Surface Modification Chemistry Based on the Electrostatic Adsorption

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Surface Modification Chemistry Based on the Electrostatic Adsorption of Poly-L-arginine onto Alkanethiol Modified Gold Surfaces Se´rgio V. P. Barreira* and Fernando Silva Departamento de Quı´mica da Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal (European Union) Received June 28, 2003. In Final Form: August 26, 2003 The reaction between the arginine’s guanidino group and R-dicarbonyl functionalities was used to develop a novel surface modification chemistry. The first modification step consists of the electrostatic adsorption of a poly-L-arginine layer onto ionizable alkanethiol modified gold surfaces. The strongly basic character of the guanidino group of the arginine residues (pKa > 12) guarantees the robust attachment of the polypeptide to negatively charged gold surfaces until very high pH. By varying the pH of the solution from which poly-L-arginine is electrostatically adsorbed, it is possible to control the amount deposited. The availability of the surface guanidino groups of the poly-L-arginine layer for further derivatization with R-dicarbonyl reaction probes, yielding stable heterocyclic condensation adducts, is demonstrated. In addition, the reaction with the heterobifunctional reagent p-azidophenyl glyoxal (APG) provides a surface terminated with a photosensitive aryl azide group which was employed for the photochemical immobilization of proteins to the surface. The application of this surface modification chemistry to immobilize antibodies is demonstrated.

Introduction The control of surface properties and sensor development were extended one step further by the introduction, 10 years ago, of the concept of electrostatically driven layer-by-layer assembly.1,2 This experiment can lead to the formation of multilayer films onto surfaces simply by alternately dipping a charged substrate into solutions of oppositely charged polyelectrolytes, the number of times depending on the number of layers desired. Since the pioneering work of Decher, this methodology has been employed to construct functional films of the most varied materials, for example, DNA,3 proteins,4 conducting polymers,5 dyes,6 metal and semiconductor nanoparticles,7 and clay microplates.8 * To whom correspondence should be addressed. Address: Departamento de Quı´mica da Faculdade de Cieˆncias da Universidade do Porto, Rua do Campo Alegre 687 4169-007 Porto, Portugal; phone: (351) 22 6082934; fax: (351) 22 6082959; e-mail: [email protected]. (1) Decher, G.; Hong, J.-D.; Schmidt, J. Thin Solid Films 1992, 210/ 211, 831-835. (2) Decher, G. Science 1997, 277, 1232-1237. (3) Lvov, Y.; Decher, G.; Sukhorukov G. Macromolecules 1993, 26, 5396-5399. (b) Shi, X.; Sanedrin, R. J.; Zhou, F. J. Phys. Chem. B 2002, 106, 1173-1180. (c) Jin, Y.; Shao, Y.; Dong, S. Langmuir 2003, 19, 4771-4777. (4) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427-3433. (b) Franchina, J. G.; Lackowski, W. M.; Dermody, D. L.; Crooks, R. M.; Bergbreiter, D. E.; Sirkar, K.; Russel, R. J.; Pishko, M. V. Anal. Chem. 1999, 71, 3133-3139. (c) Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel, J.-C. Langmuir 2001, 17, 878882. (5) Lukkari, J.; Saloma¨ki, M.; Viinikanoja, A.; A ¨ a¨ritalo, T.; Paukkunen, J.; Kocharova, N.; Kankare, J. J. Am. Chem. Soc. 2001, 123, 6083-6091. (b) Baba, A.; Park, M.-K.; Advincula, R. C.; Knoll, W. Langmuir 2002, 18, 4648-4652. (c) Li, W.; Hooks, D. E.; Chiarelli, P.; Jiang, Y.; Xu, H.; Wang, H.-L. Langmuir 2003, 19, 4639-4644. (6) Laschewsky, A.; Wischerhoff, E.; Kauranen, M.; Persoons, A. Macromolecules 1997, 30, 8304-8309. (b) Linford, M. R.; Auch, M.; Mo¨hwald, H.; J. Am. Chem. Soc. 1998, 120, 178-182. (7) Malikova, N.; Pastoriza-santos, I.; Schierhorn, M.; Kotov, N. A.; Liz-Marza´n, L. M. Langmuir 2002, 18, 3694-3697. (b) Sennerfors, T.; Bogdanovic, G.; Tiberg, F. Langmuir 2002, 18, 6410-6415. (c) He, J.A.; Mosurkal, R.; Samuelson, L. A.; Li, L.; Kumar, J. Langmuir 2003, 19, 2169-2174. (8) Glinel, K.; Laschewsky, A.; Jonas, A. M. Macromolecules 2001, 34, 5267-5274.

