Protein Immobilization to a Partially Cross-Linked Organic Monolayer

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Langmuir 2000, 16, 4953-4961

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Protein Immobilization to a Partially Cross-Linked Organic Monolayer T. Viitala,*,†,‡ I. Vikholm,§ and J. Peltonen† Department of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, 20500 Turku, Finland, and Technical Research Centre of Finland, Chemical Technology, P.O. Box 14021, 33101 Tampere, Finland Received June 24, 1999. In Final Form: December 22, 1999 The covalent attachment of Fab′ fragments of polyclonal anti-human IgG to a polymerizable lipid with a terminal linker group (N-(-maleimidocaproyl)dilinoleoylphosphatidylethanolamine) was examined by means of quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and atomic force microscopy (AFM). The linker lipid was embedded in a monolayer of dilinoleoylphosphatidylethanolamine. Both monomeric and cross-linked biofunctionalized monolayers were studied. Atomic force microscope images showed that the monomeric monolayer consisted of large holes when it was deposited on a solid substrate, while the cross-linked monolayer appeared as a planar two-dimensional film. The ability of the biofunctionalized monolayer to bind proteins decreased with UV-irradiation time. However, an increase in the linker lipid concentration in the lipid matrix increased the protein-binding efficiency. A comparison between QCM and SPR measurements indicated that the QCM measurements overestimated the binding efficiency of immobilized Fab′ fragments toward hIgG. AFM images visualized the topographical changes of the different stages of the monolayer incubation in Fab′, BSA, and hIgG protein solutions.

Introduction The conventional immunoassays used in clinical laboratories require labeling techniques for the quantification of a complementary binding reaction between a ligand and its complementary substance. In these systems the quantification of the immunological reaction relies on a marker molecule, such as a radioisotope, an enzyme, or a fluorescent probe. In most cases, the result is not obtained until several incubations, washings, and separation steps have been performed. Therefore, there is an increasing interest toward immunosensors that can monitor antigen concentrations in real time with the aid of an immobilized antibody not requiring any labeled species. Quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) are techniques suitable for the in situ detection of immunoreactions of nonlabeled species.1-7 They have attracted great interest as measuring techniques in development of biosensors owing to their simplicity, small sensor size and small sample volumes needed. When using conventional immobilization methods, the lack of control of the orientation of the biomolecules limits the proportion of available binding sites.8-10 Monolayers deposited on a solid substrate from an air/water interface, * Corresponding author. E-mail: [email protected]. Fax: +358-2215 47 06. Phone: +358-2-215 42 52. † Åbo Akademi University. ‡ Graduate School of Materials Research, Turku, Finland. § Technical Research Centre of Finland. (1) Toyama, S.; Shoji, A.; Yoshida, Y.; Yamauchi, S.; Ikariyama, Y. Sens. Actuators, B 1998, 52, 65. (2) Vikholm, I.; Albers, W. M. Langmuir 1998, 14, 3865. (3) Sakai, G.; Saiki, T.; Uda, T.; Miura, N.; Yamazoe, N. Sens. Actuators, B 1997, 42, 89. (4) Silin, V.; Plant, A. TIBTECH 1997, 15, 353. (5) Nakanishi, K.; Muguruma, H.; Karube, I. Anal. Chem. 1996, 68, 1695. (6) Ko¨βlinger, C.; Uttenthaler, E.; Drost, S.; Aberl, F.; Wolf, H.; Brink, G.; Stanglmaier, A.; Sackmann, E. Sens. Actuators, B 1995, 24-25, 107. (7) Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1998, 110, 8623. (8) Caruso, F.; Rodda, E.; Furlong, D. N. J. Colloid Interface Sci. 1996, 178, 104. (9) Attili, B.; Suleiman, A. Microchem. J. 1996, 54, 174.

i.e., Langmuir-Blodgett (LB) or Langmuir-Schaeffer (LS) films, are often characterized by a high degree of molecular orientation. These films may offer unique applications when using lipid matrices with electrically, optically, and biologically active substances.11 Many groups have used the LB technique to better control the orientation and surface density of antibodies.10,12,13 Several different approaches have been utilized to biofunctionalize monolayers, such as the use of biotin/(strept)avidin chemistry,14,15 embedding of single-chain antibodies in phospholipid monolayers,16 direct deposition of an IgG monolayer on a protein A sublayer,10 and using linker lipids,17-20 However, during deposition the monolayer structure may undergo changes, and the film on a solid support may have lost a significant fraction of its activity through phase separation of matrix components and/or disruption of the monolayer. A promising approach to overcome this problem is to use polymers or polymerizable surfactants as the main matrix component.21 An even distribution of the biofunctional lipids can be obtained by polymerizing the monolayer in the fluid phase. In other words, the risk (10) Tronin, A.; Dubrovsky, T.; Radicchi, G.; Nicolini, C. Sens. Actuators, B 1996, 34, 276. (11) Swalen, J. D.; Allara, D. L.; Andrade, E. A.; Chandross, S. G.; Israelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (12) Wang, H.; Brennan, J. D.; Gene, A.; Krull, U. J. Appl. Biochem. Biotechnol. 1995, 53, 163. (13) Petty, M. C. J. Biomed. Eng. 1991, 13, 209. (14) Herron, J. N.; Mu¨ller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 1413. (15) Xiao, C.; Yang, M.; Sui, S.-F. Thin Solid Films 1998, 327-329, 647. (16) Vikholm, I.; Gyo¨rvary, E.; Peltonen, J. Langmuir 1996, 12, 3276. (17) Hoffmann, M.; Mu¨ller, W.; Ringsdorf, H.; Rourke, A. M.; Rump, E.; Suci, P. A. Thin Solid Films 1992, 210/211, 780. (18) Egger, M.; Heyn, S.-P.; Gaub, H. E. Biochim. Biophys. Acta 1992, 1104, 45. (19) Ebato, H.; Gentry, C. A.; Herron, J. N.; Mu¨ller, W.; Okahata, Y.; Ringsdorf, H.; Suci, P. A. Anal. Chem. 1994, 66, 1683. (20) Reichert, A.; Nagy, J. O.; Spevak, W.; Charych, D. J. Am. Chem. Soc. 1995, 117, 829. (21) Lowack, K.; Helm, C. A. Adv. Mater. 1995, 7, 156.

