Polyelectrolyte-Assisted Immobilization of Active Enzymes on Glass

polycation significantly enhances biosensor sensitivity. Colm P. McMahon , Gaia Rocchitta , Pier A. Serra , Sarah M. Kirwan , John P. Lowry , Robe...
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Langmuir 2001, 17, 5361-5367

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Polyelectrolyte-Assisted Immobilization of Active Enzymes on Glass Beads John P. Santos, Eric R. Welsh, Bruce P. Gaber, and Alok Singh* Center for Biomolecular Science and Engineering, Code 6930, Naval Research Laboratory, Washington, DC 20375 Received February 19, 2001. In Final Form: June 12, 2001 Using alternating layers of charged polymers, we have constructed reactive thin films by incorporating enzymes, specifically alkaline phosphatase (AP) and glucose oxidase (GOD), into multilayers of a polycation, branched polyethylenimine (PEI), and a polyanion, poly(styrenesulfonate) (PSS), supported on a glass substrate. Experiments using a quartz crystal microbalance (QCM) demonstrated that the films grew sequentially on a solid support, while X-ray photoelectron spectroscopy (XPS) further confirmed the growth and deposition of successive enzyme layers. For both enzymes, the reactive films demonstrated increased activity with the successive number of deposited enzyme layers. In hybrid films, consisting of alternating layers of AP and GOD, both enzymes retained activities similar to those of their corresponding films of either enzyme alone. The effect of elevated temperature was also investigated for these reactive films. Increased thermal stability was found to be associated with the increase in the number of deposited enzyme layers.

Introduction Multifunctional thin films have attracted a great amount of attention due to their burgeoning scientific applications in the areas of biotechnology and biomaterials science. Ideally, films should be impervious to changes in environmental conditions such as organic solvents, change in pH, ionic strength, and temperature. Several methods, including Langmuir-Blodgett,1-4 covalent binding,5-12 and spontaneous adsorption from solution,13-15 have been used to form catalytic thin films. While each of these methods is a valid construction technique, they generally are used to fabricate two-dimensional films and thus are limited in the amount of substance deposited by the surface area of the chosen substrate. However, another method involving alternating layers of oppositely charged polyelectrolytes, denoted layer-by-layer self-assembly, has recently emerged as a successful alternative for the controlled formation of highly ordered, three-dimensional, * To whom correspondence should be addressed. (1) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1991, 205, 113. (2) Ahluwalia, A.; De Rossi, D.; Monici, M.; Schirone, A. Biosens. Bioelectron. 1991, 6, 133. (3) Barraud, A.; Perrot, H.; Billard, V.; Martelet, C.; Therasse, J. Biosens. Bioelectron. 1993, 8, 39. (4) Tronin, A.; Dubrovsky, T.; De Nitti, C.; Gussoni, A.; Erokhim, V.; Nicolini, C. Thin Solid Films 1994, 238, 127. (5) Brandow, S. L.; Chen, M.-S.; Aggarwal, R.; Dulcey, C. S.; Calvert, J. M.; Dressick, W. J. Langmuir 1999, 15, 5429-2432. (6) Rozsnyai, L. F.; Benson, D. R.; Fodor, S. P. A.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1992, 31, 759-761. (7) Bruening, M. L.; Zhou, Y.; Aguilar, G.; Agee, R.; Bergbreiter, D. E.; Crooks, R. M. Langmuir 1997, 13, 770-778. (8) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (9) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498-1511. (10) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2272-2276. (11) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2267-2271. (12) Leggett, G. J.; Roberts, C. J.; Williams, P. M.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. Langmuir 1993, 9, 2356. (13) Lin, J. N.; Crake, B.; Lea, A. S.; Shansma, P. K.; Andrade, J. D. Langmuir 1990, 6, 509-511. (14) Wahlgren, M.; Arnebrant, T.; Paulsson, M. A. J. Colloid Interface Sci. 1993, 158, 54-63. (15) Blomberg, E.; Claesson, P.; Froberg, J.; Tilton, R. Langmuir 1994, 10, 2325-2334.

