES buffer, pH 6.0 (a), and POD was assembled from a 50 mM HEPES buffer, pH 6.8 (b). For multicomponent enzyme layers, GOD was assembled from 50 mM Tris, pH 8.0, and POD from 50 mM NaAc, pH 5.2 (c). In (a) and (b), the odd layer numbers correspond to polyelectrolyte adsorption and the even layer numbers to enzyme deposition. For (c), the layer sequence is (PSS/POD)2/ PSS/(PEI/GOD)2/PEI (the PS particles were first precoated with PAH). The coated PS particles were redispersed in airequilibrated pure water (pH ∼ 5.6) prior to measurement of the microelectrophoretic mobilities.

from the pI of the enzyme (conditions employed in the current work) the main driving force for the assembly would be electrostatics, hydrophobic interactions may still play a significant role.1 GOD was assembled under conditions where it was negatively charged in solution (pH 6.0 or 8.2) since it has an isoelectric point (pI) of 4.2. Therefore, GOD was deposited alternately with the polycations PEI or PAH. In contrast, POD (pI ) 8.8) was assembled at pH values of 5.2 or 6.8 (making it positively charged) with anionic PSS as the intermediate layer. In all cases, the enzyme concentration for assembly was 2 mg mL-1, which is more than required for saturation coverage of the particles, according to adsorption isotherm data derived from SPLS measurements. Microelectrophoresis experiments were conducted to qualitatively follow the formation of the enzyme multilayers on the colloids. The ζ-potentials of the coated particles were calculated from the mobilities that were measured after deposition of each layer. Figure 1 shows the ζ-potential of PS particles coated with multilayers of PAH/GOD (a), PSS/POD (b), and those sequentially coated with PSS/POD followed by PEI/GOD multilayers (c). When the positively charged PAH or PEI polyelectrolytes formed the outermost layer, positive ζ-potentials were measured (see (a) and (c)). The slight differences in the measured values can be attributed to the different polyelectrolytes or contributions from the layer underneath, arising due to different conformations and distribution of the polymers at the surface. The ζ-potentials measured for the coated particles when GOD formed the outermost layer

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Figure 2. ζ-Potential of multilayers of POD-PSS complexes alternating with PAH (squares) and GOD-PAH complex/PSS multilayers (circles) on 470-nm PS particles as a function of layer number. The POD-PSS complex was assembled from 50 mM HEPES, pH 6.8, and the GOD-PAH complex from 50 mM MES, pH 6.0. The odd layer numbers correspond to complex adsorption and the even layer numbers to polyelectrolyte deposition. The coated PS particles were redispersed in airequilibrated pure water (pH ∼ 5.6) prior to measurement of the microelectrophoretic mobilities.

were mostly close to zero and occasionally slightly positive (a) or negative (c). Under the measuring conditions (in air-equilibrated water, pH ca. 5.6), a negative ζ-potential would be expected for a homogeneous covering of the particles. The values obtained most probably reflect variations in surface coverage of GOD on the polyelectrolyte surface. As expected from the solution conditions under which the microelectrophoresis experiments were conducted, negative ζ-potentials were observed when POD was the outerlayer (ca. -20 to -30 mV), and more negative values were obtained for the PSS/POD multilayer-coated particles with a PSS final layer (ca. -40 to -50 mV). Suffice it to say, an alternating trend in the ζ-potential was observed for all multilayer films (both single and multicomponent enzyme films), depending on whether the polyelectrolyte or the enzyme formed the outer layer. This is qualitative evidence for the stepwise deposition of polyelectrolyte and protein and suggests multilayer growth of charged macromolecules on colloid particles.27-33,37-41 An alternative approach, based on the adsorption of preformed enzyme-polyelectrolyte complexes, was employed to form enzyme multilayer films. The formation of enzyme-polyelectrolyte aggregate structures has been extensively studied by several research groups.45-47 In the current work, the proportions of enzyme and polyelectrolyte (by weight) used to form the complexes were 1:2, respectively. Measurement of the ζ-potential of GODand POD-polyelectrolyte complexes in solution yielded values close to those observed for colloids coated with the same polyelectrolyte used to form the complex, indicating that the polyelectrolyte charge dominates the observed value. Particles coated with multilayers of enzymepolyelectrolyte complexes and oppositely charged polyelectrolyte showed regular and alternating ζ-potentials (in sign) depending on the outermost layer type (Figure 2). In agreement with the overall charge of the enzymepolyelectrolyte complex, negative and positive values were observed for POD/PSS and GOD/PAH complexes adsorbed onto particles, respectively. The data in Figure 2 suggest (45) Margolin, A. L.; Sherstyuk, S. F.; Izumrudove, V. A.; Zezin, A. B.; Kabanov, V. A. Eur. J. Biochem. 1985, 146, 625. (46) Zezin, A. B.; Izumrudov, V. A.; Kabanov, V. A. Makromol. Chem., Macromol. Symp. 1989, 26, 249. (47) Izumi, T.; Hirata, M.; Takahashi, K.; Kokufuta, E. J. M. S.-Pure Appl. Chem. 1994, A31, 39.

