Langmuir 1996, 12, 4411-4416
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Enzymatic Modification of a Chemisorbed Lipid Monolayer† David C. Turner, Brian M. Peek,‡ Thomas E. Wertz, Douglas D. Archibald, Robert E. Geer,§ and Bruce Paul Gaber* Laboratory for Molecular Interfacial Interactions, Center for Bio/Molecular Science and Engineering, Code 6930 and Code 6950, Naval Research Laboratory, Washington, DC 20375-5348 Received December 8, 1995X The selectivity and specificity of enzymes may be exploited to create chemically complex surfaces which are difficult or impossible to achieve using classical synthetic chemistry. In this paper we discuss the preparation of a chemisorbed lipid film on a silicon wafer and explore the activity of free phospholipase C (PLC) on that film. A carboxylic acid derivative of the lipid dimyristoylphosphatidylcholine (DMPC) was attached to an amino-terminal silane (EDA) via amide bond formation to create an immobilized lipid layer (EDA-DMPC). Films were characterized using X-ray photoelectron spectroscopy (XPS), secondary-ion mass spectrometry (SIMS), atomic force microscopy (AFM), X-ray reflectivity, and ellipsometry. Following treatment with the enzyme phospholipase C (PLC), which catalyzes the cleavage of the lipid headgroup at the glycerol-phosphate ester bond, the lipid film was reanalyzed using the above techniques. Before analysis, nonspecifically adsorbed PLC was removed with a 25% trifluoroethanol rinse. XPS and SIMS results of the cleaned films show nearly complete removal of the phosphate from the lipid layer, indicating enzymatic activity of the PLC on the chemisorbed lipid layer.
Introduction Enzymatic chemical modification of chemisorbed organic thin films offers a potentially attractive alternative to conventional synthetic chemical modification of surface films. Enzyme-based catalysis combines mild reaction conditions in aqueous media along with a high degree of chemical specificity. By taking advantage of these properties, chemically complex surfaces may be fabricated which might be difficult or impossible to fabricate using conventional methods. For example, one may want to use a suite of enzymes to produce several different chemical moieties on a single immobilized thin film for biopatterning or sensing applications. Early work in this area by Ringsdorf and co-workers demonstrated that the enzyme phospholipase A2 is enzymatically active upon lipids in a Langmuir film at the air-water interface.1 More recently, Wilson and co-workers demonstrated that subtilisin BPN′ could be used to hydrolyze an amide bond in a peptide chemisorbed to a polymerized Langmuir-Blodgett film which was physisorbed to a hydrophobic glass slide.2 In this paper we make the next natural step and demonstrate the enzymatic activity of phospholipase C (PLC) against a phospholipid film which has been covalently attached to a silica surface. Many silane layers have been modified using chemical synthesis to incorporate biological materials, such as antibodies,3 peptides, proteins,4,5 and nucleotides,6-8 as * Author to whom correspondence should be addressed. † A preliminary account of this work appeared as: Gaber, B. P.; Peek, B. M.; Turner, D. C.; Brandow, S. L.; Leach-Scampavia, D. Polym. Prepr. 1993, 34, 108-109. ‡ Current Address: Georgia Pacific Resins, Inc., 2883 Miller Road, Decatur, GA 30035. § Code 6950. X Abstract published in Advance ACS Abstracts, July 15, 1996. (1) Ahlers, M.; Muller, W.; Reichert, A.; Ringsdorf, H.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1990, 29, 1269-1285. (2) Wilson, T. E.; Spevak, W.; Charych, D. H.; Bednarski, M. D. Langmuir 1994, 10, 1512-1516. (3) Bhatia, S. K.; Shriver-Lake, L. C.; Prior, K. J.; Georger, J. H.; Calvert, J. M.; Bredehorst, R.; Ligler, F. S. Anal. Biochem. 1989, 178, 408-413. (4) Hong, H.; Jaing, M.; Sligar, S. G.; Bohn, P. W. Langmuir 1994, 10, 153-158.
