Sustained Enzyme Activity of Organophosphorus ... - ACS Publications

Sustained Enzyme Activity of Organophosphorus. Hydrolase in Polymer Encased Multilayer Assemblies. Yongwoo Lee,† Ivan Stanish,† Vipin Rastogi,‡ ...
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Langmuir 2003, 19, 1330-1336

Sustained Enzyme Activity of Organophosphorus Hydrolase in Polymer Encased Multilayer Assemblies Yongwoo Lee,† Ivan Stanish,† Vipin Rastogi,‡ Tu-chen Cheng,‡ and Alok Singh*,† Center for Bio/Molecular Science and Engineering, Code 6930, Naval Research Laboratory, 4555 Overlook Avenue S.W., Washington, DC 20375, and US Army ERDEC, Aberdeen Proving Ground, Maryland 21010 Received August 12, 2002. In Final Form: November 22, 2002 Organophosphorus hydrolase (OPH), immobilized within polyelectrolyte multilayers deposited on glass beads (30-50 µm), showed sustained catalytic activity under ambient conditions for several months. OPH multilayers consist of two priming layers, branched poly(ethylene imine) (BPEI) and polystyrenesulfonate (PSS), followed by five alternating layers of BPEI and OPH. OPH multilayers coated on glass beads demonstrate catalytic activity similar to that of free enzyme, but over a much larger time scale (i.e., months vs hours). Stability of OPH multilayers coated on glass beads were also evaluated after depositing poly(acrylic acid) (PAA) as an outer priming layer for monomer assembly via electrostatic interactions. Three polymerizable monomers, 1,2-dihydroxypropyl methacrylate (DHPM), 1,2-dihydroxypropyl 4-vinylbenzyl ether (DHPVB), and N-[3-(trimethoxysilyl)propyl]ethylenediamine (TMSED), were anchored to the outer PAA layer and subsequently polymerized by UV irradiation or base-catalyzed hydrolysis. Polymerized DHPVB and TMSED rendered robust multilayer systems without affecting OPH activity. In aqueous sodium chloride (2 M for 2 h), OPH multilayers stabilized with an outer coating of TMSED remained 27% active as opposed to 0% activity for uncapped systems.

1. Introduction Current awareness about the effects of hazardous chemicals on human health and the environment has provided impetus to develop the means and methods to protect people (from these agents) consequent to routine handling or accidental spills. Pesticides are the most widely used chemicals outside laboratory environments and are structurally similar to known chemical nerve agents. Because of their extensive use, pesticides pose a real threat to the general public’s health. Prolonged exposure to dilute pesticide levels, especially in closed environments, may be more dangerous than a one-time exposure at a higher dose. Our interest in developing materials for continual degradation and removal of low level toxins led us to investigate the role of catalytic enzymes embedded within multilayer assemblies. For long-term application, chemical catalysts such as metal-ligand complexes1,2 are preferred over enzymes despite their relative low catalytic activity. Although enzyme shelf-life is generally limited, they do have a higher turnover rate relative to synthetic catalysts in addition to being more selective. Thus, enzyme immobilization either by covalent bonding3-5 or by physical entrapment6-10 is an ongoing scientific endeavor. In terms of enzyme immobilization, electrostatic adsorption is an attractive process because of its nonintrusive nature. Selection of weak polyelectrolytes and appropriate choice of pH can facilitate the surface modification step and reduce the complexities associated with covalent immobilization. * Corresponding author. Telephone: (202) 404-6060. Fax: (202) 767-9594. E-mail: [email protected]. † Naval Research Laboratory. ‡ US Army ERDEC. (1) Smolen, J. M.; Stone, A. T. Environ. Sci. Technol. 1997, 31, 1664. (2) Hartshorn, C.; Singh, A.; Chang, E. L. J. Mater. Chem. 2002, 12, 602. (3) Ogawa, K.; Wang, B.; Kofukuta, E. Langmuir 2001, 17, 4704. (4) Martinek, K.; Mozhaev, V. V. Pure Appl. Chem. 1991, 63, 1533. (5) Wasserman, B. P.; Hultin, H. O.; Jacobson, B. S. Biotechnol. Bioeng. 1980, XXII, 271.

