Novel Photoactive Self-Assembled Monolayer for Immobilization and

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Novel Photoactive Self-Assembled Monolayer for Immobilization and Cleavage of DNA Salvatore Sortino,*,† Salvatore Petralia,† Guglielmo G. Condorelli,† Sabrina Conoci,‡ and Giuseppe Condorelli† Dipartimento di Scienze Chimiche, Universita` di Catania, I-95125 Catania, Italy, and Si Optoelectronics, Bio- and Nano-Systems, Corporate Technology R&D, ST Microelectronics, I-95121 Catania, Italy Received August 20, 2002. In Final Form: December 11, 2002 This contribution reports on a novel self-assembled monolayer (SAM) on a gold surface, able to immobilize double-strand DNA and to induce its cleavage upon light excitation. DNA binding with the SAM is mainly driven by a highly favored electrostatic interaction, and it is shown that DNA immobilization takes place without impairing its intrinsic high order. SAM irradiation leads to an uncommon defluorination reaction, and it is unambiguously demonstrated that DNA breakage is strictly interrelated to this photochemical pathway. The photocleavage process is very efficient, is exclusively controlled by illumination conditions, seems to be non-base-specific, and does not require external additives. These features, together with the low excitation energy required, the prolonged thermal stability under physiological conditions, and the ease of preparation, make the SAM presented herein an appealing model system for designing nanodevices for applications where efficient, non-base-specific, and externally tunable cleavage of nanoscaled DNA arrays is required.

Introduction There is currently growing interest in self-assembled monolayers (SAMs) capable of performing specific functions exclusively controlled by external stimuli. Such organized assemblies are important for designing new generation nanocomposite devices for applications in multidisciplinary fields encompassing molecular electronics, photonics, biology, and medicine.1-12 Light is a very appealing trigger. Its easy manipulation makes lightcontrolled systems very intriguing in view of advantages such as multiplexity and multifunctionality. The knowledge of photophysical and photochemical properties of the so-called “photoactive unit” of SAMs is, of course, crucial for fabricating photoactive thin films. In this regard, for instance, our studies on the photoreactivity of the anticancer flutamide in solution spurred us to prepare the * To whom correspondence should be addressed. E-mail: [email protected]. † Universita ` di Catania. ‡ ST Microelectronics. (1) (a) Hickman, J. J.; Ofer, D.; Laibibis, P. E.; Whitesides, G. M.; Wrighton, M. S. Science 1991, 252, 688. (b) Abbott, L.; Whitesides, G. M. Langmuir 1994, 10, 1493. (2) (a) Hodneland, C. D.; Mrksich, M. J. Am. Chem. Soc. 2000, 122, 4235. (b) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121, 4286. (3) Wollman, E. W.; Kang, D.; Frisbie, C. D.; Lorkovic, I. M.; Wrighton, M. S. J. Am. Chem. Soc. 1994, 116, 4395. (4) Sellergren, B.; Auer, F.; Arnebrant, T. Chem. Commun. 1999, 2001. (5) Seki, T.; Sekizawa, H.; Tanaka, K.; Matsuzawa, Y.; Ichimura, K.; Supramol. Sci. 1998, 5, 373. (6) Itamar, W.; Amihood, D.; Eugenii, K. J. Phys. Org. Chem. 1998, 11, 546. (7) Chuping, L.; Guldi, D. M.; Maggini, M.; Menna, E.; Mondini, S.; Kotov, A. N.; Prato, M. Angew. Chem., Int. Ed. 2000, 39, 3905. (8) Chia, S.; Cao, J.; Stoddart, J. F.; Zink, J. I. Angew. Chem., Int. Ed. 2001, 40, 2447. (9) Collier, A.; Matterseig, G.; Wong, E. W.; Luo, Y.; Beverly, K.; Sampaio, J.; Raymo, F. M.; Stoddart, J. F.; Heath, J. R. Science 2000, 289, 1172. (10) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1998, 394, 868. (11) Poirier, G. E. Chem. Rev. 1997, 97, 1117. (12) Guiomar, A. J.; Evans, S. D.; Guthrie, J. T. Supramol. Sci. 1997, 4, 279.