The versatility of this approach resides in the nature of the interactions involved: the electrostatic attraction between oppositely charged molecules is nonspecific and has the least steric demand of all chemical bonds. The only requirement, for spontaneous assembly and robust attachment, is the existence of multiple electrostatic binding points between the molecule and the substrate.2 In addition to providing an electrostatic nonspecific surface modification path via electrostatic layer-by-layer (LBL) assembly, the functional groups of an electrostatic adsorbed molecule can be derivatized by chemical reaction. The flexible architectures that polyelectrolyte chains are supposed to adopt on the surface with loops and tails extending into the solution phase9,10 can constitute an advantage by minimizing stereochemical hindrance for surface reactions relative to the self-assembled monolayers (SAMs) ordered and closed packed architectures. The strategy of functionalizing a surface through electrostatic assembly has long been used for the purpose of biomolecule immobilization onto surfaces.11-16 For example, the electrostatic adsorption of poly-L-lysine is commonly employed as a facile way to introduce easily targeted amine groups onto a surface.11,12 The -amino group of lysine can be reacted with a bifunctional crosslinker containing an NHS ester13 or isothiocyanate14 functionalities. Biomolecule attachment is effected in a second reaction step with the second reactivity of the linker molecule. In this way, DNA has been attached to gold13-15 or silicon16 surfaces in a robust and stable fashion. (9) Lowack, K.; Helm, C. A. Macromolecules 1998, 31, 823-833. (10) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 42134219. (11) Jordan, C. E.; Frey, B. L.; Kornguth, S.; Corn, R. M. Langmuir 1994, 10, 3642-3648. (12) Frey, B. L.; Corn, R. M. Anal. Chem. 1996, 68, 3187-3193. (13) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (14) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-4956. (15) Gillmor, S. D.; Thiel, A. J.; Strother, T. C.; Smith, L. M.; Lagally, M. G. Langmuir 2000, 16, 7223-7228. (16) Strother, T. C.; Cai, W.; Zao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205-1209.

10.1021/la035158m CCC: $25.00 © 2003 American Chemical Society Published on Web 10/17/2003

Surface Modification Chemistry

Electrostatically adsorbed polylysine modified with biotin has also been used to control the specific adsorption of Avidin17 onto surfaces and a polycationic copolymer of poly-L-lysine and PEG-biotin was recently proposed as a means to produce biotinylated surfaces, for bioaffinity sensing, that minimize nonspecific adsorption.18 We investigated the electrostatic adsorption of poly-Larginine both as a route for the construction of multilayer films via layer-by-layer assembly and for producing a guanidino-functionalized surface for subsequent covalent modification chemistry. The δ-guanidino group of the arginine residues is strongly basic (pKa > 12) and in principle, polyarginine should behave as a strong polycation that adsorbs spontaneously onto negatively charged surfaces even in very alkaline conditions. The guanidino group is also known to react readily with R-dicarbonyl reagents yielding heterocyclic condensation products.19-21 This reaction is employed to block the arginine residues in proteins22-24 and has already been used as a way to create an ordered monolayer of cytochrome c at the air-water interface via the reaction between the outer arginine residue of the protein and an anchoring long alkyl-chain R-diketone.25 The present work demonstrates that the guanidino groups, introduced via polyarginine electrostatic adsorption onto ionizable alkanethiol modified gold surfaces, can participate in condensation reactions with several R-dicarbonyl compounds. The combination of pArg electrostatic adsorption and guanidino reactivity toward R-dicarbonyl functionalities is further explored for the development of a surface attachment strategy to be used for proteins. The immobilization of proteins onto surfaces has been widely studied and used as a tool for revealing protein interactions and function in immunoassays,26 biosensors,27 or affinity chromatography.28 One of the approaches utilized for protein immobilization has been the photoimmobilization using photoactivable surfaces, that is, benzophenone or aryl azide terminated surfaces.29-32 The photoreactive surface may be obtained by in-situ transformation of the (17) Frey, B. L.; Jordan, C. E.; Kornguth, S.; Corn, R. M. Anal. Chem. 1995, 67, 4452-4457. (18) Huang, N.-P.; Vo¨ro¨s, J.; De Paul, S. M.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 220-230. (19) Creighton, T. E. Proteins, Structures and molecular properties; Freeman: New York, 1984. (20) Pathy, L.; The´sz, J. Eur. J. Biochem. 1980, 105, 387-393. (21) Glomb, M. A.; Lang, G. J. Agric. Food. Chem. 2001, 49, 14931501. (22) Konishi, K.; Fujioka, M. Biochemistry 1987, 26, 8496-8502. (23) Yamasaki, R. B.; Shimer, D. A.; Feeney, R. E. Anal. Biochem. 1981, 111, 220-225. (24) Pathy, L.; Smith, E. L. J. Biol. Chem. 1975, 250, 557-564. (25) Riccio, A.; Lanzi, M.; Antolini, F.; De Nitti, C.; Tavani, C.; Nicolini, C. Langmuir 1996, 12, 1545-1549. (26) Jones, V. W.; Kenseth, J. R.; Porter, M. D.; Mosher, C. L.; Henderson, E. Anal. Chem. 1998, 70, 1233-1241. (b) Avseenko, N. V.; Morozova, T. Y.; Ataullakhanov, F. I.; Morozov, V. N. Anal. Chem. 2002, 74, 927-933. (c) Inerowicz, H. D.; Howell, S.; Regnier, F. E.; Reifenberger, R. Langmuir 2002, 18, 5263-5268. (d) Fall, B. I.; Eberlein-Koˇnig, B.; Behrendt, H.; Niessner, R.; Ring, J.; Weller, M. G. Anal. Chem. 2003, 75, 556-562. (27) Hoshi, T.; Anzai, J.-I.; Osa, T. Anal. Chem. 1995, 67, 770-774. (b) Fragoso, A.; Caballero, J.; Almirall, E.; Villalonga, R.; Cao, R. Langmuir 2002, 18, 5051-5054. (28) Hermanson, G. T.; Krishna Mallia, A.; Smith, P. K. In Immobilized Affinity Ligand techniques; Academic Press: New York, 1992. (29) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 91-93. (30) Delamarche, E.; Sundarabadu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (31) Yang, S.; Pe´res-Luna, V. H.; Lo´pez, G. P. In Protein Architecture, Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000. (32) Baas, T.; Gamble, L.; Hauch, K. D.; Castner, D. G.; Sasaki, T. Langmuir 2002, 18, 4898-4902.