10.1021/la0263965

Our previous efforts demonstrate that enzymes immobilized in polyelectrolyte multilayer assemblies retain their catalytic activity under relatively harsh conditions over long periods of time.11 While multilayer assemblies12 provide a versatile platform to fabricate multifunctional materials in research areas such as patterning,13 sensors,14,15 nonlinear optical materials,16-18 and electrochromic devices,19 there are a few drawbacks. For example, upon exposure at high salt concentration, multilayers swell and ultimately delaminate.20 In addition, prolonged enzyme exposure at high salt concentration leads to denaturation, hence loss of activity. Therefore, our current focus is to retain enzyme activity for working environments by preserving the structural integrity of multilayer assemblies. In this paper, we investigate the immobilization of organophosphorus hydrolase (OPH)21,22 within polyelectrolyte multilayers and the stabilization of OPH (6) Hong, J. D.; Lowack, K.; Schmidt, J.; Decher, G. Prog. Colloid Polym. Sci. 1993, 93, 98. (7) Lvov, Y.; Ariga, K.; Kunitake, T. Chem. Lett. 1994, 2323. (8) Anzai, J.-I.; Kobayashi, Y.; Nakamura, N. J. Chem. Soc., Perkin Trans. 2 1998, 461. (9) Forzani, E. S.; Solis, V. M.; Calvo, E. J. Anal. Chem. 2000, 72, 5300. (10) Caruso, F.; Trau, D.; Mohwald, H.; Rennenberg, R. Langmuir 2000, 16, 1485. (11) Santos, J. P.; Welsh, E. R.; Gaber, B.; Singh, A. Langmuir 2001, 17, 5361. (12) Decher, G. Science 1997, 277, 1232. (13) Zheng, H.; Lee, I.; Rubner, M. F.; Hammond, P. T. Adv. Mater. 2002, 14, 569. (14) Lee, S.-H.; Kumar, J.; Tripathy, S. K. Langmuir 2000, 16, 10482. (15) Decher, G.; Lehr, B.; Lowack, K.; Lvov, Y.; Schmidt, J. Biosens. Bioelectron. 1994, 9, 677. (16) Lvov, Y.; Yamada, S.; Kunitake, T. Thin Solid Films 1997, 300, 107. (17) Shimazaki, Y.; Ito, S. Langmuir 2000, 16, 9478. (18) Wang, X.; Balasubraminian, S.; Li, L.; Jiang, X.; Sandman, D. J.; Rubner, M. F.; Kumar, J.; Tripathy, S. K. Macromol. Rapid Commun. 1997, 18, 451. (19) Liu, S.; Kurth, D. G.; Mohwald, H.; Volkmer, D. Adv. Mater. 2002, 14, 225. (20) Dubas, S. T.; Farhat, T. E.; Schlenoff, J. B. J. Am. Chem. Soc. 2001, 123, 5368.

This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society Published on Web 01/23/2003

Enzyme Activity of OPH in Multilayer Assemblies

Figure 1. Schematic (not drawn to scale) representation of enzyme multilayer stabilization via an outer layer polymer net. (A) Formation of polymer net on enzyme-polyelectrolyte multilayers. (B) Hydrogen bonding association between a poly(acrylic acid) layer and end-capping monomers.

