Covalent Immobilization of Antibody Fragments onto Langmuir

Langmuir 2002, 18, 4953-4962

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Covalent Immobilization of Antibody Fragments onto Langmuir-Schaefer Binary Monolayers Chemisorbed on Gold P. Ihalainen* and J. Peltonen Department of Physical Chemistry, A° bo Akademi University, Porthaninkatu 3-5, 20500 Turku, Finland Received January 3, 2002. In Final Form: April 4, 2002 Binary solid-supported monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycolipoate (DPPGL) and 1-palmitoyl-2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine (DSDPPC) have been studied by Langmuir isotherm measurements at the air-water interface and by scanning probe microscopy (SPM) of the solid-supported films. The pendant disulfide ring of the lipoic acid moiety of DPPGL was used as a linker to covalently bind pepsin-cleaved antibody Fab′ fragments via a thiol-disulfide interchange reaction. The disulfide group of the matrix lipid DSDPPC was used to covalently bind the binary monolayer onto an ultraflat gold substrate by Langmuir-Schaefer (LS) deposition. The aim is to combine high immunological sensitivity and specificity with improved adhesion properties and hence mechanical, thermal, and chemical stability of the film. SPM was used to demonstrate the binding of Fab′ fragments of polyclonal anti-human IgG to two different lipid matrixes with DPPGL concentrations of 5 and 20 mol %. The subsequent antibodyantigen complex formation was further demonstrated by SPM. Fab′ fragments bound to the matrix in a slightly tilted orientation, and also some aggregates were formed. Aggregate formation was more pronounced in the film with the higher linker concentration, increasing the unspecific binding of human IgG (hIgG).

Introduction The demand is increasing for simple, selective, accurate, and easy-to-use biosensors for use as diagnostic immunoassays. A biosensor consists of a molecular recognition site (bioreceptor) and a transducer, which transforms a biochemical reaction into a measurable signal. A promising method for producing bioreceptors is the application of Langmuir-Blodgett (LB) or Langmuir-Schaefer (LS) techniques for the preparation of solid-supported highly organized and functionalized organic thin films. The functionalized lipids of the monolayer can covalently bind biomolecules in a highly oriented manner such that the bound proteins serve as binding sites for further molecular recognition.1 The mechanical stability of LB films can be improved by using preformed polymers2-4 or reactive amphiphiles polymerizable at the air-water interface5-7 or as deposited multilayers.8 However, the weakness of the system rests on the fact that the polymer film is normally only quite weakly physisorbed to the solid support, especially in case of a film LS-deposited onto a hydrophobic surface. In this article, we report the preparation and properties of a biofunctional film intended for bioaffinity measurements, where the film is covalently chemisorbed onto a gold-coated solid support. The structure is made biofunctional by incorporating synthesized lipids capable of * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +358-2-215-4706. Phone: +358-2-215-4616. (1) Powner, E. T.; Yalcinkaya, F. Sens. Rev. 1997, 17, 107. (2) Collins, S. J.; Mary, N. L.; Radhakrishnan, G.; Dhathathreyan, A. J. Chem. Soc., Faraday Trans. 1997, 93, 4021. (3) Peng, J. B.; Barnes, G. T. Colloids Surf. A 1995, 102, 75. (4) Niwa, M.; Hayashi, T.; Higashi, N. Langmuir 1990, 6, 263. (5) Rolandi, R.; Dante, S.; Gussoni, A.; Leporatti, S.; Maga, L.; Tundo, P. Langmuir 1995, 11, 3119. (6) Peltonen, J. P. K.; He, P.; Linde´n, M.; Rosenholm, J. B. J. Phys. Chem. 1994, 98, 12403. (7) Bodalia, R. R.; Duran, R. S. J. Am. Chem. Soc. 1993, 115, 11467. (8) Peltonen, J. P. K.; He, P.; Rosenholm, J. B. Langmuir 1993, 9, 2363.

covalently binding fragmented antibodies. Such a biofunctional surface can specifically bind a predetermined antigen, yielding a biosensor surface from homogeneously distributed and highly oriented proteins on a stable solidsupported thin organic film. Lipids were chosen as monolayer-forming molecules because they provide excellent matrix molecules for biofunctional thin films with amphiphilic nature. A binary solid-supported monolayer of 1,2-dipalmitoyl-sn-glycero3-phosphoglycolipoate (DPPGL) and 1-palmitoyl-2-(16(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine (DSDPPC) was formed. The pendant disulfide ring of the lipoic acid moiety of DPPGL was used to covalently bind pepsin-cleaved antibody Fab′ fragments via a thioldisulfide interchange reaction. The host lipid DSDPPC was used to covalently bind the binary monolayer onto an ultraflat gold substrate during LS deposition via disulfide groups, which are well-known to react strongly with gold and other metals in a self-assembly manner.9 Scanning probe microscopy (SPM) was used to study the attachment of fragmented proteins onto the solid-supported films. The results show that covalent coupling of antibody fragments to DPPGL embedded in a host monolayer matrix of DSDPPC was successful. The surface activity of both molecules is demonstrated through Langmuir isotherm studies. Furthermore, DSDPPC and DPPGL appeared to be miscible in the mixed monolayers. SPM measurements of DSDPPC films on the solid support revealed a homogeneous monolayer. The structure retained after the film was rinsed with ethanol indicated that the monolayer was coupled covalently to the gold substrate. SPM measurements showed that Fab′ fragments were bound to the matrix in a slightly tilted orientation. Some aggregates were also seen. Aggregate formation was pronounced in the film with the higher (9) Kada, G.; Riener, C. K.; Gruber, H. J. Tetrahedron Lett. 2001, 42, 2677.