first SAM that releases nitric oxide exclusively by light excitation.13 With the recent and revolutionary development of the Human Genome Project, DNA immobilization on solid surfaces is of great interest in DNA sensors, DNA chips, studies of DNA itself, and many other applications.14-17 Besides, DNA cleavage is of paramount importance from a chemical, biological, and clinical standpoint. The past decade has indeed witnessed an impressive growth of research activities devoted to the development of either specific or nonspecific DNA photocleaving compounds in solution, as “photosequencing or photofootprinting” agents activated by visible/near-UV light.18 From this picture, it emerges that the fusion of both themes of “DNA immobilization” and “DNA photocleavage” can be stimulating for designing photoactive DNA-cleaving nanodevices. In this context, while the use of SAMs with cationic end groups (i.e., protonated amino groups) is nowadays a welltested strategy to successfully anchor DNA on twodimensional surfaces,19-23 the photocleavage of immobilized DNA is an almost unexplored subject. The few examples reported to date have elegantly demonstrated that DNA photocleavage can be achieved by SAMs (13) Sortino, S.; Petralia, S.; Compagnini, G.; Conoci, S.; Condorelli, G. Angew. Chem., Int. Ed. 2002, 41, 1914. (14) Su, H.; Kallury, M. R.; Thompson, M.; Rach, A. Anal. Chem. 1994, 66, 679 and references therein. (15) Xu, X. H.; Yang, H. C.; Mallouk, T. E.; Bard, A. J. J. Am. Chem. Soc. 1994, 116, 8386. (16) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128. (17) (a) Brockman, J. F.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044. (b) O’Brien, J. C.; Stickey, J. T.; Porter, M. D. J. Am. Chem. Soc. 2000, 122, 5004. (c) Beier, M.; Hoheisel, J. D. Nucleic Acids Res. 2000, 28, 111. (18) For related reviews, see for example: (a) Armitage, B. Chem. Rev. 1998, 98, 1171. (b) Pratviel, G.; Bernodou, J.; Meunier, B. Angew. Chem., Int. Ed. Engl. 1995, 34, 746. (19) Caruso, F.; Rodda, E.; Furlong, D. F.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043. (20) Sukhorukov, G. B.; Mohwald, H.; Decher, G.; Lvov, Y. M. Thin Solid Films 1996, 284, 220. (21) Lang, J.; Liu, M. J. Phys. Chem. 1999, 103, 1193. (22) Higashi, N.; Inoue, T.; Niwa, M. Chem. Commun. 1997, 1507. (23) Zhang, Y.; Yang, S.; Liu, C.; Dai, X.; Cao, W.; Xu, J.; Li, Y. New J. Chem. 2002, 5, 617.

10.1021/la0264365 CCC: $25.00 © 2003 American Chemical Society Published on Web 01/04/2003

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containing fullerene as the photoactive unit.22,23 Nevertheless, the DNA breakage was not very efficient. In general, due to the small amount of photons absorbed by SAMs the yield of the photoinitiation step is a critical parameter for designing efficient photoresponsive DNAcleaving systems. In this letter, we report on a novel SAM on a gold surface, able to immobilize double-strand DNA and to induce efficiently its photocleavage. To the best of our knowledge, this represents the first example of a SAM that triggers DNA photocleavage through an uncommon photodefluorination reaction.