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ω-terminal group of self- assembled monolayers to a photosensitive group thereby obtaining a photoactivable SAM on gold30 or using the affinity of an avidin-modified surface for the ligand biotin functionalized with an aryl azide group (also known as photobiotin).29 In the present work, the polyarginine layer is reacted with a heterobifunctional reagent containing an R-dicarbonyl reactivity and a photoactivable aryl azide moiety. Protein covalent attachment is achieved afterward, when the resulting photoreactive surface, in contact with the protein solution, is exposed to UV light. The photolysis of the aryl azide results in the formation of a short-lived aryl nitrene31,33,34 which is extremely reactive toward a variety of groups of the protein.29,33-36 This immobilization scheme thus has the advantage of being not specific in the sense that the protein to be immobilized does not have to include a specific functionality. Experimental Section I. Materials. The chemicals 11-mercaptoundecanoic acid (MUA, Aldrich), 3-mercaptopropanesulfonic acid sodium salt (MPSA, Aldrich), poly-L-arginine hydrochloride (Mw, 38 300) (pArg, Sigma), poly-L-tyrosine (Mw, 16 500) (pTyr, Sigma), glyoxal (Sigma), phenylglyoxal (Fluka), p-azidophenyl glyoxal (APG, Pierce), poly(sodium 4-styrenesulfonate) (Mw, 70 000) (pSS, Aldrich), ethanol (absolute, AGA, Portugal), methanol (Merck, p.a.), boric acid (SIF, Portugal), HCl (Merck), NaOH (Pronalab, Portugal), H2SO4 (Pronalab, Portugal), hydrogen peroxide 30% (Pronalab, Portugal), NH3 solution 25% (Merck), NaCl (Merck), Na2HPO4 (Merck), KH2PO4 (Merck), KCl (Merck), aceton (RiedeldeHae¨n), acetic acid 100% (Pronalab, Portugal), poly-L-lysine hydrobromide (Mw, 32 600) (pLys, Sigma), human IgG (HIgG) (Sigma), goat anti-human IgG fluoroscein isothiocyanate (FITC) labeled (Sigma), and swine anti-rabbit IgG FITC labeled (DAKO, Denmark) were all used as received. Commercial gold slides (5 nm Cr and 100 nm Au) were purchased from Evaporated Metal Films (New York) and used as substrates after being degreased by a brief wash with acetone and then cleaned by soaking in an oxidizing solution (a 70:30 mixture of sulfuric acid and hydrogen peroxide (30%)) for about 1 min followed by thorough washing with large amounts of Millipore water. Millipore filtered water (resistivity >18 MΩ‚cm) was used to prepare all aqueous solutions and for rinsing.

II. Surface Modification Chemistry The MUA and MPSA monolayers were chemisorbed onto the gold surfaces from 1 mM ethanolic solutions for at least 24 h. After self-assembly, the surfaces were thoroughly rinsed with ethanol and water and then dried in a stream of nitrogen. a. Electrostatic Adsorption. Poly-L-arginine was electrostatically adsorbed onto the ionizable alkanethiol modified gold surfaces by dipping the substrate for 10 min in a 1 mg/mL pArg solution buffered at pH 8.2-13 (depending on the application). The buffers employed were borate (pH 8.2-10), ammonia (pH 10 and 11.2), and solutions of NaOH (0.01 M (+0.09 M NaCl) and 0.1 M). No detectable increase in the amount adsorbed was observed after 10 min indicating that the adsorption equilibrium was attained. The surface was then rinsed thoroughly with water and dried with a nitrogen stream. The adsorption of pSS was performed by dipping the polycationic terminated pArg surface in a pSS solution 1 mg/mL (prepared using the same buffer) for 10 min. (33) Ji, T. H. Biochim. Biophys. Acta 1979, 559, 39-69. (34) Brunner, J. Annu. Rev. Biochem. 1993, 62, 483-514. (35) Ji, T. H. J. Biol. Chem. 1977, 252, 1566-1570. (36) Sgro, J.-Y.; Jacrot, B.; Chroboczek, J. Eur. J. Biochem. 1986, 154, 69-76.

