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J. Phys. Chem. C 2010, 114, 10512–10519
Photooxidation of Amine-Terminated Self-Assembled Monolayers on Gold Szu-Hsian Lee,† Wei-Chun Lin,‡ Che-Hung Kuo,† Manuel Karakachian,‡,§ Yu-Chin Lin,‡ Bang-Ying Yu,‡ and Jing-Jong Shyue*,†,‡ Department of Materials Science and Engineering, National Taiwan UniVersity, Taipei 106, Taiwan, Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan, and Department of Materials Science and Engineering, Polytech Paris-Sud, Paris, France ReceiVed: February 16, 2010; ReVised Manuscript ReceiVed: March 29, 2010
Amine-terminated self-assembled monolayers (SAMs) on Au surfaces are commonly used to immobilize various types of molecules, including DNA and proteins. However, little is known about the stability of these types of surfaces. In this work, it was observed that the surface potential (as well as the isoelectric point) of amine-bearing SAMs on flat gold substrates changed significantly with time, indicating that the surface functional group is not stable under ambient conditions (standard temperature and pressure). Contact angle analysis indicated that after degradation, the polar component of the interfacial force decreased and the dispersion component increased. These results indicate that the surface has undergone a chemical transformation. X-ray photoelectron spectroscopy (XPS) was used to detect changes in the chemical state of the surface nitrogen atoms. The chemical shift of the binding energy indicates that the nitrogen is partially oxidized. Using time-of-flight secondary ion mass spectrometry (ToF-SIMS), the oxidation of the amino groups to nitroso groups was evident, as was the previously reported oxidation of the thiol groups to sulfonate groups. Two methods for retarding the oxidation of the amine functional group are presented in this work. By isolating the SAM from either light or air, the oxidation is suppressed and the surface properties are preserved. In other words, the shelf life of the amine-modified gold substrates is prolonged. This result suggests that the oxidation is either photocatalyzed or photoinduced. Introduction Self-assembled monolayers (SAMs) on solid surfaces are involved in many promising applications in the fields of nanotechnology and biotechnology. A variety of biomolecules (including proteins, peptides, DNA, antibodies and therapeutics) have been attached to SAMs. Over the past few decades, researchers have found that SAMs on gold surfaces exhibit excellent properties for use in biological applications.1 Gold is the most commonly used metal for studying the science and bioengineering aspects of SAM coatings because of the strong Au-thiol chemical bond (184 kJ/mol)2 that binds interested functional groups on the Au surface. In addition, Au nanoparticles are harmless to cells. For example, 18 nm Au nanoparticles modified with citrate or biotin show no cytotoxicity to the human K562 cell at a concentration of 250 µM.3 Recently, SAMs on gold have been used for DNA, drug, and protein delivery.4-10 However, many researchers have found that the stability of SAMs is an issue for long-term biomedical applications.11-14 Numerous researchers have investigated the stability of functional groups on Au.15,16 For example, the photooxidation of thiol groups to sulfonate groups is well documented.17,18 For the bifunctional Stern layer forms on amine-SAM in aqueous environment, amine-terminated SAMs are useful for promoting the deposition of both positively and negatively charged films.19-21 The surface potential can also be tailored by mixing amine- and carboxylic acid-terminated SAMs.22,23 In addition, The amine-terminated SAMs provide specific * To whom correspondence should be addressed. Telephone +866(2)27898000#69. Fax +886(2)2782-6680. E-mail:
[email protected]. † National Taiwan University. ‡ Academia Sinica. § Polytech Paris-Sud.
electrostatic interactions between the monolayer and the biomaterial24,25 that help the modified Au enter living cells.26 For instance, amine-terminated SAMs are used in DNA array applications.27,28 In addition, the amino groups in amineterminated SAMs are used as handles for modifying the surface with other functional groups (EGn groups) to generate SAMs with new properties, such as resistance to protein adsorption or specific adsorption of a protein of interest.25 Furthermore, using positively charged aminothiol-modified gold nanowires, both DNA and probing molecules were delivered into cells without compromising the viability of the cells.29 However, the stability of amine-terminated SAMs on Au surfaces is understudied, and this information will be valuable to future research pursuits. Since one of the main reasons for using SAMs is to tailor the properties of a surface, studying the surface potential of the SAMs is critical. For nanoparticles, electrophoresis light scattering (ELS) can be used.22 For planar substrates, streaming potential and streaming current that are generated in a microchannel consisting of two planar sample surfaces (electrokinetic analysis) can be used.30 Contact angle analysis is also frequently used to determine the interfacial force. X-ray photoelectron spectroscopy (XPS)31 and secondary ion mass spectroscopy (SIMS)32 that have shallow probing depths are also used to study the chemical properties of the surface. In this work, these tools are employed to determine the changes in the physical and chemical properties of aminebearing SAMs on Au that were stored under different conditions for various lengths of time. It was found that both the surface potential and the isoelectric point were markedly changed for specimens that were stored under ambient conditions for six days. XPS and SIMS analyses revealed that the amino groups were oxidized to nitroso groups, indicating that amine-modified
10.1021/jp101426h 2010 American Chemical Society Published on Web 05/18/2010
Photooxidation of Amine-Terminated SAMs on Gold
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Figure 1. Photo of samples stored (a) under ambient condition, (b) in ethanol, and (c) in darkness.
