pubs.acs.org/Langmuir © 2009 American Chemical Society
Photoactive Diazoketo-Functionalized Self-Assembled Monolayer for Biomolecular Patterning Ramakrishnan Ganesan,†,§ Hyun-Jung Lee,† and Jin-Baek Kim* Department of Chemistry and School of Molecular Science (BK21), Korea Advanced Institute of Science and Technology (KAIST), 373-1, Guseong-Dong, Yuseong-Gu, Daejeon, 305-701, Republic of Korea. †These authors contributed equally to this work. §Current address: Center for Biomaterial Development and Berlin Brandenburg Center for Regenerative Therapies (BCRT), Institute of Polymer Research, GKSS Forschungszentrum Geesthacht GmbH, Kantstr. 55, 14513 Teltow, Germany. Received May 26, 2009. Revised Manuscript Received June 25, 2009 A diazoketo-functionalized alkoxysilane compound was synthesized, and its self-assembled monolayer (SAM) formation was studied on glass and silicon substrates. Infrared spectroscopy was employed to follow the photoreaction in this system, which proved that carboxylic groups were generated from diazoketo groups in the surface of the substrate. Photopatterning of biotin/streptavidin on the diazoketo-functionalized SAM shows the potential of this novel platform for biomolecular patterning.
Introduction Site-selective immobilization of biomolecules is of great interest because of the applications found in several fields, including biosensors, biomaterials, and surface-biomolecular interaction studies.1 Several patterning techniques that are used to selectively immobilize biomolecules, such as photolithography, soft-lithography, dip-pen nanolithography, and spot arraying, have been reported.2 One particular promising technique is photolithography, which is well-established in the semiconductor industries. Several photolithography-based biomolecular patterning strategies have been reported, many of which involve a photoresist processing step.3 Recently, there has been a growing interest in developing photoresists and lithographic processes that do not require harsh processing conditions in order to minimize denaturation of biomolecules.4 One promising mild approach is the photoactive self-assembled monolayer (SAM) system, where biomolecular patterning can be achieved directly on the substrate without the use of a photoresist. Several photoactive SAMs have been reported on gold, glass, or silicon substrates, although SAMs on glass (or silicon) substrates are preferable for certain studies.5 For example, when a deep UV exposure system is used while using gold substrates, the Au-S bond could be dissociated, whereas the siloxane bond, which is present in glass substrates, is considerably robust.6 As the majority of photoactive *Corresponding author. E-mail:
[email protected]. Fax: þ82-42-350-2810
(1) (a) Crooks, R. M.; Ricco, A. J. Acc. Chem. Res. 1998, 31, 219. (b) Zhang, G. J.; Tanii, T.; Funatsu, T.; Ohdomari, I. Chem. Commun. 2004, 7, 786. (c) McGall, G. H.; Barone, A. D.; Diggelmann, M.; Fodor, S. P. A.; Gentalen, E.; Ngo, N. J. Am. Chem. Soc. 1997, 119, 5081. (2) (a) Feng, C. L.; Vancso, G. J.; Schonherr, H. Adv. Funct. Mater. 2006, 16, 1306. (b) Healy, K. E.; Thomas, C. H.; Rezania, A.; Kim, J. E.; McKeown, P. J.; Lom, B.; Hockberge, P. E. Biomaterials 1996, 17, 195. (c) Lee, K. B.; Lim, J. H.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 5588. (3) (a) Falconnet, D.; Koenig, A.; Assi, F.; Textor, M. Adv. Funct. Mater. 2004, 14, 749. (b) Sorribas, H.; Padeste, C.; Tiefenauer, L. Biomaterials 2002, 23, 893. (4) (a) Douvas, A.; Argitis, P.; Misiakos, K.; Dimotikali, D.; Petrou, P. S.; Kakabakos, S. E. Biosens. Bioelectron. 2002, 17, 269. (b) Doh, J.; Irvine, D. J. J. Am. Chem. Soc. 2004, 126, 9170. (5) (a) Onclin, S.; Ravoo, B. J.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 2005, 44, 6282. (b) del Campo, A.; Boos, D.; Spiess, H. W.; Jonas, U. Angew. Chem., Int. Ed. 2005, 44, 4707. (c) Yang, Z.; Frey, W.; Oliver, T.; Chilkoti, A. Langmuir 2000, 16, 1751. (6) Ryan, D.; Parviz, B. A.; Linder, V.; Semetey, V.; Sia, S. K.; Su, J.; Mrksich, M.; Whitesides, G. M. Langmuir 2004, 20, 9080.