10.1021/la990817+ CCC: $19.00 © 2000 American Chemical Society Published on Web 05/05/2000

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Scheme 1. Idealized Picture of the Different Stages of Protein Immobilization to a Deposited Cross-Linked Monolayer

of phase separation being quite common for composite monolayers compressed to a solid phase can be eliminated. To obtain an oriented and sensitive layer of antibodies, our approach has been to mix a synthesized maleimide linker lipid in a phospholipid monolayer with or without cholesterol. Hence, the monolayer is able to covalently immobilize antibodies through the reaction with a free sulfhydryl group in the hinge region of Fab′ fragments.2,22 The choice of the specific linker (N-(-maleimidocaproyl)succinimide, EMCS) was due to the following reasons: (i) the maleimide group is stable over a wide pH range (pH ) 5-8),23 (ii) the linker does not produce unstable disulfide bonds with the antibodies in serum,24 and (iii) the specific binding of antibodies has been reported to be slightly higher for EMCS than for N-succinimidylpyridyl dithiopropionate (SPDP).2,25 Our earlier measurements with QCM, SPR, radioimmunoassay (RIA), and atomic force microscopy (AFM) have demonstrated that Fab′ fragments could be coupled to a maleimide linker embedded in a monolayer matrix of phosphatidylcholine with high antigen-binding efficiency.22 We have, furthermore, studied the UV-induced polymerization of an unsaturated phospholipid to be used as the main matrix compound in a biofunctionalized monolayer.26 The aim of the present study was to demonstrate the functionality of a bioprobe produced from polymerizable materials by using the LB technique. Our earlier studies have shown that the biofunctionalized monolayers made (22) Vikholm, I.; Viitala, T.; Albers, W. M.; Peltonen, J. Biochim. Biophys. Acta 1999, 1421, 39. (23) Kitagawa, T.; Shimozono, T.; Aikawa, T.; Yoshida, T.; Nishimura, H. Chem. Pharm. Bull. 1981, 29, 1130. (24) Martin, F. J.; Papahadjopoulos, D. J. Biol. Chem. 1982, 257, 286. (25) Shriver-Lake, L. C.; Donner, B.; Edelstein, R.; Breslin, K.; Bhatia, S. K.; Ligler, F. S. Biosens. Bioelectron. 1997, 12, 1101. (26) Viitala, T.; Peltonen, J. Biophys. J. 1999, 76, 2803.

up of monomeric lipids tend to form bilayers and aggregates.22 Therefore, the idea was to obtain a cross-linked biofunctionalized monolayer with enhanced mechanical stability. Comparative studies of protein immobilization to monomeric and cross-linked lipids was carried out. The lipid monolayer in this study consisted of a mixture of a synthesized unsaturated linker lipid,27 N-(-maleimidocaproyl)dilinoleoylphosphatidylethanolamine (DLiPE-EMC), and a commercially available unsaturated phospholipid, dilinoleoylphosphatidylethanolamine (DLiPE). The synthesis, characterization and basic monolayer properties of DLiPE and DLiPE-EMC have been reported elsewhere.27 QCM and SPR were used to demonstrate the biofunctionality of the monomeric and UV-irradiated monolayers, both as floating and deposited monolayers. AFM was used to image the layers of the antibody fragments and human IgG (hIgG) and to demonstrate the enhanced mechanical homogeneity and stability of the UV-irradiated monolayer. Scheme 1 shows an idealized picture of the different processes involved in the protein immobilization onto a deposited cross-linked monolayer. Materials and Methods Lipids. The host matrix lipid 1,2-dilinoleoylphosphatidylethanolamine (DLiPE, >99% purity) was purchased from Avanti Polar Lipids. The linker lipid N-(-maleimidocaproyl)dilinoleoylphosphatidylethanolamine (DLiPE-EMC) was synthesized as previously described.27 Briefly, DLiPE-EMC was prepared by the reaction of N-(-maleimidocaproyl)succinimide (EMCS, Fluka, purity >98%) with DLiPE using triethylamine (Baker, purity >98%) as a homogeneous catalyst. Both the synthesis and purification steps were performed under argon and avoiding (27) Viitala, T.; Albers, W. M.; Vikholm, I.; Peltonen, J. Langmuir 1998, 14, 1272.