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Layer-by-layer assembly of the multilayer polypeptide films of pArg/pTyr was performed by repeated sequential dipping of the ionizable modified gold surface into alternating solutions of pArg (1 mg/mL, NaOH 0.01 M) or pTyr (0.5 mg/mL NaOH 0.01 M) for 10 min. Between each dip, the surface was rinsed with Millipore water and blown dry in a nitrogen stream. b. Surface Reactions. The reaction of glyoxal with the pArg layer (adsorbed from a borate buffer solution 0.1 M pH 9) was performed by soaking the pArg-terminated surface into 10 mM solution of the dicarbonyl reagent buffered at pH 9, 0.1 M borate buffer, for 4 h. In phenylglyoxal, the solvent was a 1:1 methanol/borate buffer mixture. It has been shown37that borate stimulates the kinetics of adduct formation of glyoxal compound derivatives that both enhances the amount of adduct formation and suppresses the formation of diaddition products. After every reaction, the surface was thoroughly rinsed with buffer and water and dried under a nitrogen stream before FT-IRRAS spectra was collected. The photoreactive surface was obtained by immersing the pArg terminated surface in a 6 mM APG solution (borate buffer pH 8.5) for at least 4 h. This reaction was carried out in the dark and the resulting surface was manipulated in subdued light conditions to prevent undesirable activation. Protein photochemical attachment was accomplished by exposing the photoreactive surface immersed in 1 mg/ mL protein solution (borate buffer pH 8.5, phosphatebuffered saline (PBS, 10 mM phosphate buffer, 3 mM KCl, 140 mM NaCl, pH 7.4) in the case of the antibodies) to UV light (λ ) 312 nm) for 5 min (Vilber Lourmat TFX-20M lamp, (power ∼180 W)). The activation interval of APG is between 240 and 350 nm.38 After the photochemical immobilization, the surface was thoroughly washed with buffer and water to remove physisorbed protein. III. FT-IRRAS Surface Characterization All the surface modification steps were characterized using Fourier transform infrared reflection-absorption spectroscopy, FT-IRRAS. The FT-IRRAS spectra in the mid-IR region were recorded on a Perkin-Elmer 2000 FTIR with its sample area modified to accommodate an external reflection sampling geometry. The spectra were obtained using the p-polarized component of the radiation at an incident angle of approximately 77° relative to the surface normal, with a narrow-band MCT detector liquid nitrogen cooled. The sample area was purged by dry air delivered from a CDA series MTI-Puregas purge gas generator. Each spectrum was obtained with a resolution of 4 cm-1. A bare gold slide, rigorously cleaned according to the procedure described in the Materials section, was used as a background surface. Background spectra consisting of 200 averaged scans were taken before collecting each sample spectra. The sample spectra reported consists of about 1000 averaged scans. IV. Contact Angle Measurements Contact angles were determined with a Dataphysics Contact Angle System OCA (Dataphysics Instruments GmbH Hamburg, Germany). The measurements were made at 25 °C and 100% humidity using Millipore water (37) Expert-Besancon, A.; Hayes, D. Eur. J. Biochem. 1980, 103, 365-375. (38) Ngo, T. T.; Yam, C. F.; Lenhoff, H. M.; Ivy, J. J. Biol. Chem. 1981, 256, 11313-11318.

Barreira and Silva

as the probing liquid. The contact angles were measured by lowering a 4 µL drop onto the surface from a bluntended needle attached to a 500 µL syringe. The contact angle was recorded immediately after the drop detached from the needle tip, using ellipse fitting included in the SCA 202 software. The contact angles reported are the average of at least five measurements. V. Immunoassay The retention of the biological activity of the immobilized antibodies was assessed through an immunoassay. Following the photoimmobilization of the primary antibody HIgG, the slide was washed with PBS buffer and then immersed in 50 mL PBS buffer for 1 h, after which the surface was again washed briefly with PBS buffer and exposed to the secondary antibody FITC labeled (10 µg/ mL in PBS). Incubation was allowed to proceed for 30 min in a humidity chamber. After incubation, the surface was first washed with PBS before being immersed for 10 min more in 50 mL PBS buffer. Fluorescence images of these samples were acquired using the Typhoon 8600 variable mode imager from Molecular Dynamics. The samples were placed face down on top of the glass scanner tray with a droplet of PBS between the gold slide and tray and scanned. The samples were protected from light during all preparation steps prior to imaging. Results and Discussion A. Electrostatic Adsorption. The FT-IRRAS spectrum of the MUA-modified gold surface is depicted in Figure 1. This spectrum was taken after rinsing with Millipore water (pH ∼6) and shows bands at 1717 cm-1 with a shoulder at 1740 cm-1 that correspond, respectively, to the hydrogen-bonded carboxylic acid, laterally dimerized,39 and to the free COOH. The small band at 1416 cm-1 is attributed to a combination of the methylene scissors deformation, δ(CH2), and to the symmetric carboxylate stretch, νs(COO-), (see Table 1 for band assignments). This spectrum coincides with those reported for this self-assembled monolayer onto gold surfaces.40 The electrostatic adsorption of pArg (schematically represented) is verified by the appearance of two prominent bands at 1679 cm-1 and at 1551 cm-1. The band at 1679 cm-1 is asymmetric because of several contributions: guanidino CN stretch, amide I (CO carbonyl stretch), and possibly the CO stretch of the remaining protonated MUA. The amide II band at 1551 cm-1 is partially overlaid by the asymmetric stretching vibration of COO- because of ionized MUA. Two other bands at 1455 and 1405 cm-1 correspond to the methylene scissors deformation of the three hydrophobic methylene groups of every arginine residue and to the symmetric carboxylate stretch of MUA, respectively. The resistance of pArg to desorb from the surface after thorough washing with Millipore water reflects the fact that the polycation is strongly held to the surface by multiple ion-pair interactions between the positively charged guanidino groups (pKa > 12) and the surface carboxylates. Partial removal of pArg (about 60%) can only be accomplished by dipping the surface for 2 h in an acidic solution (pH 3, acetic acid). The inset in Figure 1 displays the dependence of the absorption intensity at 1679 cm-1, on the pH of the solution (39) Smith, E. L.; Alves, C. A.; Anderegg, J. W.; Porter, M. D. Langmuir 1992, 8, 2707-2714. (40) Sun, L.; Crooks, R. M.; Ricco, A. J. Langmuir 1993, 9, 17751780. (b) Jiang, P.; Liu, Z.-F.; Cai, S.-M. Langmuir 2002, 18, 44954499.

