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at the air-water interface was tested by two different methods: the compression-decompression cycle isotherms and kinetics measurements. There was no ...
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Surface Chemistry and Spectroscopic and Microscopic Properties of Organophosphorus Hydrolase Langmuir and Langmuir-Blodgett Films Xihui Cao, Sarita V. Mello, Guodong Sui, Mustapha Mabrouki, and Roger M. Leblanc* University of Miami, Department of Chemistry, Coral Gables, Florida 33124-0431

Vipin K. Rostogi, Tu-Chen Cheng, and Joseph J. DeFrank U.S. Army Edgewood Chemical & Biological Center, Aberdeen Proving Ground, Maryland 21010-5423 Received April 5, 2002. In Final Form: July 3, 2002 The composition of the subphase to obtain a stable organophosphorus hydrolase (OPH) monolayer was reexamined. The surface pressure-area (π-A) isotherms show that a subphase at pH 7.6 with 0.5 M KCl can lead to a limiting molecular area close to the X-ray crystal data. The stability of the OPH monolayer at the air-water interface was tested by two different methods: the compression-decompression cycle isotherms and kinetics measurements. There was no change in the limiting molecular area during the compression-decompression cycle isotherms and only a very small change over a period of 120 min at constant surface pressure of 20 mN m-1 was observed, which are good indications for a stable OPH monolayer formation at the air-water interface. The surface potential-area isotherm was measured for OPH at the air-water interface, indicating the orientation of the polar moiety of the macromolecules during the compression. The UV-vis absorption spectra of the monolayer were recorded in situ at the air-water interface to investigate the organization of the OPH molecules. Topographies of OPH molecules at the air-water and the air-solid interfaces were studied by Brewster angle microscopy (BAM) and atomic force microscopy (AFM), respectively. The size of the molecules measured from the AFM image agrees with the limiting molecular area per molecule.

Introduction Organophosphorus (OP) compounds are widely used as pesticides and chemical warfare agents.1-3 The toxicity of the OP compounds varies considerably, and some, such as sarin, soman, and VX, are among the most toxic substances known to man. The combination of high toxicity4,5 and worldwide misuse have increased public concerns, creating an urgent need for highly sensitive methods of detection and reliable ways to detoxify these substances. One of the most recommended methods is enzymatic catalysis destruction, as it has been shown to be highly sensitive to detection and successful at detoxification. Organophosphorus hydrolase (OPH) is wellknown to be capable of catalyzing the hydrolysis of wide spectrum organophosphorus neurotoxins containing P-O, P-F, P-CN, and P-S bonds.6-11 It has extremely high * To whom correspondence should be addressed. Tel: (305) 2842194. Fax: (305) 284-6367. E-mail: [email protected]. (1) Compton, J. A. Military Chemical and Biological Agents: Chemical and Toxicological Properties; Telford Press: NJ, 1988; p 135. (2) Food and Agricultural Organization of the United Nations, FAO Product Yearbook; FAO Statistics Series, Rome, 1989; 43, p 320. (3) United States Department of Agriculture, Agricultural Statistics,United States Government Printing Office: Washington, D.C., 1992; p 395. (4) Capalamadugu, S.; Chaudhry, G. S. Crit. Rev. Biotechnol. 1992, 12, 357. (5) Donarski, W. J.; Dumas, D. P.; Heitmeyer, D. P.; Lewis, V. E.; Raushel, F. M. Biochemistry 1989, 28, 4650. (6) Omburo, G. A.; Kuo, J. M.; Mullins, L. S.; Raushel, F. M. J. Biol. Chem. 1992, 267, 13278. (7) Dave, K. I.; Miller, C. E.; Wild, J. R. Chem.-Biol. Interact. 1993, 87, 55. (8) Dumas, D. P.; Caldwell, S. R.; Wild, J. R.; Raushel, F. M. J. Biol. Chem. 1989, 264, 19659. (9) Dumas, D. P.; Wild, J. R.; Raushel, F. M. Biotechnol. Appl. Biochem. 1989, 11, 235.