Protein Immobilization to an Organic Monolayer Scheme 2. Structure of DLiPE-EMC and DLiPE

heating. The structures of DLiPE and DLiPE-EMC are shown in Scheme 2. Monolayer Formation, Polymerization, and Deposition. Monolayers were prepared in a homemade Teflon trough with dimensions of 50 × 200 × 10 mm3 using a commercial KSV 2000 LB instrument (KSV Instruments Ltd., Helsinki, Finland). Chloroform (HPLC-grade, Sigma-Aldricht) was used as the spreading solvent. Two mixing ratios of the lipid matrices were used: 10 mol % DLiPE-EMC and 20 mol % DLiPE-EMC mixed with DLiPE. The lipid matrix solution was spread onto aqueous subphases of 20 mM HEPES (Sigma), 0.9 wt % NaCl (PA-grade, Fluka) and 1 × 10-5 or 1.25 × 10-5 M uranyl acetate (UAc, PAgrade, Merck) at a pH ) 6.8 for the 10 or 20 mol % matrices, respectively. The reason for the slightly higher UAc concentration in the latter case will be discussed later. High-purity water (18.2 MΩ cm) from a Millipore Milli-Q filtering system was used for the preparation of the subphase and other buffer solutions. NaOH solution (1 M) prepared from Titrisols (Merck) was used to adjust the pH for the saline/buffer and subphase solutions. Monolayers were horizontally transferred, Langmuir-Schaeffer (LS) films, at a surface pressure of 30 mN/m onto solid substrates either before or after UV irradiation. The substrates were pressed into the subphase through the interface covered with the monolayer. The remaining lipids were removed from the interface and the substrates were lifted up, however, keeping them in buffer until further processing or analysis. Polymerization of the monolayer was carried out by UV irradiating with a 30 W low-pressure mercury lamp (maximum emission at 254 nm) placed ca. 0.2 m above the monolayer. The UV-irradiation experiments were performed at a constant surface pressure of 30 mN/m. Glass slides used for SPR measurements were coated with thin layers of chromium (5 nm, to increase the adhesion of gold) and gold (45 nm) by vacuum evaporation. These substrates were then soaked in a 1 mM solution of octadecylmercaptan (ODM, Aldrich, purity 98%) in ethanol for 24 h, rinsed with ethanol, and air-dried before deposition (Au/ODM substrates). Silicon substrates used for AFM measurements (dimensions, 6 × 9 mm2) were first peroxide-treated to generate a maximum amount of silanol groups on the surface. Thereafter, the slides were coated with octadecyltrichlorosilane (ODTCS, Aldrich, purity 95%) from toluene solutions, rinsed with toluene, and air-dried before deposition (Si/ODTCS substrates). Model Antibodies. Human IgG (hIgG) and F(ab′)2 fragments of polyclonal goat anti-human IgG (Jackson ImmunoResearch) were used as model proteins. F(ab′)2 was split into Fab′ fragments with dithiotreitol (DTT, Merck) before use.28 The molecular weight of Fab′ and hIgG were taken to be 47 and 150 kDa, respectively. (28) Ishikawa, E. J. Immunoassay 1983, 4, 209.

Langmuir, Vol. 16, No. 11, 2000 4955 Methods of Protein Immobilization Monitoring. QCM and SPR were used to monitor the protein immobilization. QCM is a mass-sensitive method, while SPR is based on an optical measuring technique. The experimental setups for the QCM and SPR techniques have been given elsewhere.2,22 but a short description is given here. QCM. The change in frequency due to the attachment of proteins to the lipid layer was measured with a 10 MHz QCM. The mass of the proteins was estimated according to Sauerbrey’s equation, where the observed decrease in resonant frequency is proportional to the change in mass of the quartz resonator. The resonators were purchased from Universal Sensors Inc. (Louisiana). The edges of the resonators were covered with a ring of silicone rubber (Dow Corning Co.), which prevented corrosion of the wires and degradation of the electrical contacts when submerged in solution. The gold electrode was cleaned with chromosulfuric acid, rinsed with water, and air-dried before the measurements. The same crystal was used repeatedly. A Hewlett-Packard 4195A spectrum/network analyzer connected to a computer was used to collect the near-resonance admittance phase spectra with a sample time of 1 min. The system automatically traced the resonant frequency at zero phase angle and collected a frequency range of 200 Hz around it. This range was rapidly sampled 10 times. The resonant frequency was calculated from a mean value and a linear regression estimate of the samples. The unirradiated monolayer containing the linker lipid was compressed to the surface pressure of 30 mN/m, and one side of the QCM was horizontally lowered to make contact with the lipid monolayer. Buffer solution (NaCl/Hepes, pH ) 6.8) was pumped through a small flow cell of Teflon placed below the QCM with a flow rate of 1 mL/min for about 3 min by means of a peristaltic pump. The pump was stopped, and when a stable baseline was reached the protein solution was pumped into the cell. The resonant frequency after each protein injection was collected as a function of time (ca. 30 min per each protein solution: Fab′, BSA, and hIgG). All experiments were performed at a temperature of 21 °C. In the case of the polymerized monolayers, the UV irradiation was performed at 30 mN/m before the QCM was lowered to make contact with the monolayer. SPR. Linearly p-polarized light of a wavelength of 632.8 nm from a He-Ne laser was directed through a prism onto a slide coated with a thin gold film positioned according to the so-called Kretschmann configuration.29 The intensity of the reflected light is measured as a function of the angle of incidence. At a critical angle of excitation of surface plasmons, a minimum in intensity of the reflected light is observed. The angle position of this minimum depends on the thickness and optical properties of the layer on the gold film.30 The monolayer matrix containing the linker lipid was compressed to the surface pressure of 30 mN/m after which it was transferred horizontally to an Au/ODM substrate. These substrates where then kept in buffer solution (NaCl/Hepes, pH ) 6.8) until they were attached to the prism via an index-matching oil. A flow cell was placed over the measurement area of the film and was filled with buffer. After a stable baseline was obtained, the protein solution was pumped into the cell and changes in the intensity of the light due to the protein adsorption onto the lipid film were detected as a function of time at the angle of incidence slightly above the resonance minimum. The surface was rinsed with buffer between every injection of protein in order to determine the amount of bound protein. The intensity was allowed to stabilize, and the difference between the levels was recorded. Atomic Force Microscopy. The samples for AFM imaging were prepared in the following way. The monolayers containing the linker lipids were transferred onto the Si/ODTCS substrates after which the slides were kept in a solution of Fab′ fragments (CFab′ ) 25 µg/mL) for 2 h, followed by 18 h in a BSA solution (CBSA ) 100 µg/mL) and 2 h in a hIgG solution (CIgG ) 100 µg/ mL). Samples were taken after each coating step, rinsed with high-purity water, and air-dried. The slides were stored dry at room temperature until the imaging was performed. (29) Kretschmann, E.; Raether, H. Phys. 1968, 241, 313. (30) Sadowski, J.; Korhonen, I.; Peltonen, J. Opt. Eng. 1995, 34, 2581.

