Arylsilanated SiOx Surfaces for Mild and Simple Two-Step Click

Aug 23, 2012 - Functionalization with Small Molecules and Oligonucleotides. Ehow H. Chen, Stephanie R. Walter, SonBinh T. Nguyen, and Franz M. Geiger*...
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Arylsilanated SiOx Surfaces for Mild and Simple Two-Step Click Functionalization with Small Molecules and Oligonucleotides Ehow H. Chen, Stephanie R. Walter, SonBinh T. Nguyen, and Franz M. Geiger* Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States S Supporting Information *

ABSTRACT: The conversion of surface-bound aminophenyl groups to azidophenyl moieties on SiOx surfaces was investigated as part of a mild, simple two-step strategy for “click”-based” surface functionalization with acetylene-functionalized reagents. Small terminal alkynes (phenylacetylene, 1-hexyne) and acetylene-modified single-stranded DNA 20mers (T20) were then used as model compounds to test the efficiency of the 1,3-dipolar cycloaddition reaction. The identities of surface species were verified, and their coverages were quantified using X-ray photoelectron spectroscopy in the C 1s, N 1s, F 1s, Cl 2p, and P 2p regions. Depending on conditions, the yield of the azidification was in the 30−90% range, and the efficiency of triazole formation depended significantly on the rigidity of the acetylene reactant. Vibrational sum frequency generation was applied to probe the C−H stretching region and test the platform’s viability for minimizing spectral interference in the C−H stretching region. Fluorescence spectroscopy was also performed to verify the presence of fluorescein-tagged DNA single strands that have been coupled to the surface, while labelfree DNA hybridization studies by vibrational sum frequency generation spectroscopy readily show the occurrence of duplex formation. Our results suggest that the two-step azidification−click sequence is a viable strategy for readily functionalizing silica and glass surfaces with molecules spanning a wide range of chemical complexity, including biopolymers.

1. INTRODUCTION Research into DNA-based biosensors has been expanding in the past decade as the applications for such devices become ubiquitous in the medical, forensic, agricultural, and environmental fields.1,2 Molecular-level insights into the hybridization event at the liquid/solid interface have been gained through various techniques,3−6 including nonlinear optical techniques such as second harmonic generation (SHG) and vibrational sum frequency generation (SFG), where the high sensitivities and unique capabilities of the experimental techniques including the ability to determine thermodynamic binding parameters7 and molecular orientation8have enabled labelfree investigations.9 An important aspect in such studies is how the linker used to covalently attach DNA oligonucleotides to a surface can significantly impact the structure of the surfacehybridized molecular layer: it has been shown that short and rigid linkers can lead to better ordering of the surface species.10 We have previously utilized N-hydroxysuccinimide (NHS)ester-terminated trichlorosilane to covalently attach DNA to SiOx surfaces and studied them with vibrational SFG to deduce the orientation of thymine methyl groups (CH3) in the C−H stretching region and gain further understanding into the hybridization of DNA at interfaces.9,11 Herein, we demonstrate the applicability of an in situ “click”-based strategy that enables improved densities, reactivities, and functionality ranges of tailored organosilane surfaces.12 Over the past two decades, click chemistry13,14 has emerged as a highly useful tool for © 2012 American Chemical Society

achieving such goals with high yields, chemical specificity, and under mild reaction conditions.15 The acetylene and azide functionalities required for copper-catalyzed 1,3-dipolar cycloaddition are easily elaborated in small molecules or biopolymers, and the triazole product is regioselective, stable, and nontoxic.14 While there is already precedence for click reactions at interfaces between alkyne-functionalized DNA and azidesubstituted alkylsilane surface layers,15−18 the limited commercial availability of azide-substituted alkylsilane precursors has prompted us to explore a new click-based strategy that can be easily integrated into a readily accessible silanized platform for deploying DNA and other molecules at interfaces. Specifically, we introduce a new simple two-step method to covalently attach acetylene-modified DNA strands to silanized SiOx surfaces using aryl azides. We first functionalize our surfaces using p-aminophenyltrimethoxysilane, a workhorse platform extensively used in our model atmospheric studies,19−21 and then transform the arylamines to azides through a mild oxidation process that was reported for solution-phase reactions.22 The resulting azidified surface is then coupled to alkyne-functionalized small molecules and DNA. Using X-ray photoelectron spectroscopy (XPS), we quantify the conReceived: June 29, 2012 Revised: August 16, 2012 Published: August 23, 2012 19886

