Analysis of Self-Assembled Monolayer Interfaces by Electrospray

Anal. Chem. 2003, 75, 6741-6744

Analysis of Self-Assembled Monolayer Interfaces by Electrospray Mass Spectrometry: A Gentle Approach Wenrong Yang, Rui Zhang, Gary D. Willett, D. Brynn Hibbert, and J. Justin Gooding*

School of Chemical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia

A general mass spectrometry technique for the characterization of alkanethiol-modified surfaces is presented. Alkanethiol self-assembled onto a gold surface (in this case, peptides were attached to the gold surface via a thiolate bond) was reductively desorbed in 0.05 M KOH in the presence of octadecyl-derivatized silica gel. The peptide adsorbed onto the silica gel, whereupon it could be filtered, washed to remove any salts, and then eluted using a mixture of 4:1 v/v methanol/water. The eluant containing the peptide was injected into a Fourier transform ion-cyclotron resonance mass spectrometer (FTICR/ MS) via electrospray ionization. The spectrum showed no fragmentation of the peptide, demonstrating the gentleness of the technique. This simple procedure is not limited to FTICR/MS and could be adapted to other mass spectrometers. The molecular-level control over surface modification afforded by alkanethiol self-assembled monolayers (SAMs) makes them attractive for the nanofabrication of useful devices such as biosensors. Such nanofabrication usually requires multistep processing of a surface. Characterization of the ultimate modified surface, or of each step of the modification, is one of the enduring challenges in molecular-scale fabrication. X-ray photoelectron spectroscopy (XPS) is commonly used for surface analysis because it is sensitive to elemental and functional group composition and can be quantified. However, XPS suffers from a lack of chemical resolution and is often unable to determine the overall chemical structures of materials adsorbed on surfaces.1 Mass spectrometry, on the other hand, is ideal for providing chemical structure,2 but the requirement of removing the SAM from the surface, and then presenting it to the mass spectrometer, in a manner compatible with biological molecules is problematical.1,3-8 * To whom correspondence should be addressed. Fax: +61 2 9385 6141. E-mail: [email protected]. (1) Trevor, J. L.; Mencer, D. E.; Lykke, K. R.; Pellin, M. J.; Hanley, L. Anal. Chem. 1997, 69, 4331-4338. (2) Cole, R. B. Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications; Wiley: New York, 1997. (3) Li, Y.; Huang, J.; McIver, R. T., Jr.; Hemminger, J. C. J. Am. Chem. Soc. 1992, 114, 2428-2432. (4) Offord, D. A.; John, C. M.; Griffin, J. H. Langmuir 1994, 10, 761-766. (5) Offord, D. A.; John, C. M.; Linford, M. R.; Griffin, J. H. Langmuir 1994, 10, 883-889. (6) Trevor, J. L.; Lykke, K. R.; Pellin, M. J.; Hanley, L. Langmuir 1998, 14, 1664-1673. (7) Cooper, E.; Leggett, G. J. Langmuir 1998, 14, 4795-4801. 10.1021/ac0345897 CCC: $25.00 Published on Web 10/28/2003