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Figure 1. Change in resonant frequency due to the immobilization of Fab′, BSA and hIgG to a 1:9 DLiPE-EMC:DLiPE floating monolayer: monomer monolayer (s) and partially polymerized (30 s UV-irradiated) monolayer (- - -). A Nanoscope IIIa (Digital instruments, Inc., Santa Barbara, CA) atomic force microscope in tapping mode was used for imaging the sample surfaces in ambient air. A J-scanner (150 × 150 µm2 scan range) with silicon cantilevers (TESP) supplied by the manufacturer (Nanoprobes TM) was used for the tapping-mode imaging. The free amplitude of the cantilever (off contact) was chosen to be about 100 nm. The engage procedure will cause a shift in the resonance frequency of the cantilever. This was taken into account, and the new resonance frequency for the tip in contact was determined and used as the operating frequency. Light or moderate tapping (damping ratio: contact amplitude/ free amplitude ≈ 0.6-0.9) was used for imaging.

Results and Discussion The synthesis of DLiPE-EMC and the monolayer characteristics of pure DLiPE and DLiPE-EMC and their mixtures have been previously reported.27 The main conclusions from that study were that the mixed monolayers showed ideal miscibility behavior and that a UVinduced cross-linking reaction of a mixed monolayer was not possible until a minor amount of UAc was added to the subphase. UAc has a condensing effect on the phospholipid monolayers,26,31 and from a monolayer crosslinking reaction point of view a sufficiently condensed monolayer is a prerequisite.26,32 Because the main aim of this study was to use polymerized monolayers for the production of stable biofunctionalized thin films, we used Hepes buffer solutions with a small amount of UAc as subphases. Quartz Crystal Microbalance. Figure 1 shows the decrease in the resonant frequency of the QCM during the immobilization of antibody fragments and subsequent immobilization of BSA and hIgG onto a monomeric and partially polymerized monolayer of 9:1 DLiPE/DLiPEEMC. The binding equilibrium for each protein was achieved within 20-30 min. The jump in the resonant frequency after each protein injection is due to the disturbance of the peristaltic pump used to inject the protein solutions. The interpretation that the majority of the Fab′ fragments were specifically bound to the lipid layer is supported by QCM measurements where the monolayer was exposed to an excess of uncleaved F(ab)2 before subsequent injection of Fab′, BSA, and hIgG. From these measurements (not shown) it was estimated that ca. 40% (31) Gorwyn, G.; Barnes, G. T. Langmuir 1990, 6, 222. (32) Viitala, T. J. S.; Peltonen, J.; Linde´n, M.; Rosenholm, J. B. J. Chem. Soc., Faraday Trans. 1997, 93, 3185.

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Figure 2. Change in resonant frequency due to the immobilization of Fab′, BSA, and hIgG to a 2:8 DLiPE-EMC:DLiPE floating monolayer: monomer monolayer (s) and partially polymerized (30 s UV-irradiated) monolayer (- - -).