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The reaction vial was lightly purged with a stream of nitrogen gas for 5 s, sealed with a Teflon-lined cap, and left to stir at room temperature for 16 h. After the reaction was completed, the sample was rinsed with acetone and water, sonicated briefly (∼1 min) in water using an Aquasonic laboratory bath sonicator (Model 75T, VWR Scientific, Batavia, IL), dried with nitrogen gas, and stored in a vacuum desiccator until further analysis. For XPS analysis, 4-ethynyl-α,α,α-trifluorotoluene and 6-chloro-1-hexyne were used in place of phenylacetylene and 1-hexyne, respectively, for additional verification of the triazole reaction. DNA. An azidophenyl-functionalized sample was submerged in DMF (2.0 mL) in a 20 mL scintillation vial equipped with a magnetic micro stirbar. While the mixture was stirred, tris[(1benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 5.3 mg, 10 μmol) was added. 5′-Hexynyl-TTT TTT TTT TTT TTT TTT TT (5′-hexynyl-T20, 1.5 mg, 220 nmol), CuSO4·5H2O (2.5 mg, 10 μmol), and sodium bicarbonate (0.8 mg, 10 μmol) were dissolved in H2O (0.5 mL) separately, and this mixture was then added to the stirring DMF solution. Next, an aliquot of tris(2-carboxyethyl)phosphine solution (TCEP, 20 μL of a 0.5 M solution in H2O, 10 μmol) was added. The reaction vessel was lightly purged with a stream of nitrogen gas for 5 s, sealed with a Teflon-lined cap, and left to stir at room temperature for 16 h. After the reaction was completed, the sample was rinsed with acetone and water, sonicated briefly (∼1 min) in water using an Aquasonic laboratory bath sonicator, dried with nitrogen gas, and stored in a vacuum desiccator until further analysis. For the fluorescence spectroscopy experiment, the DNA was also functionalized with 3′-fluorescein amidite (6FAM) in addition to the 5′-hexynyl linker. DNA Hybridization. To hybridize T20-functionalized samples, substrates were exposed to a solution (3 mL) of A20 (100 μmol) in aqueous NaCl (250 mM, pH 7) at room temperature for 2 h without stirring. The surfaces were then rinsed with aqueous NaCl (250 mM, pH 7) solution to remove any physisorbed DNA strands and analyzed immediately. C. XPS, Fluorescence, and SFG Experiments. XPS. XPS data were collected in the C 1s, N 1s, F 1s, Cl 2p, and P 2p binding energy regions using either an Omicrometer Electron Spectroscopy for Chemical Analysis (ESCA) probe (500 μm spot, 5 scans, 75 eV pass energy) equipped with Omicron EIS software or a Thermo Scientific ESCALAB 250Xi (900 μm spot, 5 scans, 75 eV pass energy) equipped with Avantage Data System software. The XPS spectra shown in Figures 2, 3, 4, and 6 were obtained from the Thermo Scientific ESCALAB 250Xi system given its superior signal-to-noise ratio; however, data from both systems were used in the quantitative analysis. Binding energies were calibrated to the alkyl carbon response at 284.5 eV. Fluorescence. Fluorescence data were collected with an excitation wavelength at 490 nm (1 scan, 3 nm slit width) using a HORIBA Jobin Yvon Fluorolog fluorometer. Spectra were background corrected with a spectrum of a blank glass slide. SFG. Detailed descriptions of our SFG system have been previously published.24,25 Briefly, the SFG laser setup consists of a 1 kHz, 120 fs amplified Ti:sapphire system that generates 800 nm laser light. The beam is split 50/50: one half to be used as the visible upconverter with narrow bandwidth as adjusted using an etalon and the other half to pump an optical parametric amplifier (OPA) producing 3−4 μm radiation with ∼150 cm−1 bandwidth. The narrow-bandwidth visible (2 μJ/ pulse) and broadband IR (1.5−2.0 μJ/pulse) pulses are focused