10.1021/la020017q CCC: $22.00 © 2002 American Chemical Society Published on Web 05/15/2002

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linker concentration, increasing the unspecific binding of human IgG (hIgG).

Ihalainen and Peltonen

Materials and General Methods. 1-Palmitoyl-2-hydroxysn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) were obtained from Avanti Polar Lipids. Methyl methanethiosulfonate (Sigma), hydrobromic acid (49%, Sigma), 16-hydroxyhexadecanoic acid (Sigma), sodium hydride (60% dispersion in mineral oil, Aldrich), pyridine (Sigma), thioacetic acid (Lancaster), sodium hydroxide (J. T. Baker), ammonia solution (25%, Riedel-de Hae¨n), fuming hydrochloric acid (37%, Merck), sodium chloride (J. T. Baker), and dehydrated calcium chloride (Fluka Chemika) were used as obtained. Dicyclohexylcarbodiimide (DCC, Acros Organics) and 4-(dimethylamino)pyridine (4-DMAP, Sigma) were recrystallized once from toluene (J. T. Baker) prior to use. Chloroform (Lab Scan) and carbon tetrachloride (J. T. Baker) were dried over phosphorus pentoxide (Sigma). Ethanol (Primalco), hexane (Sigma), methanol (Merck), acetone (Shell), and diethyl ether (Fluka Chemika) all were of at least 99% purity. The model antibody was polyclonal goat anti-human F(ab′)2 (Jackson ImmunoResearch, chromatically purified) with minimized cross section to bovine, horse, and mouse serum proteins. The antigen was chromatically purified human IgG (Jackson ImmunoResearch). F(ab′)2 was cleaved into Fab′ fragments with dithiotreitol (DTT, Acros Organics) under argon overnight in a microdialysis tube prior to use.10 According to a previous report, more than 95% of the F(ab′)2 fragments is converted to Fab′ during such a reduction.11 HEPES-EDTA buffer [10 mM HEPES (Sigma), 150 mM NaCl (Fluka), 5 mM EDTA (Sigma), pH ) 6.8] was used in all antibody-antigen systems. Chromatographic separations were carried out by using precoated Merck 0.25-mm silica gel 60 TLC plates and Merck 70-230 ASTM silica gel. Detection on TLC plates was achieved using iodine vapor. 1H NMR spectra were measured with an Oxford Instruments 400-MHz JEOL JNM-GX-4000 NMR spectrometer. UV spectra were recorded using a Shimano UV 240/ visible spectrophotometer fitted with an OPI-4 computer interface. IR spectra were recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer. Contact angles were measured using water at ambient conditions. A Kru¨ss G-1 goniometer attached to a video camera, a television, and a video cassette recorder was used for all measurements. All angles were measured within 20 s after the water drops were placed on each sample at different locations on the surface of a gold slide. The contact angle values for the pure gold substrate (65 ( 5°) were close the value of 59 ( 1° reported by Pierrat et al.12 Syntheses. The linker lipid DPPGL was synthesized as previously described by Pax and Blume13 and characterized with 1H NMR and UV/vis spectrometry. The structure of the end product is shown in Figure 1. The synthetic route of the host matrix lipid 1-palmitoyl-2(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine (DSDPPC) is outlined in Figure 1. A detailed description of the synthesis is given below (species II-VI). 16-Bromohexadecanoic Acid (II).14 16-Hydroxyhexadecanoic acid (I) (2.38 g, 9.1 mmol) was refluxed for 49 h in a 1:1 mixture of 48% hydrobromic (15 mL) and glacial acetic acid (15 mL) in the dark. Upon cooling, the 16-bromohexadecanoic acid (II) crystallized as a white solid. The mixture was filtered through a Bu¨chner funnel, washed several times with water, and recrystallized from ice-cold hexane. The yield was 2.65 g (87%). 16-Mercaptohexadecanoic Acid (III).14 Sodium hydride (60% w/w in mineral oil, 0.7 g) was separated from mineral oil in the following way: NaH in mineral oil was dissolved in diethyl ether (30 mL). The mixture was stirred, and the large portion of ether, which contained the mineral oil, was decanted away. Remaining