of the total resonant frequency change after F(ab)2 and Fab′ injections was due to unspecifically adsorbed F(ab)2 in the case of the monomeric lipid monolayer. The corresponding value for the UV-irradiated monolayer was around 25%. These results indicate that the specific binding of Fab′ was higher to the partially polymerized monolayer as compared with the monomeric film. Figure 1 also shows that a pronounced amount of the blocker agent BSA was bound to the monolayer. This is an interesting result because we have earlier noticed that almost no unspecific adsorption of BSA occurred to EMC linker lipid matrices, where the main matrix component was dipalmitoylphosphatidylcholine (DPPC).22 It has been reported that PC layers do not readily bind proteins.33,34 The PC and phosphatidylethanolamine (PE) headgroups at pH 6.8 are zwitterions with a positive charge on the amine group and a negative charge on the phosphate group,35 whereas the effective charge of BSA at this pH is negative.36 The reason for the clear binding of BSA to the DLiPE/DLiPE-EMC matrix can be attributed to the charged PE headgroups, which are quite easily accessible to molecules in the subphase as compared with the PC headgroup where the charges are more shielded by the bulky methyl groups. Thus, an electrostatic interaction would be stronger between the amine group of PE and BSA than between PC and BSA. On the other hand, PC has more pronounced hydration forces than PE,37,38 which makes it more protein-rejecting than a PE monolayer. The monolayer packing density will also influence the nonspecific binding of proteins to the lipid film, the unsaturated matrix monolayer of this study having a looser packing than a DPPC matrix monolayer. Figure 1 shows that the UV-irradiated film has a lower efficiency to bind Fab′ fragments than the monomeric monolayer. The influence of the linker lipid concentration on the monolayers ability to bind Fab′ fragments was tested as a next step. Figure 2 shows the changes in QCM resonant frequency during the immobilization of proteins (33) Chapman, D. Langmuir 1993, 9, 39. (34) Liley, M.; Bouvier, J.; Vogel, H. J. Colloid Interface Sci. 1997, 194, 53. (35) Tocanne, J.-F.; Teissie´, J. Biochim. Biophys. Acta 1990, 1031, 111. (36) Gallinet, J. P.; Gauthier-Manuel, B. Colloids Surf. 1992, 68, 189. (37) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21. (38) Rand, R. P.; Parsegian, V. A. Biochim. Biophys. Acta 1989, 988, 351.

Protein Immobilization to an Organic Monolayer

onto a monomeric and partially polymerized monolayer of 8:2 DLiPE/DLiPE-EMC. In this case the amount of UAc in the subphase had to be increased slightly from 1 × 10-5 to 1.25 × 10-5 M as compared with the 9:1 DLiPE/DLiPEEMC monolayer in order to reach similar reaction conditions for the monolayer matrix, i.e., a sufficiently condensed monolayer that could be polymerized by UV light. The frequency change due to the Fab′ immobilization to the monomeric monolayer was within the experimental resolution independent of the amount of linker lipid in the monolayer matrix or the concentration of Fab′ injected. On the contrary, the efficiency of the UV-irradiated monolayer to bind Fab′ fragments clearly increased with doubled linker lipid concentration. The area taken by a single Fab′ (∼31 nm2)22 is very large relative to the mean lipid area in the monolayer matrix (∼0.6 nm2),27 and about 50 molecules are calculated to be covered by one Fab′ fragment. This means that in theory less than 2% homogeneously distributed linker lipids in the matrix is enough for binding of a complete monolayer of Fab′. Therefore, the resonant frequency change for the monomeric monolayer being independent of the linker lipid content was expected and refers to a lipid matrix with quite evenly distributed linker lipids. UV irradiation, on the other hand, seems to inactivate part of the linkers, which, however, could be compensated for through an increased linker lipid concentration. The sensitivity of the EMC linker to UV light was studied in more detail as a function of the UV-irradiation time. The results are shown in Figure 3a. The maximum amount of immobilized Fab′ fragments (plateau value) decreased with UV irradiation time, and after about 2.5 min the monolayer did not bind Fab′ fragments anymore (see Figure 3b). A nonlinear fit of the Langmuir adsorption isotherm to the data points was used to obtain the lines in Figure 3a. Figure 3b shows that the ability of the monolayers to bind Fab′ fragments decreases almost linearly with the UV-irradiation time. The points in Figure 3b have been obtained by relating the total amount of Fab′ fragments immobilized to the monomeric and UVirradiated monolayers to each other under the assumption that the linker lipids in the monomeric monolayer have the optimal binding efficiency toward the Fab′ fragments. In conclusion, the reason for the decreased Fab′ fragment binding efficiency is due to the decomposition of the linker by UV irradiation. This conclusion is supported by the fact that the main emission band of the used UV lamp overlaps with the broad absorption band of the active part of the linker.27 It was also interesting to notice that the association constant (Table 1) obtained from the nonlinear fit of the Langmuir adsorption isotherm for the UVirradiated monolayers (Ka ≈ 3.7 × 106 M-1) was about twice the value observed for the monomeric monolayer (Ka ≈ 1.75 × 106 M-1). This shows that the binding of Fab′ is more stable and specific for the monolayers being UVirradiated owing to the more “freezed” position of the linker lipids within the cross-linked lipid matrix monolayer. These values are between the values we have obtained for saturated, monomeric DPPC/DPPE-EMC, and DPPC/ DPPE-EMC/CHOL monolayer matrices.22 The hIgG binding efficiency (binding efficiency, η ) fraction of Fab′ fragments that bind a hIgG in percent) of the Fab′ fragments immobilized onto the monomeric monolayers varied between 50 and 90% while it varied between 70 and 80% for the UV-irradiated monolayers. This clearly shows that the reproducibility of the measurements is not so good for the monomeric film but was improved for the UV-irradiated monolayers. The binding efficiencies are quite high, but it should be noted that a

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Figure 3. (a, top) Change in total resonant frequency due to the immobilization of Fab′ fragment as a function of UVirradiation time of a 2:8 DLiPE-EMC:DLiPE floating monolayer: monomer monolayer (s), 30 s UV-irradiated monolayer (- - -), 1 min UV-irradiated monolayer (‚‚‚) and 2 min UVirradiated monolayer (- - -). (b, bottom) Fraction of active linkers as a function of monolayer UV-irradiation time. Table 1. Apparent Binding Constants for the Covalent Coupling of Fab′ Fragments to the Monolayer and the Coupling of hIgG to Fab′ in Monomer and Partially Polymerized (30 s UV-Irradiation) 20 mol % DLiPE-EMC + 80 mol % DLiPE Monolayer Matrix Measured with SPR and QCM Ka (M-1) coupling of Fab′ to monolayer monomer partially polymerized coupling of hIgG to Fab′ partially polymerized