versions of the azidification and copper-catalyzed azide− acetylene cycloaddition (CuAAC) reactions. The successful functionalization of fluorophore-tagged DNA was also corroborated using fluorescence spectroscopy. In addition, we analyze the vibrational SFG spectra of surface-bound products to characterize the compounds in the C−H stretching region and demonstrate the platform’s reduced spectral interference in the C−H stretching region compared to the previously studied NHS−ester linker. The new linker strategy opens up possibilities for using it in SFG-based molecular orientation studies of functionalized surfaces in the C−H stretching region.

2. EXPERIMENTAL METHODS Unless otherwise stated, all reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI); customized DNA compounds were synthesized by Integrated DNA Technologies (IDT, Coralville, IA); and ultrapure deionized water (18.2 MΩ·cm) was obtained from a Millipore (Billerica, MA) Milli-Q system. A. Azide Transformation. As reported in our previous work,19−21,23 glass slides (Fisher Scientific) and Si(100) wafers with a 300 nm thick native oxide layer (Virginia Semiconductor, n-doped) were cleaned and functionalized with p-aminophenyltrimethoxysilane (Gelest, 90%+ isomeric purity). In a typical preparation of an azidophenyl-functionalized sample (Figure 1), an aminophenyl-functionalized sample was trans-

Figure 1. A reaction scheme for the azidification of an aminophenylfunctionalized SiOx surface, followed by a 1,3-dipolar cycloaddition to generate a triazolyl-functionalized surface.

ferred into an inert-atmosphere drybox and placed into a 20 mL scintillation vial equipped with a magnetic micro stirbar. Anhydrous acetonitrile (ACN, 8 mL) was added to the sample vial, and the capped vial was cooled in the drybox freezer (−30 °C) for 1 h. The sample vial was then removed from the freezer, and tert-butyl nitrite (tBuNO2, 800 μL, 6.7 mmol) was added to the mixture while stirring. To achieve the best conversion, the reaction vial was kept stirring for an additional 10 min after the tBuNO2 addition. Trimethylsilyl azide (TMSN3, 600 μL, 4.6 mmol) was then added dropwise, and the reaction was stirred for 1 h before the vessel was removed from the drybox. The sample was rinsed with acetone and water, dried with nitrogen gas, and immediately used for the next step. Alternatively, we found that samples can be stored in vacuum desiccator up to a several days for further use. B. Copper-Catalyzed Azide−Acetylene Cycloaddition. Alkyne-Functionalized Small Molecules. An azidophenylfunctionalized sample was submerged in dimethylformamide (DMF, 4.5 mL) in a 20 mL scintillation vial equipped with a magnetic micro stirbar. While the mixture was stirred, the acetylene compound (phenylacetylene or 1-hexyne, 100 μL, ∼900 μmol) was added. Copper sulfate pentahydrate (CuSO4·5H2O, 9 mg, 35 μmol) and sodium ascorbate (35 mg, 175 μmol) were dissolved in water (0.5 mL) separately, and this solution was then added to the stirring DMF solution. 19887

