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Langmuir 2005, 21, 9510-9517
Self-Assembled Monolayers of Peptide Nucleic Acids on Gold Surfaces: A Spectroscopic Study E. Mateo-Martı´,† C. Briones,† E. Roma´n,‡ E. Briand,§ C. M. Pradier,§ and J. A. Martı´n-Gago*,†,‡ Centro de Astrobiologı´a (CSIC-INTA), Ctra. Ajalvir, Km. 4, 28850 Torrejo´ n de Ardoz, Madrid, Spain, Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco, 28049 Madrid, Spain, Laboratoire de Re´ activite´ de Surface, UMR CNRS 7609, Universite´ P. et M. Curie, 4, Pl. Jussieu, 75005 Paris, France Received February 9, 2005. In Final Form: July 5, 2005 We have characterized self-assembled monolayers (SAMs) of thiol-derivatized peptide nucleic acid (PNA) chains adsorbed on gold surfaces by using reflection absorption infrared spectroscopy (RAIRS) and X-ray photoemission spectroscopy (XPS) techniques. We have found that the molecular orientation of PNAs strongly depends on surface coverage. At low coverage, PNA chains lie flat on the surface, while at high coverage, PNA molecules realign their molecular axes with the surface normal and form SAMs without the need of co-immobilization of spacers or other adjuvant molecules. The change in the molecular orientation has been studied by infrared spectroscopy and it has been confirmed by atomic force microscopy (AFM). PNA immobilization has been followed by analyzing the N(1s) XPS core-level peak. We show that the fine line shape of the N(1s) core-level peak at optimal concentration for biosensing is due to a chemical shift. A combination of the above-mentioned techniques allow us to affirm that the structure of the SAMs is stabilized by molecule-molecule interactions through noncomplementary adjacent nucleic bases.
1. Introduction
* To whom correspondence should be addressed. E-mail: gago@ icmm.csic.es.. † Centro de Astrobiologı´a (CSIC-INTA). ‡ Instituto de Ciencia de Materiales de Madrid (CSIC). § Laboratoire de Re ´ activite´ de Surface.
Many studies had been previously performed on the immobilization of thiols, disulfides, and thiolated DNA molecules,9-11 but the use of thiol-modified peptide nucleic acid (PNA) has resulted in higher performance for selfassembly and bioactivity. PNA is a structural DNA mimic obtained by polymerization of monomers of N-(2-aminoethyl) glycine that replace the ribose-phosphate backbone characteristic of natural nucleic acids. In PNA, the nucleobases adenine (A), cytosine (C), guanine (G), or thymine (T) are connected by methylenecarbonyl linkages to the polyamide structure.12,13 PNA exhibits unique physicochemical properties, being an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability. Moreover, PNA is characterized by its capability to strongly and specifically bind to complementary DNA,14 and it shows higher affinity and specificity for complementary ssDNA than the corresponding ssDNA sequence.12,14,15 Hence, the possibility to generate a surface with biologically relevant functionalities is certainly one of the most exciting properties of PNAs. Previous results of our group, performed ex situ and at atmospheric conditions, have shown that PNAs assemble standing up on gold surfaces, forming locally ordered SAMs that maintain their capability for recognizing complementary nucleic acids. These results were obtained by surface characterization techniques: X-ray photoemission spectroscopy (XPS), X-ray absorption near-edge spectroscopy (XANES), and atomic force microscopy (AFM), which
(1) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498. (2) Revell, D. J.; Knight, J. R.; Blyth, D. J.; Haines, A. H.; Russell, D. A. Langmuir 1998, 14, 4517. (3) Bain, C. D.; Evans, S. D. Chem. Br. 1995, 31, 46. (4) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (5) Donhauser, Z. J.; Mantooth, B. A.; Kelly, K. F.; Bumm, L. A.; Monnell, J. D.; Stapleton, J. J.; Price, D. W.; Rawlett, A. M.; Allara, D. L.; Tour, J. M.; Weiss, P. S. Science 2001, 292, 2303. (6) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y. T.; Parikh, A. N.; Unzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152. (7) Kasemoto, B. Surf. Sci. 2002, 500, 656. (8) Briones, C.; Mateo-Marti, E.; Gomez-Rodriguez, C.; Parro, V.; Roman, E.; Martı´n-Gago, J. A. Phys. Rev. Lett. 2004, 93, 208103.
(9) Poirier, G. E.; Pylant, E. D. Science 1996, 272, 1145. (10) Ulman, A. An Introduction to Ultrathin Organic Films, From Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, 1991. (11) Schreiber, F. Prog. Surf. Sci. 2000, 65, 151. (12) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497. (13) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895. (14) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Norden, B. Nature 1994, 368, 561. (15) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365, 566.