8888 DOI: 10.1021/la901870a
SAMs used for biomolecular patterning studies contain aromatic functional groups that are released upon UV light exposure, which often require rinsing steps to be removed from the substrate, the development of a novel biomolecular patterning method that exploits stable SAMS while avoiding such byproducts or additional washing steps is of great interest. Diazonapthoquinone (DNQ) is a photoactive compound that has been used as a photosensitizer in novolac matrix resin for 365 nm (I-line) lithography.7 Upon UV exposure, DNQ undergoes Wolff rearrangement to generate carboxylic groups, thereby enabling the exposed regions to dissolve in aqueous base developer.8 Recently we have developed a diazoketo-functionalized photoresist system for deep UV lithography and have also shown its applications in biomolecular patterning.9,10 The diazoketo functional groups in this system undergo Wolff rearrangement upon UV irradiation, releasing nontoxic molecular nitrogen and generating carboxylic groups, which is a process similar to the DNQ/novolac system.11 One important limitation of using this photoresist for biomolecular patterning involves detaching the substrates during prolonged immersion in aqueous solutions, which is difficult due to adhesion failure. Therefore, formation of a stable diazoketo-functionalized SAM would be highly desirable in order to incorporate diazoketo chemistry in biomolecular patterning applications, which circumvent such detachment steps. In this paper, we report the synthesis of a new diazoketofunctionalized molecule and its SAM formation on glass and silicon substrates followed by its photopatterning for biomolecular applications. (7) (a) Willson, G. C.; Yueh, W.; Leeson, M. J.; Steinh€ausler, T.; McAdams, C. L.; Dammel, R. R.; Sounik, J. R.; Aslam, M.; Vicari, R.; Sheehan, M. T. Proc. SPIE 1997, 3049, 226. (b) Dammel, R. Diazonaphtoquinone-based Resists; SPIE Optical Engineering Press: Bellingham, WA, 1993. (8) Ganesan, R.; Yoo, S. Y.; Choi, J.-H.; Lee, S. Y.; Kim, J. B. J. Mater. Chem. 2008, 18, 703. (9) (a) Ganesan, R.; Youn, S.-K.; Kim, J. B. Macromol. Rapid Commun. 2008, 29, 437. (b) Kim, J. B.; Kim, K. S. Macromol. Rapid Commun. 2005, 26, 1412. (c) Kim, J. B.; Ganesan, R.; Choi, J.-H.; Yun, H.-J.; Kwon, Y.-G.; Kim, K.-S.; Oh, T.-H. J. Mater. Chem. 2006, 16, 3448. (10) Regitz, M.; Hocker, J. Organic Syntheses; J. John Wiley & Sons: New York, 1973; Vol. V, p 179. (11) Kim, J. K.; Shin, D. S.; Chung, W. J.; Jang, K. H.; Lee, K. N.; Kim, Y. K.; Lee, Y. S. Colloids Surf. B 2004, 33, 67.
Published on Web 07/13/2009
Langmuir 2009, 25(16), 8888–8893
Ganesan et al.