multifunctional, reactive, thin films.16 Subsequently, this method has been widely adapted in the formation of films containing biological molecules such as proteins,17-21 viruses,22 enzymes,23-27 and dendrimers.28 In most cases, the factors or driving forces that direct self-organization include steric interactions, surface tension, van der Waals forces, capillary forces, and electrostatic interactions. For polyions, either polymeric or biological, the driving force for the assembly is believed to be primarily due to the electrostatic attraction and complex formation between polyanions and polycations. The layer-by-layer self-assembly technique involves the stepwise adsorption of charged species onto a charged substrate to produce a self-assembled multilayered film. In these films, multilayers are considered to have their charged constituents ionically bound to adjacent polyelectrolytes. Generally, electrostatic interactions are believed to be less demanding sterically than covalent linkages, and this leads to more retained activity for adsorbed enzymes. Using this fact, along with the prospect of being able to assemble three-dimensional, reactive (16) Decher, G.; Hong, J. Makromol. Chem., Macromol. Symp. 1991, 46, 321-327. (17) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26, 5396-5399. (18) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117-6123. (19) Lvov, Y.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (20) Decher, G.; Lehr, B.; Lowack, K.; Lvov, Y.; Schmitt, J. B. Biosens. Bioelectron. 1994, 9, 677-684. (21) Caruso, F.; Nikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427-3433. (22) Lvov, Y.; Haas, J.; Decher, G.; Mo¨hwald, H.; Mikhailov, A.; Mtchedlishvily, B.; Morgunova, E.; Vainshtein, B. Langmuir 1994, 10, 4232-4236. (23) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163-167. (24) Caruso, F.; Trau, D.; Mo¨hwald, H.; Renneberg, R. Langmuir 2000, 16, 1485-1488. (25) Caruso, F.; Schu¨ler, C. Langmuir 2000, 16, 9595-9603. (26) Pommerscheim, R.; Schrezenmeir, J.; Vogt, W. Macromol. Chem. Chem. Phys. 1994, 195, 1557-1567. (27) Kong, W.; Zhung, X.; Gao, M. L.; Zhou, H.; Li, W.; Shen, J. C. Macromol. Rapid Commun. 1994, 15, 405-409. (28) Watanabe, S.; Regan, S. L. J. Am. Chem. Soc. 1994, 116, 88558856.

10.1021/la0102556 CCC: $20.00 © 2001 American Chemical Society Published on Web 07/27/2001

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enzyme layers, the layer-by-layer technique is a potentially promising technique for biotechnology. Schematically, in layer-by-layer self-assembly technique a charged substrate is brought into contact with an oppositely charged polyelectrolyte polymer. This process facilitates overcompensation and a reversal of surface charge, leaving substrate with oppositely charged surface. Afterward the sample is washed with water and then brought into contact with a charged polyelectrolyte polymer, where the same situation occurs except now the substrate acquires a net surface charge of the electrolyte used. The adsorption of polyelectrolytes produces a polymer brush consisting of randomly arranged coils and loops that can be further modified with successive adsorbed layers. Charges on the outermost polyelectrolyte layer were tuned to accommodate the charge of chosen biomolecule. At this point, the process of neutralization and reversal of charges on surface can be repeated to produce a film of alternating polymer and biomolecule. This process can be adapted to any system that contains a surface charge, and all subsequent components may be interchanged as needed to achieve the desired film composition. In this report, we provide a detailed investigation of the formation of alkaline phosphatase and glucose oxidase multilayers on glass beads, their catalytic activity, and their thermal stability. Quartz crystal microbalance and X-ray photoelectron spectroscopy were used to characterize the formation of enzyme multilayers on planar surfaces and glass beads, respectively. Kinetic activity and thermal stability were investigated as a function of adsorbed enzyme layers. Hybrid films containing both AP and GOD were used to investigate independent catalytic as well as the effect of overall enzyme layer separation and permeability of enzyme substrates. Experimental Section Materials. Branched, poly(ethylenimine) (PEI), Mw 60 000, p-nitrophenyl phosphate (NPP), o-dianisidine dihydrochloride, β-D-glucose, tris(hydroxymethyl]aminomethane (Tris), and alkaline phosphatase (AP) from bovine intestinal mucosa were obtained from Sigma (St. Loius, MO). Poly(sodium 4-styrenesulfonate) (PSS), Mw 70 000, 1,4-piperazinebis(ethanesulfonic acid) (PIPES), and potassium phosphate were obtained from Aldrich Chemical Co. (Milwaukee, WI). Glucose oxidase (GOD) from Aspergillus niger and horseradish peroxidase were obtained from Worthington Biochemical Corp. (Lakewood, NJ). For all adsorption procedures, the polyelectrolytes were diluted to concentrations of 1 and 3 mg/mL and adjusted to the pH of 8.0 and 6.8 using NaOH and HCl for the polycation, PEI, and polyanion, PSS, respectively. Enzyme solutions were prepared and adjusted to a final concentration of 1 mg/mL for multilayer deposition; alkaline phosphatase was prepared in 10 mM Tris (pH 8.0) while glucose oxidase was prepared in 10 mM PIPES (pH 6.8). All materials were used as obtained from the manufacturer. Water was obtained from a Millipore Milli-Q purification system and had a resistivity of 18.2 MΩ‚cm. Glass beads, 30-50 µm, were obtained from Polysciences, Inc. (Warrington, PA). Prior to use all glass beads were cleaned with the alkaline detergent RBS-35 (Pierce, Rockford, IL). Beads were placed in a solution of detergent (2% v/v) and sonicated in a water bath for a period of 15 min, washed several times with water, washed three times with ethanol, and finally dried in an oven overnight at 120 °C. Quartz Crystal Microbalance (QCM) Measurements. Thin film assemblies of alternating polyelectrolyte/protein layers were monitored using a QCM. The quartz crystal microbalance measures the deposition or depletion of substances, using the Sauerbrey equation,29 based on the tendency of a piezoelectric crystal to change its natural oscillation frequency according to the addition or loss of mass on the crystal electrode. The QCM (29) Sauerbrey, G. Z. Phys. 1959, 155, 206-222.