S0743-7463(95)01520-4 CCC: $12.00
components in their structure. The immobilization of lipids to the surfaces of polymers,9,10 solids,11-15 and silane layers has also been described previously.16-23 We chose to prepare our lipid surfaces following the method of Pidgeon and Venkataram,17-23 in which a carboxylic acid derivative of dimyristoylphosphatidylcholine (DMPC) is attached to a polished silicon wafer surface modified by an amino-terminal silane (EDA). The resulting lipid layer was characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and ellipsometry and was found to be stable and uniform in coverage. (5) Vandenberg, E.; Elwing, H.; Askendal, A.; Lundstro¨m, I. J. Colloid Interface Sci. 1991, 143, 327-335. (6) Eggers, M.; Hogan, M.; Reich, R. K.; Lamture, J.; Ehrlich, D.; Hollis, M.; Kosicki, B.; Powdrill, T.; Beattie, K.; Smith, S.; Varma, R.; Gangadharan, R.; Mallik, A.; Burke, B.; Wallace, D. BioTechniques 1994, 17, 516-524. (7) Chrisey, L. A.; Roberts, P. M.; Benezra, V. I.; Dressick, W. J.; Dulcey, C. S.; Calvert, J. M. Mater. Res. Soc. Symp. Proc. 1994, 330, 179-184. (8) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (9) Foltynowicz, Z.; Yamaguchi, K.; Czajka, B.; Regen, S. L. Macromolecules 1985, 18, 1394-1401. (10) Regen, S. L.; Kirszensztejn, P.; Singh, A. Macromolecules 1983, 16, 335-338. (11) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (12) Fabianowski, W.; Coyle, L. C.; Weber, B. A.; Granata, R. D.; Castner, D. G.; Sadownik, A.; Regen, S. L. Langmuir 1989, 5, 35-41. (13) Durrani, A. A.; Hayward, J. A.; Chapman, D. Biomaterials 1986, 7, 121-125. (14) Hayward, J. A.; Durrani, A. A.; Lu, Y.; Clayton, C. R.; Chapman, D. Biomaterials 1986, 7, 132-136. (15) Hayward, J. A.; Durrani, A. A.; Shelton, C. J.; Lee, D. C.; Chapman, D. Biomaterials 1986, 7, 126-131. (16) Kallury, K. M. R.; Ghaemmaghami, V.; Krull, U. J.; Thompson, M.; Davies, M. C. Anal. Chim. Acta 1989, 225, 369-389. (17) Pidgeon, C.; Venkataram, U. V. Anal. Biochem. 1989, 176, 3647. (18) Pidgeon, C. U.S. Patent 4,931,498, 1990. (19) Pidgeon, C.; Stevens, J.; Otto, S.; Jefcoate, C.; Marcus, C. Anal. Biochem. 1991, 194, 163-173. (20) Pidgeon, C.; Ong, S.; Chol, H.; Liu, H. Anal. Chem. 1994, 66, 2701-2709. (21) Ong, S.; Cal, S.; Bernal, C.; Rhee, D.; Qiu, X.; Pidgeon, C. Anal. Chem. 1994, 66, 782-792. (22) Markovich, R. J.; Stevens, J.; Pidgeon, C. Anal. Biochem. 1989, 182, 237-244. (23) Markovich, R. J.; Qiu, X.; Nichols, D. E.; Pidgeon, C.; Invergo, B.; Alverez, F. M. Anal. Chem. 1991, 63, 1851-1860.
© 1996 American Chemical Society
4412 Langmuir, Vol. 12, No. 18, 1996
Figure 1. Different phospholipases, which selectively hydrolyze various bonds in a phospholipid. A lipid with a phosphocholine headgroup is shown where the dashed lines represent bonds hydrolyzed by each lipase. Arrows indicate bonds which are hydrolyzed by a particular lipase. Phospholipase A1 (PLA1) and phospholipase A2 (PLA2) hydrolyze the ester bond of the sn-1 and sn-2 bond, respectively. Phospholipase C (PLC) hydrolyses the glycerol-phosphate ester bond, and phospholipase D (PLD) hydrolyses the phosphate-choline bond. The result of PLC modification of a phospholipid is a diacylglycerol and a phosphocholine.