Letter
Experimental Section Materials. Allylacetoacetate, trimethoxysilane, platinum(0)1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution in xylene (Karstedt’s catalyst), anhydrous toluene, triethylamine, anhydrous acetonitrile, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Aldrich Chemical Co. and used without further purification. p-Toluenesulfonyl azide (p-TsN3) was prepared from p-toluenesulfonyl chloride and sodium azide according to the literature.10 (þ)-Biotinyl-3,6,9-trioxaundecanediamine (biotinamine), fluorescein isothiocyanate-labeled streptavidin (SAv-FITC), and rhodamine-labeled streptavidin (SAv-Rh) were purchased from Pierce. Measurements. The 1H and 13C NMR spectra of the synthesized compounds were recorded on a Bruker AM-300 FT-NMRspectrometer in CDCl3. FT-IR data were obtained using a Bruker EQUINOX-55 spectrophotometer. UV light irradiation was carried out using a deep UV exposure system (Oriel Corporation, model 82531) with a high-pressure mercury-xenon lamp and a filter transmitting light between 220 and 260 nm. A contact angle instrument (Phoenix 300, Surface Electro Optics Co. Ltd.) was used to determine the static water contact angle of the SAM before and after the UV light irradiation. A refractive index of 1.46 was used for all the samples. A grazing incidence attenuated total reflection- infrared (ATR-IR) microspectrometer (Hyperion 3000, Bruker Optics Inc.) was used to characterize the SAMs. X-ray photoelectron spectroscopy (XPS) study was performed with a VG-Scientific ESCALAB 250 spectrometer equipped with an Al KR X-ray source. Tapping-mode atomic force microscopy (AFM) images were obtained on a MultiMode SPM (Digital Instruments). The thicknesses of the SAMs were measured with a Gaertner L116s ellipsometer (Gaertner Scientific Corporation, Chicago, IL) equipped with a He-Ne laser (632.8 nm) at a 70° angle of incidence. Digital images of fluorescent biomolecular patterns were captured with an LSM 510 confocal microscope. Synthesis. 3-(Trimethoxysilyl)propyl 3-Oxobutanoate (TMSPOB). TMSPOB was synthesized by the reaction between allylacetoacetate and trimethoxysilane. One equivalent of allylacetoacetate (5 g, 35.17 mmol) and 1.25 equivalents of trimethoxysilane (5.37 g, 43.96 mmol) were mixed together in 150 mL of anhydrous toluene. Two drops of Karstedt’s catalyst was then added into the reaction mixture. The solution was allowed to stir for 1 day at 60 °C. The reaction mixture was filtered through a Celite pad and then evaporated to yield 7.72 g (83%) of TMSPOB. 1 H NMR (CDCl3, δ, ppm): 4.10-4.05 (t, 2H), 3.53 (s, 9H), 3.41 (s, 2H), 2.23 (s, 3H), 1.75-1.70 (m, 2H), 0.65-0.60 (m, 2H). 13C NMR (CDCl3, δ, ppm): 200.4, 167.0, 67.1, 50.5, 50.0, 30.0, 21.8, 5.2. IR (NaCl), νmax 1719 cm-1 (s, CdO), 1648 cm-1 (s, CdO).
3- (Trimethoxysilyl)propyl 2-Diazo-3-oxobutanoate (TMSPDOB). TMSPDOB was synthesized as follows. A solution of p-TsN3 (5.76 g, 29.2 mmol) in 15 mL of anhydrous acetonitrile was added to a solution of TMSPOB (7.72 g, 29.2 mmol) and triethylamine (5.91 g, 58.4 mmol) in 15 mL of anhydrous acetonitrile at 0 °C. The reaction mixture was stirred under nitrogen atmosphere at 0 °C for 30 min and then warmed to 30 °C with stirring for 3 h. The solution was extracted with diethyl ether several times. After drying with magnesium sulfate and removal of the solvent, the crude mixture was precipitated into carbon tetrachloride. The precipitated p-toluenesulfonamide was removed by filtration. After filtration, the solution was evaporated to yield 8.42 g (29 mmol, 96.3%) of TMSPDOB as yellow oil. 1H NMR (CDCl3, δ, ppm): 4.20-4.16 (t, 2H), 3.54 (s, 9H), 2.44 (s,
3H), 1.80-1.74 (m, 2H), 0.66-0.60 (m, 2H). 13C NMR (CDCl3, δ, ppm): 190.0, 161.2, 67.0, 50.4, 28.0, 22.0, 5.1. IR (NaCl) νmax 2138 (CdNdN), 1719 (ester CdO), 1657 (keto CdO) cm-1. Formation of Diazoketo-Functionalized SAMs. Formation of SAMs on silicon and glass substrates was done in a manner similar to that reported in the literature.11 Piranha-treated silicon and glass substrates were used for making SAMs of TMSPDOB. A solution of TMSPDOB in 10 mL of toluene was applied over the glass/silicon substrate and kept at 65 °C for a period of 12 h, followed by sequential washing with toluene (three times), ethanol, and water. The substrate was blown dry with nitrogen and used for further experiments.