The interest on self-assembled monolayers (SAMs) of biomolecules has been increasing during the past decade due to their applicability in many areas of science including microelectronics, materials science,1 molecular recognition,2 biotechnology, and biosensor development.3-5 Selfassembly provides a rapid and easy way to modify surfaces in order to produce organic films with tailored properties, particularly those required for the development of biosensors. Most of the studies performed until now have been carried out on gold surfaces due to their highly inert nature and slow rate of oxidation and contamination from the environment.6 Understanding the properties of the adsorbed biolayers at a molecular level is of key importance for the emergent field of bionanotechnology because the design of biofunctional surfaces requires knowledge at a molecular level that combines molecular organization with bioefficiency.7 We have recently described the molecular structure of SAMs of single-stranded peptide nucleic acids (ssPNA) on gold and their use as efficient biosensors for the characterization of target DNA molecules in solution.8
10.1021/la050366v CCC: $30.25 © 2005 American Chemical Society Published on Web 09/16/2005
Monolayers of Peptide Nucleic Acids on Gold Surfaces
avoid restrictions derived from fluorescent labeling of the target molecule.8 In this contribution, we focus on the structure and characterization of the ssPNA adlayers immobilized on the surface, an important issue for their applications as biosensors. We have deeply investigated the chemical interactions that stabilize the structure of SAMs of ssPNA at different molecular coverages, discussing further the role played by the surface. The use of surface-sensitive techniques is a reliable approach to characterize PNA monolayers immobilized on gold surfaces because they provide information about both surface structure and chemistry, two qualities that are known to strongly influence the biological response of the film.7 The reflection absorption infrared spectroscopy (RAIRS) technique has been used in order to characterize in detail the biomolecular orientation on surfaces.16,17 RAIRS uses infrared light to excite internal vibrations of adsorbed molecules, the frequency of these vibrations being dependent both on the chemical groups of the adsorbate and on the molecule adsorption geometry on the surface. Furthermore, the application of the surface selection rule, “only vibrational modes with a dipole moment change normal to the surface will be observed”, leads us to distinguish possible orientations of the adsorbed molecule with respect to the surface.18 On the other hand, XPS spectra and a detailed analysis of corelevel peaks provide qualitative and quantitative information on the chemical composition of the surface. Moreover, the overall morphology and structural changes of the different biofilms have been described by means of AFM images. Here, we report the use of Fourier transform infrared spectroscopy (FT-RAIRS), AFM, and XPS to characterize the immobilization of thiol-derivatized ssPNA on gold surfaces. These complementary techniques provide detailed information about the structure of the layers of immobilized chains on the surface as well as the type of intermolecular bonds that stabilize the SAMs. We have investigated the dependence of the molecular orientation on PNA concentration and immobilization time. We will show that a structural transition occurs from lying to standing up molecules as a function of the coverage. This information allows us to determine the orientation of PNA chains self-assembled on the gold surface, to characterize the intermolecular bonding of ssPNA chains, and to determine the influence of surface-molecule versus molecule-molecule interaction in the ordering of the layers. 2. Experimental Details We have used ssPNA molecules with the sequence (written from the terminal amino to the terminal carboxyl group) CysO-O-AATCCCCGCAT, purchased (HPLC purified) from Applied Biosystems. The cysteine moiety at the N terminus of the PNAs provides the thiol group that allows immobilization on gold surfaces. The “O” spacer unit is a molecule of 8-amino-3,6dioxaoctanoic acid, used to separate the hybridization portion of the molecule from the surface. The immobilization of ssPNA on gold surfaces for coverage dependence studies was performed for 3.5 h, at concentrations of 0.01, 0.1, 1, and 10 µM in H2O (Milli-Q grade). Time-dependence studies were carried out with 1 µM solutions of ssPNA at immobilization times of 15 min, 1 h, and 3.5 h. In all cases, immobilization was performed at 22 °C in a humid chamber (more experimental details can be found in ref (16) Mateo-Marti, E.; Barlow, S. M.; Haq, S.; Raval, R. Surf. Sci. 2002, 501, 191. (17) Barlow, S. M.; Haq, S.; Raval, R. Langmuir 2001, 17, 3292. (18) Poling, G. W. J. Colloid Interface Sci. 1970, 34, 265.
Langmuir, Vol. 21, No. 21, 2005 9511 19). For this experimental environment and concentrations above 5 µM, we have determined by XPS and AFM8 that the amount of molecules immobilized on the surface is close to saturation. The adsorption was carried out on polycrystalline Au layers evaporated on glass (Arrandee, Werther, Germany), flame annealed to produce a predominant (111) faceting of the surface. The Au substrates were placed facing down over a small reservoir containing a 20 µL drop of the ssPNA solution. After the immobilization step, the crystals were vigorously rinsed in H2O with agitation, dried by blowing argon, and analyzed in the air by a polarization modulation infrared spectrometer (PM-RAIRS). The PM-RAIRS spectra were recorded on a commercial NICOLET Nexus spectrometer. The external beam was focused on the sample, with a mirror, at an optimal incident angle (see below). The incident beam was modulated between p and s polarizations using a ZnSe grid polarizer and a ZnSe photoelastic modulator (HINDS Instruments, PEM 90, modulation frequency ) 37 kHz). The light reflected at the sample was then focused on a nitrogen-cooled MCT detector. An important advantage of the PM-RAIRS technique, over the classical RAIRS mode of analysis, is that the signal is directly extracted from the ∆R/R data, avoiding a reference spectrum to be recorded on a bare sample. Also, this technique provides an enhanced sensitivity to the vibration modes normal to the surface, at a short distance from the surface (