ether was quickly removed by rotary evaporation. Bromide II was converted to thioacetate by adding 50 mL of ice-cold methanol, 16-bromohexadecanoic acid (II, 2.6 g, 7.8 mmol), and thioacetic acid (1.18 mL, 16.6 mmol) into the flask, which contained previously separated sodium hydride and refluxing 19 h. The addition of the thioacetic acid formed a turbid yellow mixture, which turned to a clear yellow solution after heating. After 19 h of refluxing, the solution had turned colorless, and white solid had been formed. After the mixture had been cooled to room temperature, the thioester was hydrolyzed by adding 60 mL of 1 M NaOH solution (degassed with N2) and refluxing for 3 h under nitrogen. The reaction mixture was then cooled with an ice bath and poured into a beaker with a magnetic stirrer containing ice-cold water (200 mL), concentrated HCl (10 mL), and diethyl ether (225 mL). The layer containing ether was separated, washed with water (3 × 100 mL) and saturated aqueous NaCl solution (100 mL, 0.38 g/mL), and then dried over dehydrated CaCl2. Finally, the mixture was filtered, and the solvent was removed by rotary evaporation. The resulting white solid substance was recrystallized from ice-cold hexane, yielding 1.4 g (63%) of 16-mercaptohexadecanoic acid. The product was stored at 0 °C in the dark. TLC (2% MeOH in CHCl3): Rf (product) ) 0.40. 1H NMR (CDCl3): δ 1.26 (s, 22H, CH2), 1.32 (t, 1H, SH), 1.57-1.67 (m, 4H, HSCH2CH2(CH2)11CH2), 2.35 (t, 2H, HOOCCH2CH2), 2.52 (t, 2H, HSCH2CH2). 16-(S-Methyldithio)hexadecanoic Acid (IV).15,16 A mixture of 16-mercaptohexadecanoic acid (1.22 g, 4.2 mmol), methyl methanethiosulfonate (0.65 mL, 6.3 mmol), and pyridine (0.51 mL, 6.3 mmol) in chloroform (13 mL) was stirred at room temperature in the dark for 23 h. 16-Mercaptohexadecanoic acid dissolved poorly in chloroform, but the solubility increased as the 16-(Smethyldithio)hexadecanoic acid was formed, and the solution was completely clear at the end of the reaction. Upon removal of the solvent by evaporation under reduced pressure, the residue was washed twice with ice-cold ethanol (the white-brown solid turned white), followed by recrystallization in ice-cold hexane. The product was dried over P2O5 to yield 0.92 g (72%) of 16-(Smethyldithio)hexadecanoic acid. The product was stored at 0 °C in the dark. TLC (2% MeOH in CHCl3): Rf (product) ) 0.45. 1H NMR (CDCl3): δ 1.26 (s, 22 H, CH2), 1.57-1.72 (m, 4H, SSCH2CH2(CH2)11CH2), 2.35 (t, 2H, HOOCCH2CH2), 2.41 (s, 3H, CH3), 2.71 (t, 2H, SSCH2CH2). 16-(S-Methyldithio)hexadecanoic Acid Anhydride (V).17 A solution of DCC (0.32 g, 1.52 mmol) in dry CCl4 (5 mL) was added to a solution of 16-(S-methyldithio)hexadecanoic acid (0.92 g, 3.04 mmol) in dry CCl4 (30 mL). The reaction mixture was kept at room temperature under N2 atmosphere and protected from light. After 18 h, the mixture was filtered by suction to remove the N,N-dicyclohexylurea that had formed. Examination of the filtrate by IR spectroscopy revealed the presence of 16-(Smethyldithio)hexadecanoic acid anhydride (νCdO 1752 and 1819 cm-1) and the absence of the parent carboxylic group (νCdO 1702 cm-1). The solvent was removed by evaporation under reduced pressure. The yield was 0.8 g (87%). The product was dried over P2O5 and stored at -5 °C in the dark. 1-Palmitoyl-2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero3-phosphocholine (VI).16,18,19 1-Palmitoyl-2-hydroxy-sn-glycero3-phosphocholine (0.33 g, 0.66 mmol) was suspended in 35 mL of dry chloroform and 4-DMAP (0.3 g, 2.45 mmol) and previously prepared anhydride (0.8 g, 1.32 mmol) was added to the mixture. The reaction mixture was stirred at room temperature under nitrogen atmosphere and protected from light. 1-Palmitoyl-2hydroxy-sn-glycero-3-phosphocholine dissolved poorly in chloroform, but its solubility increased as the 1-palmitoyl-2-(16-(Smethyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine was formed, and the solution was completely clear at the end of the reaction. After 43 h, the mixture was added to a separator funnel and diluted to 50 mL with chloroform. Then, 35 mL of methanol

(10) Ishikawa, E. J. Immunoassay 1983, 4, 209. (11) Martin, F. J.; Hubbell, W. L.; Papahadjopoulos, D. Biochemistry 1981, 20, 4229. (12) Pierrat, O.; Lechat, N.; Bourdillon, C.; Laval, J.-M. Langmuir 1997, 13, 4112. (13) Pax, H.; Blume, A. Chem. Phys. Lipids 1993, 66, 63. (14) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321.