© 2003 American Chemical Society

Mass spectrometry has seen considerable interest for the characterization of SAM-modified surfaces.1,3,4,6-15 However, many mass spectrometry techniques are too disruptive for the investigation of biomolecules on surfaces. For example, direct laser desorption mass spectrometry of SAM surfaces leads to molecular fragmentation and substrate effects, such as the generation of fragments from species on gold such as AuS+ and Au2S-.6,10,13,14,16-19 Furthermore, attempts at quantifying direct laser desorption have been hindered by large fluctuations in desorption and ionization efficiency. Similar problems exist with static secondary ion mass spectrometry (SIMS)1 and time-of-flight secondary ion mass spectrometry (TOF/SIMS).4 With TOF/SIMS, not only are Aucluster-related ions observed, but the method also suffers from relatively low secondary ion yields and matrix effects that can limit quantification, even with the use of internal standards. As an alternative, Shibue et al.8 used thermal desorption high-resolution mass spectrometry (TD/HRMS) to investigate mixed self-assembled monolayers on gold surfaces. The heating of the interface to 500-600 K in this technique, however, is not compatible with stable biomolecules.8 Therefore, biologically friendly MS techniques for the evaluation of alkanethiolate-modified surfaces are still required. This issue has recently begun to be addressed by Su and Mrksich20,21 using MALDI/TOF mass spectrometry of SAM-modified surfaces directly. Here, we present a different method for investigating alkanethiol monolayer modified surfaces (8) Shibue, T.; Nakanishi, T.; Matsuda, T.; Asahi, T.; Osaka, T. Langmuir 2002, 18, 1528-1534. (9) Scott, J. R.; Baker, L. S.; Everett, W. R.; Wilkins, C. L.; Fritsch, I. Anal. Chem. 1997, 69, 2636-2639. (10) English, R. D.; Van Stipdonk, M. J.; Sabapathy, R. C.; Crooks, R. M.; Schweikert, E. A. Anal. Chem. 2000, 72, 5973-5980. (11) English, R. D.; Van Stipdonk, M. J.; Diehnelt, C. W.; Schweikert, E. A. Rapid Commun. Mass Spectrom. 2001, 15, 370-372. (12) Zhong, W. Q.; Nikolaev, E. N.; Futrell, J. H.; Wysocki, V. H. Anal. Chem. 1997, 69, 2496-2503. (13) Kornienko, O.; Ada, E. T.; Hanley, L. Anal. Chem. 1997, 69, 1536-1542. (14) Kornienko, O.; Ada, E. T.; Tinka, J.; Wijesundara, M. B. J.; Hanley, L. Anal. Chem. 1998, 70, 1208-1213. (15) McCarley, T. D.; McCarley, R. L. Anal. Chem. 1997, 69, 130-136. (16) Brockman, A. H.; Shah, N. N.; Orlando, R. J. Mass Spectrom. 1998, 33, 1141-1147. (17) Fukuo, T.; Monjushiro, H.; Hong, H. G.; Haga, M. A.; Arakawa, R. Rapid Commun. Mass Spectrom. 2000, 14, 1301-1306. (18) Hanley, L.; Kornienko, O.; Ada, E. T.; Fuoco, E.; Trevor, J. L. J. Mass Spectrom. 1999, 34, 705-723. (19) Warren, M. E.; Brockman, A. H.; Orlando, R. Anal. Chem. 1998, 70, 37573761. (20) Su, J.; Mrksich, M. Langmuir 2003, 19, 4867-4870. (21) Su, J.; Mrksich, M. Angew. Chem., Int. Ed. 2003, 41, 4715-4718.