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eV that corresponds to the nitrogen of the NH2 group.23 The minimal response at 401 eV in this spectrum suggests a lack of amines hydrogen-bonded with adjacent surface-bound amines, indicating that the silane deposition formed a well-ordered layer.23 After being exposed to tBuNO2, the XPS spectrum of the azidophenyl-functionalized surfaces now exhibits peaks at 401 and 404 eV, corresponding to the sp2-hybridized and sphybridized nitrogens of the N3 moiety, respectively.17,27,28 As shown in Table 1, the efficiency of the azidification ranges from 35% to 90% (calculated using eq S1 in the Supporting Information). Delaying the addition of TMSN3 after tBuNO2 by ∼10 min tends to increase the amount of amines converted, which is not surprising as the reaction was initially cooled to 30 °C below the reported solution-phase reaction temperature of 0 °C.22 The conversion may partially be limited by the isomeric purity (90+%) of the p-aminophenyltrimethoxysilane used for the functionalization, as tBuNO2 may be too bulky to react with o- and m-substituted aminophenyl moieties bound to the surface. The reduced number of accessible degrees of freedominherent to surface reactionswould additionally lower the yield below the reported 93% for the solution-phase azidification of aminophenyl.22 While these hindrances prevent us from achieving complete surface azidification, it is possible to optimize this reaction to generate a surface comprising of orthogonally reactive amines and azides, similar to work recently reported by Gibbs-Davis and co-workers,27 but without the use of mixed organosilane solutions. Cu-Catalyzed 1,3-Cycloaddition of Phenylacetylene and 1-Hexyne to Azidophenyl-Functionalized Surfaces. XPS spectra were collected in the C 1s, N 1s, F 1s, and Cl 2p regions for azidophenyl-functionalized samples after exposure to CuAAC conditions in the presence of either 4-ethynyl-α,α,αtrifluorotoluene (Figure 3) or 6-chloro-1-hexyne (Figure 4). For substrates reacted with 4-ethynyl-α,α,α-trifluorotoluene, the XPS spectrum shows a clear decrease of the sp-hybridized azide nitrogen peak at 404 eV when compared to that for the unreacted azidophenyl-functionalized surface (Figure 3A). A shift of the sp2-hybridized nitrogen peak from 401 to 400 eV is also observed, indicating the presence of both sp3- and sp2hybridized triazole nitrogen atoms. The formation of a triazole is further verified by CF3-related signals at 698 and 292 eV in the F 1s and C 1s regions, respectively (Figure 3B,C). Similarly, 6-chloro-1-hexyne reacts quantitatively with surface-bound azidophenyl, given the complete disappearance of sp-hybridized azide nitrogen peak at 404 eV (Figure 4A) and the appearance of a new chloride peak at 200 eV in the Cl 2p region (Figure

Figure 2. XPS spectra of aminophenyl- and azidophenyl-functionalized SiOx surface in the N 1s region (gray and black, respectively). Spectra are vertically offset for clarity.

and overlapped spatially and temporally to generate the SFG signal, and a spectrograph/CCD assembly (Princeton Instruments) serves to record the SFG spectra after spectral isolation of the SFG signal using the appropriate spatial and optical filters. Spectra were collected for 3 min and averaged over 10 acquisitions for each center frequency of the broadband IR beam; three different IR center frequencies were employed over the course of the experiment. SFG spectra were collected in ssp polarization, a commonly used polarization combination with a good signal-to-noise ratio for initial studies, provided the optimal angles for the incident visible and IR beams in the experimental setup.26 The ssp-polarized SFG spectra yield information primarily on those vibrational modes that have their transition dipole moment components orientated along the surface normal. Following background subtraction, the SFG intensities were normalized to the IR power distribution and the frequencies were calibrated to the symmetric methylene C− H stretch of a polystyrene standard at 2849.5 cm−1.

3. RESULTS AND DISCUSSION A. XPS Analysis of Surface-Bound Arene and Triazole Species That Result in the 1,3-Dipolar Cycloaddition of Small Molecules. Aminophenyl- and Azidophenyl-Functionalized Surfaces. The XPS spectrum of the aminophenylfunctionalized surface (Figure 2) exhibits a major peak at 399.5

Figure 3. XPS spectra of azidophenyl-functionalized SiOx surface before (black) and after reaction with 4-ethynyl-α,α,α-trifluorotoluene (purple) in the (A) N 1s, (B) F 1s, and (C) C 1s regions. Spectra are vertically offset for clarity. 19888

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Figure 4. XPS spectra of azidophenyl-functionalized SiOx surface before (black) and after reaction with 6-chloro-1-hexyne (green) in the (A) N 1s and (B) Cl 2p regions. Spectra are vertically offset for clarity.