(15) Samuel, N. K. P.; Singh. M.; Yamaguchi, K.; Regen, S. L. J. Am. Chem. Soc. 1985, 107, 42. (16) Runquist, E. A.; Helmpamp, G. M., Jr. Biochim. Biophys. Acta 1988, 940, 10. (17) Selinger, Z.; Lapidot, Y. J. Lipid Res. 1966, 7, 174. (18) Mason, J. T.; Broccoli, A. V.; Huang, C.-H. Anal. Biochem. 1981, 113, 96. (19) Mangroo, D.; Gerber, G. E. Chem. Phys. Lipids 1988, 48, 99.

Experimental Section

Covalent Immobilization of Antibody Fragments

Langmuir, Vol. 18, No. 12, 2002 4955

Figure 1. Synthetic route for the preparation of DSDPPC and molecular structure of DPPGL. and 20 mL of 0.1 M HCl were added, and the lower phase was isolated. The upper phase was re-extracted with 2 × 20 mL of chloroform, the extracts were combined, and the solvent was removed by rotary evaporation. The residue was precipitated by the addition of acetone-chloroform (95:5) and purified by column chromatography. The column was eluted with chloroform, chloroform-methanol (9:1), and chloroform-methanol-NH3 (25%) (1:1:0.1). The product was precipitated by the addition of acetone-chloroform (95:5) and filtered by suction to yield 300 mg (60%) of pure 1-palmitoyl-2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine. The product was identified by 1H NMR and UV spectroscopy. TLC (CHCl3-MeOH-NH3 (25%) (65:25:4): Rf (product) ) 0.35. UV (CHCl3): λ1 ) 250 nm. 1H NMR (CDCl ): δ 0.88 (t, 3H, CH (CH ) , 1.25 (s, 46H, CH ), 3 3 2 12 2 1.54-1.60 (m, 4H, OOCCH2CH2), 1.65-1.72 (m, 2H, SSCH2CH2), 2.26-2.33 (m, 4H, OOCCH2), 2.41 (s, 3H, CH3SS), 2.70 (t, 2H, SSCH2), 3.39 (s, 9H, N+(CH3)3), 3.88 (m, 2H, POCH2CH2), 3.934.03 (m, 2H, CHCH2OP), 4.10-4.15 (m, 1H, CH2CHCH2OP), 4.36-4.40 (m, 3H, POCH2CH2 and CH2CHCH2OP), 5.18-5.23 (m, 1H, CHCH2OP). Surface Pressure Measurements. A commercially available computer-controlled KSV LB-5000 Langmuir trough (KSV Instruments, Helsinki, Finland) with a Wilhelmy balance was

used. A Milli Q filtration system (Millipore Corp.) was used to purify the water for the subphase having a resistance of 18 MΩ cm. DSDPPC was mixed in various ratios with the linker lipid (DPPGL) in chloroform to produce a solution with a concentration of 1 mg/mL and spread onto the surface of the subphase using a microsyringe. The solvent evaporated within 10-20 min, leaving a monomolecular layer of lipid on the subphase surface. Films of this material were compressed at 5 mm/min to produce surface pressure vs mean molecular area isotherms. The experiments were carried out at selected temperatures between 10 and 31 °C. Surface Potential Measurements. The surface potential measurements for the pure lipids (DSDPPC, DPPGL, and DPPC) were performed with a LB-5000 Langmuir trough. The experiments were carried out at 20 ( 0.4 °C. The surface potential, ∆V, of the monolayer was measured simultaneously with the surface pressure by the vibrating plate method. The upper, vibrating Pt electrode was positioned ca. 2 mm above the subphase surface, and it was perforated to minimize the noise. LS Deposition of the Lipid Monolayers onto a Gold Substrate. Ultraflat gold surfaces for use as substrates for horizontal deposition were prepared following the procedure described by Wagner et al.20 The mixed film of DSDPPC and

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Figure 2. Simplified schematic illustration of LS horizontal deposition of a binary DSDPPC-DPPGL film onto a gold substrate utilizing the specific reaction between gold and the disulfide group. DPPGL was prepared as described in surface pressure measurements, and the monolayer was compressed to a predetermined pressure. The surface pressure was kept constant at a predetermined value of 30 mN/m for 30 min. The stabilization resulted in only very minor changes in the mean molecular area (∼3%), showing the high stability of the Langmuir monolayer. The gold substrate was then brought into contact with the floating monolayer for a certain period of time. After being lifted, the lipid-coated substrate was washed with high-purity water and absolute ethanol and dried with nitrogen. A schematic illustration of the horizontal deposition procedure is shown in Figure 2. Scanning Probe Microscopy (SPM). The samples for SPM imaging were prepared in the following way. The gold substrates with the LS-deposited binary lipid monolayers were kept in a HEPES-EDTA buffer solution of Fab′ fragments for 2 h, followed by 18 h in a BSA solution at 4 °C, and subsequently 2 h in a hIgG solution. Samples were gathered from each coating step, and they were rinsed with buffer and high-purity water and dried with nitrogen prior to SPM imaging. A Nanoscope IIIa (Digital Instruments, Inc., Santa Barbara, CA) SPM in tapping mode was used to image the sample surfaces in ambient air. A J-scanner (150 × 150 µm2 scan range) and silicon cantilevers (TESP) supplied by the manufacturer (Nanoprobes TM) were used for imaging. The free amplitude of the oscillating cantilever (off contact) was 60 nm. The engage procedure, which caused a shift in the resonance frequency, was taken into account, and the new resonance frequency for the tip in contact was determined and used as the operating frequency. Light tapping with a damping ratio (contact amplitude/free (20) Wagner, P.; Hegner, M.; Gu¨ntherodt, H.-J.; Semenza, G. Langmuir 1995, 11, 3867.