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that employs electrospray ionization. Electrospray ionization mass spectrometry (ESIMS) is an important tool for the analysis of peptides and proteins because it is a soft ionization method, yielding little molecular fragmentation (unless induced at the ESI atmosphere-vacuum interface). A key attraction of ESIMS is its capability for analyzing extremely small volumes of liquids,2 but a potential drawback is the suppression of ionization in solutions of high salt concentration.22 The purpose of this paper is to demonstrate a gentle method of removing alkanethiol SAMs from electrode interfaces that is compatible with retaining the integrity of biomolecules, as well as the subsequent analysis of the desorbed molecules using electrospray mass spectrometry. The alkanethiol SAM is electrochemically reductively desorbed from an electrode surface into electrolyte containing octadecyl-derivatized silica gel (C18). The idea of reductively desorbing the alkanethiolate from an electrode surface followed by analysis by MS is not completely new, as it was suggested by McCarley and McCarley,15 but to the best of our knowledge, this is the first demonstration of the principle. Desorbed hydrophobic molecules adsorb onto the C18, which can be separated by filtration and subsequently desorbed once more into a solution compatible with electrospray ionization (ESI) mass spectrometry. The analysis is completed using a combination of ESI with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The advantages of FTICR are high resolution and accuracy,23 allowing unambiguous molecular identification. The procedure is used to investigate peptide-modified interfaces for the detection of metal ions in the environment.24,25 To establish a protocol for characterizing peptide-modified electrodes using ESI/ FTICR/MS, Cys-Gly was selected as a model peptide. Importantly Cys-Gly has a thiol side chain on the cysteine that allows it to self-assemble on gold surfaces. Further demonstrations of the power of this new technique include assessments of an alkanethiol(3-mercaptoproprionic acid, MPA) modified electrode and then, after the tripeptide Gly-Gly-His has been attached to the MPA, a peptide used for the detection of Cu(II).25,26 EXPERIMENTAL SECTION 3-Mercaptopropionic acid (MPA), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Gly-Gly-His, Cys-Gly, and 2-(N-morpholino)ethanesulfonic acid (MES) were obtained from Sigma Chemical Co. (Sydney, Australia). All reagents were used without further purification. All solutions were prepared with purified water (18 MΩ cm, Millipore, Sydney, Australia). Glassware was soaked in 6 M HNO3 and carefully cleaned before use to avoid contamination by metal ions. The self-assembly of Cys-Gly was achieved by immersing a clean gold electrode in a 1 mM Cys-Gly aqueous ethanol solution for 12 h and then rinsing it with absolute ethanol. Because GlyGly-His contains no cysteine side chain, the gold surface was first modified by 3-mercaptopropionic acid (MPA) by soaking the electrode in 1.0 mM solution of MPA in ethanol for 12 h. The (22) Brockman, A. H.; Dodd, B. S.; Orlando, N. Anal. Chem. 1997, 69, 47164720. (23) Smith, R. D. Int. J. Mass Spectrom. 2000, 200, 509-544. (24) Yang, W.; Gooding, J. J.; Hibbert, D. B. Analyst 2001, 126, 1573-1577. (25) Yang, W.; Jaramillo, D.; Gooding, J. J.; Hibbert, D. B.; Zhang, R.; Willett, G. D.; Fisher, K. J. Chem. Commun. 2001, 1982-1983. (26) Yang, W.; Chow, E.; Willett, G. D.; Hibbert, D. B.; Gooding, J. J. Analyst 2003, 128, 712-718.