Table 1. Quantification of the Azidification and 1,3-Dipolar Cycloaddition Reactions Using XPS Analysis in the N 1s Region

that the entries in Table 1 are representative of the many trials that were performed for these experiments. Since the yields for the solution-based 1,3-cycloadditions between phenylacetylene and 1-hexyne with azidophenyl are comparable and quantitative,30 our aforementioned results suggest that phenylacetylene reacts with the surface-bound azidophenyl moieties in a less consistent manner than 1-hexyne. At the higher azidophenyl densities of ∼60% to 90% (Table 1, entries 1b, 1c), this inconsistency may be due to steric hindrance induced by “clicked” phenylacetylene, which prevents other acetylene molecules from reaching the remaining surface azidophenyl sites. Studies on similarly reactive silane surfaces demonstrated comparable scenarios of sterically hindered surface groups. 31,32 However, steric hindrance is less of an issue when the amine-to-azide conversion is lowered to ∼35% (Table 1, entry 1a): as the density of surface-bound azidophenyl groups is lowered, reacted surface sites do not restrict access to other azide sites. This result may be important if this azidophenyl-functionalized platform is used with other rigid and bulky molecules or if the unconverted aminophenyl surface sites are utilized in a bifunctional organosilane surface.

4B). Experiments with the corresponding non-halogenated compounds were also performed, and the XPS results were comparable to the reactions with the halogenated compounds, suggesting that halogenation of the alkyne substituent does not significantly change the reactivity of the alkyne moiety and that the halogenated compounds can be used to confirm product formation. While XPS can, in theory, afford relative elemental compositions,29 the integrated areas obtained from the halide elemental responses far exceeded the expected amount of nitrogen, even after correction with the respective sensitivity factors. This suggests that integration across discrete XPS regions is not a suitable way to estimate conversions. However, we may solely analyze the N 1s region to determine the amount of triazole formed by comparing the ratios of the 401 eV sp2hybridized nitrogen peak to the 404 eV sp-hybridized nitrogen peak before and after the reaction, as shown by eqs S2−S6 in the Supporting Information. The calculated triazole yield for phenylacetylene varies greatly from 50% to 100% (Table 1, entries 1a, 1b, 1c), though the cycloaddition seems to be more favorable with a lower azidophenyl density. In contrast, 1hexyne consistently affords 78−100% yield, regardless of the surface azide density (Table 1, entries 2a, 2b, and 2c). We note 19889