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that additional layers were deposited with each alternate deposition of polyelectrolyte and oppositely charged enzyme-polyelectrolyte complexes. This finding is in agreement with that reported for the formation of polyelectrolyte/enzyme-polyelectrolyte complexes on planar surfaces.22 We employed the technique of SPLS to quantitatively determine the thickness of the enzyme multilayers. SPLS allows the layer thickness of adsorbed macromolecules on particles to be measured with high precision (several nanometer changes in diameter can be discerned).30-32 The combined thickness of the first enzyme and polyelectrolyte layer pair was 3.4 ( 0.8 nm, regardless of the enzyme or buffer solutions used (in this work) for immobilization of the enzyme layer.48 The thicknesses were measured for particles having an outermost polyelectrolyte layer, as this increased the stability of the coated colloids. Monolayer coverage of GOD is expected to yield a thickness of about 6 nm (diameter is approximately 6 nm, from protein data bank). For POD, a monomolecular layer is expected to give a thickness of approximately 3.5 nm.25 The layer thicknesses obtained correspond to submonolayer coverage (the polyelectrolyte layer in the polymer-enzyme bilayers is likely to have a thickness of 0.5-1.5 nm). SPLS experiments on particles coated with a single enzyme outermost layer (deposited on polyelectrolyte-modified PS particles) revealed that up to 80% of the originally adsorbed enzyme was removed as a result of adsorption of a subsequent polyelectrolyte layer. This indicates that although a moderate amount of enzyme is adsorbed initially, a large fraction of this enzyme is adsorbed through interactions that are weaker than those resulting from subsequent association with the polyelectrolyte in solution. Similar removal processes have recently been observed for dye-polyelectrolyte multilayer films fabricated on planar surfaces.19,21 However, despite the removal of enzyme with polyelectrolyte adsorption, the remaining immobilized enzyme was successfully coated with polyelectrolyte (as confirmed by microelectrophoresis), hence facilitating deposition of the next enzyme layer. In accordance with the microelectrophoresis experiments, SPLS revealed stepwise growth of GOD- and PODpolyelectrolyte multilayer films on colloid particles under different solution conditions. Thicknesses of 2-3 nm were measured for each subsequently deposited GOD/polyelectrolyte or POD/polyelectrolyte bilayer for films comprising up to 10 layer pairs (i.e., 5 enzyme layers). The bilayer thicknesses were found to be independent, within experimental error, of the buffer solutions (with different salt contents and pH) employed in this work. It should also be noted that the systems were formed with different intermediate polyelectrolyte layers. It was also possible to assemble PAH/PSS/POD/PSS on the latex particles, which yielded a layer increase of 4 nm, followed by PEI/ GOD/PEI, further increasing the film thickness by 5 nm (measured by SPLS). This verifies that it is possible to assemble multiple enzymes on the same particle. For particles coated with more than about 5 enzyme layers, aggregation was often observed. This may be explained by an increase in surface roughness with increasing layer number, resulting from the submonolayer enzyme coverage. Evidence for this was provided by atomic force microscopy, which clearly showed significant surface roughness and patchiness for the enzyme multilayercoated particles, compared with the smooth and uniform surfaces for the uncoated particles (data not shown). (48) Details on how the thickness measurements were calculated can be found in refs 39 and 40.