Figure 1 shows several of the possible enzyme decomposition schemes for a phosphocholine lipid, including removal of phosphocholine with phospholipase D (PLD), removal of phosphocholine with phospholipase C (PLC), and removal of the acyl chains with phospholipase A1 and A2 (PLA1 and PLA2). Since the presence of phosphate can be followed using XPS, we treated the immobilized DMPC monolayer with phospholipase C to hydrolyze the lipid headgroup at the glycerol-phosphate ester bond and release phosphocholine from the surface (Figure 1). After the PLC treatment, XPS and negative-ion SIMS spectra indicated a surface significantly depleted in phosphorous, in agreement with the expected PLC hydrolysis scheme. The main experimental difficulty encountered in this work was nonspecific adsorption of PLC to the lipid interface during the enzyme treatment step. This is a common problem observed in aqueous protein systems interacting with solid surfaces.24 A recipe for minimizing this adsorption is discussed as well as the implications of protein fouling with regard to enzyme-based surface modification. Experimental Section Materials. Solvents were obtained from commercial sources and used without further purification. [3-(2-Aminoethyl)amino)propyl]trimethoxysilane (EDA) was obtained from Hu¨ls America (Piscataway, NJ), (1-benzotriazolyloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), from Advanced ChemTech (Louisville, KY), 1-myristoyl-2-(13-carboxytridecanoyl)sn-3-glycerophosphocholine (DMPC-COOH), from Regis Chemical Co. (St. Louis, MO), phospholipase C (PLC), from Boehringer Mannheim (Indianapolis, IN), and polished native Si 〈100〉 n-type wafers from WaferNet, Inc. (San Jose, CA). Water was was triply distilled and deionized with a resistivity greater than 15 MΩ cm. EDA Film Formation. Silicon wafers were cleaned by immersion in 1:1 HCl/methanol for 30 min, followed by a distilled water rinse, immersion in concentrated H2SO4 for 30 min, a second distilled water rinse, and finally heating in distilled water, between 80 and 100 °C, for 5-15 min. Clean wafers were immersed in a solution of 94% 1 mM acetic acid/methanol, 5% distilled water, and 1% EDA for 15 min, rinsed three times with methanol, and baked on a 120 °C hot plate for 3 min. Lipid Layer Formation. EDA-modified silicon wafers were immersed in 50 mL of a 9:1 CHCl3/DMF solution which contained DMPC-COOH (180 mg, 0.25 mmol), PyBOP (130 mg, 0.25 mmol), (24) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-340.
Turner et al. and diisopropylethylamine (DIEA; 44 mL, 0.25 mmol). The reaction was run overnight at room temperature, after which the wafers were washed three times each with dimethylformamide (DMF) and CHCl3, sonicated for 10 min each in 9:1 CHCl3/ DMF and distilled water, and then dried under a stream of nitrogen. Phospholipase C Treatment. The specific activity of PLC (expressed as micromoles of lipid hydrolyzed per minute per milligram of PLC) is pH dependent with a maximum between pH 7.5 and 8.025 and is reduced by the presence of monovalent anions.26 Therefore, conditions used to hydrolyze the phosphocholine headgroup from the EDA-DMPC layer used a pH of 7.8 and a minimum of anionic salts. A stock PLC solution was made by adding 5 mL of a 1.77 mg/mL solution of PLC to 3.0 mL of 10 mM PO4 buffer at pH 7.8. The final concentration of PLC in this solution was 3 mg/mL. The lipid wafers were treated with the above PLC solution for eight 15 min segments. To minimize nonspecific adsorption of enzyme to the film surface, the wafers were washed with buffer followed by 3:1 distilled water/ trifluoroethanol (TFE) washes between each PLC solution treatment segment. Approximately 1.5 mL of this solution was required to cover the surface of a 1 in. × 1 in. square of EDADMPC-coated wafer. After the final treatment with the PLC solution, the wafer was rinsed with buffer and then sonicated for 5 min in 3:1 distilled water/TFE. Surface Measurements. Film thickness was measured using a Gaertner Model 114C ellipsometer (Chicago, IL) equipped with a helium-neon laser (632.8 nm), using a 70° angle of incidence and a compensator setting