QCM

SPR

1.75 × 106 3.7 × 106

1.15 × 106

2.1 × 107

2.8 × 107

linear correlation between the decrease in resonant frequency and the mass of bound proteins is assumed. This does not necessarily hold when measuring with the QCM in liquid. Much higher frequency changes have been measured in liquid as compared with those measured in air.2,39,40 This has also been shown to be dependent on the protein.36 Usually the higher frequency response in liquid media than in air has been attributed to changes in the acoustic properties of the interfacial layer (viscoelasticity (39) Geddes, N. J.; Paschinger, E. M.; Furlong, D. N.; Caruso, F.; Hoffmann, C. L.; Rabolt, J. F. Thin Solid Films 1995, 260, 192. (40) Rickert, J.; Brecht, A.; Go¨pel, W. Biosens. Bioelectron. 1997, 12, 567.

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Viitala et al. Table 2. Binding Efficiency of Fab′ Fragments Attached to Monomeric and Partially Polymerized (30 s UVIrradiated) Linker Lipid Matrices (20 mol % DLiPE-EMC + 80 mol % DLiPE) at a Concentration of 50 µG/mL to Bind hIgG, As Measured with QCM, SPR, and AFM ηa (%) monomer partially polymerized

Figure 4. Change in reflection intensity as a function of time when Fab′ (C ) 25 µg/mL), BSA (C ) 100 µg/mL) and hIgG (C ) 100 µg/mL) are injected on the monomer monolayer (s) and 30 s UV-irradiated monolayer (- - - ) of 2:8 DLiPE-EMC:DLiPE. The solid lined arrows indicate the injection of protein, and the dashed lined arrows indicate rinsing with buffer. The inset shows the change in SPR signal when Fab′ or F(ab)2 were immobilized to a 30 s UV-irradiated monolayer (2:8 DLiPEEMC:DLiPE).

changes), slipping, or entrapped water molecules in the protein layer.41,42 The acoustic contribution may be marked in the present case were viscoelastic layers are adsorbed. Furthermore, the viscoelasticity changes from that of the more or less covalently bound Fab′ layer to that of the more loosely immobilized antigen layer and changes the acoustic response between these layers. In conclusion, the changes observed in resonance frequency are not directly proportional to changes in mass of the adsorbed layer. The association constant (Table 1) for the immunological reaction was estimated to be 2.1 × 107 M-1. Similar values were obtained for saturated DPPC/DPPE-EMC and DPPC/ DPPE-EMC/CHOL lipid matrices with the same commercial polyclonal proteins.22 This indicates that the immunological complex is able to form and is equally stable on these three matrices as long as the Fab′ fragments are properly immobilized. The bad reproducibility of the monomeric monolayers might be due to insufficient mechanical stability when the quartz crystal is lowered onto the monolayer before the measurements, which presumably increases the fraction of unspecifically bound Fab′ fragments. This is also indicated by the data of Figure 3b where the fraction of active linkers for the monomeric monolayer (0 min UVirradiated) does not coincide with the line drawn through the points obtained for the UV-irradiated monolayers. In fact, the value for the monomeric monolayer is ca. 20% higher than the value obtained by extrapolating the line characteristic for the irradiated films to t ) 0 min. The difference hence refers to a higher unspecific binding of Fab′ to the monomeric monolayer. Surface Plasmon Resonance. Immobilization of proteins to the deposited monomeric and UV-irradiated LS layers was determined by SPR. Figure 4 shows the change in reflection intensity as a function of time when Fab′, BSA, and hIgG were injected on the deposited film (2:8 DLiPE-EMC/DLiPE). The solid lined arrows indicate the time when the protein was injected, and the dashed lined arrows indicate where a buffer rinse was performed. The insert in Figure 4 shows the immobilization of Fab′ (41) Meccea, V. M. Sens. Actuators, A 1994, 40, 1. (42) Albrecht, O.; Gruler, H.; Sackmann, E. J. Colloid Interface Sci. 1981, 79, 319.