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Figure 1. FT-IRRAS spectra of the electrostatic adsorption of poly-L-arginine (solid line) onto the carboxylic acid-terminated alkanethiol monolayer (MUA) (dotted line), as schematically represented. The inset depicts the absorbance at 1679 cm-1 as a function of the pH of the dipping solution. Table 1. FT-IRRAS Band Assignments surface

wavenumber

(cm-1)

assignment

figure

MUA

1740 1717 1585 1416

ν(CdO), free carboxylic acid stretch ν(CdO), H-bonded carboxylic acid νas(COO-), asymmetric carboxylate stretch νs(COO-) + δ(CH2), symmetric carboxylate stretch and methylene scissors deformation

1, 2, 4, 5A

pArg

1676 1620-1560 1545 1452 1408 1350

ν(CdO) amide I (carbonyl stretch) + ν(CdN) guanidino stretch δ(N-H+), -NH2+ deformation vibration ν(C-N) + δ(N-H), amide II (CN stretch and NH bending) δ(CH2), methylene scissors deformation ω(CH2), methylene wagging deformation ν(C-N) + τ(CH2), amide III band (C-N stretch) and methylene twistinga

1, 2, 4, 5

pTyr

1670 1617, 1596, 1518 1540 1450 1348 1260, 1175 1110

ν(CdO), amide I carbonyl stretch ν(CdC), ring stretch ν(C-N) + δ(N-H), amide II (CN stretch and NH bending) δ(CH2) methylene scissors deformation ν(C-N) + ω(CH2), amide III band (C-N stretch) and methylene wagging interaction of O-H deformation and C-O stretching vibrations of the phenolic group )C-H, in-plane ring deformation vibration

2

a

From ref 41.

from which pArg is electrostatically adsorbed, in the range of 8.2-13. The analysis of the MUA/pArg FT-IRRAS spectra shows that there is practically no effect until pH 11. This may reflect the strongly basic character of the δ-guanidino group with pArg adsorbing in a fully charged state until this pH. The increase in the amount deposited beyond pH 11 suggests that the pArg chains become only partially ionized and consequently more pArg molecules are needed to neutralize the same surface charge. In addition, the surface packing of pArg in a partially charged state may be favored, relative to the fully ionized pArg, by an increase in the interstrand hydrogen bonding and hydrophobic interactions following the reduction in the electrostatic repulsion between chains. Some authors10,42 associated the increase in film thickness as the charge density of the assembling polyelectrolytes is diminished to a change in the conformational arrangement of the polyelectrolytes strands on the surface, that is, an increase in the number of loops and tails (weakly bound segments) relative to the number of train segments (strongly bound to the surface). A similar conformational change may be (41) Nissink, J. W. M.; Van der Maas, J. H. Appl. Spectrosc. 1999, 53, 33-39. (42) Dubas, S. T.; Schlenoff, J. B. Macromolecules 2001, 34, 37363740.

expected as the charge density of the depositing pArg chains is reduced by increasing the pH of the dipping solution above pH 11. By manipulating the pH of the pArg solution in the electrostatic assembly process, it may be possible to achieve some degree of control over the extent of guanidino group functionalization and even on the conformational arrangement adopted by the polypeptide chains on the surface. Another conclusion that can be drawn from these results is that a pH induced desorption may be easily prevented by combining pArg with an alkanethiol monolayer having a strong acid functionality (such as the sulfonic acid) as the terminal ionizable group. Recent studies43 have demonstrated the existence of a threshold value of charge density below which surface charge reversal is not high enough for the sequential LBL assembly. The ability of pArg to retain a high charge density even when assembled from high pH solutions was utilized in the construction of polypeptide multilayer films of pArg and poly-L-tyrosine (pTyr) as the polyanionic species. The tyrosine residue contains a phenolic hydroxyl group with a pKa of about 9.7-10.1.19 This means that pTyr will be sufficiently ionized for electrostatic assembly (43) Steitz, R.; Jaeger, W.; Klitzing, R. V. Langmuir 2001, 17, 44714474. (b) Glinel, K.; Moussa, A.; Jonas, A. M.; Laschewsky, A. Langmuir 2002, 18, 1408-1412.

