Biocolloids with Ordered Urease Multilayer Shells as Enzymatic

Anal. Chem. 2001, 73, 4212-4217

Biocolloids with Ordered Urease Multilayer Shells as Enzymatic Reactors Yuri Lvov† and Frank Caruso*,‡

Institute for Micromanufacturing, Louisiana Tech University, Ruston, Louisiana 71272, and Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany

The preparation of biocolloids with organized enzymecontaining multilayer shells for exploitation as colloidal enzymatic nanoreactors is described. Urease multilayers were assembled onto submicrometer-sized polystyrene spheres by the sequential adsorption of urease and polyelectrolyte, in a predetermined order, utilizing electrostatic interactions for layer growth. The catalytic activity of the biocolloids increased proportionally with the number of urease layers deposited on the particles, demonstrating that biocolloid particles with tailored enzymatic activities can be produced. It was further found that precoating the latex spheres with nanoparticles (40-nm silica or 12-nm magnetite) enhanced both the stability (with respect to adsorption) and enzymatic activity of the urease multilayers. The presence of the magnetite nanoparticle coating also provided a magnetic function that allowed the biocolloids to be easily and rapidly separated with a permanent magnet. The fabrication of such colloids opens new avenues for the application of bioparticles and represents a promising route for the creation of complex catalytic particles. Largely due to its simplicity and versatility, the layer-by-layer (LbL) self-assembly process has in recent years been widely exploited to form a diverse range of nanocomposite thin films.1 The method involves the sequential adsorption of oppositely charged species, and relies on the reversal of the terminal surface charge of the thin film after deposition of each layer. The LbL process, although first demonstrated by Iler in the mid-1960s2 for the alternate adsorption of charged colloid particles on solid substrates, has only received considerable attention following the pioneering work of Decher and Hong on polyelectrolytes a decade ago.3 This strategy offers the possibility of designing ultrathin ordered films with nano- to micrometer thickness, with a precision better than a few nanometers, and with defined molecular composition. A large variety of polyelectrolytes are amenable to the process, as are nanoparticles and proteins.1,4-6 The assembly of protein/polyion multilayers in this way provides new possibili* Author for correspondence. E-mail: [email protected]. † Louisiana Tech University. ‡ Max Planck Institute of Colloids and Interfaces. (1) Decher, G. Science 1997, 227, 1232-1237. (2) Iler, R. J. Colloids Interface Sci. 1966, 21, 569-594. (3) Decher, G.; Hong, J. D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 14301434.

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ties to create organized enzyme layers with complex architectures. The LbL assembly process elaborated for planar solid supports has also been applied to colloid particles with diameters ranging from 10 nm to 5 µm (e.g., gold nanoparticles, latex spheres, biocrystals, lipid tubules, and other colloid templates).7-10 In this process, a polycation solution is added to a suspension of colloid particles, and after adsorption saturation, the particles are separated from the unadsorbed polycation in solution (usually by centrifugation or filtration). At this stage the particles are coated with a polycation layer. Thereafter, a polyanion layer is deposited in the same manner, and if desired, additional polycation/ polyanion layers are deposited by further alternate adsorption cycles. The LbL deposition of organized biomolecule multilayered shells on latex particles is a promising approach for creating colloids for biocatalysis applications,11-13 as has recently been demonstrated for glucose oxidase, peroxidase, and β-glucosidase.12,13 Such organized enzyme/polyion nanocomposite multilayers, with enzyme layers arranged in a predetermined order within semipermeable polymeric shells, present a new class of bionanoreactors. The degree of order and complexity that can be imparted to these systems via the flexible LbL technique makes them distinct from the more traditional liposome or copolymer shelled enzymatic reactors.14 In this work, as part of our efforts to create unique and complex biocolloids with tailored enzymatic activity, we report the LbL assembly of ordered urease multilayer shells on 470-nm-diameter (4) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117-6123. (5) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. J. Ferment. Bioeng. 1996, 82, 502-506. (6) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427-3433. (7) Caruso, F.; Caruso, R. A.; Mo ¨hwald, H. Science 1998, 282, 1111-1114. (8) Gittins, D. I.; Caruso, F. Adv. Mater. 2000, 12, 1947-1949. (9) Caruso, F.; Trau, D.; Mo¨hwald, H.; Renneberg, R. Langmuir 2000, 16, 1485-1488. (10) Lvov, Y.; Price, R.; Singh, A.; Selinger, J.; Spector, M.; Schnur, J. Langmuir 2000, 16, 5932-5936. (11) Caruso, F.; Mo ¨hwald, H. J. Am. Chem. Soc. 1999, 121, 6039-6046. (12) Caruso, F.; Fiedler, H.; Haage, K. Colloids Surf., A: Physicochem. Eng. Aspects 2000, 169, 287-293. (13) (a) Caruso, F.; Schuler, C. Langmuir 2001, 16, 9595-9603. (b) Schu ¨ ler, C.; Caruso, F. Makromol. Rapid Commun. 2000, 21, 750-754. (14) (a) Nardin, C.; Thoeni, S.; Widmer, J.; Winterhalter, M.; Meier, W. Chem. Commun. 2000, 1433-1434. (b) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier Science Publishers: Amsterdam, 1993. 10.1021/ac010118d CCC: $20.00