QCM

SPR

AFM

50-90% 70-80%

20% 55%

52%

and F(ab)2 to a UV-irradiated monolayer (2:8 DLiPE-EMC/ DLiPE). The insert demonstrates that the Fab′ fragments have a clearly higher specific interaction with the deposited layer than the F(ab)2, which indicates covalent binding of Fab′ to the UV-irradiated film. Clear differences between the monomeric and UVirradiated LS films to bind proteins can be seen in Figure 4. The monomeric film binds a large amount of Fab′ and BSA but a relatively small amount of hIgG as compared with the UV-irradiated film, which, however, binds a relatively large amount of hIgG with respect to the relatively low binding of Fab′ and BSA. The explanation for this data becomes obvious when studying the morphology of the films. Figure 5 shows tapping-mode images of the horizontally deposited monomeric and UV- irradiated monolayers. The monomeric monolayer has large holes, and it has evidently not been stable enough to remain as a true monolayer during the deposition. The UV-irradiated monolayer appears more clearly as a planar film. We may hence conclude, in conjunction with the SPR measurements, that a large amount of Fab′ and BSA was unspecifically bound to the monomeric layer because of the large holes in the monolayer. The unspecifically immobilized Fab′ fragments are randomly oriented, which is suggested to explain the low efficiency of Fab′ to bind hIgG as shown in Figure 4 (η ) 20%, see Table 2). On the other hand, for the UV-irradiated monolayer the binding efficiency was clearly higher (η ) 55%, see Table 2). This further supports the conclusion drawn from the QCM measurements that most of the Fab′ fragments are specifically bound to the irradiated monolayer, partially because of its enhanced mechanical stability and simultaneously owing to the “freezed” position of the molecules in the monolayer. The binding efficiency of Fab′ toward hIgG as determined by SPR was lower than the corresponding values obtained by QCM (see Table 2). This indicates the tendency of QCM to overestimate the binding efficiency, as we already concluded earlier. However, a lowering of the binding efficiency due to deposition of the monolayer is not completely ruled out. The Ka value (Table 1) for the reaction between Fab′ and the LS-deposited UV- irradiated monolayer as determined by SPR was clearly lower than that determined by QCM, whereas the Ka value for the Fab′-hIgG conjugate is of the same order of magnitude as that estimated from the QCM measurements. The same trend was observed for a DPPC/DPPEEMC matrix22 indicating that the immunological reaction was successful as long as a proper immobilization of the Fab′ fragments was achieved. Atomic Force Microscopy. The depth of the holes in the monomeric film in Figure 5 was 2.0-2.5 nm, which is consistent with AFM measurements on DLiPE films made in a liquid environment43 and in air.26 The high edges of the holes (3-5 nm from the hole bottom level) indicated a folding of the monolayer. Even though the UV- irradiated monolayer (Figure 5b) did not contain large (43) Hui, S. W.; Viswanathan, R.; Zasadzinski, J. A.; Israelachvili, J. N. Biophys. J. 1995, 68, 171.

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c) Figure 5. AFM tapping-mode images (1×1 µm2) of (a) monomeric and (b) 30 s UV-irradiated 2:8 DLiPE-EMC:DLiPE monolayers deposited on Si/ODTCS substrates at 30 mN/m. (c) Schematic figure of the proposed structure of the monolayer in Figure 5b. The light to dark height scale in (a) and (b) is 5 nm.

holes, it was not as compact and defect-free as, for example, a monolayer of a saturated phospholipid or fatty acid. It mainly consisted of relatively homogeneously distributed short networks of slightly folded regions with thicknesses of 1.5-3 nm. This structure is quite similar to what we have previously reported for UV-irradiated linoleic acid (LA) LB films.44 However, the folded region still retains as a planar structure, which was not the case for the LA LB films. A suggested structure for the UV-irradiated monolayer is shown in Figure 5c, where the hole boundaries have curved to resemble a partial hemimicellar-like structure. Figure 6 shows tapping-mode AFM images of UVirradiated LS monolayers with two different concentrations of the linker lipid in the monolayer matrix and incubated for 2 h in a Fab′ solution. The increase of the linker lipid concentration in the lipid matrix clearly increased the amount of Fab′ fragments immobilized to the surface. This result fully coincides with the QCM measurements and supports the conclusion that part of the linker lipids is inactivated by the UV irradiation. Besides the evidently higher amount of Fab′ fragments bound to the 20 mol % linker lipid matrix, there is a clear difference between the heights and the structure of the Fab′ fragments immobilized to the lipid film. In the case of the low concentration of the linker lipid (Figure 6a), two different types of aggregates can be distinguished: small globular objects and large clusters much bigger in size than the globular objects. The height of the globular (44) Peltonen, J.; Viitala, T. Scanning Probe Microscopy of Polymers; Ratner, B. D., Tsukruk, V. V., Eds.; ACS Symposium Series 694; American Chemical Society: Washington, DC, 1998, p 231.

objects was within 5-8 nm with a width of 40-100 nm, and the larger clusters had a height of 10-17 nm and width of 100-150 nm. Histogram analysis gave a mean aggregate height of 6.8 nm. For the higher linker lipid concentration (Figure 6b), the aggregates resembled globular objects with a more uniform size distribution. The globular objects could be divided into two groups according to specific height distribution: aggregates with a height of 3-6 nm and width of 30-80 nm and aggregates with a height of 8-10 nm and width of 75-100 nm. In this case the histogram analysis gave a mean aggregate height of 4.2 nm. Comparison of the height of the aggregates in the low and high linker concentration case reveals that the Fab′ fragments have a standing position in the first case and a slightly slanted or lying position in the latter case. Fab′ fragment dimensions have been taken to be 7 × 5 × 4 nm3 (height × width × length).45 The reason for the appearance of high aggregates in the monolayer containing less linker lipid is not clear. One explanation could be that the proteins on the sample containing more linker lipid cover the whole lipid layer. Hence, the height differences probably refer to antibodies of different orientations, i.e., not all the bound antibodies are vertically oriented with respect to the planar lipid layer. Addition of BSA to a 20 mol % linker lipid matrix incubated in a Fab′ fragment solution made the sample surface quite smooth, see Figure 7a. Some holes were clearly visible with a depth of 2-6 nm, indicating that (45) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753.