Enzyme Multilayers on Colloids

Figure 3. Thickness of enzyme-polyelectrolyte complex/ polyelectrolyte multilayer films assembled in alternation with oppositely charged polyelectrolyte onto 470-nm PS particles, as determined by SPLS: (a) GOD-PAH complex/PSS; adsorption conditions for complexes were 50 mM MES buffer, pH 6.0, and (b) POD-PSS complex/PAH; adsorption conditions were 50 mM HEPES buffer, pH 6.8. The values shown correspond to bilayer thicknesses (i.e., complex and polyelectrolyte).

When the enzymes were deposited from pure water solutions, no significant thickness increase was observed by SPLS, suggesting that at best a low fraction of monolayer coverage occurred. However, the assembly of GOD or POD multilayers on macroscopically flat substrates, where both GOD and POD were adsorbed from pure water solutions, was recently reported.22,25,26 In this context, it is important to note that after deposition of enzyme or polymer layers on planar surfaces, a drying step is commonly conducted. In some instances, such drying may induce adsorption via nonspecific interaction of enzyme and polyelectrolyte. In our systems, drying is not desirable, as it would most probably lead to irreversible aggregation of the coated particles, particularly when the enzyme forms the outermost layer. The loss of immobilized enzyme from the particle surface with subsequent polyelectrolyte adsorption was overcome by premixing the enzyme with a polyelectrolyte of opposite charge to form an enzyme-polymer complex in solution prior to adsorption. It was found that the complex could be successfully deposited in alternation with oppositely charged polyelectrolyte, leading to stepwise assembled multilayer films. In this method, the enzyme complex was not removed by the next polymer layer (within the sensitivity of SPLS). The layer thicknesses were larger than those for the films fabricated using uncomplexed enzymes (as described above, see Figure 3). The average thickness for the bilayers of the GOD-PAH complex and PSS was about 5 nm, whereas that for bilayers of the complex POD-PSS and PAH was 4.2 nm. The individual polyelectrolyte layer (i.e., the intermediate layer) thicknesses were approximately 1 nm. The larger thickness values for the adsorbed complexes (compared to the polyelectrolyte layers) indicate that a greater amount of material is adsorbed, which is due to immobilization of enzyme in the layers. TEM was employed to examine the regularity of the enzyme-complex/polyelectrolyte multilayers on the particles. The layers, which exhibited some roughness, could be clearly distinguished, and a fairly uniform coating was observed on individual PS particles (Figure 4). Enzyme Stability. The practical application of immobilized enzymes depends on their stability under various conditions, e.g. temperature and pH. The stability of some enzymes with respect to temperature and pH can

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Figure 4. TEM micrograph of PS particles coated with 4 bilayers of GOD-PAH complex/PSS. The presence of enzyme/ polymer on the surface is evident from the increase in roughness seen for the coated particles. The uncoated particles have a smooth, nontextured surface (images not shown). Image (b) is a higher magnification of image (a).

Figure 5. Thermostability of (a) GOD in PEI/GOD layers and (b) POD in PSS/POD layers immobilized on PS particles. The corresponding data for GOD and POD in free solution are also shown. The solid lines correspond to the enzymes in solution and the dotted lines to the immobilized enzymes.

be enhanced upon their immobilization. For example, GOD has been found to be more stable when immobilized in microcapsules49 and multilayer films.22 In the current work, the thermostability of GOD and POD was examined when immobilized on PS particles. The corresponding solution experiments were also performed. A known amount of enzyme or number of coated particles was exposed to a certain temperature for 10 min, and, immediately following the activity, assays were performed. Figure 5 shows the activity versus temperature profiles for GOD (a) and POD (b) immobilized on PS particles and free in solution. GOD in solution shows maximum activity at around 35 °C, whereas GOD immobilized on particles has a maximum activity at 25 °C. Apart from the differences seen in the 25 to 45 °C region, a sharp reduction in activity with temperature is observed for both free and immobilized GOD, with a sharper drop for the enzyme in solution. GOD in solution loses all of its activity after 10 (49) Komori, T.; Muramatsu, N.; Kondo, T. J. Microencapsulation 1986, 3, 219.