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carboxylic acid terminated SAM was activated using a mixture of 15 mM N-hydroxysuccinimide (NHS) and 75 mM 1-ethyl-3 (3dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 2-(Nmorpholino)ethanesulfonic acid (MES) buffer solution (pH 5.5) for 30 min. The resultant succinimide ester monolayers were reacted for 30 min in a solution of Gly-Gly-His (50 mg mL-1) in MES buffer to give a Gly-Gly-His-modified surface where the tripeptide is attached through the amino group of the first glycine and terminates with the carboxylic acid of the histidine. The SAM was quantitatively desorbed from gold surfaces by linear sweep voltammetry (-200 to -1200 mV, 20 mV s-1), leading to electrochemical reduction of the sulfur-gold bond at about -700 mV (potentials vs Ag|AgCl|0.3 M Cl-) in 0.05 M KOH to yield the thiolate.27 The area under the reduction peak allows quantification of thiol at the electrode surface. The desorption for mass spectrometry studies was performed in a stirred solution of 1 mL of 0.05 M KOH solution and 0.2 mL of 50% acetonitrile in water that was mixed well with 0.2 mg octadecyl-derivatized silica gel (C18) (particle size, 40 µm). The peptide accumulates on the C18, which was subsequently filtered with a Durapore membrane and rinsed with Milli-Q water. Peptide was then eluted from the C18 by two aliquots of 0.5 mL of 4:1 v/v methanol and water. The eluted solution was analyzed by ESI/FTICR/MS in the negative ion mode. The negative ion spectrum was preferred to the positive ion mass spectrum, which, for SAMs, contains few structurally specific molecular species.28-30 RESULTS AND DISCUSSION Before analysis of electrode products by mass spectrometry, it was important to quantify the amount of alkanethiol or peptide adsorbed on the electrode. The coverage was quantified via reductive desorption as described previously.31-33 A cyclic voltammogram of the desorption of the peptide Gly-Cys is shown in Figure 1. The position of the reductive desorption peak at - 870 mV (vs Ag/AgCl) is consistent with the desorption potentials for amino acids27 and aliphatic alkanethiols33 on polycrystalline gold surfaces. The surface concentration of Cys-Gly was 7.48 ×10-10 mol cm-2, which is below the theoretical maximum surface coverage for an alkanethiol on Au(111) of 7.6 × 10-10 mol cm-2.31 Desorbing Cys-Gly in the presence of C18 silica gel, rinsing to remove salts, and eluting with 4:1 v/v methanol and water allowed the sample to be analyzed by electrospray mass spectrometry. The resultant mass spectrum shows only the dimer ([Cys-Gly]2 - H)- with the predicted isotope pattern (Figure 2), thus indicating no molecular fragmentation during removal from the gold surface or with the voltage settings used in the electrospray. The absence of fragments shows that the removal procedure is gentle compared with other methods of removal of alkanethiols from surfaces.30 It is unclear whether the disulfides are formed (27) Yang, W.; Gooding, J. J.; Hibbert, D. B. J. Electroanal. Chem. 2001, 516, 10-16. (28) Tarlov, M. J.; Newman, J. G. Langmuir 1992, 8, 1398-1405. (29) Leggett, G. J.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. J. Phys. Chem. 1993, 97, 5348-5355. (30) Leggett, G. J.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. J. Chem. Soc., Faraday Trans. 1993, 89, 179-180. (31) Walczak, M. M.; Popenoe, D. D.; Deinhammer, R. S.; Lamp, B. D.; Chung, C.; Porter, M. D. Langmuir 1991, 7, 2687-2693. (32) Walczak, M. M.; Alves, C. A.; Lamp, B. D.; Porter, M. D. J. Electroanal. Chem. 1995, 396, 103-114. (33) Losic, D.; Shapter, J. G.; Gooding, J. J. Langmuir 2001, 17, 3307-3316.

Figure 1. Linear sweep reductive desorption of Cys-Gly from a gold electrode in 0.05 KOH (starting potential at -500 mV). Scan rate ) 100 mV/s.

Figure 3. Plot of the peak height of the FTICR/MS negative ion spectrum of the disulfide of Cys-Gly as a function of concentration of Cys-Gly in solution. Error bars are the (95% confidence intervals from five replicate measurements.

Figure 2. Electrospray FTICR/MS negative ion spectrum of the dipeptide Cys-Gly desorbed from a gold electrode. Figure 4. Electrospray FTICR/MS negative ion spectrum of MPA desorbed from a gold electrode.

on the gold surface or during either the reductive desorption or the electrospray step. Knoll and co-workers have observed desorption dimerization from TPD experiments with hexanethiol and hexadecanethiol SAMs.34 Dimers are the most prevalent desorbed species of thiols because of favorable energetics. The energy required to break two thiol-surface bonds (RS-Au), 167.2 kJ mol-1, is mostly gained back by formation of the disulfide bond (RS-SR ) 309.3 kJ mol-1).35 To assess the new procedure as a possible tool for the quantitative analysis of SAM-modified interfaces, it was important to ascertain the recovery, i.e., the percentage of the monolayer from the surface that is ultimately analyzed in the spectrometer. To measure the recovery, a calibration curve was constructed of peak height of ([Cys-Gly]2 - H)- versus concentration of CysGly introduced into the ESI/FTICR/MS (Figure 3). Between 0 and 0.25 µM, the response follows a linear relationship, but above 0.25 µM, the peak height reaches a plateau. This result is very similar to that reported by Padley et al.,36 who observed a linear response for lysine in ESI up to 10-8 M, above which the signal flattened out. Using this calibration curve of disulfide to quantify the concentration of Cys-Gly in the reductively desorbed sample, a recovery of 92% was determined. The high recovery indicates (34) Nishida, N.; Hara, M.; Sasabe, H.; Knoll, W. Jpn. J. Appl. Phys. 1 1997, 36, 2379-2385. (35) Zhong, C. J.; Porter, M. D. J. Am. Chem. Soc. 1994, 116, 11616-11617. (36) Padley, H. R.; Bashir, S.; Wood, T. D. Anal. Chem. 1997, 69, 2914-2918.