Santos et al. device was fabricated in our laboratory and built upon the Hewlett-Packard 53131A Universal counter. All the electrodes utilized had polished gold surfaces, and their resonant frequency was stable for several hours at (2 Hz. For all QCM measurements the assembly of multilayers was monitored at ambient conditions, ca. 22 °C. Prior to the deposition of the desired enzyme, a polyion precursor layer was assembled on the resonator by first depositing a few layers of PEI and PSS. Resonators were initially suspended in a 1 mg/mL solution of PEI at pH 8.0, which was stirred for 15 min, after which samples were washed three times with water and then dried in a stream of nitrogen gas. Once the resonator equilibrated, usually within 5 min, the frequency shift was recorded. Next, a layer of PSS was deposited following the same method outlined above. This procedure was repeated until a precursor layer of (PEI-PSS)2PEI was formed. At this stage, resonators were modified with the desired enzyme. Procedures for enzyme deposition were identical to those outlined for the polyelectrolytes except that the enzymes were deposited at a concentration of 1 mg/mL and the incubation time was increased to 20 min. With QCM an estimation of the mass increase due to absorption is found according to the frequency shift. For our system, the apparent area of the quartz microbalance was 0.196 cm2 per side. Putting this number to Sauerbrey equation, we calculated a 1 Hz frequency shift, ∆F (Hz), from a mass of 2.14 ng.29-31 Furthermore, frequency shift permitted calculation of deposited film thickness according to the following relationship:

d (nm) )

0.027(-∆F (Hz)) F

(1)

where F is the density of the adsorbed material: 1.2 g/cm3 for polyions32 and 1.3 g/cm3 for proteins using a spherical approximation.33 Enzyme Multilayer Formation on Glass Beads. The procedure for the modification of the glass beads is similar to that described above and has also been reported for the modification of colloidal particles.34-37 Essentially, 1 g of clean beads was placed in a vessel and suspended in 30 mL of PEI and mixed in vortex modified to accommodate 50 mL tubes for 15 min at room temperature. Then the beads were allowed to settle, the supernatant was decanted, and the beads were washed three times with approximately 30 mL of water. PSS was then deposited according to this method, and deposition was repeated until a precursor layer of (PEI-PSS)2-PEI was achieved. At this point, the beads were amenable to the deposition of the enzyme, either AP or GOD. Thirty millilitersof 1 mg/mL enzyme was added to the beads and mixed for 20 min and then washed three times with water. Next, a layer of PEI was deposited, and multilayer films were assembled by alternating between PEI and the enzyme until the desired number of enzyme layers was achieved to give a final film composition of (PEI-PSS)2-(PEI-Enz)x (x ) 2, 5, 8, 11). Hybrid beads, those containing both AP and GOD, were prepared in the same manner except GOD and AP layers were alternated in the deposition process until films of the nature (PEI-PSS)2-[(PEI-GOD)-(PEI-AP)]5 were fabricated. All samples were freeze-dried and stored at ambient conditions until needed. X-ray Photoelectron Spectroscopy (XPS). Samples were prepared by dusting powders onto double-stick tape and removing excess silica powder with a blast of dry N2. Spectra were obtained with a Perkin-Elmer Physical Electronics 5100 spectrometer using Mg KR radiation from a standard dual anode, nonmono(30) Ebara, Y.; Okahata, Y. Langmuir 1993, 9, 574−576. (31) Ebara, Y.; Okahata, Y. J. Am. Chem. Soc. 1994, 116, 1120911212. (32) Density of 1.2 g/cm has been applied for calculating the polyion layer thickness (see ref 18). (33) Creighton, T. E. Protein Structure, a Practical Approach; IRL Press: New York, 1990; p 43. (34) Voigt, A.; Lichtenfeld, H.; Sukhorukov, G. B.; Zastrow, H.; Donath, E.; Ba¨umler, H.; Mo¨hwald, H. Ind. Eng. Chem. Res. 1999, 38, 4037-4043. (35) Sukhorukov, G. B.; Donath, E.; Moya, S.; Susha, A. S.; Voigt, A.; Hartmann, J.; Mo¨hwald, H. J. Microencaps. 2000, 17, 177-185. (36) Chen, K. M.; Jiang, X.; Kimerling, L. C.; Hammond, P. T. Langmuir 2000, 16, 7825-7834. (37) Caruso, F.; Mo¨hwald, H. Langmuir 1999, 15, 8276-8281.

