Anal. Chem. 1997, 69, 4331-4338
Surface Mass Spectrometry of Biotinylated Self-Assembled Monolayers Jennifer L. Trevor,†,‡ Donald E. Mencer,†,§ Keith R. Lykke,†,| Michael J. Pellin,† and Luke Hanley*,‡
Materials Science and Chemistry Divisions, Argonne National Laboratory, Argonne, Illinois 60439, Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, and Department of Chemistry, The Pennsylvania State UniversitysHazelton, Hazelton, Pennsylvania 18533
Biotin and biotinylated self-assembled monolayers (SAMs) on gold have been investigated using time-of-flight secondary ion mass spectrometry, direct laser desorption, laser desorption with 193 nm photoionization of ion- and laser-desorbed species, and laser desorption with vacuum ultraviolet (VUV, 118 nm) photoionization. Our results indicate that direct laser desorption and laser desorption combined with 193 nm multiphoton ionization can detect a chromophoric molecule like biotin that is covalently bound to a SAM. However, secondary ion mass spectra were dominated by fragmentation, and ion desorption/ 193 nm photoionization detected no species related to biotin. The dominant features of the laser desorption/ VUV mass spectra were neat and Au-complexed dimers of intact and fragmented biotinylated SAM molecules. Multiphoton and single-photon ionization of laser-desorbed neutrals from biotinylated SAMs both led to the production of ions useful for chemical analysis of the monolayer. Multiphoton ionization with ultraviolet radiation was experimentally less challenging but required a chromophore for ionization and resulted in significant fragmentation of the adsorbate. Single-photon ionization with VUV radiation was experimentally more challenging but did not require a chromophore and led to less fragmentation. X-ray photoelectron spectra indicated that the biotinylated SAM formed a disordered, 40-60 Å thick monolayer on Au. Additionally, projection photolithography with a Schwarzschild microscope was used to pattern the biotinylated SAM surface and laser desorption/photoionization was used to detect biotinylated adsorbates from the ∼10 µm sized pattern. Over the past decade, there has been considerable interest in studying the adsorbate-surface interface for technological applications.1-3 Self-assembled monolayers (SAMs) of alkanethiolates and other organic compounds have been used to form surface-bound molecular arrays that are stable, chemically well†
Argonne National Laboratory. University of Illinois at Chicago. § The Pennsylvania State UniversitysHazelton. | Present address: Optical Technology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899. (1) Rojas, M. T.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 5883-5884. (2) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113-158. (3) Sundberg, S.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 12050-12057. ‡
S0003-2700(97)00283-7 CCC: $14.00
© 1997 American Chemical Society
defined, and (sometimes) ordered.4 The novelty of using SAMs stems from their ability to be further modified into surface layers with complex chemical functionality: a variety of interesting materials can be anchored to the surface by starting with simple aliphatic alkanethiols and incorporating proteins,5 antibodies,6 polymers,7 and C60.8,9 While many of these chemically tailored surfaces have yet to find industrial application, one use for functionalized SAMs is molecular recognition on a biosensor surface.10,11 Biosensors typically operate by converting the binding of a ligand to a receptor surface into an electrical signal. Most development strives to control the geometric environment of the surface-bound molecules and avoid steric hindrances between the ligand molecule and the receptor.12 There are many reports of techniques that tether molecules in a site-specific fashion to the surface for this purpose.3,13,14 Photopatterning with a mask is one such way that has been successful at immobilizing SAMs on various portions of the surface to create arrays of host molecules.15 This method allows for freedom of design in surface structuring because the choice of pattern depends upon the ability to pattern the mask. Construction of biosensing devices usually requires multistep processing of the surface. Quartz crystal microbalance, surface plasmon resonance, and cyclic voltammetry are often used to monitor the adsorbate-surface interaction, but these methods do not provide direct chemical identification of the adsorbate.16,17 X-ray photoelectron spectroscopy (XPS) is commonly used for surface analysis since it is sensitive to elemental and functional group (4) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (5) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 43834385. (6) Willner, I.; Blonder, R. Thin Solid Films 1995, 266, 254-257. (7) Ford, J.; Vickers, T. J.; Mann, C. K.; Schlenoff, J. B. Langmuir 1996, 12, 1944-1946. (8) Arias, F.; Godinez, L. A.; Wilson, S. R.; Kaifer, A. E.; Echegoyen, L. J. Am. Chem. Soc. 1996, 118, 6086-6087. (9) Tsukruk, V.; Everson, M. P.; Lander, L. M.; Brittain, W. J. Langmuir 1996, 12, 3905-3911. (10) Henke, C.; Steinem, C.; Janshoff, A.; Steffan, G.; Luftman, H.; Sieber, M.; Galla, H.-J. Anal. Chem. 1996, 68, 3158-3165. (11) Spinke, J.; Liley, L.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7013-7019. (12) Knoll, W.; Angermaier, L.; Fritz, T.; Batz, G.; Furuno, T.; Guder, H. J.; Hara, M.; Liley, M.; Niki, K.; Spinke, J. Mater. Res. Soc. Symp. Proc. 1994, 330, 165-170. (13) Delmarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (14) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chem. 1995, 67, 36053607. (15) Morgan, H.; Pritchard, D. J.; Cooper, J. M. Biosens. Bioelectron. 1995, 10, 841-846. (16) Dickert, F. L.; Haunschild, A. Adv. Mater. 1993, 5, 887-895. (17) Piehler, J.; Brecht, A.; Gauglitz, G. Anal. Chem. 1996, 68, 139-143.