Au surfaces are not stable in ambient environments. Two methods were investigated for protecting amine-bearing SAMs from degradation. These methods isolate the samples from air or light and were shown to preserve the physical and chemical properties of the amine-terminated SAM surface. Experimental Section Deposition of SAMs. A gold substrate was prepared by deposition onto a 20 mm ×10 mm glass slide in a high vacuum e-beam evaporator. First, a 5 nm thick chromium layer was deposited to enhance adhesion, and then a 150 nm thick layer of high purity gold (99.99%, Summit-Tech Resource Corp., TW) was deposited on the Cr adhesion layer. The substrates were then rinsed with pure ethanol, and were exposed to a vacuum UV (193 nm) for 10 min in air to remove contaminations. The deposition solution consisted of 30 mL of 1 mM solution of 8-amino-1-octanethiol hydrochloride (HS(CH2)8NH2 · HCl) (98.9% purity, Dojindo, Japan) in ethanol (Fisher Scientific, HPLC grade) mixed with 5 mL of 1 M HCl. The gold substrates were immersed overnight in the deposition solution for the growth of SAM. After SAM deposition, the substrates were flushed with absolute ethanol and sonicated for 3 min to remove residual HCl and excess surfactants. The samples were then dried with a stream of nitrogen. Sample Handling. The referencing “freshly deposited” specimens were measured within 1 min after preparation. Samples stored under ambient condition were put into a covered glass dish and stored on a desktop in an office (Figure 1a). The environment was air-conditioned at 25 °C, the humidity was 40%, and the office air was filtered with a Honeywell 17450 HEPA air purifier. The luminance was ∼1000 Lux as provided by fluorescent lighting and was applied during the entire experiment. For a sample stored in ethanol (Figure 1b), about 5 mL of HPLC grade ethanol was added to the dish to cover the specimen. The height of the ethanol was about 2 mm above the specimen so that the pressure difference can be neglected. For samples stored in darkness (Figure 1c), the glass dish was covered by aluminum foil to isolate from the light. All the glass dishes were placed side-by-side; hence, they were exposed to identical environments in addition to the controlled exposure to light and air. Electrokinetic Measurements. The ζ potential of the solid substrate surface was measured by the streaming current of planar interfaces using a cell with an adjustable channel in an electrokinetic analyzer (SurPass, Anton Paar GmbH, Graz, Austria). Two planar samples were separated by a 100-150 µm gap and were aligned in parallel to form a microchannel. A solution of 0.1 mM NaCl electrolyte was pumped through the microchannel at 0-300 mbar with a syringe pump, and the current across the channel was measured using Ag/AgCl electrodes. The pH of the electrolyte was titrated to ap-
proximately 11.5 or to approximately 2.5 by adding NaOH (0.1 M) or HCl (0.1 M), respectively. The sample was rinsed thoroughly with the electrolyte before each measurement so that the acid-base reaction between the surface functional group and the electrolyte reached equilibrium at a given pH. The results of the streaming current measurements were converted to ζ potentials by using the Helmholtz-Smoluchowski equation and the Fairbrother-Mastin approach. Each ζ potential at a given pH value represents an average of at least four individual measurements. Contact Angle Measurement. A Rame-Hart goniometer was used for contact angle measurements. Advancing contact angles were determined by placing a drop of liquid (∼3 µL of 0-100% aqueous ethylene glycol solutions) on the sample with a micro syringe and then increasing the volume (by adding an additional ∼2 µL of the solution), keeping the area in contact with the substrate constantly and leaving the syringe in the drop. The data presented are averages of at least three distinct spots on two different samples. The surface energy was then calculated using the OWRK model.33 X-ray Photoelectron Spectrometry (XPS) Measurements. The chemical element spectra were detected by a PHI 5000 VersaProbe ULVAC-PHI, Chigasaki, Japan) system using a microfocused, monochromatic Al KR X-ray (25 W, 100 µm). The takeoff angle (with respect to the surface) of the photoelectron was 45° unless otherwise mentioned. The base pressure of the system is below 10-8 Pa using oil-less pumping systems. A dual beam charge neutralizer (Ar+ gun and flooding electron beam) was used to compensate for the charge-up effect. Spectra were collected with the pass energy set to 58.7 eV, and the binding energy was referenced to and normalized against the Au 4f peak at 84.1 eV.34 The energy resolution at this condition was 0.64 eV as measured by the fwhm of Ag 3d5 peak. The typical data acquisition time was ∼5 min to minimize the possible damage to the monolayer; the ratio of peak intensity was converted to atomic percentage using the sensitivity factors built into Multipak software package. Time-of-Flight Secondary Ion Mass Spectrometry (ToFSIMS) Measurements. The experiments were performed with a PHI TRIFT V nanoTOF ToF-SIMS system. The pulsed primary ion source was Bi32+ and was operated at 25 kV (5.1 nA DC, 0.043 pA pulsed) with a rastering area of 200 µm × 200 µm and an incident angle (with respect to the surface normal) of 40°. A flooding electron beam was used for charge compensation, and the mass was calibrated with CH-, OH-, C2H- and CH3+, C3H5+, C4H7+ for negative and positive spectra, respectively. Results and Discussion It is known that the quality of amine-terminated SAMs is hard to control. Using a simple ethanol solution, oxidized sulfur
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Figure 2. XPS spectra of amine-SAMs freshly deposited with (solid lines) and without (broken lines) the addition of HCl.