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chromatic source (400 W, 15.0 kV). All spectra were taken at 10-20°, to maximize signal while avoiding the tape as much as possible. Enzymatic Activity Assays. To measure the activity of the alkaline phosphatase modified beads, a requisite amount, about 20 mg, was placed in a quartz cuvette, and 0.95 mL of 0.1 mM NPP in 10 mM Tris (pH 8.0) was added to the beads. The cuvette was inverted several times to disburse the beads, and once the beads had settled to the bottom of the cuvette the absorbance was monitored at 405 nm for a period of 20 min. For the GOD modified beads the activity was measured by placing approximately 5 mg of beads in a quartz cuvette containing 100 µL of 18% (w/v) β-D-glucose, 33 µL of peroxidase of approximately 43 units/mL, and 833 µL of 0.26 mM o-dianisidine that had been oxygenated via gas sparging immediately prior to use. The reaction was mixed and the absorbance, after bead settling, at 460 nm was monitored for a period of 5 min. Initial reaction velocities, herein referred to as activity, are reported as absorbance units per minute per milligram of sample. Temperature-Dependent Measurements. Freeze-dried samples were placed in an oven overnight at the desired temperature, and the activity was determined following the procedures outlined above.

Results and Discussion QCM. The quartz crystal microbalance was utilized to monitor the sequential deposition of alternating polyelectrolyte and enzyme layers on a surface. Figure 1 demonstrates the growth of thin films on QCM resonators. A precursor film, consisting of five alternating layers of PEI and PSS [(PEI-PSS)2-PEI], was used for all QCM, as well as functional film formation, experiments. Therefore, even numbered layers denote the deposition of enzyme while the odd-numbered layers correspond to that of PEI after the fifth layer. This precursor layer was used to provide a stable film base for subsequent layer deposition. Since it is suspected that polyions grow upon a surface starting as patches that eventually grow into a complete film,36 the presence of a precursor layer helps alleviate the presence of defects that may occur in the initial stages of film growth. In the case of alkaline phosphatase, after the precursor layer was deposited onto the gold-coated QCM resonator surface, six layers of enzyme were deposited, and a progression of increased mass was noted (Figure 1a). For every subsequent layer of alkaline phosphatase deposited, compared to the previously deposited enzyme layer, there was an observed mass increase. It was also noticed that the first three layers did not increase linearly as did the fourth through sixth layers. Even though a precursor layer was originally deposited on the resonator, this discrepancy could be due to partial coverage or incomplete layer formation and supported the findings of van Duffel et al.38 However, since there was a general increase in deposited mass, the increase in mass may be attributed to the deposition of alkaline phosphatase. The effect of the suspected defects in the nascent film was neutralized as a linear increase in mass was observed after the fourth layer. Glucose oxidase deposition onto a resonator was likewise monitored using the quartz crystal microbalance (Figure 1b). Once again, the precursor layer was deposited onto the surface followed by 13 alternating layers of enzyme and PEI. In the case of glucose oxidase, a linear growth occurred after the second deposited enzyme layer, indicating uniform growth after the second layer of enzyme deposition, compared to after the third layer, as was the case for the alkaline phosphatase. This points to the (38) van Duffel, B.; Schoonheydt, R. A.; Grim, C. P. M.; De Schryver, F. C. Langmuir 1999, 15, 7520-7529.