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Figure 6. Relative activity versus pH profile for GOD immobilized on PAH-coated PS particles.

min at 65 °C. In contrast, GOD immobilized on the particles still retains about 15% of its activity after the same treatment. These data compare favorably with those found for GOD immobilized on planar substrates.22 The effect of temperature on the activity behavior of POD is more complex. POD in solution shows a rather high activity from 25 to 65 °C, after which a rapid drop occurs, and all of its activity is lost at 85 °C. Immobilized POD, however, loses most of its activity earlier (at 65 °C, 20% of activity retained) but then essentially plateaus off to retain 10% activity even at 85 °C. The activity of enzymes can drastically change in different pH environments. In solution, GOD shows the highest activity at pH 5.8; at pH 3 the activity is about 25%, and at pH 8.5 it loses all of its activity.50 When immobilized on particles, GOD is much more resistant to higher pH values (Figure 6). At pH 8, 50% of its activity is retained, and even at extreme conditions such as pH 11, about 10% of the original activity is preserved. The data obtained are very similar to those reported for GOD immobilized on planar substrates.22 From the above data, it is clear that improved stability (with respect to temperature and pH) occurs upon immobilization of the enzyme GOD on particle surfaces. Various explanations have been given for the increased stability of immobilized enzymes over those free in solution, including changes in the microenvironment and restricted conformational mobility.45,46 The activity of POD was also measured as a function of pH (see Experimental Section), and it was found to be virtually pH-insensitive over the pH range 2-9.5, in agreement with other studies.51 Enzymatic Catalysis. The activity of GOD in the multilayer films on the particles was measured by following the coupled enzymatic reaction of GOD and POD, whereas the activity of POD was determined by the standard assay (see Experimental Section). Each series of activity measurements was normalized for particle number, which was determined by SPLS. To examine whether GOD or POD lost activity upon immobilization, we performed control experiments where the same amount of enzyme that was immobilized on the particles was added to solution. (The amount adsorbed on the particles was determined by UV-vis spectroscopy, i.e., by monitoring the difference in solution between the protein tryptophan absorbance at 280 nm before and after adsorption.) Activity tests were then conducted in the same way for both the enzyme in solution and that immobilized. Within experi(50) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (51) Sanders, S. A.; Bray, R. C.; Smith, A. T. Eur. J. Biochem. 1994, 224, 1029.

Figure 7. Activity (∆A500/min) of enzyme multilayers prepared under different conditions on negatively charged PS particles: (a) (squares) PEI/GOD bilayers, GOD assembled from 50 mM Tris, pH 8.0; (circles) PAH/GOD bilayers, GOD assembled from 50 mM MES, pH 6.0. In all cases, the outer layer was GOD. (b) PSS/POD bilayers; POD was assembled from 50 mM HEPES, pH 6.8. In all cases, the outer layer was POD. The data for each series of experiments were normalized for particle number, corresponding to samples containing 0.8 × 107 particles.

mental error, no loss of activity was observed for the immobilized GOD or POD enzymes. An earlier report showed that at least 80% of GOD activity was retained in GOD-polymer multilayer films, the 20% loss most probably originating from the severe conditions used to remove the protein from the film in that work.22 Figure 7 shows the rate of change of absorbance at 500 nm for PS particles coated with one to four PEI/GOD layers (a, top curve), one to five PAH/GOD layers (a, bottom curve), or one to five PSS/POD layers (b). The absorbance corresponds to samples that contain the same total number of particles. For the PEI/GOD layers, an increase in the enzymatic activity (per particle) is seen with layer number. The PAH/GOD layers assembled on particles also show an increase in activity with layer number; however, the relative increase diminishes with each additional enzyme layer. The differences in the data are attributed to differences in the enzyme/polymer layer structure (confirmed by atomic force microscopy measurements). Some protein multilayers formed by the sequential adsorption of protein and polyelectrolyte on planar surfaces have been reported to have an open, porous-type structure and to be permeable to proteins larger than 5 nm in diameter.52 It is well established that diffusion effects become important as the thicknesses of multilayer films increase.22 Since the actual thickness of the enzyme layers is not precisely known (because the adsorbing polyelectrolyte removes a significant amount of preadsorbed enzyme), we take the similar thicknesses for the enzyme/polyelectrolyte pairs (2-3 nm, see earlier) to be an indication that essentially equal amounts of enzyme remain immobilized in each layer for a given system. Assuming this, the PEI/GOD (52) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 4559.