© 2001 American Chemical Society Published on Web 08/08/2001

latex spheres. Urease catalyzes the hydrolysis of urea [(NH2)2CO + 3H2O f CO2 + 2NH4OH, and the reaction rate may be monitored by following the increase of the solution pH.15-19] Urease (Mw 480 000) has an isoelectric point at pH 5 and is stable and active between pH 4 and 8.5.15-17 It was employed in the LbL assembly either as a negative “nanocolloid” at pH 8 deposited in alternation with polycations or as a positive “nanoparticle” at pH 4.5 and consecutively deposited with polyanions. The catalytic activity of the resulting biocolloids was found to be proportional to the number of urease layers deposited on the particles. As an added feature, prior to enzyme adsorption, the colloid particles have been coated with an additional layer of silica or magnetite nanoparticles in order to enhance their total surface area and promote further enzyme deposition. In traditional bioreactors, urease has been immobilized by covalent bonding or with acrylamide gels on different substrates (glass beads, wool, nylon netting, or nitrocellulose).16-18 Unlike the LbL technique employed here, these approaches do not allow control over the amount of enzyme deposited (and hence total enzyme catalytic activity), nor the possibility to assemble enzyme in ordered layers on the microto nanometer scale. EXPERIMENTAL SECTION Urease (U-4002, type IX from jack bean, Sigma) was used at a concentration of 2 mg/mL in 20 mM Tris buffer at pH 8 or at pH 4.5 in 20 mM acetate buffer. Poly(dimethyldiallylammonium chloride) (PDDA; Aldrich, medium molecular weight) and poly(styrenesulfonate) (PSS; Aldrich, Mw 70 000) were used in water solutions at pH 6.5 at a concentration of 2 mg/mL. For the polyelectrolyte (PE) precursor assembly step, i.e., priming of the particle surfaces, a PSS solution containing 0.5 M NaCl was used. The polystyrene (PS) spheres with sulfate surface groups were synthesized following the procedure outlined in the literature.20 Transmission electron microscopy showed the latex spheres had a mean diameter of 470 nm, with a polydispersity of less than 2% in diameter. The silica particles (mean diameter of 40 nm) were obtained from Nissan Chemical Inc. and magnetite nanoparticles (Fe3O4, mean diameter of 12 ( 2 nm) from Mediport, GmbH. The pH-sensitive dye bromcresol purple (Acros), urea, and EDTA (both Sigma) were used for the urease activity assay. The polyion/urease assembly process on flat substrates was monitored by the quartz crystal microbalance (QCM) technique.4-6 In the measurements, the resonator was immersed in a polyion solution for a given period of time, removed, and dried, and the frequency change of the crystal was measured. The long-term stability (several hours) of the quartz resonator frequency was within (2 Hz. The resonators used were coated with evaporated gold electrodes (0.16 cm2) on both faces, and their resonance frequency was 9 MHz (AT-cut). (15) Moynihan, H.; Lee, C.; Clark, W.; Wang, N.-H. Biotechnol. Bioeng. 1989, 34, 34951-34963. (16) Vasudevan, P.; Ruggiano, L.; Welland, R. Biotechnol. Bioeng. 1990, 35, 1145-1149. (17) Schussel, L.; Atwater, J. Chemosphere 1995, 30, 985-994. (18) Paddeu, S.; Fanigliulo, A.; Lanzin, M.; Dubrovsky, T.; Nicolini, C. Sens. Actuators 1995, 25, 876-882. (19) Chandler, H.; Cox, J.; Harley, K.; MacGregor, A.; Premier, R.; Hurrel, J. J. Immunol. Methods 1982, 53, 187-194. (20) Furusawa, K.; Norde, W.; Lyklema, J. Kolloid-Z. Z. Polym. 1972, 250, 908916.