efficiency in the hydrolysis of many different phosphotriester and phosphothiolester pesticides (P-O bonds) such as paraoxon (kcat > 3800 s-1) and coumaphos (kcat ) 800 s-1). Enzymatic biosensors for the determination of organophosphate nerve agents have been considered to be a very promising method due to their high efficiency and sensitivity.12 Up to now, the majority of biosensors for OP substances are based on acetylcholinesterase (AChE) inhibition, using methodologies such as amperometric,13-20 potentiometric,21-25 or spectrophotometric26-31 techniques. (10) Dumas, D. P.; Durst, H. D.; Landis, W. G.; Raushel, F. M.; Wild, J. R. Arch. Biochem. Biophys. 1990, 277, 155. (11) Ashani, Y.; Rothschild, N.; Segall, Y.; Levanon, S.; Raveh, L. Life Sci. 1991, 49, 367. (12) Munnecke, D. M. Biotechnol. Bioeng. 1979, 21, 2247. (13) Skladal, P. Anal. Chim. Acta 1991, 252, 11. (14) Marty, J.-L.; Sode, K.; Karube, I. Electroanalysis 1992, 4, 249. (15) Palleschi, G.; Bernabei, M.; Cremisini, C.; Mascini, M. Sens. Actuators, B 1992, 7, 513. (16) Skladal, P.; Mascini, M. Biosens. Bioelectron. 1992, 7, 335. (17) La Rose, C.; Pariente, F.; Hernandez, L.; Lorenzo, E. Anal. Chim. Acta 1994, 295, 273. (18) Trojanowicz, M.; Hitchman, M. L. Trends Anal. Chem. 1996, 15, 38. (19) Martorell, D.; Cepedes, F.; Alegret, S.; Matinez-Faregas, E.; Alegret, S. Anal. Chim. Acta 1997, 337, 305. (20) Palchetti, I.; Cagnini, A.; Del Carlo, M.; Coppi, C.; Mascini, M.; Turner, A. P. F. Anal. Chim. Acta 1997, 337, 315. (21) Tran-Minh, C.; Pandey, P. C.; Kumaran, S. Biosens. Bioelectron. 1990, 5, 461. (22) Chuna Bastos, V. L. F.; Chuna Bastos, J.; Lima, J. S.; Castro Faria, M. V. Water Res. 1991, 25, 835. (23) Kumaran, S.; Tran-Minh, C. Anal. Biochem. 1992, 200, 187. (24) Kumaran, S.; Morita, M.Talanta 1995, 42, 649. (25) Danzer, T.; Schwedt, G. Anal. Chim. Acta 1996, 318, 275. (26) Rogers, K. R.; Cao, C. J.; Valdes, J. J.; Eldefrawi, A. T.; Eldefrawi, M. E. Fundam. Appl. Toxicol. 1991, 16, 810.

10.1021/la020326g CCC: $22.00 © 2002 American Chemical Society Published on Web 09/06/2002