Figure 3. Pressure-area isotherms of DSDPPC on a pure water subphase at different temperatures. amplitude) of about 0.7-0.8 was used for imaging. The SPM imaging was accomplished within 30 min after sample preparation.

Results and Discussion Pressure-Area and Surface Potential Isotherms of Pure DSDPPC and DPPGL. The surface pressurearea isotherms of pure DSDPPC on a pure water subphase at different temperatures diplayed in Figure 3 clearly show that DSDPPC forms a monolayer with a characteristic


Langmuir, Vol. 18, No. 12, 2002 4957 Table 1. Surface Potentials and Effective Dipole Moments of Phospholipid Monolayers and Their Head Group Regions at the Water-Air Interface at 20 ( 0.4 °C monolayer

Aext (nm2)

∆Vmax (mV)

∆VR (mV)

µn (D)

µR (D)

DSDPPC DPPC

0.45 0.49

DPPGL

0.42

+402 +625 (+669)a +462

+109 +85 (+99)a -161

+0.48 +0.81 (+0.82)a +0.52

+0.13 +0.11 (+0.12)a -0.18

a

Values taken from Vogel and Mo¨bius.19

of DSDPPC (0.45 nm2) and DPPC (0.49 nm2) in Table 1 shows that the disulfide group enhances the condensation of the monolayer. This indicates that the space filling of DSDPPC is more effective than that of DPPC, most probably because of the unequal lengths of the alkyl chains of DSDPPC. This presumably leads to a more perpendicular orientation of the lipids as compared to DPPC. This also means that DSDPPC reaches a preferable orientation considering the gold-sulfur adsorption process. The surface potential isotherms nicely followed the changes in the compression isotherms and were reproducible throughout the entire area range studied. Typical ∆V-A isotherms for DSDPPC, DPPC, and DPPGL are shown in Figure 4. The values of ∆Vmax obtained for DSDPPC, DPPC, and DPPGL were 402, 625, and 462 mV, respectively. The surface potential for a monolayer at the air-water interface can, in its very simplest form, be expressed by the Helmholtz equation as

∆V ) µn/0A

Figure 4. Pressure-area and surface potential isotherms of (A) pure DSDPPC, (B) DPPC, and (C) DPPGL monolayers on a pure water subphase at 20.0 ( 0.4 °C.

temperature-dependent liquid-expanded to liquid-condensed phase transition (LE-LC). This LE-LC phase transition is apparent between temperatures 12.2 and 23.7 °C. For comparison, the phase transition temperature for DPPC is 41.5 °C.13 As reported earlier,21 DPPGL is also surface active and has an LE-LC phase transition below the crystal melting point Tm (22.9 °C). Above Tm, the monolayer remains in the LE state throughout the compression.18 At 20.0 ( 0.4 °C, DSDPPC (Figure 4A) collapsed at a surface pressure of 43 mN/m. This value is lower than those for DPPC (Figure 4C) and DPPGL (Figure 4B), which both collapsed at a surface pressure of 55 mN/ m. The results show that the terminal disulfide group introduced in the alkyl chain decreases the collapse pressure. However, the comparison of extrapolated mean molecular area (Aext) values of compressed monomer films (21) Ihalainen, P.; Peltonen, J. Langmuir 2000, 16, 9571.

(1)