Enzyme Multilayers on Colloids

data suggests that the film is permeable to the lowmolecular weight substrate, even after 4 enzyme layers have been deposited. In contrast, even though the total film activity increases slightly with layer number, the average activity per GOD layer decreases for PAH/GOD films, indicating that as the film becomes thicker substrate diffusion limitations begin to play a significant role. This is consistent with results previously reported.22 In addition, it is worth noting that another important difference in the preparation of the two films was the salt content in the solution used to deposit the intermediate polyelectrolyte layers. PEI was deposited from pure water, whereas PAH was adsorbed from a 0.5 M salt solution. The presence of salt is known to yield thicker adsorbed polyelectrolyte layers, as it causes the polyelectrolyte to adopt a more coiled conformation as a result of electrostatic screening.12,53 The thicker PAH layers (than PEI) may actually coat the surface of the immobilized enzyme such that substrate accessibility is restricted to a greater extent than the case for PEI-coated enzyme surfaces. (This issue will be addressed in more detail later.) SPLS showed that the PAH/GOD bilayers were marginally thicker than the PEI/GOD layers (3.5 vs 2.5 nm). A further explanation may be differences in the enzyme distribution (i.e., isolated or aggregated enzyme) and degree of conformational change upon immobilization of the GOD on the polyelectrolyte surfaces. These factors are also known to influence the activity of immobilized enzymes.22 The submonolayer coverage of GOD obtained for each adsorption step may yield an enzyme distribution that is readily accessible by the substrate. It should be pointed out that simply increasing the amount of enzyme immobilized does not lead to enhanced activities because of reduced accessibility and conformational changes.22 The PSS/POD multilayers on PS particles (b) display a similar trend to the PEI/GOD layers (a). An increase in activity is seen with increasing layer number. Similar factors outlined above may also apply to this system. In summary, an increase in total activity was observed for both GOD and POD singlecomponent enzyme systems with increasing layer number, reflecting that enzyme is immobilized in a stepwise manner. This confirms the microelectrophoresis and SPLS data and shows that the enzymatic activity can be tailored through judicious choice of enzyme multilayer systems and assembly conditions. For certain applications of enzyme multilayer-coated particles, multi-enzyme systems are required. To this end, we created multi-enzyme films of two inner PSS/POD bilayers and two outer PEI/GOD bilayers on PS particles. Exposing the coated particles to an aqueous solution of glucose and o-dianisidine resulted in oxidation of the dye (Figure 8). The enzymatic activity is somewhat low, perhaps due to the restricted amount of POD in the film (i.e., POD not being in excess); however, the result demonstrates that the coupled enzymatic reaction between GOD and POD was successfully achieved using this multienzyme film and that the immobilized enzymes are active. The effect of film structure, enzyme deposition order, and spacing between enzyme layers have all been extensively shown to influence the efficiency of sequential catalysis reactions for enzyme multilayers assembled on filters.22,25,26 Enhanced reaction efficiencies and enzyme activities can be achieved by optimization of such parameters.25,26 The activity of enzyme multilayers constructed from enzyme-polyelectrolyte complexes (deposited in alternation with oppositely charged polyelectrolyte) is shown as (53) Decher, G.; Schmitt, J. Prog. Colloid Polym. Sci. 1992, 89, 160.

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Figure 8. Time course of the conversion (absorbance at 500 nm) of the sequential enzymatic reaction catalyzed by GODPOD multi-enzyme films assembled onto PS particles. POD was assembled from 50 mM NaAc, pH 5.1, GOD from 50 mM Tris, pH 8.1.