Figure 1. QCM profile of deposition of alkaline phosphatase and glucose oxidase multilayers. (A) Six layers of AP deposited on the gold resonator. Filled circles are AP and open circles are polyelectrolytes. (B) Thirteen layers of GOD deposited on a gold resonator. Open circles are polyelectrolytes and filled circles are GOD. The filled diamond represents the 10th layer that had a 1 h deposition time. The filled triangle is 5 min incubation time in the 12th layer, and the filled square corresponds to an additional 15 min of GOD deposition.

possibility that a precursor of more than two and probably on the order of five alternating layers of PEI and PSS are needed for the linear growth of enzyme layers when using the layer-by-layer deposition technique. In both experiments there appeared to be some mass lost upon the addition of PEI during the enzyme deposition stages. This is not uncommon in these types of experiments and is often attributed to the possible disruption of polyelectrolytes and enzymes from the underlying layers due to either ion displacement or charge neutralization in the underlying layers.39 According to these measurements, the PEI/PSS layer thickness, as calculated from equation 1, was determined to be 6-8 nm, which is consistent with other published data.18,37,40-50 Using (39) Schlenoff, J. B.; Ly, H.; Li, M. J. Am. Chem. Soc. 1998, 120, 7626-7634. (40) Cheung, J. H.; Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2712-2716. (41) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo¨hwald, H. Macromolecules 1999, 32, 2317-2328. (42) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 4559-4565. (43) Delcorte, A.; Bertrand, P.; Wischerhoff, E.; Laschewsky, A. Langmuir 1997, 13, 5125-5136. (44) Pfau, A.; Schrepp, W.; Horn, D. Langmuir 1999, 15, 3219-3225. (45) Leporatti, S.; Voigt, A.; Mitlo¨hner, R.; Sukhorukov, G.; Donath, E.; Mo¨hwald, H. Langmuir 2000, 16, 4059-4063.

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the Sauerbrey equation, the deposited mass can be calculated from the measured frequency shift, and it was observed that the change in mass for successive layers was 16.0 ( 4.2 ng of alkaline phosphatase and 4.05 ( 1.3 µg of glucose oxidase. Overcompensation of charge and, therefore, a reversal of surface charge upon adsorption of successive layers is essential for layer-by-layer self-assembly. Previous work determined the conditions needed for this to occur with various polyelectrolytes using UV-vis absorption spectroscopy.40,51 In the earlier study,40 adsorption of polyelectrolytes occurred rapidly, with maximum adsorption happening in about 5 min for more concentrated solutions and about 25 min for the dilute solution (1 × 10-4 M). No additional adsorption was observed in either case even when the adsorption time was increased up to 24 h; thus, it is a self-limiting or self-assembling adsorption process. In the later study,51 assembly was demonstrated to occur on the order of 1-2 min and be independent of the polyelectrolyte molecular weight. On the basis of the work performed on the immobilization of immunoglobulin G,21 a 20 min deposition time was utilized. In this study, the 20 min time frame was supported by QCM. In Figure 1b, the 10th layer of GOD was incubated for a period of approximately 1 h, and a deposited mass of 4959 ng was observed. When the resonator was incubated for a 5 min time period, as seen in the 12th layer (Figure 1b), significantly less GOD was deposited, 2660 ng. This sample was further incubated for 15 min to bring the total incubation time up to the standard 20 min, and the amount deposited, 5415 ng, was demonstrated to be in line with the preceding and following results. Hence, the amount of glucose oxidase deposited when using extended time allotments was not significantly higher than the amount observed for the normal 20 min time period. Hence, incubation times greater than 20 min were not needed for the successful deposition of enzymes in the layer-by-layer self-assembly technique, and additional adsorption onto a partially formed layer was possible and produced a saturated or complete film, as observed from the additional incubation time experienced in the 12th layer. XPS. In an attempt to map enzyme deposition on glass beads, alkaline phosphatase-modified glass beads were analyzed using X-ray photoelectron spectroscopy. Glass beads are a sodium silicate and thus contain sodium, silicon, and oxygen. Both polyelectrolytes, PEI and PSS, are sources of carbon, and PEI also contains nitrogen; however, sulfur is only present in PSS. Alkaline phosphatase contains oxygen, nitrogen, and carbon but no silicon, sodium, or sulfur. While PEI and PSS contain aliphatic and aromatic carbon atoms, they do not contain any distinctive carbon functional groups. However, the alkaline phosphatase does contain carbonyl functionalities, and the signal arising from these can be deconvoluted from the total carbon signal acquired. On the basis of the elemental constituents, the sulfur (2p), sodium (1s), carbon (1s), silicon (2p), nitrogen (1s), and oxygen (1s) signals (46) Caruso, F.; Nikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422-3426. (47) Dubas, S. T.; Schlenoff, J. B. Macromolecules 1999, 32, 81538160. (48) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1996, 12, 3038-3044. (49) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 285, 797-801. (50) Ruths, J.; Essler, F.; Decher, G.; Riegler, H. Langmuir 2000, 16, 8871-8878. (51) Wang, L.; Fu, Y.; Wang, Z.; Fan, Y.; Zhang, X. Langmuir 1999, 15, 1360-1363.