multilayer assemblies via polymer net encasement (Figure 1). Our strategy is to have an additional polyelectrolyte layer deposited to cushion the OPH layer from the endcapping monomers, which through polymerization produces a robust polymer net. 2. Experimental Section 2.1. Materials and Methods. Unless otherwise stated, all chemicals and reagents were used as received. Enzyme organophosphorus hydrolase (OPH, EC 3.1.8.1) was received as freezedried powder from Aberdeen Proving Ground, MD. Methyl parathion (MPT) was purchased from Chem Service (West Chester, PA). Glass beads (30-50 µm) were purchased from Polysciences, Inc. Bis-tris propane (BTP), CHES, branched poly(ethylene imine) (BPEI, MW 60,000), poly(acrylic acid) (PAA, MW 750 000), poly(sodium 4-styrenesulfonate) (PSS, MW ∼70 000), N-[3-(trimethoxysilyl)propyl]ethylenediamine (TMSED), ω-mercaptohexadecanoic acid (HMA), and p-nitrophenol (pNP) were purchased from Aldrich Chemical Company. Biuret and Folin & Ciocalteu’s phenol reagents were purchased from Sigma. RBS 35 detergent was purchased from Pierce (Rockford, IL). All solutions were prepared using Millipore, Milli-Q ultrapure water (18.2 MΩ‚cm). The quartz crystal microbalance (QCM) was a USI-100 microbalance from Sanya Tsusho Co. (Tokyo, Japan) linked with a Hewlett-Packard 53131A, 225 MHz universal counter. QCM measurements were carried out using a 0.340 in. diameter goldplated crystal of 9 MHz frequency. A frequency change was linked directly to the mass of polyelectrolyte deposited and was calculated using the Sauerbrey equation. A Beckman DU 650 UV-vis spectrophotometer was used for colorimetric enzyme assays. IR and NMR spectra were recorded on a Nicolet Impact 400 D IR spectrophotometer and a 400 MHz Bruker DRX-400 nuclear magnetic resonance spectrophotometer, respectively. Agitating beads for deposition was conducted using a Laboratory Rotator (Model 099A RD 4512, Glas-Col, Terre Haute, IN). The surface morphology of beads was examined using environmental scanning electron microscopy (ESEM) Electroscan Model E3 microscope (Wilmington, MA), operated at 20 keV. Samples were attached to a refrigerated Peltier stage maintained at 4 °C and imaged in an environment of water vapor (2-5 Torr) in a hydrated state. 2.2. Synthesis of End-Capping Monomers. Three endcapping monomers, 1,2-dihydroxypropyl methacrylate (DHPM), 4-vinylbenzyloxy-3-(1,2-dihydroxy)propane (DHPVB), and TMSED, were used in the study. Synthesis of DHPM was carried (21) Hill, C. M.; Li, W.-S.; Cheng, T.-C.; DeFrank, J. J.; Raushel, F. Bioorg. Chem. 2001, 29, 27. (22) Singh, A. K.; Flounders, A. W.; Volponi, J. V.; Ashley, C. S.; Wally, K.; Schoeniger, J. S. Biosens. Bioelectron. 1999, 14, 703.