where µn is the normal component of the molecular dipole moment, is the relative permittivity of the monolayer, and 0 is the permittivity of vacuum. According to Vogel and Mo¨bius,22 the total effective dipole moment µn can be divided into two parts, µR and µω. All contributions associated with the polar headgroup, including the reorganization of water molecules near the lipid monolayer interface, are represented by µR, whereas µω represents the effective dipole moment of the hydrophobic part of the monolayer. If we assume that ) 1 and use the effective dipole moment value +0.35 D for a CH3 group of a hydrocarbon chain aligned parallel to the normal of the surface within a close-packed monolayer,22 we can estimate the effective dipole moment of the hydrated polar headgroups of the phospholipids studied here from surface potential data. The contributions of the monolayer-air and the monolayer-water interfaces to the total surface potential are compiled in Table 1. Aext represents the extrapolated mean molecular area, where the monolayer appears in the most condensed state. The CH2 groups of the hydrocarbon chains do not contribute to the total dipole moment.23 The zwitterionic phosphocholines have relatively small effective dipole moments of +0.13 D for DSDPPC and +0.11 D for DPPC, whereas the value is -0.18 D for DPPGL. The effective dipole moment of the SSCH3 group was calculated within the limits of error to be close to zero. Pressure-Area Isotherms of Binary Monolayers. Surface pressure-area isotherms of binary mixtures of DSDPPC and DPPGL measured at 20.0 ( 0.4 °C are shown in Figure 5. The clear LE-LC phase transition present in the isotherms of the pure components disappeared when (22) Vogel, V.; Mo¨bius, D. J. Colloid Interface Sci. 1988, 126, 408. (23) Habib, M.; Bockris, J. O. M. Langmuir 1986, 2, 388.

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Figure 5. Pressure-area isotherms of DSDPPC-DPPGL mixed monolayers on a pure water subphase at 20.0 ( 0.4 °C.

Figure 6. Mean molecular area for the mixed monolayer of DSDPPC-DPPGL as a function of the molar fraction of DSDPPC at 20.0 ( 0.4 °C. The dotted lines (drawn between data points for pure DSDPPC and DPPGL and representing ideal mixing between the lipids) are added to demonstrate the deviation of the data points from the linear dependence of the molecular area on the mixing ratio of the film components.

the linker molecule was introduced in the mixture, but it reappeared at higher linker concentrations and was clearly present at the mixture ratio of 50:50 mol %. At low DPPGL concentrations, the mixed monolayers collapsed at the same surface pressure as pure DSDPPC (43 mN/m), but at higher linker mole ratios (over 5 mol %), the mixed monolayers collapsed at the same surface pressure as pure DPPGL (55 mN/m). The extrapolated mean molecular areas of the compressed binary monolayers followed the same trend. At low linker mole ratios, Aext was the same as that of pure DSDPPC (0.45 nm2), whereas for higher linker concentrations (over 5 mol %), Aext was equal to that of pure DPPGL (0.42 nm2). This indicates that the linker is miscible with DSDPPC at low mole ratios only, whereas at higher mole ratios, the two molecules tend to phase separate. The excess area criterion24,25 was also tested to study the miscibility of the components in the binary monolayers. Figure 6 shows the average molecular area of the mixed DSDPPC-DPPGL monolayer as a function of the molar fraction of DSDPPC at surface pressures of 5, 15, 25, and 35 mN/m. The dotted lines in Figure 6 demonstrate the deviation of the data points from the linear dependence of molecular area on the mixing ratio of the film compo(24) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley-Interscience: New York, 1966 (25) Birdi, K. S. Lipid and Biopolymer Monolayers at Liquid Interfaces; Plenum Press: New York, 1989.


nents. The lines are drawn between data points of pure DSDPPC and DPPGL and represent ideal mixing behavior. The large deviations from the linear dependence at higher surface pressures when over 20 mol % of linker component is present in the mixture indicates that the components in the binary monolayer are miscible only when the linker lipid is in a clear minority (e20 mol %). The deviation from the additivity rule could also arise from the fact that pure DSDPPC and DPPGL have very different phase transition surface pressures, as seen in Figure 5. Mole ratios of 95:5 and 80:20 DSDPPC/DPPGL were chosen for antibody immobilization studies because of the good miscibility and stability properties of the corresponding binary films. SPM Measurements of Pure DSDPPC Monolayers on a Gold-Coated Substrate. Figure 7 shows typical SPM height images of the (A) pure gold substrate and (B-F) DSDPPC monolayer deposited onto the gold-coated substrate using LS deposition at a surface pressure of 30 mN/m at 20.0 ( 0.4 °C (B-F). Gold substrates were kept in contact with the Langmuir monolayer for (B) 20 s, (C) 1 min, (D) 3 min, (E) 15 min, and (F) 2 h. The images show that the gold substrate is almost fully covered with lipid molecules already after 20 s of adsorption. The dark holes in the height image represent the gold surface with no DSDPPC molecules adsorbed. In the 20-s, 1-min, and 3-min samples, there are more holes in the monolayer and they are larger than those in the 15-min and 2-h samples. A cross-section analysis gave an intermediate depth of the holes of 1.6-2.9 nm. In the 2-h sample, there were practically no visible holes; the substrate was fully covered with a lipid monolayer. However, the locally darker, i.e., lower, areas visible on the surface indicate that the density of the monolayer was not homogeneous over the surface. The most probable reason for this is the locally varying molecular tilt angle of the adsorbed lipids. The intermediate monolayer thickness value of 1.6-2.9 nm corresponds to the reported experimental thickness value of 1.77 nm for a self-assembled monolayer of 1-palmitoyl-2-(16-mercaptohexadecanoyl)-sn-glycero-3phosphocholine spontaneously adsorbed on gold and to the predicted maximum length of 2.8 nm for the same phosphocholine calculated from space-filling models.26 The root-mean-square roughness (Rrms) value over a 1 × 1 µm2 area of the lipid monolayer on the gold substrate varied with adsorption time, as shown graphically in Figure 8. These results show clearly that, although the monolayer adsorption is very fast as expected, the monolayer reaches its final homogeneity only after 2 h. On the basis of these results, it was decided to use an adsorption time of 2 h for further protein adsorption studies. The measured contact angle values of the gold substrate coated with a DSDPPC monolayer varied slightly with adsorption time, as shown in Figure 8. Although the DSDPPC-coated gold substrate was somewhat more hydrophilic than the pure gold surface, the contact angle values showed that DSDPPC molecules were adsorbed in a somewhat tilted orientation with respect to the surface normal, thus decreasing the hydrophilicity of the surface, especially for short adsorption times. The contact angle value of the gold substrate coated with a DSDPPC monolayer was close to the reported value of 47 ( 6° measured for 1-palmitoyl-2-(16-mercaptohexadecanoyl)sn-glycero-3-phosphocholine on gold.26 SPM Measurements of Protein Immobilization to the Binary Films. Figure 9 shows typical SPM height (26) Diem, T.; Czajka, B.; Weber, B.; Regen, S. L. J. Am. Chem. Soc. 1986, 108, 6094.