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Figure 2. XPS data for 11 deposited AP layers on glass beads. (A) Circles are the Si (2p), triangles are O (1s), diamonds are C (1s), and the squares are N (1s). (B) Triangles are S (2p) and hexagons are Na (1s).

were measured on plain glass beads, beads with a deposited precursor layer, and 2, 5, 8, and 11 layers of alkaline phosphatase (Figure 2). As expected, the plain glass beads contain some nominal amount of silicon, oxygen, sodium, and carbon but no sulfur or nitrogen signal. As consecutive layers were adsorbed, the signal for silicon, oxygen, and sodium decreased by 96%, 64%, and 95%, respectively. Since XPS only probes a specific depth, depending on the takeoff angle, these results demonstrate a loss in glass signal due to the buildup of layer thickness. Both the carbon and nitrogen signals increased by 74% and 92%, respectively, where nitrogen was calculated from its initial layer of detection, which was the precursor layer. The carbon signal started out at a relative amount of 42.3%, which could be associated with adsorbed hydrocarbons on the glass surface, and increased to 73.4% and appeared to plateau at about this level after the fifth deposited enzyme layer. Since the carbon signal is attributed to both the PEI and alkaline phosphatase, it appeared that there was a steady state of adsorbed polyion and enzyme at this stage in the film formation. Nitrogen initially started out at below detectable levels and increased up to values above 10% after the fifth layer, and it was first detected in the precursor layer, as expected, when there was some appreciable nitrogen due to the deposited PEI. The nitrogen signal leveled off like the carbon signal, again suggesting a steady-state signal originating from both the enzyme and polyelec-

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trolyte. Sulfur was the only element that did not follow either a generally increasing or decreasing trend and can only have its signal ascribed solely to one source. Poly(styrenesulfonate) was the only source of sulfur in the outlined procedure; therefore, it can serve as a tracer for the deposition of PSS and the precursor layer as a whole. It was observed that the sulfur signal began at levels below the detection limit of the system and increased to a measurable amount of 1.24% in the precursor layer. This is definitive proof that there was some PSS and thus a precursor layer deposited on the glass beads. Upon further addition of layers, the sulfur signal fell to a value of less than 0.5% and dropped off a total of 75% from it first detected amount in the precursor layer. This was also definitive proof that substances were being deposited on the surface of the glass beads, after the precursor layers. Signals acquired via X-ray photoelectron spectroscopy are an accumulation from all the functionalities of a single atom. A carbonyl group produces a signature that is different and distinguishable from the signal produced by an aliphatic carbon, and this fact was used to distinguish between substances in the current study. Neither polyelectrolyte contains a carbonyl group while the alkaline phosphatase does contain acidic carbons in its structure; thus, a signal attributed to this functional group would originate only from the deposition of the enzyme. Figure 3 shows a series of deconvoluted carbon (1s) signals. In the first two spectra, representing the plain glass beads and beads with a precursor layer adsorbed, Figure 3, a and b, respectively, peaks centered around 288 and 289 eV are evident. However, in the spectra for 5 (Figure 3c) and 11 (Figure 3d) alkaline phosphatase layers deposited onto glass beads, there appears to be a slight peak shift where the original peaks are centered around 286 and 288 eV, and there is also a third, higher energy peak that is centered around 289 eV. This third, higher energy peak was attributed to the acidic carbons, and since the only source of a carbonyl is found in alkaline phosphatase, this provided proof that the enzyme was actually deposited and present on the sample. Activity. Recently Caruso et al. demonstrated, using glucose oxidase and peroxidase, the deposition and activity of enzymes on polystyrene latex beads that were less than 1 µm in diameter by the layer-by-layer self-assembly technique.25 In the current work, glass beads of significantly larger diameter, 30-50 µm, were modified with either AP or GOD using the layer-by-layer self-assembly technique, producing samples of 2, 5, 8, and 11 layers of enzymes. Parts a and b of Figure 4 demonstrate the activities obtained for the AP and GOD, respectively, with both demonstrating similar characteristics. In Figure 4a, a linear increase in activity, as a function of deposited layer of alkaline phosphatase, was evident and pronounced. As successive layers of enzymes were deposited, there was an added amount of enzyme per milligram of glass beads to produce more activity per bead and, consequently, progressively higher activity. Another characteristic observed in Figure 4a,b was that, as the number of layers increased, so did the error involved in measuring the activity. This was interpreted to mean that as more layers were deposited there was more discrepancy in the overall structure of the films as was previously proven by TEM,34,37,52 atomic force microscopy,42,45 and scanning electron microscopy.37,42 In the layer-by-layer self-assembly technique, it was demonstrated that, initially, the first layers do not cover the (52) Caruso, F.; Lichtenfeld, H.; Giersig, M.; Mo¨hwald, H. J. Am. Chem. Soc. 1998, 120, 8523-8524.