Scheme 1. Photoreactivity of LM in Neutral Aqueous Solution

Experimental Section Chemicals. 11-Mercapto undecanoic acid (Aldrich), lomefloxacin (LM), calf thymus DNA (ct-DNA), poly[(dGdC)2], poly[(dAdT)2], and Tris (Sigma) were used as received. All solvents used were spectrophotometric grade. Monolayer Preparation. All glassware used to prepare monolayers was immersed in piranha solution at 70 °C for 1 h. Warning: piranha solution should be handled with caution. Next, the glassware was rinsed with large amounts of high-purity water and dried. Au substrates (1 × 2 cm2, ca. 800 Å thick) were sonicated with CH2Cl2 and then soaked in a 5 mM CH2Cl2 solution of 11-mercapto-1-undecanol for 20 h at room temperature. The above thiol concentration was chosen on the basis of the lowest value of the contact angle (θa) obtained from θa measurements in the concentration range of 10-4-10-2 M. The thiol-functionalized substrate was then rinsed several times with CH2Cl2 to remove the free thiol and dried at room temperature (step i). After that, SAM 1 was soaked in an anhydrous dimethylformamide (DMF) solution of LM for 48 h under argon flux in the presence of 1% H2SO4 as a catalyst, rinsed several times with DMF and then with water, and eventually dried at room temperature (step ii). The degree of conversion of SAM 1 into SAM 2 (vide infra) did not change significantly for longer reaction times, whereas it was lower for shorter reaction times. Irradiation Conditions. Irradiation of SAM 2 was carried out in a 3 mL quartz fluorescence cuvette (10 mm path length) using a Rayonet photochemical reactor equipped with 8 RPR lamps with an emission in the 320-390 nm range with a maximum at 350 nm. Wavelengths below 330 nm were filtered through a cutoff. The incident photon flux on the sample was ca. 5 × 1016 quanta s-1. Irradiation experiments were performed under the same experimental conditions for all the samples. Instrumentation. X-ray photoelectron spectroscopy (XPS) measurements were performed with a PHI 5600 Multy Technique System equipped with an Al standard X-ray source operating at 14 kV and a hemispherical analyzer. The electron takeoff angle (θ) was 45°. The binding energies of the collected spectra were calibrated against the Au 4f7/2 photoelectron signal originating from the underlying substrate. Absorption spectra were carried out with a Beckman 650 DU spectrophotometer. Contact angles were measured using a goniometer (KERNCO) under ambient conditions.

Results and Discussion Photophysical, photochemical, and photobiological studies carried out in solution by others and by us on the antibacterial drug LM in the absence and in the presence of DNA represented a decisive starting point for this work. In brief, it has been demonstrated that (a) LM induces a remarkably efficient DNA cleavage upon light excitation;24 (b) the photocleavage is directly related to a very uncommon photodefluorination reaction25 occurring selectively from position 8 with an extraordinarily high quantum yield (Φ ) 0.6-0.9), leading to a carbene as the key (24) Martinez, L. J.; Chignell, C. F. J. Photochem. Photobiol., B 1998, 45, 51. (25) Photodefluorination is a quite uncommon reaction for fluoroaromatics due to the strength of the C-F bond (125 kcal/mol). Actually, there are only a few precedents in the literature; see for example: (a) Yang, N. C.; Huang, A.; Yang, D. H. J. Am. Chem. Soc. 1989, 111, 8060. (b) Zhang, G.; Wan, P. Chem. Commun. 1994, 19.

Scheme 2

a

a Conditions: (i) CH2Cl2, 25 °C, 20 h; (ii) DMF, 1% H2SO4, 55 °C, 48 h.

intermediate in the photodecomposition (Scheme 1);26-28 (c) LM may bind to DNA through a favored electrostatic interaction between the positively charged piperazinyl group and the negatively charged DNA backbone;29 (d) the LM-DNA complex undergoes photodefluorination as efficiently as free LM.29 The above scenario is very attractive and stimulated in us the idea of developing a LM-based SAM on a gold surface able to act as both a DNA-immobilizing and a DNAphotocleaving nanosystem. The two-step synthetic approach to the synthesis of the LM-based SAM is reported in Scheme 2. It involves (i) chemisorption of 11-mercapto-1-undecanol, a coupling layer, on the Au substrate to give SAM 1 and (ii) reaction with LM to obtain SAM 2. Aqueous contact angle (θa) measurements after step (i) reveal a θa value less than 10° for the thiol-functionalized surface. This finding is consistent with the presence of hydroxyl headgroups, in accordance with analogous results obtained for alkanethiol SAMs on bulk Au substrates.30 XPS measurements provided unequivocal evidence that the condensation reaction occurred. The signal of the sulfur 2p at 162 eV was indeed accompanied by the presence of the diagnostically important and unambiguous signal related to the fluorine 1s at 688 eV. On the basis of the fluorine/sulfur ratio, the degree of conversion of SAM 1 into SAM 2 was estimated to be ca. 15%. (26) Fasani, E.; Mella, M.; Caccia, D.; Fagnoni, M.; Albini, A. Chem. Commun. 1997, 1329. (27) Martinez, L. J.; Li, G.; Chignell, C. F. Photochem. Photobiol. 1997, 65, 599. (28) Monti, S.; Sortino, S.; Fasani, E.; Albini, A. Chem.sEur. J. 2001, 7, 2185. (29) Sortino, S.; Condorelli, G. New. J. Chem. 2002, 26, 250. (30) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: New York, 1991.