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Figure 2. FT-IRRAS spectra of the layer-by-layer assembly of the pArg/pTyr multilayer film (the pArg layers correspond to the dotted lines), assembled on a MUA-modified gold surface from pH 12 solutions. The inset shows the increase in absorbance of the amide I band, with the number of layers deposited.

only in very alkaline conditions (pH 12). Figure 2 depicts the evolution of the FT-IRRAS spectrum during the layerby-layer buildup of a pArg/pTyr polypeptide multilayer film, up to 10 layers. The presence of pTyr in the film contributes to the absorbance at 1670 cm-1 (amide I band), 1540 cm-1 (amide II band), and is responsible for the additional bands observed at 1518 cm-1 (sharp peak, assigned to the ring CdC stretching vibrations) at 1260 and 1175 cm-1 (because of the interaction of the O-H deformation and C-O stretching modes of the phenol group). The inset shows that the intensity of the amide I band increases in a reproducible fashion from layer to layer indicating regular film growth. Films obtained by the LBL assembly method involving other synthetic polypeptides, namely, the polylysine/poly(glutamic acid) pair have already been proposed to fabricate electroactive films onto electrode surfaces44,45 or as model systems to study the secondary structures adopted by proteins on polyelectrolyte multilayers.46 The charge density of polylysine changes profoundly near pH ∼9.5,44 as a consequence of the weak base character of the lysine residues. The pKa of the -amino groups of poly-L-lysine is about 10.5 and consequently this polycation does not assemble onto a negatively charged surface at pH 10.644 and desorbs upon exposure to a pH 12 solution.11 The use of pArg opens the way to successful electrostatic assembly and resistance to desorption at higher pH. B. r-Dicarbonyl Reactions. The electrostatic binding of pArg onto the ionizable SAM-modified gold surface is a route to create a guanidino-terminated surface, Figure 1. The guanidino group on arginine’s side chain of proteins can be specifically targeted by the use of R-dicarbonyl compounds: R-keto aldehydes and R-diketones. Under alkaline conditions, this type of group can condense with the guanidino to form a stable Schiff base-like complex.19,21,22,25,47 Previously, Riccio et al.25 employed the reaction between a long chain R-diketone and the outer arginine residue of cytochrome c to create monolayers of this metalloprotein at the air-water interface. (44) Cheng, Y.; Corn, R. M. J. Phys. Chem. B 1999, 103, 8726-8731. (45) Cheng, Y.; Murtoma¨ki, L.; Corn, R. M. J. Electoanal. Chem. 2000, 483, 88-94. (46) Boulmedais, F.; Schwinte´, P.; Gergely, C.; Voegel, J.-C.; Schaaf, P. Langmuir 2002, 18, 4523-4525. (47) Pande, C. S.; Pelzig, M.; Glass, J. D. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 895-899.

Figure 3. Reaction scheme for the formation of the stable heterocyclic condensation adducts (dihidroxyimidazolidines) between the guanidino group of the electrostatically bound polyL-arginine and several R-dicarbonyl probes used. The possibility of borate stabilization is considered. A, glyoxal; B, phenylglyoxal; and C, p-azidophenyl glyoxal (APG).

The availability of guanidino groups for reaction with species from solution was assessed by making the pArg layer to react with several R-dicarbonyl reagents: glyoxal, phenylglyoxal, and p-azidophenyl glyoxal (APG). According to the literature,20,21,48 the reactions of arginine with R-dicarbonyl reagents yield double carbinolamines as products, as represented schematically in Figure 3. Evidence for the occurrence of reaction between the probes and the pArg monolayer may be obtained from the analysis of FT-IRRAS spectra, Figure 4. The spectra are consistent with the formation of the products represented in Figure 3. In the glyoxal reaction, Figure 4A, the spectrum does not show any strong bands apart from those previously assigned to the MUA/pArg assembly; nonetheless, spectral changes induced in the MUA/pArg bands are clearly evident and may be interpreted as the occurrence of glyoxal modification as will be explained in the following paragraphs. These spectral changes are also observed as a consequence of the reaction of the two other probes, although in these cases the surface spectra reveal other features. In phenylglyoxal, Figure 4B, a new band appears around 1100 cm-1 which may be attributed to a combination of ring deformation vibrations and C-O stretch. The very weak bands at ∼1300 cm-1 may be assigned to a combination of O-H deformation and B-O stretch (48) Werber, M. M.; Moldovan, M.; Sokolovsky, M. Eur. J. Biochem. 1975, 53, 207-216.