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Figure 6. AFM tapping-mode images (1×1 µm2) of UV-irradiated (30 s) monolayers deposited on Si/ODTCS substrates at 30 mN/m after incubation in a Fab′ fragment solution: (a) 1:9 DLiPE-EMC:DLiPE, CFab′ ) 50 µg/mL and (b) 2:8 DLiPE-EMC:DLiPE, CFab′ ) 25 µg/mL. The light to dark height scale in (a) and (b) is 15 and 10 nm, respectively.

Figure 7. AFM tapping-mode images (1×1 µm2) of UV-irradiated (30 s) 2:8 DLiPE-EMC:DLiPE monolayers deposited on Si/ ODTCS substrates at 30 mN/m after (a) immobilization of Fab′ fragments (25 µg/mL) and BSA (0.1 mg/mL), and (b) immobilization of Fab′ fragments (25 µg/mL), BSA (0.1 mg/mL), and hIgG (0.1 mg/mL). The light to dark scale in (a) and (b) is 5 and 20 nm, respectively.

BSA had only filled the surface free of Fab′ fragments. This conclusion is based on the fact that BSA has an ellipsoidal form with the dimensions of 11.6 × 2.7 × 2.7 nm3 46 and height differences of 2-3 nm could be measured between the highest points of the globular objects and the dominant plane of the image. This also indicates that BSA had adsorbed in a lying orientation. When the sample was further incubated in a hIgG solution, the adsorbed hIgG formed a networklike struc(46) Riddiford, C. L.; Jennings, B. R. Biochim. Biophys. Acta 1966, 71, 126.

ture consisting of large close-packed globular domains; see Figure 7b. The height of the individual domains could not be determined, but the thickness of the overall layer could be estimated through the larger holes seen in the image. The thickness of the antigen layer was 7-12 nm, which corresponds to the dimensions of hIgG when bound to the Fab′ fragments in a slightly slanted or end-on orientation. This interpretation is also supported by the fact that the average height value did not exceed 14 nm, which is the maximum length of an IgG molecule. The network structure formed by hIgG seems to be an

Protein Immobilization to an Organic Monolayer

intermediate form of the hIgG topography seen in our earlier study on protein immobilization to lipid matrices of DPPC/DPPE-EMC and DPPC/DPPE-EMC/CHOL.22 In that study it was found that the DPPC/DPPE-EMC/CHOL lipid matrix with hIgG formed only large globular objects with no network structure while the DPPC/DPPE-EMC lipid matrix with hIgG formed a network structure without any visible globular objects. The hIgG-binding efficiency of the Fab′ fragments estimated from the AFM images was ∼52%. The surface coverage of the proteins was roughly estimated from a mean area per protein. An area of ∼31 and ∼64 nm2 was calculated for one Fab′ fragment (dimensions; 7 × 5 × 4 nm3)45 and one IgG molecule (dimensions: 14 × 8.5 × 4 nm3),47 respectively. The binding efficiency value corresponds well to that observed by SPR. The SPR and AFM measurements gave binding efficiencies slightly lower than that obtained from the QCM measurements, further supporting the earlier conclusions concerning the QCM data. It is also necessary to bare in mind that structural changes of proteins due to the adsorption to a solid substrate may take place,48-51 which also can have an influence on the binding efficiency. Conclusions The sensitivity of a biofunctional thin film is directly coupled to parameters such as homogeneity, stability, and reproducibility of the lipid matrix and the ability to covalently immobilize proteins. We have successfully demonstrated the biofunctionality of monomeric and partially cross-linked monolayers with embedded linker (47) Silverton, E. W.; Navia, M. A.; Davies, O. R. Proc. Natl. Acad. Sci. 1977, 74, 5140. (48) Buijs, J.; Norde, W. Langmuir 1996, 12, 1605. (49) Kondo, A.; Oku, S.; Muramaki, F.; Higashitani, K. Colloids Surf., B 1993, 1, 197. (50) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. (51) Soderquist, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386.

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lipids. It was observed that the ability of the lipid monolayer to bind proteins decreased with increasing UVirradiation time, which was attributed to the UV-induced decomposition of the maleimide part of the linker. A sufficient retention of the binding efficiency for the proteins was achieved by increasing the linker lipid concentration in the lipid matrix and by UV-irradiating the monolayer for only a very short time. Furthermore, the enhanced mechanical stability of the UV-irradiated monolayer was seen by comparing the topography of the monomeric and partially cross-linked monolayers. SPR and QCM measurements revealed that the monomeric monolayer had a slightly higher tendency for unspecific protein binding as compared with the partially cross-linked monolayer. AFM and SPR measurements gave a binding efficiency of the biofunctional monolayer that was somewhat lower than that determined by QCM. This difference is due to the false interpretation of the QCM data for adsorbed viscoelastic protein layers for which the frequency change is not directly proportional to the change in mass. The partially cross-linked deposited monolayer retained the protein-binding efficiency better than the monomeric monolayer, which is attributed to the higher mechanical stability and more homogeneous structure of the crosslinked monolayer. The results demonstrate that the covalent coupling of Fab′ fragments to a polymerizable monolayer matrix is a promising approach to achieve a controlled immobilization of antibodies with high antigen-binding efficiency. However, the quite high level of unspecific BSA adsorption and the decomposition of the linker lipid due to UV irradiation may limit the use of the studied monolayer matrix in selective immunodetection. These problems could be overcome by using matrix compounds with low unspecific protein binding (for example, phosphatidylcholines) and using other types of monomeric materials polymerizable without UV irradiation. LA990817+