Properties of Organophosphorus Hydrolase Films

In the AchE-based biosensors, the signal is inversely proportional to the concentration of organophosphate because the presence of organophosphate blocks the enzyme activity. Although these biosensors were well investigated and developed, they have limitations such as a long incubation time prior to analysis and treatment with pyridine-2-aldoxime after analysis for partial regeneration of the enzyme. In addition, neurotoxins other than OP compounds, for example, carbamate pesticides, can also inhibit AChE, making these analytical methods less selective. Recent developments in organophosphorus hydrolase based biosensor research have demonstrated their many advantages over the AChE biosensors. Since the signals produced by the hydrolysis reaction are directly proportional to the concentration of OP compounds present, OPH biosensors provide the advantages of a simpler, quicker, and more selective method of detection. The products of the enzyme-induced hydrolysis of the organophosphate molecule can be monitored electrochemically or spectrophotometrically, leading to OPHbased potentiometric electrodes32-35 and optical biosensensors.36,37 To design an enzymatic biosensor, the performance is largely dependent on a sufficient contact between the enzyme active site and the OP compounds, regardless of whether the potentiometric or spectrophotometric signal is detected. In the biosensors previously investigated, the enzymes were either free in buffer solution or immobilized in nylon microporous or silica gel membranes. The thicklayer diffusion problems in the conventional immobilization techniques limited the performance and the response time of the biosensors. Chemical adsorption can partially reduce the thick-layer diffusion problems but results in a loss of the enzyme activity. Besides, some substrates, such as electrodes, piezoelectric crystals, or optical fibers, do not have developed chemically active surfaces. The Langmuir-Blodgett (LB) film deposition technique has been proposed, and the feasibility of biosensors using the LB technique has been demonstrated.38,39 Compared to other methods used to immobilize the enzyme, the LB technique provides advantages such as ultrathin film deposition, a highly ordered molecular array, ease of packing and stacking molecules, low temperature, and biomimetic membrane fabrication. The quantity and orientation of the adsorbed molecules on the thin film can be controlled by choosing proper parameters, such as pH of the subphase, and surface pressure of the monolayer. To obtain the LB monolayer, the surface chemistry investigation is essential. The composition of the subphase, (27) Hobel, W.; Polster, J. J. Anal. Chem. 1992, 343, 101. (28) Trettnak, W.; Reiinger, F.; Zinterl, E.; Wolfbeis, O. S. Sens. Actuators, B 1993, 11, 87. (29) Garcia De Maria, C.; Munoz, T. M.; Townhend, A. Anal. Chim. Acta 1994, 295, 287. (30) Moris, P.; Alexandre, I.; Roger, M.; Remacle, J. Anal. Chim. Acta 1995, 302, 53. (31) Andres, R. T.; Narayanaswamy, R. Talanta 1997, 44, 1335. (32) Mulchandani, P.; Mulchandani, A.; Kaneva, I.; Chen, W. Biosens. Bioelectron. 1999, 14, 77. (33) Rainina, E.; Efremenco, E.; Varfolomeyev, S.; Simonian, A. L.; Wild, J. Biosens. Bioelectron. 1996, 11, 991. (34) Muchandani, A.; Chauhan, S.; Mulchandani, P.; Kaneva, I.; Chen, W. Electroanalysis 1998, 10, 733. (35) Mulchandani, A.; Mulchandani, P.; Kaneva, I.; Chen, W. Anal. Chem. 1998, 70, 4140. (36) Mulchandani, A.; Pan, S.; Chen, W. Biotechnol. Prog. 1999, 15, 130. (37) Rogers, K. R.; Wang, Y.; Mulchandani, A.; Mulchandani, P.; Chen, W. Biotech. Prog. 1999, 15, 517. (38) Sriyudthsak, M.; Yamagishi, H.; Moriizumi, T. Thin Solid Films 1988, 160, 463. (39) Choi, J. W.; Bae, J. Y.; Min, J.; Cho, K. S.; Lee, W. H. Sens. Mater. 1996, 8, 493.