Surface Modification Chemistry

Langmuir, Vol. 19, No. 24, 2003 10329 Table 2. FT-IRRAS Band Assignments

surface APG

wavenumber (cm-1) 2130 (doublet) 1610 1509 1292

assignment azidea

figure 4C, 5C

1130, 1010 1100

νas (NdNdN), asymmetric stretch of the aryl ν (CdC), ring stretch ν(CdC), ring stretch νs(NdNdN) + δ(O-H) + ν(B-O), symmetric stretch of the aryl azidea, OH deformation vibration of the double carbinolamine and BO stretch because of borate stabilizationa )C-H, in-plane ring deformation vibrations of the para-substituted benzene ring ν(C-O), CO stretch

MPSA

1215 1041

νas(SO3-), asymmetric sulfonate stretch νs(SO3-), symmetric sulfonate stretch

5B, 5C

pSSb

1600 1455 1411 1200 (doublet) 1128, 1009 1036

ν(CdC), ring stretch δ(CH2), methylene scissors deformation ω(CH2), methylene wagging deformation νas(SO3-), asymmetric sulfonate stretch )C-H, in-plane ring deformation vibrations of the para-substituted benzene ring νs(SO3-), symmetric sulfonate stretch

5A, 5B

a

From ref 49. b From ref 50.

Figure 4. FT-IRRAS spectra corresponding to the R-dicarbonyl surface reactions (solid lines). A, glyoxal; B, phenylglyoxal; C, APG. Noticeable is the shift, COO- f COOH, in the ionization equilibrium of the MUA SAM, as a consequence of the surface reactions, as indicated by the decrease in the COO- bands and increase in the COOH band (stars (/)).

(because of borate stabilization).25 Differences between this spectra and the one corresponding to the APG reaction (Figure 4C) are the two prominent bands at 2130 cm-1 (doublet) and 1292 cm-1, corresponding to the asymmetric νas(NdNdN) and symmetric νs(NdNdN) stretch of the aryl azide group respectively, and a sharp peak at 1509 cm-1 corresponding to the CdC para-substituted ring stretch (see Table 2 for band assignments).

Additional evidence for the occurrence of surface reaction is provided by contact angle measurements. Specifically, the contact angle of the pArg-terminated surface increased from 42.8° ( 1.8° to 62.3° ( 1.0°, 64.1° ( 1.1°, or 63.1° ( 0.5° upon reaction with glyoxal, phenylglyoxal, or APG, respectively. The decrease in the hydrophilicity of the surface indicates that part of the hydrophilic guanidino groups reacted with the R-dicarbonyl reagents. The values of contact angle measured after reaction of the three probes are similar which may be related to the fact that the same kind of product is obtained, a dihydroxyimidazolidine borate stabilized (Figure 3). As referred, all the FT-IRRAS spectra obtained after the reaction of the pArg layer with the R-dicarbonyls reveal changes in the bands at 1680, 1551, and 1405 cm-1, previously assigned as MUA/pArg bands. To explain the origin of these variations, the following observations were taken into account. These spectral changes are also observed as a consequence of the electrostatic assembly of a polyanionic layer of pSS over the MUA/pArg bilayer, as shown in Figure 5A (see Table 2 for pSS bands assignments), suggesting that the variations, namely, the increase in absorbance at 1680 cm-1, are not due to unreacted carbonyl groups; in contrast when pArg is adsorbed onto a sulfonic acid-terminated monolayer (MPSA SAM), no change is observed in the pArg bands (MPSA does not absorb in the region considered) both in the electrostatic assembly of pSS (depicted in Figure 5B) and in the surface reactions (depicted in Figure 5C for the APG reaction). According to these results, it may be safe to attribute these band variations to the MUA layer. The decrease in the intensity of the bands at 1405 and 1551 cm-1, which correspond to the COO- symmetric and asymmetric stretch, respectively, is accompanied by an increase, and broadening, of the band at 1680 cm-1, the region of the COOH absorption. It seems to exist a connection between chemical changes affecting the pArg outer layer and the ionization equilibrium of the ω-COOH group of the MUA internal layer. Recent studies51,52 reported similar phenomena. When a polycarboxylic acid was embedded within a multilayer of strong polyelectrolytes, its degree of (49) Socrates, G. Infrared Characteristic Group Frequencies; John Wiley & Sons: Chichester, U.K., 1980. (50) Gregoriou, V. G.; Hapanowicz, R.; Clark, S. L.; Hammond, P. T. Appl. Spectrosc. 1997, 51, 470-476. (51) Xie, A. F.; Granick, S. J. Am. Chem. Soc. 2001, 123, 3175-3176. (b) Xie, A. F.; Granick, S. Macromolecules 2002, 35, 1805-1813. (52) Kharlampieva, E.; Sukhishvili, S. A. Langmuir 2003, 19, 12351243.