that the majority of Cys-Gly that is electrochemically desorbed from the gold surface adsorbs onto the C18, despite the fact that the peptide is relatively hydrophilic. The high recovery also shows that the peptides are only weakly bound to the silica gel, as the eluting solvent is also reasonably hydrophilic. A second example of this technique was modification of a gold surface of the alkanethiol MPA, followed by reductive desorption in the presence of the C18 silica gel; rinsing to remove salts and eluting with 4:1 v/v methanol and water allowed the sample to be analyzed by electrospray MS (Figure 4). The only species observed in the negative ion mass spectrum was the dimer ([MPA]2 - H)-. In the case of MPA, a very water-soluble alkanethiol, the recovery was poor. The poor recovery with hydrophilic alkanethiols might suggest a limitation to the general applicability of the method. However, as a range of silica gel modifications are available from HPLC and solid-phase extraction, achieving the correct balance between the polarity of the solvent during adsorption, the modification on the silica gel, and the elution solvent should allow this to become a generic method. It must be noted, however, that any adsorption chemistry must be able to be reversed in an appropriate solvent to give the final solution for analysis. The final example of the technique is to investigate SAMmodified surfaces where another molecule has been coupled to the SAM, a common scenario in the development of modified Analytical Chemistry, Vol. 75, No. 23, December 1, 2003

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Figure 5. Electrospray FTICR/MS negative ion spectrum of modified Gly-Gly-His desorbed from a gold electrode.

electrodes and our main interest.37 As a model of this sort of electrode, we investigated the attachment of the tripeptide GlyGly-His to an MPA-modified electrode using carbodiimide coupling. The spectrum (see Figure 5) of the desorbed species from the Gly-Gly-His electrode showed a parent ion peak at m/z 711.12 attributed to the disulfide ([His-Gly-Gly-(CH2)2S]2 - H)-. Again, there was no evidence of fragmentation during the removal of the peptide from the gold surface. (37) Gooding, J. J.; Mearns, F.; Yang, W.; Liu, J. Electroanalysis 2003, 15, 8196. (38) Jiang, L.; Glidle, A.; Griffith, A.; McNeil, C. J.; Cooper, J. M. Bioelectrochem. Bioenerg. 1997, 42, 15-23.

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CONCLUSIONS A gentle strategy has been developed using a combination of high-resolution mass spectrometry (ESI/FTICR/MS) with electrochemical reductive desorption to characterize alkanethiol SAMs on gold surfaces. Peptides attached to a gold surface via a thiolate bond are reductively desorbed in 0.05 M KOH in the presence of octadecyl-derivatized silica gel. The peptide adsorbs onto the silica gel. The gel is filtered, washed to remove any salts, and then eluted in a methanol/water mixture for injection into an electrospray FTICR/MS. As distinct from other methods of characterizing SAM-modified surfaces, the combination of reductive desorption and high-resolution mass spectrometry is a unique analytical method for SAMs, allowing desorbed peptides to be identified clearly, with little or no fragmentation. It is conceivable that this procedure could be applied to other biomolecules, although it might be necessary to use different silica gels depending on the properties of the analyte. It is important to emphasize, however, that the method could be combined with other mass spectrometers. The approach allows unambiguous determination of molecular structures that have been fabricated on electrode surfaces, which has important implications for the characterization of molecular-level constructs so important in nanotechnology. ACKNOWLEDGMENT We thank the Australian Research Council for funding this research. Received for review June 2, 2003. Accepted September 25, 2003. AC0345897