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Figure 3. Deconvolution of the XPS C (1s) signal: (A) bare glass beads; (B) precursor layer of (PEI-PSS)2-PEI; (C) five layers of AP deposited on glass beads; (D) 11 layers of AP deposited on glass beads. High-energy peak is observed in only in the alkaline phosphatase-modified beads.

whole substrate, and as subsequent layers were deposited gaps were filled in until a state existed where the other layers consist of whole and continuous layers of polyelectrolytes or other substances.38 While the precursor helped alleviate this problem, it was never eliminated and led to the formation of rougher surfaces compared to the substrate. Addition of either nanoparticles or macromolecules such as proteins and enzymes, which have molecular dimensions greater than the depth of a deposited polyelectrolyte, only helped perturb the system and introduced more discrepancy in the amount of deposited

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Figure 4. Activity of AP- and GOD-modified glass beads: (A) AP-modified beads; (B) GOD-modified beads. Both show and increase in activity as the number of layers is increased.

enzyme as the number of layers increased. This facet of the deposition scheme led to the greater error involved in the measurement of the activity as more enzymes were deposited. However, it was without a doubt that there was an overall increase in deposited enzyme, and thus the activity increased as more layers were added onto the glass beads. Despite the exact nature of the assembly, activity increased in proportion to the number of layers. Similar results for glucose oxidase-modified beads were observed. Once again, an apparently linear increase in activity was observed as the number of enzyme layers increased and was similar to trends previously measured using GOD;25 however, in the previous work, only five layers of GOD were deposited in the reported uncomplexed case. In the current work, there was still an increase in activity in the 8- and 11-layer systems for both the APand GOD-modified beads. This demonstrated that diffusion of the enzyme substrates through consecutive layers was not hindered as the number of PEI-enzyme layers increases. This was consistent with the observation that compounds with a molecular weight less than 5000 can rapidly penetrate polyelectrolyte multilayers.53 Hybrid beads, consisting of both alkaline phosphatase and glucose oxidase, (PEI-PSS)2-[(PEI-GOD)-(PEIAP)]5, were determined to contain activity for both AP and GOD separately as well as retain activity after the assay for one enzyme was performed. Beads were first checked for the activity of either AP or GOD (parent (53) Radtchenko, I. L.; Sukhorukov, G. B.; Leporatti, S.; Khomutov, G. B.; Donath, E.; Mo¨hwald, H. J. Colloid Interface Sci. 2000, 230, 272-280.

Santos et al.

Figure 5. Relative activity of GOD modified beads as a function of temperature. A) 8 layer beads at temperatures from22 to 130 °C. B) Relative activity of the 5, 8, and 11 layer GOD beads as a function of temperature. Triangles are 5-layer, circles are 8-layer, and squares are 11-layer beads.