For the multilayer shell formation, ∼1011 latex particles were added to a 2-mL Eppendorf centrifuge tube followed by the addition of polyions, nanoparticles, and urease to give shell architectures of the following sequence: {PDDA/PSS/PDDA/ 40-nm silica/PDDA/(urease/PDDA)1-4} or {PDDA/PSS/PDDA/ 12-nm magnetite/PDDA/(urease/PDDA)1-4}. After addition of each species, 15 min was allowed to elapse so that saturation adsorption of the components on the colloid particles was reached. The coated latex spheres were then centrifuged at 10600g, and the supernatant containing the unadsorbed species was removed. This procedure was repeated four times after the adsorption step to avoid admixing of the sequentially deposited components, similar to the approach outlined previously.11-13 To determine the amount of adsorbed polyanion or protein, UV spectra of the supernatant were compared with those of the standard polyion or enzyme solutions prior to exposure to the particles. A Malvern Zetasizer-4 (microelectrophoresis), a Philips-CM12 transmission electron microscope operated at 120 kV, and a Hewlett-PackardAgilent 8453 UV-visible spectrophotometer were used to follow the growth of the enzyme multilayers on the colloid particles. The electrophoretic mobility of coated particles was obtained by taking the average of five measurements at the stationary level in airequilibrated pure water (pH ∼5.6) without added electrolyte. The mobilities (u) were converted to the electrophoretic (ζ) potential using the Smoluchowski relation ζ ) uη/, where η and  are the viscosity and permittivity of the solution, respectively. TEM samples were prepared by depositing a diluted suspension of the coated particles onto a carbon-coated copper grid. The mixture was allowed to air-dry for 1 min and the extra solution was then blotted-off. Urease Activity Assay. A colorimetric assay based on the hydrolysis of urea was used for the activity control of free and immobilized urease, as monitored by the pH-sensitive dye bromcresol purple. The enzymatic reaction was monitored by following the dye absorption at 588 nm.18,19 A control solution containing 25 mM urea, 0.015 mM bromcresol purple, and 0.2 mM EDTA was adjusted to pH 5.8. This solution was placed in a 3-mL UV cell and magnetically stirred. A known amount of the urease multilayer-coated PS spheres (or free urease for the calibration) was then added, and the kinetics were monitored by following the absorption at 588 nm. The solution changed from yellow to dark purple during the reaction, corresponding to a change in solution pH from 5.8 to 7.5. The increment of the absorbance with time was used to characterize the urease activity in the sample.18 RESULTS AND DISCUSSION Assembly of Urease Multilayers on QCM Electrodes. Urease multilayers were first constructed on QCM electrodes in order to establish the conditions for suitable multilayer growth. The QCM frequency shift, caused by the deposition of material on the electrode surface, can be related to the adsorbed mass and layer thickness of the material via the Sauerbrey relation.21 For the 9-MHz QCM electrodes used in this work, the following relationships between QCM frequency shift (∆F, Hz) and enzyme/ polyion mass (M, g) and thickness (L, nm), ∆M ) -0.87 × 10-9∆F and ∆L ) -0.017∆F, apply and were thus (21) Sauerbrey, G. Z. Phys. 1959, 155, 206-214.