Figure 9. Activity (∆A500/min) of enzyme-polyelectrolyte complexes assembled in alternation with oppositely charged polyelectrolyte on PS particles: (squares) GOD-PAH complex/ PSS bilayers, GOD-PAH complex was assembled from 50 mM MES, pH 6.0; (circles) POD-PSS complex/PAH bilayers, PODPSS complex assembled from 50 mM HEPES, pH 6.8. In all cases, the outer layer was the enzyme-polyelectrolyte complex. The data for each series of experiments were normalized for particle number, corresponding to samples containing 0.8 × 107 particles.

a function of enzyme layer number in Figure 9. Particles coated with the preformed complexes show a lower total enzymatic activity than the corresponding enzyme multilayer films fabricated from the uncomplexed enzymes (the rate of change of absorbance at 500 nm is an order of magnitude less). The largest activity increase for both the GOD and POD systems was observed for the first layer. Thereafter, only small increases in total enzymatic activity were seen with increasing enzyme layer number. Substrate diffusion limitation obviously plays an important role as the layers grow in thickness (from 3 to 20 nm). The substrate binding sites on the enzyme may also become increasingly blocked or less accessible as a result of the additional polyelectrolyte layers deposited (see below). An explanation for the low enzymatic activity may be the enhanced aggregation of enzyme that occurs when it is complexed with polyelectrolyte in solution. Aggregation of the enzyme is known to reduce its activity.22 Therefore, the effect of enzyme complexation on its activity in solution was studied. Enzyme (GOD or POD) that was precomplexed with oppositely charged polyelectrolyte (enzyme-to-polymer mass ratio of 1:10) in solution had 60-70% less activity than the corresponding free enzyme

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Figure 10. Relative activity of PS particles coated with one layer of GOD as a function of additionally deposited PAH/PSS layers. The activity of the first GOD layer after adsorption of the first polyelectrolyte layer on top of it was normalized to 100% as the polymer adsorption step caused removal of some of the enzyme. Data points for repeat experiments are shown. GOD was assembled from 50 mM MES, pH 6.0.

in solution. This decrease in activity of the complexed enzyme is likely to be due to blocking of the substrate binding sites of the enzyme by the polyelectrolyte and/or any induced enzyme aggregation. Varying the enzymeto-polyelectrolyte mass ratio beyond 1:10 (i.e., 1:2) produced the same result with respect to activity loss. Several investigations have shown that enzymes lose a proportion of their activity when complexed with polyelectrolyte in solution.45,47 The above data shows that although stable and regular enzyme multilayers can be produced using complexes of enzyme and polyelectrolyte, a significant loss in enzyme activity occurs. Hence, the resulting multilayer films are less efficient in terms of catalysis than those formed by the sequential adsorption of enzyme and polyelectrolyte. Nonetheless, this strategy represents an alternative method to construct multilayer films of enzymes that undergo removal from surfaces with subsequent polyelectrolyte deposition. Influence of Polyelectrolyte Multilayers on Enzymatic Catalysis. In some cases, it may be desirable to vary the spacer distance, that is, the number of polyelectrolyte interlayers separating each enzyme layer. For example, insufficient separation between enzymes on a filter was reported to cause considerable inhibition of the sequential reaction between the two enzymes.26 To investigate the effect of spacer layers on enzymatic activity, single-component enzyme films consisting of one enzyme (GOD) layer immobilized in the interior with multiple PAH/PSS polyelectrolyte layers deposited on top were assembled on PS particles. The GOD activity was measured as a function of increasing polyelectrolyte layer number (Figure 10). The activity measured after deposition of the first polyelectrolyte layer was set at 100%, since the adsorption of the first polymer layer caused removal of some preadsorbed enzyme. After two additional polyelectrolyte layers, the enzymatic activity decreased ca. 10%. Deposition of a further two layers (i.e., total of 5 layers) resulted in a sharp drop in the enzymatic activity to 50%. The reduction in activity continued until about a total of 7-9 layers (30%) was reached, after which only a slight decrease was observed with deposition of additional polyelectrolyte layers. The decrease in activity most likely reflects diffusion phenomena; however, it is also possible that some of the enzyme catalytic centers may be blocked by deposition of additional polyelectrolyte layers. It is well-known that polyelectrolytes within polyelectrolyte multilayer films interpenetrate one an-