Langmuir, Vol. 19, No. 4, 2003 1331 out by reacting equimolar amounts of solketal and methacryloyl chloride in dichloromethane at 7 °C. The resulting ester was isolated by column chromatography on silica gel (Rf 0.55 in 80:20 hexane:ethyl acetate) followed by acid hydrolysis to yield the desired monomer in 61% yield (Rf 0.34; 80:20 ethyl acetate: hexane). Similarly, DHPVB was synthesized by reacting a toluene solution of solketal with NaH. Slow addition of vinyl benzyl chloride to sodium alkoxide led to the formation of the intermediate, which upon acid hydrolysis afforded DHPVB in 39% yield (Rf 0.32 in 80:20 ethyl acetate:hexane). Both monomers were characterized by recording 1H NMR spectra. Monomers were protected from heat and light to avoid unwanted polymerization. 2.3. Deposition of OPH-Polyelectrolyte Multilayers. Conditions for multilayer deposition were first optimized on goldcoated QCM resonators. Before initiating layer deposition, the QCM resonators were cleaned by washing with ethanol, rinsing with deionized water, and thorough drying under a steady stream of nitrogen. For polyelectrolyte deposition, 1 mM BPEI solution and 3 mM PSS solution were prepared in deionized water and the pH was adjusted to pH 8.0 and pH 6.8, respectively, by addition of aqueous NaOH or HCl. After each polyelectrolyte deposition (10 min), gold resonators were rinsed with deionized water and dried with a gentle steady stream of nitrogen, and then the frequency shift was measured. Polyelectrolyte multilayers were built on glass beads (30-50 µm) by sequential immersion in their respective polyelectrolyte solution. All three polyelectrolytes used in this study were dissolved in water, and their pH was adjusted by adding dilute solution of hydrochloric acid or sodium hydroxide. After treatment with each polyelectrolyte, the substrates were briefly washed with deionized water and the supernatant was decanted to remove extraneous polyelectrolyte. Both glass beads and gold resonators were first modified by putting an initial BPEI layer, followed by deposition of three alternating layers of PSS-BPEI to make a BPEI-(PSS-BPEI)3- assembly, serving as a precursor layer. Thereafter, five alternating enzyme-polyelectrolyte layers were deposited. For glass beads the final configuration was silica(BPEI-PSS)3-(BPEI-enzyme)5. In a typical experiment, 2-3 g of glass beads was immersed in a 10 mL deposition solution of polyelectrolytes placed in a 20 mL centrifuge tube. The centrifuge tubes were mounted on a Laboratory Rotator spinning wheel at 35 rpm and agitated for 10 min. The same protocol was followed for the washing cycles subsequent to each deposition. Upon completion, the OPH multilayered beads were freeze-dried and stored at room temperature. 2.4. Determination of Total Protein Content and Enzyme Activity on Glass Beads. Protein assays were carried out to determine the amount of OPH immobilized within the multilayer assemblies on glass beads. Typically, 100 mg of glass beads was soaked in 1.5 mL of 2 M NaCl solution and agitated for 2 h using a vortex mixer and centrifuged for 3 min at 5000 rpm, and the supernatant was decanted. To a 0.2 mL aliquot from this supernatant, 2.2 mL of Biuret reagent and 0.1 mL of Folin and Ciocalteu’s phenol reagent were added and the contents were mixed. Absorbance of the resulting solution was monitored at 725 nm to calculate the protein content on the beads from the standard curves prepared using known concentrations of OPH. OPH activity in multilayers deposited on glass beads was determined by monitoring MPT hydrolysis. A 0.25 mM MPT solution was prepared in CHES buffer with methanol (at 15% v/v). In a typical run, the OPH-coated glass beads (50 mg) were placed in a 20 mL scintillation vial with 4 mL of 10 mM BTP (pH 7.8) containing 50 µM CoCl2‚2H2O and 1 mL of 10 mM CHES (pH 8.6) containing 0.25 mM methyl parathion. Beads in buffer solution were agitated using a vortex mixer. A 0.5 mL aliquot was withdrawn periodically, filtered through a sintered glass filter to remove particulates, and the absorbance measured at 405 nm for pNP. Absorbance values recorded at each time interval within the first 20 min were plotted against time to calculate the initial velocity of MPT hydrolysis. For determining OPH activity as a function of MPT concentration, 50 mg of catalytic material (i.e., glass beads containing OPH in multilayers) was placed separately in five scintillation vials. Each vial having a total volume of 5 mL contained 0.025, 0.05, 0.075, 0.1, and 0.2 mM MPT in CHES buffer at pH 8.6. A total of seven data points for