Streptavidin Patterning on Diazoketo-Functionalized SAMs. The diazoketo-functionalized SAMs were irradiated with UV light under a photomask. The patterned diazoketo-functionalized SAM samples were immersed in a solution containing 15 mM NHS, 45 mM EDC, and 10 mM biotin-amine for 6 h at room temperature.8 The substrates were then rinsed well with a copious amount of deionized water to obtain biotin-amine patterned surfaces. These substrates were then incubated with fluorescently tagged streptavidin (Pierce) (0.1 mg mL-1) in phosphatebuffered saline (PBS, pH 7.4) containing 0.1% (w/v) bovine serum albumin and 0.02% (v/v) Tween 20 at room temperature. After 1 h, the samples were removed, washed several times with PBS and distilled water, and dried. For control experiments to measure the nonspecific binding, the biotin-amine patterned substrates were immersed in 0.2 mM solutions of Rh-SAv (in PBS, pH 7.4) that had been presaturated with 200 μM biotin-amine for 1 h at room temperature. Fluorescence images were viewed with an LSM 510 confocal microscope.
Results and Discussion The synthetic route for preparation of TMSPDOB is shown in Scheme 1. Hydrosilylation reaction of allylacetoacetate with trimethoxysilane in the presence of Karstedt’s catalyst gave TMSPOB in 83% yield. This was further reacted with p-TsN3 in the presence of triethylamine to give TMSPDOB in 96% yield. Formation of SAMs on silicon and glass substrates was done on freshly piranha-cleaned samples. The silyl ethers of TMSPDOB were hydrolyzed to produce silanols, which further reacted with the surface silanols as well as the silanols from the neighboring TMSPDOB to form a network structure that is anchored to the substrate covalently. Ellipsometry was used to characterize the thickness of the SAMs formed on the silicon substrate. An average thickness of 11.6 ( 1 A˚ was observed when a solution of 15 mg of TMSPDOB in 10 mL of toluene was applied over the silicon substrate and kept at 65 °C for a period of 12 h, which corresponds to the formation of a monolayer of TMSPDOB. This substrate was used for further studies and is represented as a diazoketo-functionalized SAM in this paper. AFM was employed to observe the homogeneity of the diazoketo-functionalized SAM. Figure 1 shows the AFM images of the piranha-treated silicon wafer as well as the diazoketofunctionalized SAM. The 1 and 3 μm scan samples of piranhatreated silicon substrates showed an average root-mean-square roughness of 0.31 and 0.28 nm, respectively, whereas the 1 and 3 μm scan samples of diazoketo-functionalized SAMs showed an average roughness of 0.35 and 0.32 nm, respectively. This shows that the roughness of the diazoketo-functionalized SAM is
Scheme 1. Synthetic Route for Preparation of TMSPDOB
Langmuir 2009, 25(16), 8888–8893
DOI: 10.1021/la901870a
8889
Letter
Ganesan et al.
Figure 1. AFM micrographs of (a) 1 μm and (b) 3 μm scan samples of piranha-treated silicon substrate; (c) 1 μm and (d) 3 μm scan samples of a diazoketo-functionalized SAM on a silicon substrate.