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Figure 1. QCM monitoring (frequency change vs adsorption steps) of urease/PDDA assembly: {PDDA/PSS/PDDA/(urease/PDDA)5}.

used.4,6 Figure 1 shows the QCM results for the construction of a PDDA/PSS/PDDA/(urease/PDDA)5 multilayer film on a QCM electrode. The adsorption time for each deposition step was 15 min, which was previously determined to be sufficient for saturation adsorption of polyions at the concentrations used.4,5 The first three polyelectrolyte layers serve as a precursor film to provide a uniform charge and a smooth surface for subsequent urease deposition.4 A regular stepwise decrease in the QCM frequency was observed. The average ∆F was 235 ( 15 Hz for urease layer formation (deposition at pH 8) and 60 ( 7 Hz for the PDDA layer. These values correspond to a urease coverage of 6.4 mg/m2 and thickness of 4.0 nm and a PDDA layer thickness of 1.5 nm. The PDDA thickness calculated from the QCM data is in excellent agreement with values obtained for PDDA layers by other techniques.1,6 An increase in the urease adsorption time from 15 to 20 min did not yield thicker layers, indicating that 15 min was sufficient to achieve saturation adsorption of urease. The urease layer thickness of 4.0 nm in the urease/PDDA multilayers corresponds to the X-ray crystallographic dimensions of urease isolated from jack beans.22 The thickness of a urease monolayer adsorbed on a cationic poly(allylamine) surface (3.8 nm), determined by surface plasmon resonance,23 is in close agreement with the value found in this work. Multilayer growth similar to that found for the urease/PDDA system, as assessed by QCM (data not shown), was observed for the assembly of urease in alternation with poly(ethyleneimine) (PEI). The assembly of urease at pH 4.5 in alternation with PSS resulted in a frequency decrease of 520 ( 20 Hz for each urease layer. This corresponds to a thicker enzyme layer (8.8 nm) adsorbed at each deposition step, compared with that obtained when deposited at pH 8 in alternation with PDDA or PEI (4.0 nm). Both the urease/PSS multilayers and urease/PEI multilayers yielded very low catalytic activities compared with the urease/ PDDA multilayers (most likely due to substrate diffusion effects

and blocking of active sites of the enzyme13). Hence, the assembly of urease in alternation with PDDA is considered in detail in this work. Urease Multilayer Formation on PS Spheres. The conditions established for the successful assembly of urease multilayers on the planar QCM substrates were subsequently employed to form enzyme multilayer shells on microparticle templates (470nm PS spheres). The precursor film (PDDA/PSS/PDDA) with an additional outermost silica or magnetite nanoparticle layer provided a better surface for the formation of stable urease multilayer shells. Attempts to deposit urease onto PDDA/PSS/ PDDA-modified PS particles yielded a low enzyme amount in the shells. This effect was also observed in our earlier studies: polyion/protein films assembled on QCM electrodes with a roughness of ∼0.1 µm facilitated the formation of multilayers, whereas in some cases, it was only possible to form one layer on a (smoother) glass slide.4 For example, weakly attached enzyme layers can be removed from the substrate surface by the next incoming polyion via the formation of water-soluble polyelectrolyte-enzyme complexes, as was found for histone/DNA interactions.24 An improved stability with respect to adsorption (i.e., more strongly bound) of glucose oxidase multilayers deposited onto gold nanoparticle layers, compared with glucose oxidase deposited on a less rough substrate, was also observed.25 Hence, a primer nanoparticle layer was deposited on the PS spheres to increase the surface roughness and improve the adsorption stability of urease. The growth of the urease multilayers on the PS particles was first followed by microelectrophoresis. Figure 2 gives the zeta (ζ) potential of the latex particles coated with polyions, nanoparticles, and urease {PDDA/PSS/PDDA/40-nm silica or 12-nm magnetite/ PDDA/(urease/PDDA)1-4}. The ζ-potential alternates between negative and positive values, corresponding to the sequential adsorption of cationic and anionic species, respectively. This

(22) Jabri, E.; Lee, M.; Hausinger, R.; Karplus, P. J. Mol. Biol. 1992, 227, 934937. (23) Nabok, A.; Ray, A.; Hassan, A.; Yets, R.; Majeed, R. Proc. SPIE Conf. Smart Electron. MEMS 1999, 3673, 230-238.