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Barreira and Silva

Figure 6. Surface modification scheme for the photochemical attachment of proteins onto surfaces. Some of the guanidino groups of the electrostatically bound pArg are reacted with the R-dicarbonyl moiety of APG to create a photoreactive aryl azide terminated surface (A). Subsequent photolysis with UV light, in the presence of the target protein, effects the immobilization (B).

Figure 5. FT-IRRAS spectra that resulted from the electrostatic adsorption of the polyanion pSS (solid lines) on the MUA/ pArg assembly (A) or MPSA/pArg assembly (B). Spectrum C corresponds to the APG reaction when pArg is electrostatically adsorbed onto a MPSA SAM.

ionization oscillated in response to each polycationic (increasing) or polyanionic (decreasing) added layers during the multilayer buildup. These oscillations were, as in the present case, easily detected because of the distinct infrared absorption frequencies of the protonated and deprotonated carboxylic acid species. A simple explanation can be that in the MUA/pArg bilayer the majority of COOH groups of MUA are ionized as a consequence of being involved in ion-pair formation with the pArg guanidino groups. When part of the guanidino groups are targeted with R-dicarbonyl probes or form ion-pairs with the sulfonate groups of pSS, some of the surface carboxylates are displaced and become free to form ion-pairs with small ions (the reaction media is basic) in a first stage. These small ions are easily washedoff during the rinsing step with Millipore water, pH 6. The final balance is an increase in the number of protonated MUA molecules relative to the preexistent MUA/pArg situation. Granick et al.51 predicted the use of a weak polyacid embedded in a multilayer film for the design of surface sensors for adsorbed polyions. This work demonstrates likewise that by following the spectroscopic changes resulting from the shift in the ionization equilibrium of the MUA SAM it is possible to detect the occurrence of covalent modification on the pArg layer. C. Protein Immobilization. The steps employed in the protein immobilization chemistry are shown in Figure 6. In the first step, p-azidophenyl glyoxal (APG), the derivative of glyoxal containg the photosensitive phenyl

Figure 7. Difference FT-IRRAS spectra of the photochemical immobilization of the proteins poly-L-lysine (pLys) (bottom) and HIgG (top). The reference spectrum was that of the APG surface.

azide group, is reacted with the pArg monolayer. This results in the formation of the heterocyclic condensation product, as described in the previous section, leaving the photoreactive group on the surface, available for subsequent cross-linking by photoactivation. In the second step, the photoreactive surface immersed in the protein solution is exposed to UV light effecting the protein photochemical attachment. The highly reactive nitrene, that results from photolysis of the aryl azide, can undergo several reactions.33-35 The most likely route, however, is ring expansion to a dehydroazepine intermediate;34 this group is highly reactive toward nucleophiles, especially amines. This is the reaction path illustrated in Figure 6. The aryl azide photoreactive group was employed previously for micron-scale patterning of antibodies onto gold and SiO2 surfaces, using photobiotin-avidin chemistry.29 Figure 7 shows the difference spectra corresponding to the covalent immobilization of poly-L-lysine (pLys) (bottom) and the antibody HIgG (top). The photoimmobilization of both proteins is evidenced, namely, by the increase in the amide I at 1670 cm-1 and amide II at 1547

Surface Modification Chemistry

Figure 8. Immunoassay fluorescence images; A, HIgG surface probed with anti-HIgG FITC labeled; B, control experiment demonstrating the negligible nonspecific interaction. The dark spot corresponds to the solution containing the specific secondary as in image A. A drop of a solution containing a nonspecific secondary (swine anti-rabbit IgG FITC labeled) was spotted in the lower half of the slide.

cm-1 bands accompanied by the decrease in the aryl azide bands at 2130, 1509, and 1292 cm-1. There is a difference in the intensity of the amide bands which may reflect the greater extension of HIgG immobilization. One possible reason for this may be some degree of electrostatic repulsion between unreacted surface guanidino groups and the positively charged lysine residues of pLys. The unreacted guanidino groups bear positive charge and should assist in the binding of negatively charged proteins and make the process more difficult for positively charged ones.32 Additionally, the structures of the two proteins may influence the way the molecules pack on the surface leading to different coverages: HIgG is a very large molecule (∼150 kDa) with a Y-like structure, while pLys is a “linear” and smaller (∼30 kDa) molecule. The immobilization of HIgG provided a useful test of the coupling strategy presented.30 Immunoglobulins are capable of high affinity binding ( 12) and its ability to form stable heterocyclic adducts with R-dicarbonyl reagents. The first property confers resistance to desorption making the surface modification with pArg robust, while the condensation with R-dicarbonyl-containing reagents provides a way to covalently attach molecules to the pArg-modified surface. This last modification path can be employed to create an aryl azide functionalized surface for immobilizing proteins without loss of their biological activity. Acknowledgment. This research is partially funded by the Portuguese government through the Fundac¸ a˜o para a Cieˆncia e a Tecnologia (FCT) CIQ-L4. Se´rgio Barreira acknowledges FCT for a Ph.D. grant. The authors wish to acknowledge Dr. Monica Castro for her assistance during the immunoassay experiments at the Institute of Molecular and Cellular Biology (IBMC) Porto. LA035158M