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Figure 2. QCM profile for deposition of OPH and polyelectrolytes on gold-coated resonators. The deposition sequence is bare gold-(BPEI-PSS)5-(BPEI-OPH)12. (Only every third data point/layer is presented.) each MPT concentration were collected over a period of 20 min by monitoring the absorption of pNP formed upon MPT hydrolysis. Absorbance values were converted to pNP concentration using standard curves prepared by plotting absorbance at 405 nm against known amounts of pNP. Enzyme activity measurements using diisopropyl fluorophosphate (DFP) substrate were made by monitoring F- release with an ion-specific electrode. The reaction mixture consisted of 50 mM (NH4)2CO3, 0.1 mM MnCl2, 3 mM DFP, and 0.3-0.4 U of enzyme or enzyme matrixes in a total volume of 2.5 mL. One unit (U) of enzyme catalyzes the release of 1.0 mmol of F- per minute at 25 °C. Specific activity is expressed as U per mg of protein. 2.5. Deposition and Polymerization of End-Capping Monomers on OPH-Polyelectrolyte Multilayers. QCM gold resonators were used to quantitatively confirm the deposition of end-capping monomers. Deposition of end-capping monomers was facilitated by depositing an additional PEI-PAA layer on the outermost OPH layer. The PAA layer (1 mM, pH 4.8) produced excess negative charges on the surface to facilitate electrostatic attraction of the end-capping monomers. Aqueous solutions of three monomers DHPM (1 mM), DHPVB (1.5 mM, pH 9.8), and TMSED (1 mM) were used for end-capping deposition. The gold resonators had the following multilayer configuration: goldHMA-(PEI-PSS)3-PEI-PAA-end-capping monomer. Stability of the multilayers deposited on QCM resonators was monitored via a change in frequency after immersing them in methanol, dichloromethane, acetonitrile, and 1 M NaCl solution. OPH multilayers on glass beads were prepared by the following protocol described in the previous section. Beads containing PAA as an outermost layer were treated with a 10 mL aliquot of endcapping monomer solution in a centrifuge tube mounted on a Laboratory Rotator at 35 rpm for 10 min and rinsed with water. Water was removed from the beads by freeze-drying. Glass beads had the following multilayer configuration: silica-(PEI-PSS)3(PEI-OPH)5-PEI-PAA-end-capping monomer. Polymerization of monomers deposited on gold resonators and glass beads was carried out by UV irradiation or by raising solution pH. Glass beads having the outer DHPVB monomer layer were mixed and photopolymerized (254 nm for 3 min). Glass beads having the outer TMSED monomer layer were polymerized by immersing them in 0.15% NH4OH solution with gentle agitation (30 s). Thereafter, the beads were rinsed with water, freeze-dried overnight, and assayed for enzyme content and activity.

3. Results 3.1. Deposition of Enzyme in Polyelectrolyte Multilayers on Flat Gold Surfaces. Figure 2 shows the weight increase of OPH and polyelectrolytes deposited on

Lee et al.

a gold-coated quartz crystal. Using QCM, a persistent linear increase in weight was observed with the addition of polyelectrolyte and enzyme layers. In this case, we built a total of 35 layers to confirm the trend in layer deposition. Our results show a large weight increase in the deposition of PSS as opposed to BPEI. However, enzyme deposition generally showed a greater weight increase than that of the BPEI polyelectrolyte. For the multilayer configuration gold-(BPEI-PSS)3-(BPEI-OPH)5, OPH deposition averaged 267 ( 25 ng per layer. For three separate priming laying experiments, an average weight gain per layer of 11.2 ( 4 ng was observed. In select cases, we used ω-mercaptohexadecanoic acid to form self-assembled monolayer (SAM) as a priming layer to build multilayers. Mass change in polyelectrolytes deposition was not noticeable between SAM-modified and bare gold substrates. Upon longer contact time or higher enzyme concentration, we did not observe an increase in enzyme weight. Enzyme multilayers were deposited using 0.1, 1.0, and 10.0 mg/mL OPH concentration. Increasing the polyelectrolyte surface charge by adjusting solution pH was not conducted due to the concern that pH-induced conformational changes may lead to enzyme inactivation regardless of the amount adsorbed to the multilayer glass beads. On average, 2.7 ( 1.3 mg of OPH per 100 mg of glass beads was deposited, based on three separate experiments. 3.2. Enzyme Activity in Polyelectrolyte Multilayers Immobilized on Glass Bead Substrates. Several enzyme-catalyzed reactions follow the Michaelis and Menten formulas23 (eq 1)

velocity (M s-1) ) Vmax[MPT] MPT

[MPT] + Km

)

kcat[enzyme][MPT] [MPT] + KmMPT

(1)

where Vmax signifies the maximum reaction rate (i.e., at saturation) and Km (i.e., the half-saturation constant) is a value at which half the maximal velocity rate occurs. Alternatively, Vmax can be expressed as a function of the total enzyme concentration and the irreversible forward rate of reaction (eq 1). For our enzyme multilayered system, we observe a hyperbolic profile against initial MPT concentration (Figure 3). However, due to mass transfer considerations (i.e., boundary layer, substrate diffusion through the multilayer, etc.), a more appropriate expression for MPT catalysis is given in eq 2:

initial TON (s-1) ≡

|

a[MPT]0 velocity (M s-1) ) [enzyme] b + [MPT]0 0 (2)