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the stability of the monolayer, and the microscopic topography of the LB film must be investigated. The main goal of our work is to design a sensitive and simple biosensor using the LB immobilization technique. Our later report shows that the ultrathin film biosensor prototype has a rapid response time and a broad linear response range. An investigation of the Langmuir film at the air-water interface is required prior to the LB monolayer deposition. Mello et al.40 have reported the first investigation on the surface properties of OPH. To observe the interaction between the monolayer and the paraoxon substrate, a stable monolayer was obtained at the airwater interface, and the limiting molecular area per molecule was measured as 320 Å2 molecule-1 reported by Mello et al. in their work. However, this value does not agree with the molecular size reported by X-ray crystal data (a ) 80.3 Å, b ) 93.4 Å, and c ) 44.8 Å).41 The cause of a much smaller limiting molecular area compared to the X-ray crystal data could be the low purity of the sample. In this paper, a new one-step protocol for OPH purification was applied, and the purity of the OPH sample was improved up to 90% (the highest purity to date). The surface chemistry properties of the Langmuir film were reexamined, and the subphase composition was reinvestigated. Brewster angle microscopy (BAM) and atomic force microscopy (AFM) techniques were applied to study the topography of OPH molecules at the air-water and air-solid interfaces, respectively. Experimental Section Materials. Organophosphate hydrolase (E.C.3.1.8.1) was extracted and purified by the U.S. Army Laboratory (Edgewood Chemical and Biological Center, MD) with a purity of 85-90%. The purification procedure was submitted for patent. OPH stock solution (1.8 mg/mL) was prepared in 100 mM bis-tris-propane (BTP), pH ) 7.5, containing 10 µM Co2+. The stock solution was kept in the refrigerator at 4 °C. The spreading solution was freshly made on the day of the experiment at a concentration of 0.18 mg/mL. The water used as a subphase was purified by a Modulab 2020 water purification system (Continental Water Systems Corp., San Antonio, TX). The pure water has a specific resistance of 18 MΩ cm and a surface tension of 72.6 mN m-1 at 20 ( 1 °C. Buffer solutions with different pH values were prepared: pH 3.0 (0.1 M KH phthalate and 0.1 M HCl), pH 5.0 (0.1 M KH phthalate and 0.1 M NaOH), pH 6.0, 7.5, and 8.0 (0.1 M KH2PO4 and 0.1 M NaOH), and pH 9.0 (0.1 M Tris and 0.1 M HCl). KCl was added into the buffer solution as an electrolyte. All the chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Methods. All isotherms were measured in a clean room class 1000, at a constant temperature of 20.0 ( 0.5 °C and a relative humidity of 50 ( 1%. For surface pressure-area isotherms, we used a KSV minitrough (KSV Model 2000 Instrument Ltd., Helsinki, Finland) with an area of 225 cm2 (7.5 cm × 30 cm). The trough is supplied with an electronic balance that uses a Wilhelmy plate as the pressure sensor and has a sensitivity of 0.02 mN m-1. For monolayer compression, two symmetrically movable barriers controlled by a computer were used. This trough has a quartz window fitted in the middle for in situ UV-vis spectroscopic measurements. Surface potential measurements were obtained in the KSV trough, using the Kelvin method: a vibrating plate is set 1 mm above the monolayer, and a platinum foil is dipped into the pure subphase as the counter electrode. It consists of a capacitor-like system. The in situ UV-vis absorption spectra of the Langmuir monolayer were performed with a HP Spectrophotometer model 8452 A, settled on a rail close to the KSV trough, suitable for approach toward the quartz window. For the Brewster angle microscopic studies, a Nippon trough, equipped with a moving wall system (NL-LB140S-MWC, Nippon Laser & Electronics Lab., Nagoya, Japan) was connected to a Brewster angle microscope (EMM633S, Nippon Laser & Electronics Lab.), (40) Mello, S. V.; Coutures, C.; Leblanc, R. M. Talanta 2001, 55, 881. (41) Benning, M. M.; Kuo, J. M.; Raushel, F. M.; Holden, H. M. Biochemistry 1994, 33, 15001.

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Figure 1. Surface pressure-area isotherms of the OPH monolayer obtained with different buffer subphases at different pH values, i.e., pH ) 3.0, 5.0, 6.0, 7.6, 8.0, and 9.0.

Figure 2. Surface pressure-area and surface potential-area isotherms of the OPH monolayer measured on a pH ) 7.6 buffer subphase in the presence of 0.5 M KCl.

a helium-neon laser (wavelength ) 632.8 nm and power 10 mW), and a CCD camera. The images from the CCD were captured and digitized into a computer using a digital video capture mode (Snappy Video Snapshot, Rancho Cordova, CA) for further analysis or printout. The Nippon trough had dimensions of 0.8 × 5 × 44 cm3. A PicoSPM microscope (Molecular Imaging, Inc., Phoenix, AZ) was used to probe the LangmuirBlodgett OPH monolayer which was scanned in air mode AFM and using Mac mode. The OPH monolayer was transferred at a surface pressure of 20 mN m-1 on a freshly cleaved graphite substrate, highly oriented pyrolytic graphite (HOPG). A typical compression speed of 20 Å2 molecule-1 min-1 and a spread volume of 20-100 µL were used to measure the isotherms. The purity of the subphase solutions was confirmed by compressing the surface area free of enzyme samples, leading always to a nil surface pressure after complete compression. The surface area was always expanded to its maximum and the surface pressure was set at 0 mN m-1 before starting any experiment.