(24) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 284, 797-801. (25) Decher, G. University Louis PasteursInstitute Charles Sadron, personal communication.

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Figure 2. ζ-Potential of the coated PS particles vs the number of adsorption steps for shell compositions of {PDDA/PSS/PDDA/40-nm silica/PDDA/(urease/PDDA)1-4} (circles) and {PDDA/PSS/PDDA/12nm magnetite/PDDA/(urease/PDDA)1-4} (squares). The coated PS latex particles were re-dispersed in air-equilibrated pure water (pH ∼5.6) prior to measurement of the electrophoretic mobilities.

indicates a reversal in surface charge of the PS particles. At step four, after silica or magnetite nanoparticle adsorption, a slightly less negative ζ-potential was observed than when PSS formed the outermost layer, but the subsequent adsorption of PDDA restored the higher positive ζ-potential of about +45 mV. At the next (sixth) step, urease was adsorbed and the ζ-potential decreased to -12 mV. This value is close to what would be expected since the measurements were performed at pH ∼5.6, and given that the isoelectric point of urease is 5. The seventh PDDA adsorption step again recharged the particle surface to about +40 mV. Repeatable and alternating ζ-potentials were observed with further deposition of urease and PDDA. Overall, Figure 2 qualitatively indicates the alternate adsorption of the deposited species with an associated change in the surface charge. This is a key aspect of the LbL assembly technique.1 UV-visible measurements were conducted in order to examine whether removal of adsorbed urease occurred from the particle surface (modified with the precursor nanoparticle layer) during the LbL buildup of the multilayers. The amount of adsorbed urease on the latex surface was determined by monitoring the UV absorption of the protein at 275 nm for the initial (standard) enzyme solution and the supernatant after adsorption was completed. (Urease layer formation onto the PS spheres was performed by adding a 2 mg/mL urease solution to the particles coated with the precursor film.) The absorption spectrum of the standard/blank solution is shown in Figure 3, spectrum 1. The measured UV-visible spectra of the supernatant of this urease solution after addition to the particles and centrifugation is displayed in Figure 3, spectrum 2. The spectrum reflects the nonadsorbed urease in the supernatant. From the difference in these two spectra, it is estimated that ∼20% of the total available urease adsorbed on the modified PS spheres. The next step involved the adsorption of the polycation PDDA. As reported previously, subsequent polycation adsorption can interact with adsorbed protein and remove it from the particle surface.13a However, the supernatant after PDDA was added to the ureasemodified PS particles did not show any maximum at 275 nm (Figure 3, spectrum 3). This indicates that no significant amount of urease was removed. Similar UV-visible spectra were recorded after each deposition step.

Figure 3. UV-visible monitoring of urease adsorption onto PS particles for the buildup of the shell architecture {(PDDA/PSS/PDDA/ 40-nm silica/PDDA/urease/PDDA}: UV-visible spectra of the (1) initial urease solution that was added to the latex suspension, (2) supernatant containing nonadsorbed urease, and (3) supernatant after the subsequent adsorption of PDDA.

Figure 4. Transmission electron microscopy image of a PS particle coated with {PDDA/PSS/PDDA/40-nm silica/PDDA/(urease/PDDA)3}.