comparable to that of the bare silicon substrate, and the diazoketo-functionalized SAM is homogeneous. The schematic diagram of the lithographic process used for biomolecular patterning on the diazoketo-functionalized SAM and its photoreaction are shown in Figure 2. Selected regions of the diazoketo-functionalized SAM surface were exposed to deep UV light using a photomask, and the irradiated regions were used for immobilization of biomolecules. Upon UV light irradiation, the diazoketo-functional groups present in the SAM underwent Wolff rearrangement to give carboxylic acid. This was confirmed by IR spectral analysis of TMSPDOB liquid as well as ATR-IR analysis of the thin TMSPDOB film formed on silicon substrate as shown in Figure 3. The unexposed TMSPDOB shows a characteristic peak of diazo stretching at 2138 cm-1, and the ester carbonyl and keto carbonyl peaks appeared at 1719 and 1657 cm-1, respectively. After 20 min of UV exposure, a complete disappearance of diazo peak was observed, and the ester carbonyl and keto carbonyl peaks merged to form a broad peak at 1733 cm-1. A new broad peak between 3100 and 3600 cm-1 appeared which is characteristic of carboxylic group. These changes prove 8890 DOI: 10.1021/la901870a
that the Wolff rearrangement took place in this system. To confirm that the Wolff rearrangement proceeds on the surface, ATR-IR was employed. Due to the sensitivity limitation of the ATR-IR, a 58 ( 4 A˚ thick film of TMSPDOB was used, which was formed on a silicon wafer by applying 100 mg of TMSPDOB in 10 mL of toluene and keeping the substrate at 65 °C for 12 h. The unexposed film shows the characteristic peaks of diazo, ester carbonyl, and keto carbonyl at 2143, 1723, and 1653 cm-1, respectively. Upon UV light exposure, the diazo peak intensity reduced with increasing the exposure dose, whereas the intensity of the newly formed broad peak at 3100-3600 cm-1 increased upon increasing the exposure dose. This further confirms the occurrence of the Wolff rearrangement on the surface. Water contact angle measurements before and after UV light irradiation with neutral water and an aqueous NaOH solution (pH=11.8) were done on a diazoketo-functionalized SAM, and the values are shown in Figure 4. The contact angle of the piranha-treated silicon wafer was found to be 26 ( 2°. It can be seen from Figure 4 that the contact angle of the diazoketofunctionalized SAM surface was found to be 57 ( 2°. This clearly Langmuir 2009, 25(16), 8888–8893
Ganesan et al.
Letter
Figure 2. (I) Schematic illustration of the lithographic process used for the biomolecular patterning on the diazoketo-functionalized SAM: (a) UV light exposure under photomask, (b) immobilization of biotin-amine, and (c) binding with streptavidin. (II) Photoreaction of diazoketo-functionalized SAM.
Figure 3. (I) IR spectra of TMSPDOB (a) before and (b) after UV light exposure. (II) ATR-IR spectra of a thin TMSPDOB film formed on a silicon wafer under different irradiation times.
Figure 4. Contact angle measurements of a diazoketo-functionalized SAM with various UV irradiation times using water and an aqueous NaOH solution (pH=11.8).
upon increasing the exposure time. However, the difference between before exposure and after exposure was only about 6°. This follows the same trend with our previous report.12 To gain additional information, contact angle measurements with NaOH solution (pH 11.8) on a diazoketo-functionalized SAM were done.13 There was seen a clear trend of reduction in contact angle upon increasing the irradiation time. More than 20° difference in contact angle was observed when the diazoketo-functionalized SAM was exposed for 60 s, above which further reduction in contact angle was almost negligible. This proves that, after UV light exposure, the surface of the diazoketo-functionalized SAM became acidic, which is in agreement with the IR data. XPS has been employed for further characterization of photoreaction of the diazoketo-functionalized SAM. The wide-angle XPS of a diazoketo-functionalized SAM is given in Figure 5, which shows the characteristic peaks of O, N, and C at ∼535, 400, and 288 eV, respectively. The N 1s peak intensity of the diazo group has been measured before and after UV light irradiation, and the corresponding XPS spectra are given in the inset of Figure 5. A reduction of 61% in the N 1s peak intensity was observed after irradiation with UV light for 60 s, which shows that
shows the presence of diazoketo-functionalized SAM on the silicon wafer. After irradiation with UV light, the water contact angle on the diazoketo-functionalized SAM gradually decreased
(12) Kim, J. B.; Ganesan, R.; Yoo, S. Y.; Choi, J.-H.; Lee, S. Y. Macromol. Rapid Commun. 2006, 27, 1442. (13) Zhao, B.; Moore, J. S.; Beebe, D. J. Anal. Chem. 2002, 74, 4259.