monolayer was compressed at nil surface pressure to a larger surface area per molecule and a lower collapse surface pressure than those obtained at the other pH values. The isotherms in Figure 1 showed that the pH of the subphase has a significant effect on the solubility of the enzyme, and the most insoluble OPH monolayer can be obtained at the pH value of the isoelectric point (7.6). However, at pH values below and above the isoelectric point, a smaller limiting molecular area per molecule was observed indicating a higher solubility of the enzyme. The reason for the variation of solubility of OPH is that the amide functional group of the enzyme can act as a proton acceptor in acidic solutions and a proton donor in alkaline solutions, both cases resulting in a higher solubility of the enzyme. The addition of an electrolyte into the subphase is also known to reduce the solubility of the protein in the subphase and consequently to promote formation of a stable monolayer at the air-water interface.40,43 We reexamined the effect of the ionic strength of the subphase on the packing and orientation of the OPH, and our results agree with the report from Mello et al.40 The π-A isotherms showed that the surface area per molecule of OPH changes markedly with the addition of KCl electrolyte into the buffer subphase. The effect of concentration of the KCl in the subphase was investigated at 0, 0.1, 0.3, 0.5, and 0.6 M at pH 7.6. It was found that the limiting molecular area of OPH in the presence of KCl is larger than that in its absence. And the largest limiting molecular area was obtained with 0.5 M KCl addition. The variations in the isotherms may be due to the lower solubility of the enzyme in the subphase in the presence of the electrolyte. As reported in earlier work,43,44 the macromolecules alter their conformation in response to the changes in their energy environment, including interactions with the interface. The enzyme at the interface may unfold to a twodimensional conformation to maximize the polar side chains interacting with the subphase and the nonpolar side chains exposed to air. The unfolding may result in the improved adsorption of the enzyme at the interface.45

Results and Discussion Experiments were selected to determine the optimum conditions for developing a stable monolayer of OPH at the air-water interface. As is known for protein studies, one of the biggest difficulties in obtaining monolayers of proteins at the air-water interface is that they are water soluble. The solubility of the enzyme varies with the pH of the subphase, and this can be shown in the surface pressure-area isotherms. If the enzyme is soluble in the subphase, some enzyme molecules will dissolve into the subphase and this will result in a smaller surface area per molecule in the π-A isotherm. The effect of pH on the OPH monolayer was determined by compressing the monolayer on a subphase at pH values of 3.0, 5.0, 6.0, 7.6, 8.0, and 9.0. Among these pH values, pH 7.6 represents the isoelectric point of OPH.42 The different pH values of the subphase were chosen in order to study the behavior of the enzyme on a pH subphase below or above the isoelectric point. The surface pressure-area (π-A) isotherms at different pHs are shown in Figure 1. We can see that at pH values below and above 7.6, smaller limiting molecular areas were measured. No collapse surface pressure was observed at a pH value of 3.0. At pH values of 5.0, 6.0, 8.0, and 9.0, a collapse surface pressure of about 30 mN m-1 was observed. At pH 7.6, the OPH (42) Rowland, S. S.; Speedie, M. K.; Pogel, B. M. Appl. Environ. Microbiol. 1991, 57, 440.

(43) Dziri, L.; Puppala, K.; Leblanc, R. M. J. Colloid Interface Sci. 1997, 194, 37. (44) Chudinova, G. K.; Polrovskaya, O. N.; Savitskii, A. P. Russ. Chem. Bull. 1995, 44, 1958. (45) MacRitchie, F. Adv. Colloid Interface Sci. 1986, 25, 341.

Properties of Organophosphorus Hydrolase Films

Figure 3. The compression/decompression cycle of the OPH monolayer on a pH ) 7.6 buffer subphase in the presence of 0.5 M KCl from nil to 5, 10, 15, and 20 mN m-1 surface pressure.

Figure 4. The surface area of the OPH monolayer on a pH ) 7.6 buffer subphase in the presence of 0.5 M KCl when the surface pressure was held at 20 mN m-1 for 2 h.

The phenomenon of decreasing solubility in the bulk in the presence of an electrolyte was observed with other protein monolayers such as bull serum albumin (BSA)44 and AChE.43 On the basis of the effect of pH and concentration of an electrolyte in the subphase, we have found the optimal experimental conditions to form a stable OPH monolayer at the air-water interface, namely, pH 7.6 and 0.5 M KCl concentration. The isotherm of OPH under these conditions represents a stable protein monolayer adsorption at the air-water interface. From Figure 2, an apparent limiting molecular area of 7000 ( 400 Å2/molecule (the experimental error is between 5 and 8%) and a collapse surface pressure at around 25 mN m-1 were observed. By comparison of this result to the X-ray crystal study, which reported that an OPH unit cell (one molecule per asymmetric unit) has dimensions of a ) 80.3 Å, b ) 93.4 Å, and c ) 44.8 Å by single-crystal X-ray diffraction analysis,41 a surface area of 7500 Å2/molecule can be calculated (80.3 Å (a) × 93.4 Å (b)). We have measured a surface area of 7000 ( 400 Å2/molecule from the surface pressure-area isotherm. This experimental value agrees well with the X-ray crystal data. Furthermore, the molecular size in two dimensions and the thickness of the monolayer will