TABLE 1: Angle-Resolved XPS on Amine-SAMs Freshly Deposited with and without HCl sample with HCl without HCl
TOA (deg)
Au (%)
O (%)
C (%)
N (%)
S (162.3 eV) (%)
S (168.7 eV) (%)
10 80 10 80
13.2 30.7 18.5 43.5
1.3 0.9 19.4 11.7
71.9 56.4 53 37.1
7.8 6.6 5.4 4.2
5.7 5.4 2.1 2.4
0.1 0 1.6 1.1
without HCl top layer layer 2 layer 1 substrate
with HCl
thickness (nm)
composition
thickness (nm)
composition
0.32 0.81 0.16 ∞
62.2% O, 23.6% N, 14.4% S (168.7 eV) C 55.1% O, 19.4% N, 25.4% S (162.3 eV) Au
0.13 0.77 0.19 ∞
5.1% O, 94.8% N, 0.1% S (168.7 eV) C S (162.3 eV) Au
can be clearly observed right after SAM deposition.35,36 Various protocols such as adding triethylamine in the deposition solution35 followed by washing with acidic ethanolic solution36 are developed to improve the quality. In its amine form, the lone electron pair can donate electrons to Au to form covalent Au-N bonds.37 Such an undesired bonding group will distort the alignment of molecules on the surface, and the exposed sulfur can oxidize easily.38 In this work, HCl was added to the deposition solution. It protonized the amine to form ammonium ions which would not bond covalently with Au. In addition, while dimers with amino groups were easy to form through hydrogen bonds between the H and the lone-pair on N, the positively charged ammonium groups repelled each other to prevent multilayers from forming. As a result, the quality of the deposited amine-SAM could be improved. Figure 2 shows the amine-SAMs deposited on Au with (solid lines) and without (broken lines) the addition of HCl. While significant O was found on the SAMs deposited without HCl, only a trace amount of the O was observed with that deposited with HCl. In addition, the majority of the N is in amine form (399 eV).39 Furthermore, the oxidized S (168.7 eV) was not observed with that deposited with HCl. On the other hand, the majority of the N shifted to
a higher binding energy when deposited without HCl. It is known that extremely low pH is required to fully protonize amine-SAM.31 Compared with the SAM deposited with HCl, only a minor shoulder is observed at 401 eV that exists with ammonium group31 where N has a formal charge of +1. When the SAM was deposited without HCl, the amine group would be protonized to a less extent, and thus the significant peak at 401 eV could not be simply attributed to the presence of ammonium. Instead, the chemical shift suggested the existence of Au-N bonding where the Au cation pulls the electron from an N atom and gives N a formal charge of +1 that increases the binding energy of the N 1s when the SAM is deposited without HCl. To further investigate the quality of the SAM, angle-resolved XPS was carried out with takeoff angles (TOAs) of 10° and 80° (Table 1). The angle-resolved spectrum was analyzed with the Multipak software package. The algorithm was based on Hill’s method40 and was extended to multi-elements.41 The best structural fit indicated that some of the molecule was bonded to Au through the N group and the oxidized S was located at the outermost layer for SAMs deposited without HCl addition. For SAMs deposited with HCl, the best structural fit indicated
Photooxidation of Amine-Terminated SAMs on Gold
J. Phys. Chem. C, Vol. 114, No. 23, 2010 10515
Figure 3. ζ potentials versus pH for amine-terminated SAMs stored under ambient conditions for 1, 3, and 6 days. The error bars indicate the standard deviation.
TABLE 2: Advancing Contact Angle between an Aqueous Solution of Ethylene Glycol and (Sample 1) a Freshly Prepared Amine-Terminated SAM on an Au Surface; (Sample 2) a Specimen Aged for 6 Days under Ambient Conditions
sample 1 sample 2
H2O (0%)
10%
30%
50%
70%
90%
ethylene glycol (100%)
86.5 74.6
83.5 71.8
74.3 67.6
73.7 62.8
62.6 57.6
60.4 52.4
58.8 52.2
good molecular alignment, and the residual oxygen located at the outermost layer could be attributed to oxygen containing adsorbates.42 On the basis of XPS observation, it could be concluded that the quality of the amine-SAM was enhanced significantly with the addition of HCl in the deposition solution. Figure 3 shows the ζ-potential as a function of pH for amineterminated SAMs stored under ambient conditions for 1, 3, and 6 days. The data shows that the surface potential dramatically decreased with increasing exposure time. The isoelectric point (IEP) of a freshly prepared amine-terminated SAM was 7.2, and the IEP of samples stored in air for 1, 3, and 6 days decreased to 5.6, 4.9, and