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tioned mode at 2920 cm−1 of the aminophenyl group to a combination band of an NH2 bend (1610 cm−1) with an aromatic C−H in-plane bend (1310 cm−1). In the SFG spectrum of surface-bound 1,4-diphenyl-1,2,3triazolyl groups, C−H aromatic stretching modes appear above 3000 cm−1, confirming the successful cycloaddition of phenylacetylene to the surface, which results in an additional phenyl group on the tethered molecule. By symmetry, the SFG response above 3000 cm−1 is attributed to just the single aromatic C−H oscillator in the para position. Vibrational modes below 3000 cm−1 are observed after triazole formation, likely due to new combination bands and overtones of the triazole compound and unreacted aminophenyl and azidophenyl sites. In contrast, the SFG spectrum of surface-bound 4-butyl-1phenyl-1,2,3-triazolyl groups displays resonances similar to those in the spectrum for the azidophenyl-functionalized surface. However, we also observe the appearance of the CH3 symmetric stretch and the CH3 Fermi resonance (coupled between the CH3 bending mode and the CH3 asymmetric mode) at 2875 and 2940 cm−1, respectively, which are associated with the CH3 group of the butyl moiety.35 We note that these responses appear to be in-phase with the combination bands of unreacted aryl sites and that the CH3 asymmetric stretch, normally expected at ∼2960 cm−1, is absent from our SFG spectra. Thus, we conclude that in spite of the presence of the aryl combination bands, the aromatic C−H, CH2, and CH3 moieties bound through the cycloaddition surface reaction are readily detected. C. Characterization of DNA-Functionalized Surfaces. Given the successful 1,3-cycloaddition of small-molecule alkynes to our azidophenyl-functionalized substrates, we explored their Cu-catalyzed functionalization with acetylenemodified DNA. Azidophenyl-functionalized Si(100) and glass surfaces were exposed to hexynyl-functionalized oligonucleotides (5′-hexynyl-T20-6-FAM-3′ and 5′ hexynyl-T20) under CuAAC conditions and then analyzed using XPS, fluorescence spectroscopy, and SFG (Figure 6). Thymine nucleotides were chosen as the CH3 group may be used as an intrinsic label for SFG experiments.9,11 The presence of functionalized DNA strands on the T20-6-FAM-functionalized Si(100) substrate were readily confirmed by XPS as a broad peak at 133.5 eV in the P 2p region (Figure 6A), which agrees with previous studies of DNA on Au surface.36 The fluorescence spectrum of the T206-FAM-functionalized glass slide (Figure 6B) exhibits a highly visible fluorescein fluorescence signal, also supporting the presence of surface-conjugated DNA groups after the CuACC reaction. That these groups resulted from the surface click reaction is evident from a control where the reducing agent TCEP was not added to the reaction: the resulting substrate displayed only a very minimal fluorescence (Figure 6B), attributed to physisorbed T20-6-FAM or small amounts of clicked single-strand DNA. To quantify the amount of DNA strands on the oligonucleotide-functionalized Si(100) substrate surface, we again utilized XPS in the N 1s region. Given that each DNA strand adds 40 additional nitrogens to the surface nitrogen content (see eqs S7−S11 in the Supporting Information), the surface population of T20 strands is estimated as 2.8% and 0.6% for 5′-hexynyl-T20 and 5′-hexynyl-T20-6-FAM-3′, respectively (Table 1, entries 3a, 3b). Assuming a surface site density of 5 × 1013 aminophenyl-functionalized sites/cm2, obtained from previous SHG experiments,37 we estimate the density of

B. Vibrational SFG Responses of Aminophenyl-, Azidophenyl-, and Triazolyl-Functionalized Surfaces. Vibrational SFG spectra at the air/solid interface were collected for glass substrates functionalized with aminophenyl, azidophenyl, 1,4-diphenyl-1,2,3-triazolyl, and 4-butyl-1-phenyl-1,2,3triazolyl groups to verify the presence of the phenyl and butyl groups and to determine the applicability of the azidophenyl click−substrate platform for vibrational SFG analysis in the C− H stretching region (Figure 5). The SFG spectrum of the

Figure 5. Vibrational SFG spectra (C−H stretching region using ssp polarization) of silica surface functionalized with aminophenyl (gray), azidophenyl (black), 1,4-diphenyl-1,2,3-triazolyl (purple), and 4-butyl1-phenyl-1,2,3-triazolyl (green) moieties. Spectra are vertically offset for clarity. The shape of the broadband IR pulse is overlaid on the bottom spectrum as reference.

aminophenyl-functionalized surface displays two vibrational modes at ∼2860 and 2920 cm−1, which have been previously assigned in a liquid/vapor/solution-phase FTIR/Raman spectroscopic study as a combination band of a ring-stretching mode (1600 cm−1) with an NH2-sensitive ring-stretching mode (1279 cm−1) and as an overtone of a ring-stretching mode (1468 cm−1), respectively.33 We note that the vibrational mode at 2920 cm−1 may need to be reassigned due to the vibrational assignments of surface-bound azidophenyl moiety (see below). The aromatic C−H stretches show negligible signal intensity, if any, which is not surprising as those modes are oriented mainly orthogonally to the SFG probe direction. In addition to residual contributions from remaining aminophenyl moieties, the SFG spectrum of the azidophenylfunctionalized surface displays a mode at ∼2900 cm−1, which can be tentatively assigned as a combination band of a ringstretching mode (∼1600 cm−1) with an N3-sensitive ringstretching mode (∼1300 cm−1), based on FTIR assignments of aminophenyl and FTIR peak values of azidophenyl (in KBr).33,34 The reduced intensity of aminophenyl-associated vibrational modes after the azidification suggests that both of these modes are probably strongly coupled to the NH2 substituent. Therefore, we tentatively reassign the aforemen19890