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Figure 1. XPS spectrum for the P 2p region of SAM 2 after treatment with ct-DNA.

Figure 2. UV spectra of aqueous solutions in which SAM 2 samples containing immobilized ct-DNA were immersed and (a) irradiated for 5 min and (b) kept in the dark for 50 min. The inset shows the total absorbance monitored at 260 nm as a function of the irradiation time.

The suitability of SAM 2 as a DNA-immobilizing system was tested by soaking the film in a Tris buffer solution (50 mM, pH 7.4) of ct-DNA (1.8 × 10-2 M in base pairs) for 3 h. The substrate was then washed several times with the same buffer to remove the free ct-DNA and dried at room temperature. The unambiguous phosphorus 2p signal revealed by the XPS analysis of the film clearly indicated the presence of ct-DNA molecules in SAM 2. As shown in Figure 1, this core level could be fit to the 2p1/2 and 2p3/2 (binding energy ) 134 eV) spin-orbit components. This binding energy agrees very well with those recently reported for DNA immobilized on a cationic SAM31 and provides a strong indication that no degradation of the biopolymer takes place upon coordination with SAM 2. Due to the presence of the quaternary nitrogen of the piperazinyl group, the top surface of SAM 2 is positively charged at neutral pH, so it is conceivable that immobilization of ct-DNA is facilitated by a highly favored electrostatic interaction with its negatively charged phosphodiester backbone. This view is in accord with recent literature reports19-23,31 and with our investigation on the LM-ct-DNA complex carried out in aqueous solution.29 The suitability of SAM 2 as a photoactive DNA-cleaving nanosystem was tested by irradiation of the monolayermodified gold plate immersed in Tris buffer solution (3 mM, pH 7.4). Figure 2 shows the UV spectrum of the aqueous solution from which the modified gold plate was (31) (a) Kumar, A.; Pattarkine, M.; Bhadbhade, M.; Mandale, A. B.; Ganesh, K. N.; Datar, S. S.; Dharmadhikari, C. V.; Sastry, M. Adv. Mater. 2001, 13, 341. (b) Higashi, N.; Takahashi, M.; Niwa, M. Langmuir 1999, 15, 11.

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Figure 3. XPS spectrum for the F 1s region of SAM 2 (a) prior to and (b) after 45 min of irradiation.