enzyme), washed, dried, and then assayed to determine the activity of the previously unchecked (counter) enzyme. As demonstrated in Table 1, there was activity for both AP and GOD when first assayed as well as after the counterassay was previously performed. While there was some decrease, 57% and 69% for AP and GOD, respectively, in activity from the initial parent assay, it was not known whether this was due to environmental conditions or the coupling of the two systems. Also, measured activities obtained for the hybrid beads were on the order and actually slightly higher than the activities measured for the corresponding beads modified solely with either AP or GOD. In the hybrid beads, the five enzyme layers were actually spread out over the space of 10 enzyme layers; there were alternating GOD and AP layers in the formation of the films. This lends proof to the idea that the activity of enzyme layers on the inner portion of the multilayer films was similar to those on the outer portion of the multilayer films and was not hindered by the presence of outer layers of polyelectrolytes and enzymes. Temperature Dependence. Thermal stability of reactive thin films is a factor that limits their use in applications such as biosensors or bioreactors. Enzymes are known to denature at elevated temperatures due to the unfolding of their secondary and tertiary structures. One advantage of the layer-by-layer self-assembly technique is that it allows multicenter interactions, which help stabilize biological molecules such as proteins and DNA.50 Using polyelectrolytes to incorporate biological

Active Enzymes on Glass Beads

Langmuir, Vol. 17, No. 17, 2001 5367

Table 1. Activity of Enzymes (GOD and AP) in Hybrid Multilayers Deposited on Glass Beads; Activities are Reported in Units of Abs/min/mg parent enzyme activity activity after counter enzyme assay activity of parent enzyme deposited in five layers

molecules affords some inherent structural stability to the molecules and thus increases the thermal stability and allows for enzymes to remain active at elevated temperatures. Eight-layer glucose oxidase-modified beads were checked for activity after more than 12 h at elevated temperatures ranging from 50 to 130 °C (Figure 5a). Data are presented as the percent of activity relative to the respective beads at ambient conditions. As expected, activity did decrease at elevated temperature and beads became inactive at temperatures at and above 100 °C. However, beads remained active up to 90 °C; these beads still retained 7% of their activity at this elevated temperature. Previously, similar experiments were performed, and it was noticed that activity of GOD immobilized in polyelectrolyte films dropped to about 10% at 65 °C;25 however, these films were only exposed to elevated temperatures for a period of 10 min, and only one layer of enzyme was incorporated in the reactive thin film. At 60 °C glass beads modified with eight layers of GOD still retained 56% of their activity, and the activity did not fall to below 10% until the temperature was elevated to 90 °C. Increased number of deposited enzyme layers increased the thermal stability when incorporated in films of polyelectrolytes. To further investigate the added thermal stability with increased number of layers, 5- and 11-layer glass beads were also incubated for periods of greater than 12 h at elevated temperatures. Figure 5b shows further proof that there was greater thermal stability inherent to the samples with an increased number of enzyme layers. For all temperatures, the 11-layer beads demonstrated increased retained activity while the 5-layer beads retained the least amount of activity. This is conclusive evidence that increased thermal stability occurs with the increased number of deposited enzyme layers.

GOD

AP

0.0614 ( 0.0041 0.0188 0.0422 ( 0.029

2.63 × 10-4 ( 4.01 × 10-5 1.02 × 10-4 6.22 × 10-5 ( 2.89 × 10-5

Conclusions Glass beads were successfully utilized as a support for the layer-by-layer self-assembly of enzymes. The polyelectrolytes PEI and PSS were used to fabricate thin reactive films of the enzymes alkaline phosphatase and glucose oxidase. Films were grown in a manner which promoted sequential enzyme deposition, as proven by the quartz crystal microbalance results. Deposition times of greater than 20 min are not needed for the successful adsorption of enzymes, and additional enzymes can be deposited onto incomplete surfaces to produce saturated layers. XPS was utilized to demonstrate that the films were formed in a sequential manner. Films that have entrapped enzymes, both AP and GOD, of up to 11 layers demonstrated increased catalytic activity with increasing number of deposited layers. Diffusion of enzyme substrates was not affected by the presence of added polyion and enzyme layers, and multifunctional layers composed of different enzymes are possible. Assembled films demonstrated a decrease in activity with increasing temperature. However, increased thermal stability was shown to be associated with the increased number of deposited enzyme layers. It is the combination of increased catalytic and thermal stability that makes layer-by-layer self-assembly such an attractive technique. While there is some inherent loss of activity upon immobilization, with the increasing number of enzyme layers, reactive films can be constructed that approach the activity of the native enzymes in solution. Also, the increased thermal stability will aid in the formation of active, catalytic, thin films. Acknowledgment. This work was supported by the Office of Naval Research. J.P.S. is a NRC Research Associate. We thank Jacob Hirsch of the Polymer Science and Engineering Department, University of Massachusetts, Amherst for his help with XPS. LA0102556