where a and b are kinetic parameters for the enzyme immobilized system that represent the apparent maximal turnover number (TON) and the apparent half-saturation constant, respectively. A least-squares fit to the data gives initial TON values of 0.01 s-1 and 37 µM for a and b, respectively. This Km value is close to that reported previously taking into account temperature differences via van’t Hoff’s relationship.24 On the other hand, the maximum turnover rate is noticeably lower than free enzyme, indicating that substrate accessibility does play a significant role in MPT hydrolysis for our system. (23) Bailey, J. E.; Ollis, D. F. In Biochemical Engineering Fundamentals; McGraw-Hill Inc.: New York, 1986; pp 86-156. (24) Lai, K.; Stolowich, N. J.; Wild, J. R. Arch. Biochem. Biophys. 1995, 318, 59.

Enzyme Activity of OPH in Multilayer Assemblies

Figure 3. Turnover rate as a function of initial MPT concentration for OPH multilayers deposited on silica spheres.

These enzyme beads were also evaluated for their performance against a nerve agent simulant, diisopropyl flourophosphate (DFP). The observed OPH turnover rate for DFP hydrolysis was 15.38 s-1. These results were collected from OPH multilayered samples post 2 weeks of preparation and room temperature storage. This rate is calculated based on the amount of enzyme immobilized within the multilayers. 3.3. Stabilization of Polyelectrolyte Multilayer Assemblies. To render OPH enzyme active over an extended period of time under chemically and mechanically stressful conditions, a polymer net was laid on the outermost surface to protect and physically restrain the polyelectrolytes and enzymes immobilized within them. Formation of the monomer layer(s) on the outer multilayer surface was confirmed by monitoring an increase in weight using QCM. The deposition conditions optimized for laying a monomer film on gold resonators were also applied to the glass bead samples. The conditions for encasement were first optimized on gold-coated QCM resonators, which were soaked sequentially into solutions of polyelectrolytes (BPEI and PSS) terminated with an outermost layer of PAA. Two polymerizable chemical agents, DHPM and DHPVB (Figure 1B), synthesized in our laboratory were used to end-cap the multilayer assemblies. QCM was used for monitoring the physisorption of these monomers. The results show no discernible weight increase during DHPM deposition. The DHPM monomer did not bind to the PAA surface even with prolonged exposure time (16 h), with higher concentrations (10 mM), and at higher pH (pH 10). A noticeable and reproducible weight gain was observed for DHPVB end-capped multilayers, indicative of a monolayer deposition (Figure 4). The DHPVB anchored to multilayers on gold were polymerized by UV irradiation with a hand-held UV lamp (at 254 nm for 3 min). Both sides of the gold resonator were exposed to UV, and no noticeable mass change was observed before or after UV irradiation. In addition, the chemical functionality of DHPVB end-capped polyelectrolytes was confirmed by grazing angle FT-IR (refer to the Discussion section). Using ESEM, the surface morphology of glass beads was evaluated before and after deposition of the polyelectrolytes, enzyme, and monomers. Thermally pretreated bead samples displayed a rough surface (Figure 5A). Upon further deposition of polyelectrolytes and OPH, surface roughness decreased significantly as shown in Figure 5B.

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Figure 4. QCM deposition profile of end-capping monomers on enzyme-polyelectrolyte multilayers (gold-(BPEI-PSS)2BPEI-PAA-end-capping monomer). O, multilayer sequence ending with DHPM; b and 1, multilayer sequence ending with TMSED and DHPVB monomers, respectively. The seventh layer is due to end capping.

Figure 5. ESEM images of glass beads after OPH multilayer deposition. (A) Unmodified glass beads. (B) Multilayered glass beads with PAA as the outer layer. (C) Multilayered glass beads with PAA with polymerized TMSED. (D) Multilayered glass beads with PAA with polymerized DHPVB. Bar represents 10 µm.