withdrawn after 5 min irradiation. The presence of the typical absorption band around 260 nm due to the π,π* transition of nucleobases suggests unequivocally that DNA photocleavage occurs already after 5 min of light irradiation. Actually, the DNA chains are cleaved into short fragments such as oligonucleotides and nucleotides, which rapidly diffuse into the bulk solution and thus are easily detectable spectrophotometrically.22 In contrast, no significant absorption of the aqueous phase was observed when ct-DNA immobilized on SAM 2 was kept in the dark (Figure 2). This result accounts for a high thermal stability of the ct-DNA film under our experimental conditions. As illustrated in the inset of Figure 2, the amount of cleaved ct-DNA increases monoexponentially as a function of the irradiation time, reaching a plateau after ca. 40 min. Interestingly, the XPS analysis of the sample irradiated for 45 min showed that the amount of fluorine in the sample is ca. 50% lower than that found in the nonirradiated sample (Figure 3), thus providing clear and direct evidence for the almost complete loss of one of the two fluorine atoms from SAM 2. This result is remarkable since it suggests that (a) SAM 2 undergoes exclusively an uncommon light-triggered defluorination, similarly to LM,26-28 its ethyl ester,27 and the LM-ct-DNA complex,29 and (b) ct-DNA photocleavage is strictly interrelated to the photodefluorination reaction. On the basis of the electronic and molecular structure, there is no reason to believe that both the photochemical behavior and the cleavage mechanism of SAM 2 differ from those of LM and its ethyl ester in solution. Therefore, a photodefluorination pathway triggered by a short-lived charge-transfer excited singlet state and generating a carbene as the key intermediate appears to be most likely.26-28 Martinez and Chignell demonstrated that such a carbene plays a dominant role in DNA photocleavage.24 As pictorially depicted in Scheme 3, SAM 2 can thus be viewed as composed of an immobilizing unit, the piperazinyl ring, and a photocleaving unit, the aromatic chromophore, supported by the close-packed hydrophobic chains. To gain more insight on the type of photocleavage, we performed some experiments with either poly[(dGdC)2] or poly[(dAdT)2] previously immobilized on SAM 2 with the same procedure described above for ct-DNA. Figure 4 displays the total absorbance at 260 nm of the bulk aqueous solutions from which the above samples were withdrawn after different irradiation times. The experimental data related to ct-DNA were also included in the same figure for the sake of comparison. The behavior observed in these preliminary experiments suggests that the photocleavage may take place basically in a non-basespecific fashion. These results are fully consistent with

Letters Scheme 3. Pictorial View of DNA Immobilization and Photocleavage by SAM 2

Figure 4. Total absorbance monitored at 260 nm of the bulk aqueous solutions from which SAM 2 samples with previously immobilized (2) poly[(dGdC)2], (9) poly[(dAdT)2], or (O) ct-DNA were withdrawn after different irradiation times.

the photochemical properties of SAM 2. As widely documented in the literature, carbene species are known to trigger DNA breakage indiscriminately through diverse mechanistic pathways.18,32 In our case, a direct reaction of carbene with the DNA backbone may be highly favored

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due to the close proximity of the two reaction partners. Furthermore, the lack of base specificity is in accord with the following findings: (a) LM is inefficient in photogenerating singlet oxygen,27,28 which, on the contrary, was observed to induce guanine-selective cleavage in twodimensional immobilized DNA;22 (b) thermodynamically feasible photoinduced electron transfer between the LM triplet state and the DNA bases leading to base-selective cleavage through typical “hole trapping” processes18,33 appears unlikely. In fact, the negligible quantum yield of the LM triplet state28 and the improbable π-electron overlap of the photoactive unit of SAM 2 with the DNA bases, an indispensable prerequisite for the above process,34 support well this hypothesis. In summary, we have demonstrated that SAM 2 is able to immobilize DNA and to induce its photocleavage already after a few minutes of irradiation through an unusual photodefluorination reaction. The high photocleavage efficiency, the exclusive control of the photocleavage through the illumination conditions without the need of external chemical additives, the relatively low excitation energy required, the nonspecificity of the cleavage, the prolonged thermal stability under physiological conditions, and the ease of preparation are remarkable advantages offered by SAM 2. In light of this, it represents an attractive model system for designing nanodevices for potential applications where efficient, non-base-specific, and externally tunable cleavage of nanoscaled DNA arrays is required. Acknowledgment. Financial support from MURST “cofinanziamento di programmi di ricerca di rilevante interesse nazionale” and INCA (Consorzio Interuniversitario per la Chimica dell’Ambiente) is gratefully acknowledged. LA0264365 (32) (a) Nielsen, P. E.; Jeppesen, C.; Egholm, M.; Buchard, O. Nucleic Acids Res. 1988, 16, 3877. (b) Maiya, B. G.; Ramana, C. V.; Arounaguri, S.; Nagarajan, M. Bioorg. Med. Chem. Lett. 1997, 7, 2141. (33) Ly, D.; Kan, Y.; Armitage, B.; Schuster, G. B. J. Am. Chem. Soc. 1996, 118, 8747. (34) Breslin, D. T.; Coury, J. E.; Anderson, J. R.; McFail-Isom, L.; Kan, Y.; Williams, L. D.; Bottomley, L. A.; Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 5043.