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composition and can be readily quantified. However, XPS suffers from a lack of chemical resolution and is often unable to determine overall chemical structure. An alternative to the aforementioned methods is the use of surface mass spectrometry to study biosensor surfaces. Direct (single) laser desorption mass spectrometry has already been used to characterize a variety of SAMs,18 although it often leads to molecular fragmentation and there are severe matrix effects associated with it.19 Attempts at quantifying direct laser desorption have been hindered by large fluctuations in desorption and ionization yields. Time-of-flight secondary ion mass spectrometry (TOF-SIMS, hereafter referred as SIMS) has imaging capabilities and has become the standard mass spectrometric method for organic and biological surface analysis. However, SIMS suffers from relatively low secondary ion yields and matrix effects that can limit quantification, even with the use of internal standards. Laser secondary neutral mass spectrometry overcomes these limitations by separating the desorption and ionization steps and postionizing the sputtered neutrals.20 Since the neutral population is orders of magnitude greater than the secondary ion population, the measurement can reflect the true concentration of the surface. For additional selectivity, it is possible to vary the wavelength, intensity, and pulse length of the postionization laser. Laser desorption of neutrals followed by photoionization has also been shown to be particularly useful for surface analysis.21 Because of its sensitivity, selectivity, and potential to be quantitative, postionization has been applied to many different areas of research. A few examples of surface analysis of molecular species using postionization include peptides desorbed from thinlayer chromatography plates,22 polycyclic aromatic hydrocarbons from meteoritic acid residues,23 and imaging of patterned biomolecules and polymers deposited on various surfaces.24-26 It was previously suggested that postionization mass spectrometry could be used to characterize molecular biosensors.27 However, mass spectrometric studies of host-guest chemistry at surfaces have been mainly confined to matrix-assisted laser desorption and fastatom bombardment.28-32 Both of these methods often require extensive sample treatments that may not be appropriate in certain applications. For example, covalent modification of a sample with (18) Li, Y.; Huang, J.; McIver, R. T. J.; Hemminger, J. C. J. Am. Chem. Soc. 1992, 114, 2428-2432. (19) Savina, Michael R.; Lykke, Keith R. Trends Anal. Chem. 1997, 16, 242252. (20) Nicolussi, G. K.; Pellin, M. J.; Lykke, K. R.; Trevor, J. L.; Mencer, D. E.; Davis, A. M. Surf. Interface Anal. 1996, 24, 363-370. (21) Zenobi, R. Int. J. Mass. Spectrom. Ion Processes 1995, 145, 51-77. (22) Krutchinsky, A. N.; Dolgin, A. I.; Utsal, O. G.; Khodorkovski, A. M. J. Mass Spectrom. 1995, 30, 375-379. (23) Kovalenko, L.; Maechling, C.; Clemett, S. J.; Philippoz, J.-M.; Zare, R. N. Anal. Chem. 1992, 64, 682-690. (24) Wood, M.; Zhou, Y.; Brummel, C. L.; Winograd, N. Anal. Chem. 1994, 66, 2425-2432. (25) Terhorst, M.; Niehuis, E.; Benninghoven, A. Surf. Interface Anal. 1992, 19, 822-826. (26) Lykke, K. R.; Wurz, P.; Parker, D.; Pellin, M. J. Appl. Opt. 1993, 32, 857866. (27) Hagenhoff, B. Biosens. Bioelectron. 1995, 10, 885-894. (28) Chilkoti, A.; Schwartz, B. L.; Smith, R. D.; Long, C. J.; Stayton, P. S. Biotechnology 1995, 13, 1198-1204. (29) Bartsch, H.; Konig, W. A.; Strassner, M.; Hintze, U. Carbohydr. Res. 1996, 286, 41-53. (30) O’Donnell, M. J.; Tang, K.; Koster, H.; Smith, C. L.; Cantor, C. R. Anal. Chem. 1997, 69, 2438-2443. (31) Kingshott, P.; StJohn, H. A. W.; Chatelier, R. C.; Griesser, H. J. Polym. Mater. Sci. Engin. 1997, 76, 81. (32) Henke, C.; Steinem, C.; Janshoff, A.; Steffan, G.; Luftmann, H.; Sieber, M.; Galla H.-J. Anal. Chem. 1996, 68, 3158-3165.