It appears that the initial polyelectrolyte deposition sequence acted to fill in the crater regions. Figure 5C,D shows that addition of the capping layer (TMSED and DHPVB) did not provide any additional structural information relative to Figure 5B. The surface remained structurally smooth as observed with ESEM. Two sets of TMSED end-capped multilayers on gold surfaces were deposited and polymerized by alkaline hydrolysis using 0.15% ammonium hydroxide for 30 s. Mechanical stability of the multilayer polyelectrolytes (i.e., the priming layer) was monitored against exposure to organic solvents for 5 min. This step may cause a mass increase due to solvent permeation into the multilayers or a mass loss due to polyelectrolyte delamination if the polymer net dissolved into the organic solvent. Dichloromethane, methanol, and acetonitrile were chosen mainly because these are the most common solvents found in

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Figure 6. Percent activity against salt stress of noncapped OPH multilayer beads, PAA-capped multilayer OPH beads, polymerized DHPVB on PAA-capped OPH multilayer beads, polymerized TMSED on PAA-capped OPH multilayer beads, and mildly polymerized TMSED on PAA-capped OPH multilayer beads. Data are presented as normalized percent activity (relative to the highest activity obtained for the TMSED capped system) of OPH-coated glass beads exposed to 2 M NaCl for 2 h.

working environments. A weight gain due to TMSED adsorption (1.86 µg) was not significantly altered when the multilayers were exposed to dichloromethane (1.78 µg), methanol (1.80 µg), and acetonitrile (1.76 µg) at room temperature for 5 min. No mass change in TMSED endcapped polyelectrolytes illustrates that the outer polymer net is rugged and resistant to organic solvents. 3.4. Enzyme Activity of Polymer-Encased OPHCoated Glass Beads. DHPVB and TMSED end-capped OPH multilayer assemblies on glass beads were prepared by sequential adsorption of PAA and end-capping monomers. Activity of polymer-encased OPH multilayers was determined immediately after their formation and compared with their activity subsequent to exposure in sodium chloride solutions. Beads obtained after constructing a polymer net involving polymerized DHPVB or TMSED showed enzyme activity comparable to beads without a polymer net. Initial activity of 1.8 × 10-9 M s-1 observed for OPH-coated glass beads was completely lost upon their exposure to 2 M NaCl solution (2 h). Under the same salt stress condition, OPH in multilayers coated with a PAA layer showed minimal activity (3% relative activity). DHPVB end-capped OPH-coated glass beads were found to be more active showing 12% retention of original activity. TMSED-coated OPH glass beads, retaining 27% relative activity, were found to be the most effective against salt stress. This could be a result of the formation of a highly cross-linked polymer net. Normalized percent activity (relative to the highest activity obtained for the TMSED-capped system) of OPH-coated glass beads exposed to 2 M NaCl solution is presented in Figure 6. 4. Discussion Prolonged enzyme stabilization in nonnative environments has been a subject of intensive research over the past decade.25 Using a minimally intrusive method of immobilization, we embed OPH within weakly charged polyelectrolyte multilayers, which provide shelter from relatively harsh physical and chemical environments. (25) Pastoriza-Santos, I.; Scholer, B.; Caruso, F. Adv. Funct. Mater. 2001, 11, 122.

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Weakly charged polyelectrolyte multilayers consisting of BPEI and PSS at near neutral pH (pH 8.0) were capable of immobilizing OPH dissolved in buffer (pH 7.8). OPH embedded in multilayers under these conditions showed hydrolytic activity over a period of 6 months when stored dry under ambient conditions. Electrostatic interaction allowed the assembly of stacked polyelectrolyte multilayers,26 and electrostatic interaction coupled with hydrogen bonding promoted OPH immobilization within the multilayers leading to sustained enzyme activity. Furthermore, we did measure the half-life for OPH activity in aqueous media (i.e., 100% humidity). In BTP buffer containing 50 µM CoCl2 (i.e., cofactor), free OPH has a half-life of 12 h at ambient conditions and in 5 half-lives (2.5 days) OPH has lost all of its activity. OPH hydrolyzes MPT to produce p-nitrophenol (pNP) and dimethoxyphosphinothioxo-1-ol. pNP has a strong extinction coefficient and therefore allows for convenient spectrophotometric monitoring of MPT hydrolysis. OPH catalysis is linear (i.e., first order) at low MPT concentration (