Langmuir, Vol. 18, No. 12, 2002 4959

Figure 7. Height images of LS monolayers of pure DSDPPC on a gold substrate as a function of exposure time. The gold substrate was kept on the subphase surface covered by a monolayer compressed to a surface pressure of 30 mN/m. The contact time was (B) 20 s, (C) 1 min, (D) 3 min, (E) 15 min, and (F) 2 h. Also shown is typical height image of a pure gold substrate (A). The height scale is 5 nm, and the image size is 1 × 1 µm2.

images of the binary monolayers of DSDPPC-DPPGL of two different compositions, (A) 95:5 and (B) 80:20 mol %, deposited at a surface pressure of 30 mN/m, the substrate being kept in contact with the floating monolayer for 2 h. The images show that, in both cases, the gold substrate is fully covered with a monolayer and no large holes are visible, nor are there any signs of phase separation.

The contact angle value for the 95:5 mol % mixture film was 55 ( 5°, and for the 80:20 mol % monolayer, it was 63 ( 5°. The slightly higher values than those obtained for pure DSDPPC monolayer indicate that the binary monolayers are less organized and homogeneous than the pure DSDPPC monolayers. However, the smaller contact angle value for the 95:5 mol % mixture as compared to the

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Figure 8. Rrms (b) and contact angle (O) values of LS monolayers of pure DSDPPC on a gold substrate as a function of exposure time.

Figure 9. Height images of binary DSDPPC-DPPGL monolayers on gold for the mixing ratios of (A) 95:5 and (B) 80:20 mol %. The gold substrate was kept in contact with the floating monolayer for 2 h. The height scale is 10 nm, and the image size is 2 × 2 µm2.

80:20 monolayer indicates that increasing the concentration of linker in the binary system results in a more inhomogeneous surface. Also, the Rrms values over a 2 × 2 µm2 area, 0.31 nm for the 95:5 and 0.41 nm for the 80:20 mixed film, support this conclusion.


SPM was further used to image the binding of Fab′ fragments and antibody-antigen complex formation on DSDPPC-DPPGL matrixes (Figure 10) with the fractions of the lipids being 95:5 (A, C, and E) and 80:20 mol % (B, D, and F). Fab′ fragments were immobilized from a HEPES-EDTA buffer solution with a concentration of 30 µg/mL, and the Rrms values were determined to obtain comparable information about the immobilization process (Table 2). A clear change in the Rrms value occurred as a result of Fab′ fragment binding, i.e., in the 95:5 mol % mixture, the Rrms value increased by 0.90 nm to 1.21 nm, and for the 80:20 mol % film, the increase was 1.45 nm to 1.86 nm. The higher initial Rrms value of the 80:20 mol % mixture, however, contributed to the fact that the Fab′ fragments formed more and larger aggregates on the matrix system. This was apparent from the SPM height images (Figure 10A and B), where globular objects were observed. For the 95:5 mol % mixture, the diameter of the globular object varied between 40 and 86 nm, whereas for the 80:20 mol % film, the corresponding value was 80200 nm. The dimensions of the globular objects for the 95:5 mol % film correspond to the value of 20-80 nm reported recently by Vikholm et al.27 However, tip-sample convolution always results in distorted lateral dimensions of objects,29 and it is impossible to draw exact conclusions about, e.g., the amount of proteins within the observed globules. The height data, in contrast, can be regarded much more reliable, especially because of the very smooth gold substrate, which served as a good reference surface for thickness comparisons. Because the dimensions of the proteins are known theoretically, the measured height differences from the substrate to the lipid monolayer and further to the protein layer can be used to make estimations about the molecular orientations within the films. The measured height of the Fab′ fragments, with values of 4.0-6.0 nm for the 95:5 mol % mixture and 5.1-7.4 nm for the 80:20 mol % mixture, corresponded well to the Fab′ fragment dimensions of 7 × 5 × 4 nm3 measured by X-ray diffraction.28 On the basis of these findings, most the objects in the case of 95:5 mol % mixture are believed to be Fab′ fragments bound to the DSDPPC-DPPGL matrix in a side-on (4 nm), edge-on (5 nm), or standing (7 nm) but slightly tilted orientation. The exposure of the surface to 0.1 mg/mL BSA in HEPES-EDTA buffer solution decreased the Rrms value for both compositions (Table 2). This was expected because BSA has no specific affinity to lipid layers, although it does adsorb to almost any surface.30 The decrease in the Rrms values indicates that the BSA molecules had adsorbed onto the surface where the lipid matrix was free from Fab′ fragments. That is why the height images (images 10C and 10D) look much smoother than the surfaces with only Fab′ bound (images 10A and 10B). Consequently, most probably, BSA did not adsorb on the Fab′ fragments. When hIgG molecules were introduced to the surface (images 10E and 10F), the Rrms values increased considerably for both film structures as seen in Table 2. The increase in the Rrms value was smaller, however, in the 80:20 system than in the 95:5 system, indicating that the surface concentration of the immobilized antibodies was higher in the former case, as stated earlier. This decreases (27) Vikhom, I.; Albers, W. M.; Viitala, T.; Peltonen, J. Biochim. Biophys. Acta 1999, 1421, 39. (28) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753. (29) Elender, G.; Kuhnrer, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11, 565. (30) Go¨lander, C.-G.; Kiss, E. J. Colloid Interface Sci. 1988, 121, 240.