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Figure 5. UV-vis absorption spectra of the OPH monolayer at the air-water interface at surface pressures of 0, 5, 10, 15, 20, 25, 30, and 35 mN m-1. The inset shows the graph of the absorbance versus the surface pressure.

be shown to be confirmed by the AFM experiments. A point of inflection on the OPH isotherm was also observed at a surface pressure around 18 mN m-1 in the presence of KCl, which was not observed in its absence, similar to the isotherms of other proteins such as AChE (around 16 mN m-1).43 The presence of a point of inflection might be interpreted as a change of conformation of the OPH when the monolayer was compressed at the air-water interface. The surface potential technique measures the dipole moment changes during compression of the monolayer at the air-water interface. This measurement gives a good indication of the orientation of the polar moiety of the macromolecules forming the monolayer. With the subphase at pH 7.6 and the presence of KCl at a concentration of 0.5 M, the surface potential-area and the surface pressure-area isotherms are shown in Figure 2. We have observed at large surface area and at nil surface pressure important fluctuations in the signal of the surface potential, which were due to the formation of domains of enzyme at the air-water interface. These fluctuations became smaller during compression of the film, and surface potential started to increase gradually from a surface area of 8500 to 5000 Å2/molecule. This corresponds to a change in surface pressure from nil to 16 mN m-1. At this point, the hydrophobic moieties of OPH molecules are lifted up into the air and the monolayer is in a liquid expanded phase. Further compression to a surface area below 5000 Å2/molecule (surface pressures greater than 16 mN m-1) results in a smaller increase in the surface potential although the change of the surface pressure is still significant. One can notice that the point of inflection at surface pressure 16 mN m-1 is critical. The change in surface potential is significant before the point of inflection and fairly small after passing it. We conclude that this corresponds to the reorientation of the molecules and the conformation rearrangement in order to maximize the exposure of the nonpolar groups to the air. The stability of the OPH monolayer at the air-water interface was examined by two different methods under the optimal conditions of the subphase (pH ) 7.6 and 0.5 M KCl), that is, the compression/decompression cycle and the kinetic measurements. The results are shown in Figures 3 and 4, respectively. From Figure 3, no significant decrease in the apparent limiting molecular area was observed during the compression and decompression cycle

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Figure 6. 3-D BAM images recorded during the compression of the OPH monolayer at surface pressures of (a) 0, (b) 20, and (c) 25 mN m-1.

at surface pressures of 5, 10, 15, and 20 mN m-1. In the kinetic measurements shown in Figure 4, the monolayer was compressed to a surface pressure of 20 mN m-1 and held constant at this value for a period of 120 min. The area per molecule decreased by about 750 Å2/molecule over 120 min. This value corresponds to less than 11% of the limiting molecular area of the OPH monolayer. The decrease of the area is due to the rearrangement of molecules to reduce the holes or a partial solubility of the molecules at a surface pressure of 20 mN m-1. The results shown above suggest the formation of a stable OPH monolayer at the air-water interface. The UV-vis absorption spectra of the OPH monolayer were measured in situ at the air-water interface. Figure 5 shows the absorption spectra of the OPH monolayer at different surface pressures. An absorption band at 210 nm was observed, which is assigned to the amide group of the enzyme. With the compression of the monolayer, the intensity of the absorbance at 210 nm increased with the surface pressure. The inset in Figure 5 shows the relationship of the absorbance at 210 nm versus the surface pressure. In the range of surface pressure from 0 to 25 mN/m, the absorbance increases proportionally with the surface pressure of the monolayer within the experimental error. However, the linear relationship is not observed at