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verified with XPS, fluorescence spectroscopy, and vibrational SFG. The level of triazole functionalization appears to decrease when the substituents on the alkynyl derivatives is either too rigid, as in the case of phenylacetylene, or too large, as in the case of alkynylated DNA. While there is convolution in the SFG spectra due to combination bands of the aryl compounds, our azidophenyl-functionalized strategy holds promise as a potential platform for DNA SFG orientational studies in the C−H stretching region. Deuteration of the aryl hydrogen atoms in the linker platform presented here should further reduce spectral interference by minimizing C−H stretching contributions from the linker platform. Notably, the ability to control reaction yields in this strategy should readily enable its development into bifunctional surfaces possessing orthogonally reactive aminophenyl and azidophenyl surface groups.

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ASSOCIATED CONTENT

S Supporting Information *

Equation derivations and peak fitting. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 6. XPS spectrum in the P 2p region (A) and fluorescence spectrum (B) (excitation at 490 nm) of a T20-5′-6-FAM-functionalized SiOx surface. (C) SFG spectrum of a T20-functionalized SiOx surface. Spectra are vertically offset for clarity.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

DNA-functionalized sites to be ∼5 × 1011 sites/cm2. This low surface coverage is similar to those reported in previous DNAfunctionalized silane studies18,38,39 and is not surprising when one considers that coverage may be restricted by the large footprints of the DNA strands, a subject that has been investigated for DNA−thiol−gold nanoparticle systems.40 Additionally, the electrostatic repulsion between surfacebound strands would also disfavor high surface coverage. The vibrational SFG spectra for azidophenyl- and T20functionalized surfaces (Figure 6c) display very similar vibrational modes; however, the spectrum for the A20hybridized T20-functionalized surface displays two new vibrational modes at 2840 and 2930 cm−1, indicative of a CH2 symmetric stretch and CH3 Fermi resonance (coupled between the CH3 bending mode and the CH3 asymmetric mode), respectively. The observed appearance of these resonances arises from the high sensitivity of vibrational SFG toward the level of molecular ordering at the interface, as previously demonstrated in similar DNA hybridization experiments that employed an N-hydroxysuccinimide ester linker.11 Notably, the observed DNA vibrational modes appear to be in-phase with the combination bands of the aryl system, similar to the aforementioned SFG spectrum of 4-butyl-1-phenyl-1,2,3triazole. This feature will be very useful in future orientational analyses of surface-functionalized small molecules and biomolecules using the azidophenyl-functionalized platform. In addition, it will allow for the analysis of changes in the molecular distribution functions that such systems assume upon interaction with molecular and ionic target species in sensing applications.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation Environmental Chemical Sciences program under Grant CHE0950433. We acknowledge Spectra-Physics Lasers, a division of Newport Corporation, for equipment support. S.R.W. acknowledges the Achievement Rewards for College Scientists (ARCS) foundation. F.M.G. acknowledges an Irving M. Klotz Professorship. The XPS work was completed at the Keck Interdisciplinary Surface Science Center (Keck II) of Northwestern University. We also acknowledge Dr. Bong Jin Hong for his assistance with the fluorescence spectroscopy measurements as well as Drs. Ibrahim Eryazici and Mitchell H. Weston for their helpful discussions concerning the optimization of cycloaddition reaction conditions.

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REFERENCES

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4. CONCLUSIONS In summary, we reported a simple and mild surface reaction to transform surface-bound aryl amines to azides that can undergo CuAAC reactions with several acetylene derivatives. Successful azidification and triazole formation with phenylacetylene, 1hexyne, and acetylene-functionalized oligonucleotides were 19891

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The Journal of Physical Chemistry C

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dx.doi.org/10.1021/jp306437b | J. Phys. Chem. C 2012, 116, 19886−19892