Langmuir, Vol. 18, No. 12, 2002 4961

Figure 10. Height images of (A, B) Fab′ fragment, (C, D) BSA, and (E, F) hIgG-exposed (DSDPPC-DPPGL monolayers on gold for the mixing ratios of (A, C, E) 95:5 and (B, D, F) 80:20 mol %. Both monolayers were horizontally deposited from a Langmuir film kept at a surface pressure of 30 mN/m for a deposition time of 2 h. The height scale is 10 nm, and the image size is 2 × 2 µm2.

the interaction with hIgG because of steric hindrance. The average height of the adsorbed hIgG molecules was 7.0-9.0 nm for the 95:5 and 5.0-8.0 nm for the 80:20

system. Although the dimensions of both the Fab′ fragments and the hIgG molecules depend on the orientation of the Fab′ fragments in the hinge region, it was concluded

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Table 2. Rrms Values over an Area of 2 × 2 µm2 after the Different Protein Immobilization Steps Measured by SPM Rrms (nm) monolayer

95:5 mol %

80:20 mol %

phospholipid monolayer Fab′ BSA hIgG

0.31 1.21 0.52 1.60

0.41 1.86 0.67 1.10

from the height values that hIgG molecules were adsorbed in a somewhat tilted orientation on the surface. Conclusions The surface activity of the synthesized lipids DSDPPC and DPPGL was demonstrated through Langmuir isotherm studies. It was also shown that the terminal disulfide group introduced into the alkyl chain decreased the collapse pressure and enhanced the condensation of the monolayer because of the unequal lengths of the alkyl chains of DSDPPC, leading to a preferable orientation of DSDPPC considering the gold-sulfur adsorption process. The effective dipole moment of the SSCH3 group was calculated to be close to zero through surface potential measurements. The mixed monolayer of DSDPPC and DPPGL appeared to be miscible at low linker concentrations, whereas at higher mole ratios, the two molecules tended to be phase separated. SPM height images, Rrms values, and contact angle data for the DSDPPC monolayers

on gold showed that chemisorption during LangmuirSchaefer deposition was successfully carried out. The gold substrate became almost fully covered with lipid molecules already after 20 s of adsorption, but the monolayer reached its final homogeneity only after 2 h. The data for the mixed film of DSDPPC and DPPGL adsorbed on gold indicated that the binary monolayers were less organized and homogeneous than the pure DSDPPC monolayers and that increasing the concentration of the linker molecules in the binary system further increased the inhomogeneity of the surface. Antibody-antigen complex formation was demonstrated by SPM. SPM measurements showed that Fab′ fragments bind to the matrix in a slightly tilted orientation. The higher linker concentration induced more aggregate formation, increasing the unspecific binding of hIgG. The results show that the covalent coupling of antibody fragments to DPPGL embedded in a host monolayer matrix of DSDPPC is a promising way to achieve the site-directed immobilization of antibodies with high antigen-binding efficiency. Chemisorption of the monolayer onto the solid support enchances not only the mechanical but also the chemical and thermal stability of the biofunctional assay compared to that of conventional physisorped Langmuir-Schaefer films. Acknowledgment. Financial support from the National Technology Agency of Finland (Grant 40141/00) is gratefully acknowledged. LA020017Q

Covalent Immobilization of Antibody Fragments onto Langmuir

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