Figure 4 shows a transmission electron microscopy (TEM) image of a PS sphere coated with {PDDA/PSS/PDDA/40-nm silica/PDDA/(urease/PDDA)3}. A rather uniform distribution of silica nanoparticles is obtained on the surface of the PS spheres. The diameter of the coated particles is ∼575 nm. Previous work has shown that TEM can be used to evaluate enzyme multilayer deposition on particles.13a However, in this study, the presence of nanoparticles on the surface of the PS spheres makes it difficult to discern the adsorbed enzyme. PS particles prepared in the same way with 12-nm magnetic particles showed a less uniform coating of nanoparticles (data not shown). The construction of the urease multilayers on colloid particles involved a number of centrifugation steps to separate the coated particles from free enzyme and polyelectrolyte. This process inevitably resulted in the loss of particles with each step. Hence, the QCM technique, which can measure several nanograms of adsorbed material, was used to obtain an estimate of the particle concentration. A 5-µL aliquot of a latex solution was placed on the surface of a QCM electrode and dried, and ∆F was measured. ∆F was then converted to a mass change, and the initial concentration of particles was calculated (assuming the shell mass is negligible). Experiments with particles of known concentration Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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Figure 5. Decrease of the PS particle concentration as a function of the number of adsorption steps. The data are derived from QCM weighing of an aliquot of the latex solution dried on the electrode after each adsorption step.

showed this method to be reliable to within (5% of the actual particle concentration. Figure 5 demonstrates the decrease of particle concentration for the formation of a multilayer film of {PDDA/PSS/PDDA/40-nm silica/PDDA/(urease/PDDA)3}. The initial concentration of 0.5 × 1011 particles/cm3 decreased to 0.2 × 1011 particles/cm3 after 11 deposition steps. The concentration drop was ∼8% for each deposition step accomplished with quadruple centrifugation. Thus, the relationship between latex concentration (C) drop and deposition step (n) can be expressed as a geometric progression Cn ) Coqn, where q ) 0.92. This provides a means to determine the number of particles, which is important for quantitative comparison of the enzymatic data (see below). Knowing the modified latex particle concentration (Figure 5), the diameter and surface geometry from TEM (Figure 4), and the amount of adsorbed urease, the thickness of the urease layer on the latex surface can be calculated. At step five of the assembly process the latex concentration was 0.33 × 1011 particles/cm3, or 0.66 × 1011 particles for a volume of 2 mL. The added amount of urease was 2 mg/mL × 1.5 mL ) 3 mg, and 20% of this amount (i.e., 0.6 × 10-3 g) was adsorbed onto the PS spheres. Thus, the amount of urease adsorbed onto one PS particle in one adsorption step is 9.1 × 10-15 g. Taking the protein density as 1.3 g/cm3, we can estimate the enzyme volume per particle as 7 × 10-15 cm3. The surface area of a 575 nm latex sphere modified with nanoparticles is ∼80% more than that of the corresponding surface area of a smooth sphere without nanoparticles adsorbed and is equal to 1.85 × 10-8 cm2. The urease mass coverage in a layer is 9.1 × 10-15 g/1.85 × 10-8 cm2 ) 4.9 ( 1.0 mg/m2. This is only an estimation and the error in this value is about (20%. Nevertheless, the urease coverage obtained in this way is within experimental error of that measured by QCM on a planar substrate (6.4 ( 0.5 mg/m2; see earlier) and suggests that approximately one monolayer of urease is deposited on the particle surface with each deposition step. Similar values are obtained, despite the fact that a drying step was employed after deposition of urease in the QCM experiments. This implies that the drying step after deposition of each urease layer on the QCM electrode surface does not have a significant influence on the urease layer thickness. This was confirmed by drying urease/PDDA layers at every other deposition step or at every fourth deposition step in QCM experiments. 4216 Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

Figure 6. Absorbance at 588 nm for the enzymatic activity assay of PS particles coated with one, two, and three layers of urease/ PDDA. The shell architecture is {PDDA/PSS/PDDA/45-nm silica/ PDDA/(urease/PDDA)1-3}.