surface pressures greater than 25 mN m-1, which corresponds to the collapse value of the surface pressure. This result indicates the formation of a homogeneous OPH monolayer at the air-water interface before the monolayer reaches the collapse surface pressure. The surface topography of the enzyme was studied by using two microscopic techniques: BAM at the air-water interface and AFM in contact mode of a LangmuirBlodgett film of the enzyme. The 3-D BAM images are shown in Figure 6. Topographies at three different surface pressures (0, 20, and 25 mN m-1) were analyzed. The difference in contrast arises from the presence of a monolayer at the interface with a different refractive index. The areas in the image with different brightnesses are due to the different molecular density which correlates with the thickness of the monolayer at the interface. During the compression of the monolayer from a molecular area of 10 000 Å2 molecule-1 and at nil surface pressure (Figure 6a), we observed different domains with different sizes dispersed randomly on the aqueous surface. The highest peaks correspond to the presence of the enzyme clusters on the surface. While the enzyme monolayer was compressed to a higher surface pressure, the domains started growing and the patches became larger. At a surface pressure of 20 mN m-1 (molecular area of 4500 Å2

Properties of Organophosphorus Hydrolase Films

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Figure 7. AFM images of the OPH LB film deposited at a surface pressure of 20 mN m-1 onto HOPG. The image sizes are (a) 2.4 µm × 2.4 µm, (b) 1.0 µm × 1.0 µm, and (c) 250 nm × 250 nm, and the scan rate is 3.3 Hz.

molecule-1), the growth of domains was observed (Figure 6b) and a homogeneous monolayer was formed. At a surface pressure of 25 mN m-1 (Figure 6c, molecular area near 4000 Å2 molecule-1), some clusters reappeared on the surface and the thickness of part of the topography is larger, indicating an overlap of the molecules caused by the collapse of the monolayer. Overall, the BAM images agree well with the surface potential measurements to reveal the reorientation of the macromolecules at the airaqueous interface. The AFM technique is applied for the imaging of the OPH Langmuir-Blodgett film. In our measurements, the LB films were prepared by deposition of one monolayer of OPH at 20 mN m-1 onto HOPG with a deposition ratio of around 1.00. The surface topography image is shown in Figure 7a-c, at different resolutions. From Figure 7a, we can distinguish four different areas according to the brightness in contrast. These are black, red-yellow, yellow, and white areas. The black area represents the uncovered surface of HOPG, and the white area represents the top surface of the monolayer. The red-yellow area represents the enzyme molecules near the substrate, and the yellow area represents the molecules near the top surface. Figure 7a shows a homogeneous monolayer with pores between the molecules. Looking more closely in Figure 7b, we can see that the enzyme molecules are closely clustered to each other. The shapes of the particle can be seen from Figure 7c and the size of the particle can be measured. As is shown in Figure 7c, all the particles in this image can

be grouped in two different sizes, small and large particles with globular and ellipsoidal shapes, respectively. Smaller particles are in greater abundance (84%) compared to the larger particles (16%). Averaging all the sizes by measuring the dimensions with respect to the ellipsoidal axis showed that the sizes for the small and large particles are 10 nm × 7.4 nm × 3.4 and 19 nm × 13.6 nm × 7.0 nm, respectively. Referring to these data, we can attribute the dimensions of the small particles to the monomer and the large ones to the dimer. On the basis of the AFM size measurements, the OPH monomers have the two dimensions at the surface of 10 nm × 7.4 nm, which gives a surface area value of 7400 Å2 molecule-1. This value agrees very well with the surface pressure-area isotherm (Figure 2) and the X-ray crystal data.41 Furthermore, the thickness of the monolayer can be measured by using the height difference between the top of the LB monolayer and the substrate surface, which was performed by comparing the brightest area (top of the surface) and the dark area (the substrate surface). The monolayer thickness was measured as 3.6 nm. The value is less than the OPH unit cell dimensions for the c axis, that is, 4.48,41 confirming the measured value of one molecule film thickness. Conclusion A stable and homogeneous OPH monolayer was prepared at the air-water interface with a subphase of pH 7.6 and 0.5 M KCl concentration. A limiting molecular area of 7000 ( 400 Å2 molecule-1 was measured under

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optimal conditions at the air-water interface. A surface area of 7400 Å2 molecule-1 was confirmed by the AFM measurements for the LB film. Combining the AFM measurements with the surface properties at the airwater interface, we have confirmed that the Langmuir monolayer can be transferred to the solid subphase without changing its molecular topography. The LB deposition

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leads to an ultrathin, compact OPH film that can be employed as a biosensor for the detection of OP compounds. Acknowledgment. This work was supported by a grantfromtheU.S.ArmyResearchOffice(DAAD19-00-1-0138). LA020326G