Caruso and Schu¨ ler

other; a layer can interpenetrate 3-4 adjacent polymer layers.12 Hence, immobilized enzyme coated with multiple polyelectrolyte layers may have significantly less enzymatically active sites exposed for catalysis reactions than enzyme that is covered with a single polymer layer. At high layer number, the polymer multilayers effectively have the role of creating a more “closed system” for catalysis reactions. Another plausible reason may be changes in the microenvironment (e.g., slight pH changes) that may occur as a result of slow diffusion of the enzyme reaction product once formed. A similar experiment was performed for particles coated with one layer of a premixed PAH/GOD complex. An 80% loss of GOD activity was observed after two additional polyelectrolyte layers were deposited, with 100% loss after deposition of a further layer. These experiments show that the catalytic activity of enzymes on particles can be severely compromised by adsorbing additional polyelectrolyte layers and that in cases where an effective separation between two enzymes is required to facilitate an efficient sequential enzyme reaction, careful optimization of the layer spacing is required. Multifunctional Colloids. Deposition of enzyme multilayers onto carriers that can be readily separated may provide an attractive system for separations. Magnetic support materials have been widely used in biotechnology, bioseparations, immunoassays, and enzyme immobilization.54-57 We employed 200-nm PS particles that were precoated with four layers of Fe3O4 nanoparticles and poly(diallyldimethylammoniumchloride),37 followed by two (PSS/PAH) additional polyelectrolyte layers and an outer GOD layer. The particles were fabricated using the LbL approach, beginning with the magnetic nanoparticles and followed by the enzyme multilayers. The magneticfunctionalized particles could be drawn to the bottom of a reaction tube by using a magnet. The activity of the GOD layer on the particles was measured and the particles were then separated with a magnet, after which they were washed 5 times with water. The cycle was repeated five times. The measured activity was within (15% for each cycle, showing that it was possible to recover the particles and that the immobilized enzyme remains active after cycling. The above strategy opens a promising pathway for the fabrication of tailored magnetic, biocatalytic, and reusable particles. Conclusions Enzyme multilayer films were successfully assembled on PS particles using the layer-by-layer approach, a promising and highly versatile procedure that lends itself to the deposition of a large number of charged species. GOD immobilized on particles was more stable against higher temperatures and pH than free GOD in solution. The enzyme multilayer-coated particles were enzymatically active, with the general finding that the total activity of the particles increased with increasing enzyme layer number. It was also found that increasing the number of polyelectrolyte layers deposited on top of the immobilized enzymes causes a reduction in enzyme activity, most probably due to blocking of the substrate binding sites and reduced diffusion of the substrates. The coupled enzymatic reaction between GOD and POD assembled on the same particle (multi-enzyme film) was also demon(54) Dunnill, P.; Lilly, M. D. Biotechnol. Bioeng. 1974, 16, 987. (55) Hirschbein, B. L.; Whitesides, G. M. Appl. Biochem. Biotechnol. 1982, 7, 157. (56) Uhlen, M. Nature 1989, 340, 733. (57) Nakamura, N.; Hashimoto, K.; Matsunaga, T. Anal. Chem. 1991, 63, 268.

Enzyme Multilayers on Colloids

strated. Enzyme multilayers constructed by the alternating adsorption of polyelectrolyte and premixed enzyme/ polyelectrolyte complexes exhibited a lower activity than the films prepared from the uncomplexed species. The successful recovery and reuse of magnetic-functionalized particles coated with enzyme layers with a magnet was also achieved. The strategy presented is promising for the creation of particles with complex and multiple catalytic functions. Major advantages associated with such particles are the potential to increase the catalytic output in relation to the number of immobilized enzyme layers, reuse and easy separation of the particles, and their inherently high surface area for reaction. We are presently

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exploiting biospecific interactions to construct complex and biofunctional multi-enzyme catalysis systems. Acknowledgment. This work was supported by the German Federal Ministry of Education, Science, Research and Technology and the Innovationskolleg Biomolekulare Erkennungssysteme fu¨r die Biochemische Analytik. Helmuth Mo¨hwald is thanked for helpful discussions, Heinz Lichtenfeld for assistance with the SPLS experiments, Michael Giersig for help with the TEM measurements, and Andreas Fery for AFM measurements. LA000942H