In all cases, similar urease/PDDA bilayer thickness values were obtained with or without drying. Enzymatic Activity. Figure 6 shows the catalytic activity of the modified latex spheres containing one, two, and three urease/ PDDA layers {PDDA/PSS/PDDA/45-nm silica/PDDA/(urease/ PDDA)1-3}. For these data, an equal amount of urease (0.2 mL) was added to the modified latex spheres in 2.8 mL of 0.025 M urea solution. The solution pH increase due to ammonia production was monitored by the pH sensitive dye, bromcresol purple. The absorbance of this dye at 588 nm increases linearly with pH in the range pH 5-7.5. For all three samples, the same number of PS spheres was used, taking into account the concentration correction coefficient following the curve in Figure 5. The urease enzyme activity was determined from the rate of ammonium production with time.18,19 For the calculations, the data between 1000 and 1800 s were used as the slope of each curve in this region is almost constant. The differences seen in the absorbance curves reflect the different amount of urease in the multilayer shells. The activity increment for one, two, and three urease layer-coated PS spheres is 0.4 × 10-3, 0.7 × 10-3, and 1.0 × 10-3 absorbance units/ s, respectively. The mass of urease in these one-, two-, and threelayer shells is about 9 × 10-17, 17 × 10-17, and 24 × 10-17 g (error (20%), as determined from UV-visible measurements of the supernatant solutions after urease adsorption. These data reveal an approximately linear relationship between the enzymatic activity and immobilized urease content in the multilayer shell. To compare the above activity values with the activity of free urease in solution, different amounts of urease solution were added to a similar assay and the urease activity was measured. An addition of 25 µL of 0.01 mg/mL free urease corresponded approximately to the activity of 0.2 mL (0.2 × 1011 particles/cm3) of triple-layer urease shells on PS particles. Such referencing of urease/latex sphere catalytic activity to the activity of a known amount of free urease revealed that the activity of immobilized urease (in a triple-layer shell) was 25% of that of free enzyme. This is a reasonable decrease because of substrate diffusion limitations and difficulties in reaching the active centers of immobilized urease. For comparison, the activity of glucose oxidase immobilized on latex decreased 10% for the first three layers and 50% for four to five polyelectrolyte layers deposited on

top of the enzyme.13a It is interesting to note that urease multilayer assemblies with a different polycation (PEI) (at pH 8) or polyanion (PSS) (at pH 4.5) gave ∼100 times lower immobilized enzyme activities compared to the urease/PDDA system, despite being assembled in a similar fashion (according to the shell architectures). This may be due to the different compactness of the multilayers resulting from different polymer conformations on the surface (e.g., PDDA is a linear polycation and PEI is a branched polycation). Another characteristic feature of the urease catalytic reaction is a 10-min dead time, during which no product was detected. A similar dead time was observed for low concentrations of free urease and, probably, is connected to accumulation of the reaction product. Magnetic Functionalization of Particles. In a separate experiment, 12-nm-diameter Fe3O4 nanoparticles were deposited in the shell architecture to give the particles a magnetic function. The shell sequence was {PDDA/PSS/PDDA/12-nm Fe3O4/ PDDA/(urease/PDDA)1-4}. The Fe3O4 nanoparticle distribution on the surface of the latex spheres is less uniform than the silica shell (Figure 4) and more than monolayer coverage (data not shown). Nevertheless, the absolute enzymatic activity of the magnetic catalytic particles was similar to that of the corresponding particles with a layer of 40-nm silica. In addition, an approaching 3-kG permanent magnet to the tube wall resulted in the collection of all of the modified latex spheres (on a wall region closest to the magnet) in ∼30 s. Immersing the permanent magnet into the solution containing the magnetic/urease-coated particles resulted in their collection on the magnet. This added magnetic

function is particularly useful in applications where separation and reuse of such particles is required. CONCLUSIONS The preparation, characterization and application of urease multilayer-coated PS particles with defined shell architectures have been demonstrated. The inclusion of nanoparticle interlayers (40nm silica or 12-nm magnetite) yields a stable assembly of urease/ PDDA multilayer shells on the core carriers. The catalytic activity of the bioparticles increased proportionally with the amount of urease in the shells, and was controlled by varying the urease layer number. The introduction of magnetic nanoparticles in these biocolloids readily allowed their removal from the reaction zone in solution. The stable, multifunctional biocolloid nanoreactors prepared combine the benefits of high surface area with easy and rapid separation (through the magnetic function), thereby making them attractive for use in various applications. ACKNOWLEDGMENT C. Schu¨ler is thanked for discussions on the enzyme assembly regimes and M. Spasova for assistance with TEM. This work was supported by the BMBF and the Max Planck Society.

Received for review January 25, 2001. Accepted June 21, 2001. AC010118D

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