4332 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
Figure 1. Cartoon of the biotinylated self-assembled monolayer covered Au surface after photopatterning.
a matrix compound prior to functionalization may not always be possible or desirable. Addition of the matrix compound after functionalization might lead to dissolution of the functionalizing species and result in a loss of spatial resolution. Most important, both matrix-assisted laser desorption and fast-atom bombardment are difficult to quantify and can lead to preferential ionization of select species. While both methods show promise, two-step desorption/photoionization methods are clearly preferable for many surface mass spectrometry applications. Biotin (vitamin H) is involved in molecular recognition with the proteins avidin and streptavidin and has been widely exploited for many applications due to the high affinity and specificity of this interaction. Molecules that are difficult to detect can be tagged with a biotin moiety and released to probe complex solutions. Ringsdorf and co-workers found that among a variety of biotinylated alkanethiols they synthesized, the compound displayed in Figure 1 promoted the best binding to the protein streptavidin.2 We have chosen biotin and biotinylated SAMs for surface analysis because of their application in biosensor technology as the ligand half of a host-guest pair. It is also adventageous to compare multilayers with monolayers for each mass spectrometric method since the biotin head group may serve as the chromophore for photoionization of the biotinylated adsorbate. In this paper, we present experiments performed with two separate TOF-MS instruments. We focus on the best method to mass spectrometrically analyze biotin and biotinylated SAMs on a Au surface. These biotinylated SAMs form disordered monolayers on Au. We compare direct laser desorption, ion sputtering/ photoionization, and laser desorption/photoionization with the established method of SIMS. We also compare one- and twophoton ionization of species that have been laser desorbed from the biotinylated SAM. We use XPS to provide further chemical and structural information on the biotinylated SAM. In addition, we photopattern a biotinylated SAM surface and use laser
desorption/postionization to chemically analyze ∼10 µm sized features of the photopatterned surface. EXPERIMENTAL SECTION Apparatus. These studies were performed on two separate time-of-flight mass spectrometers (TOF-MS) that have been described in detail previously.20,26,33 The first instrument is a reflectron-type TOF-MS instrument known as CHARISMA (Chicago Argonne resonant ionization spectrometer for microanalysis).20 The xyz translation of the sample in the reflectron TOFMS was accomplished using inchworm motors (Burleigh) with a mechanical resolution of ∼0.01 µm. SIMS and ion sputtering of neutrals was accomplished with a 5 keV, ∼0.5 mm diameter Ar+ ion beam generated in a Colutron ion source. The ion sputtering experiments were performed in a pulsed mode with 800 ns Ar+ ion pulses, which corresponds to ∼107 primary ions/pulse. Experiments were also conducted on a linear 1.3 m TOF-MS constructed at Argonne.26 Samples were inserted into the chamber (base pressure ∼10-8 Torr) by a sample-transfer stage that facilitated the rapid exchange of samples. The target was held at 8 kV and was positioned in front of two extraction grids that served to increase the mass resolution.34 A motor-driven mirror mount preceding the microscope (described below) was used for scanning the desorption beam across the surface. On both instruments, the desorption laser beam was introduced onto the sample surface by means of a Schwarzschild microscope. The sample was viewed optically through the Schwarzschild microscope with white light illumination and a CCD camera. A N2 gas laser with a wavelength of 337 nm and a pulse length of ∼4 ns was used for laser desorption. The laser beam was focused on the sample surface to a diameter of ∼1 µm by means of the Schwarzschild microscope, allowing laser microprobe analysis. The N2 laser beam was attenuated for all laser desorption experiments, and we estimate that the incident energy on the sample surfaces was less than 100 nJ/pulse for all direct laser desorption experiments performed in combination with ArF postionization. For laser desorption experiments performed in combination with vacuum ultraviolet (VUV) photoionization, the desorption intensity at the surface was