of -45°. Optical constants of the silicon substrate were determined prior to silanization. Film thicknesses were measured using the substrate optical constants measured for the unmodified wafer, and an assumed bulk refractive index of 1.45 was used for the lipid film. Because of this assumption, the film thicknesses measured using ellipsometry may have an associated systematic error of up to 50%. Contact angles were measured using a Zisman type goniometer with a static, sessile 20 mL drop of distilled water.27 Atomic force microscope images were collected using a Digital Instruments NanoScope III atomic force microscope equipped with silicon nitride cantilevers (Digital Instruments, Santa Barbara, CA). Samples were immersed in distilled water and imaged using the fluid cell attachment in contact mode with a constant applied force of less than 10 nN. Film roughness values were obtained from AFM images and were defined as the average root-meansquare (RMS) deviation from the mean height in a 100 × 100 nm2 area. X-ray Photoelectron Spectroscopy. XPS measurements were performed on a Surface Science Instruments SSX-100 XPS spectrometer equipped with a monochromatic Al KR source, hemisperical analyzer, and multichannel detector. Spectra were collected with the analyzer at 80° with reference to the sample surface normal at an operating pressure of approximately 3 × 10-9 Torr. Data were collected with a pass energy of 150 eV and a spot size of 1000 µm. The binding energy scales for all spectra were referenced by setting the CHx peak maxima in the C 1s spectrum to 285.0 eV. Peak fitting was done using Gaussian peak shapes with commercial software supplied by Surface Science Instruments. For calculation of XPS elemental composition, the analyzer transmission function was assumed not to vary with photoelectron kinetic energy (KE), the photoelectron escape depth was assumed to vary as (KE)0.7, and Scofield’s photoionization cross sections were used.28 The surface composition was assumed homogenous within the photoelectron escape depth for simplicity when comparing enzyme treated to untreated films. The solid acceptance angle of the analyzer lens was decreased to 12° × 30° by placing an aperture over the analyzer lens to improve the depth resolution.29 Quadrupole Static Secondary Ion Mass Spectrometry (SIMS). The static secondary ion mass spectrometry (SIMS) (25) Aakre, S.; Little, C. Biochem. J. 1982, 203, 799-801. (26) Eaton, B. R.; Dennis, E. A. Arch. Biochem. Biophys. 1976, 176, 604-609. (27) Wasserman, S. R.; Whitesides, G. M.; Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Axe, J. D. J. Am. Chem. Soc. 1989, 111, 5852-5861. (28) Scofield, J. H. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129-137. (29) Tyler, B. J.; Castner, D. G.; Ratner, B. D. J. Vac. Sci. Technol. 1989, A7, 1646-1654.
Modification of a Chemisorbed Lipid Monolayer
Langmuir, Vol. 12, No. 18, 1996 4413 Table 1. Properties of Surface-Modified Layers from Ellipsometry (Film Thickness), Sessile Drop Water Contact Angle, and AFM layer
thickness contact angle RMS roughness
Figure 2. Reaction scheme for the attachment of DMPC-COOH to the EDA layer. See description in the Experimental Section. experiments were performed with a Physical Electronics 3700 SIMS system (Eden Prairie, MN) mounted on a custom ultrahigh vacuum (UHV) system. The UHV system has a turbomolecular and Ti sublimation pumped analysis chamber with an XYZΘ sample manipulator. The base pressure in the chamber is 1 × 10-10 Torr. Samples are transferred into the analysis chamber from a turbomolecular-pumped sample introduction chamber. The PHI SIMS system contains a 90° adjustable energy filter and a Balzers 0-511 amu quadrupole mass spectrometer for detection of positive and negative secondary ions emitted by the sample. A differentionally pumped Leybold Heraeus ion gun (Koln, Germany) was used to produce a 0.5 nA, 3.5 keV Xe+ primary ion beam. The ion beam was rastered over a 5 × 5 mm2 area, and the total exposure time of the sample to the ion beam including setup and data acquisition was less than 7 min. This corresponded to a total ion dose per sample of