Langmuir 2009, 25(16), 8888–8893
DOI: 10.1021/la901870a
8891
Letter
Ganesan et al.
Figure 5. XPS spectra of the diazoketo-functionalized SAM before and after 60 s of UV exposure. Inset shows the N 1s core level spectra of a diazoketo-functionalized SAM before and after 60 s of UV exposure.
Figure 6. Fluorescence micrograph images of (a) FITC-SAv and (b) Rh-SAv patterned on diazoketo-functionalized SAMs on glass substrates (scale bar = 100 μm).
a major portion of diazoketo-functional groups underwent photoreaction, while about 39% of them did not react at this 8892 DOI: 10.1021/la901870a
exposure dose. Higher exposure time leads to heating up of the samples and drying up of the environment, which does not help Langmuir 2009, 25(16), 8888–8893
Ganesan et al.
for the occurrence of Wolff rearrangement, but leads to possible side reactions of carbene. To show the applicability of this approach for biomolecular immobilization, we have chosen the well-known biotin/streptavidin patterning. For this, biotin-amine was immobilized first on the UV light irradiated regions of the diazoketo-functionalized SAM on a glass substrate using EDC/NHS coupling chemistry. As a result, biotin was patterned on the exposed regions, which was further used for binding with FITC-SAv and Rh-SAv samples. Well-defined green and red fluorescent patterns were observed under the confocal fluorescence microscope and shown in Figure 6. On the other hand, incubating the biotin-patterned SAM sample with Rh-SAv that had been presaturated with 200 μM biotinamine did not result in streptavidin patterning (data not shown). This nonspecific binding experiment has additionally proven the specificity and applicability of this approach for biomolecular patterning. Additional work on quantification of the photoproduct and kinetics of the photoreaction is currently underway. The diazoketo approach reported in this work allows direct patterning/functionalization of carboxylic acid groups on silicon surfaces. Since free carboxylic acid groups are not compatible with chloro or alkoxysilane groups, other methods to prepare carboxyl-terminated SAMs on silicon substrates may require complicated process steps. Moreover, the diazoketo system could serve as a substrate for multicomponent chemical and biomolecular patterning.14 For instance, a dual chemical patterning can (14) (a) Synytska, A.; Stamm, M.; Diez, S.; Ionov, L. Langmuir 2007, 23, 5205. (b) Petrou, P. S.; Chatzichristidi, M.; Douvas, A. M.; Argitis, P.; Misiakos, K.; Kakabakos, S. E. Biosens. Bioelectron. 2007, 22, 1994.
Langmuir 2009, 25(16), 8888–8893
Letter
be achieved by exposing UV light on selected regions that can be functionalized to possess a first type of functional group other than carboxyl group, followed by maskless flood UV exposure to render the remaining regions with carboxylic acid groups. Multicomponent biomolecular patterning can be achieved using a mask aligner or suitable patterning strategies. As molecular nitrogen is the only byproduct released, the successive UV exposure will not contaminate the surface, which is crucial for multicomponent biomolecular patterning.
Conclusions We have synthesized a novel diazoketo-functionalized photoactive compound that can form stable SAMs on silicon and glass substrates. Formation of the diazoketo-functionalized SAM and its photoreaction were characterized using ellipsometry, AFM, IR analyses, contact angle measurements, and XPS. Selective UV light irradiation on the diazoketo-functionalized SAM resulted in the formation of carboxylic groups on the UV light exposed regions. The carboxylic groups were used for the immobilization of biotin followed by binding with streptavidin. This approach shows the potential as a novel platform for patterned immobilization of various biomolecules such as DNA, protein, or synthetic biopolymers. Acknowledgment. The authors would like to acknowledge the financial support of the Brain Korea 21 (BK21) project. The authors thank Mr. Sang-Jin Cho, from the Materials Science Laboratory at Sungkyunkwan University, for ATR-IR measurements.
DOI: 10.1021/la901870a
8893
