Roles of Water, Acidity, and Surface Morphology in Surface-Assisted

Apr 8, 2008 - School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, ... M. Michele Dawley , Claire Pirim , and Thomas M. Orl...
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J. Phys. Chem. C 2008, 112, 6953-6960

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Roles of Water, Acidity, and Surface Morphology in Surface-Assisted Laser Desorption/ Ionization of Amino Acids Yanfeng Chen, Haiyan Chen, Alex Aleksandrov, and Thomas M. Orlando* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 ReceiVed: August 31, 2007; In Final Form: December 31, 2007

The roles of adsorbed water, acidity, terminal OH groups, and surface morphology in surface-assisted laser desorption/ionization (SALDI) of amino acids from porous graphite and silicon substrates are examined. The SALDI yields and relative intensity ratios of protonated arginine, tryptophan, histidine, methionine, glutamine, and glycine are found to be very similar using porous graphite and porous silicon, despite the large differences in substrate electronic structure and surface chemistry. SALDI does not occur using initially pristine borondoped Si(100) substrates. However, adsorption of water at 130 K to Si(100) containing adsorbed amino acids produces a SALDI signal similar to that observed from porous graphite and porous silicon surfaces containing aqueous amino acid solutions adsorbed at 300 K. The SALDI yields from all substrates are greatly reduced after removal of physisorbed and chemisorbed water and are completely quenched from Si(100) and porous Si once the surface terminal hydroxyls are removed via recombinative desorption. The ion yields of all amino acids increased greatly with reduction of the solution pH, indicating important roles of surface/interface layer acidity and proton affinity of the desorbing amino acid. Multiphoton-induced ionization of interfacial water and terminal-OH-derived surface states may be important in SALDI. Surface morphologies that lead to the adsorption of water matrices with dispersed analytes are most effective because they enhance and maximize protonated complex formation and escape.

Introduction The introduction of matrix-assisted laser desorption/ionization (MALDI) less than two decades ago1,2 has dramatically improved the applicability of laser mass spectrometry in analytical, biological, environmental, clinical, pharmaceutical, and material analysis. Direct laser desorption ionization (LDI) has been studied extensively since 1970,3 but it usually exhibits severe molecular degradation for low-mass analytes. This greatly hindered LDI from being widely used. By simply adding energyabsorbing organic matrix molecules during sample preparation, MALDI overcomes some problems associated with LDI and generates intact molecular ions. Coupled with time-of-flight (TOF) mass spectrometry (MS), MALDI MS is especially useful because it allows mass determination of large biomolecules and synthetic polymers of molar mass greater than 100 000 Daltons (Da).4 Soft ionization, high detection sensitivity, and relative simplicity have made MALDI a popular technique5 and revolutionized the analysis of fragile or nonvolatile large molecules such as peptides/proteins,6 carbohydrates,7 nucleic acids/oligonucleotides/DNA,8,9 oligosaccharides,10 toxins,11 and polymers.12 Although MALDI has been remarkably successful in the analysis of large molecules, it is has limitations with respect to analysis of low-molecular-weight compounds. One of the major reasons is that MALDI produces many matrix ions in the low mass range (m/z < 600). The small molecule signals are then difficult to separate and identify because of the massive obscuring background signals of the matrix. Surface-assisted laser desorption/ionization (SALDI),13,14 combined with MALDI, has created interest in utilizing the full * To whom correspondence should be addressed. Phone: (+1) 404894-4012. Fax: (+1) 404-894-7452. E-mail: thomas.orlando@chemistry. gatech.edu.

power of laser desorption ionization over the entire mass range of interest. SALDI uses substrate materials or particles that promote desorption/ionization of analytes. This helps to avoid the background of typical traditional organic matrix molecules while maintaining the advantages of MALDI. From this point of view, different active surfaces such as carbon suspended in solution,13,15 carbon nanotubes (CNTs),16,17 graphite,18-20 porous silicon,21,22 and column arrays of silicon23 were all defined as useful SALDI substates.14 The general applicability of SALDI MS has demonstrated that it is a promising and powerful technique for the detection of small/intermediate analytes.24 Despite the potential importance of SALDI, only a few systematic investigations that evaluate the performance of various substrates have been reported.25-27 Correlations between the porous structure of silicon substrates and SALDI performance have been discussed, and it has been suggested that surface morphology plays a pivotal role in the desorption/ ionization process.25,27 Contrary to this hypothesis, similar experiments on porous oxide surfaces such as sol-gel, silica glass, and alumina films did not generate SALDI signals successfully.19,28,29 Because SALDI applications typically use materials with band gaps in the visible region and high absorption coefficients, the electronic structure and optical properties of the substrate may also be crucial. For example, the signals of SALDI on porous silicon (often referred to as desorption/ionization on silicon, DIOS) has been attributed to the high ultraviolet (UV) absorptivity of silicon.21,30 However, no SALDI ions were obtained by UV irradiation on Si(100) crystals at room temperature.26 Though the importance of substrate interactions and interfacial energy exchange in SALDI has been mentioned, most detailed descriptions of the processes governing SALDI have focused

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Figure 1. Schematic of the ultrahigh vacuum apparatus and experimental arrangement for surface-assisted laser desorption/ionization mass spectrometry studies. Samples were initially prepared outside the vacuum chamber and then put in the antechamber for vacuum drying. Once the antechamber pressure reached 10-6 Torr, the samples were then transported in the UHV system (10-9-10-10 Torr) and mounted on a variabletemperature rotatable sample holder. See text for further operational details.

on thermal desorption followed by plasma and plume interactions occurring within pores or above the surface.27 In most cases, the substrate is treated mainly as a source of thermal energy. However, it is important to note that when using UV excitation, bound electron-hole pairs (i.e., excitons) and separated electrons and holes are created in the irradiated target. The excitations/charge carriers have very fast velocities, and the bound excitons can self-trap or localize at defects prior to radiative decay. In this case, well-known phenomena such as electronic energy transfer and desorption or dissociation induced by electronic transitions occurs at the interface or at defect sites.31 This is a rapid process that requires energy localization lasting only several hundred femtoseconds. It dominates in the fluence regime which is below the threshold for ablation and plume formation, and can easily dominate in SALDI applications using power densities 50 on blank porous silicon was about 2-3 times smaller than that on blank graphite. Unknown peaks most likely due to impurities in the amino acid samples were also found and labeled with asterisks. Single-crystal Si(100) was also used as a SALDI substrate to analyze the standard solution of six amino acids by laser desorption/ionization. At room temperature, Figure 2c demonstrates that no analyte signal could be obtained. However, protonated molecular ions (MH+) of all of six amino acids were detected easily by lowering the sample temperature below 200 K and by adsorbing water. The SALDI mass spectrum of amino acids coadsorbed with water at 130 K is shown in Figure 2d. Several Na+/K+ containing peaks were also observed, but the intensities remained below those of the protonated ions. The six amino acids were investigated individually using SALDI-MS on different substrates, and the ion yields did not show obvious differences relative to the mixtures, confirming

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Figure 3. A comparison of (a) ArgH+ SALDI signals as a function of substrate temperature, (b) temperature-programmed desorption (TPD) of arginine (Arg), and (c) TPD of water from porous silicon. Each data point in (a) represents a separate experiment at a specific temperature on a clean and newly deposited analyte film. In (b) and (c), the analyte was deposited as an aqueous solution at room temperature and then cooled to 100 K.

that there were no matrix effects among these analytes under the described experimental conditions. In addition, the desorbing ion masses from the mixed and single-component solutions were also determined by QMS analysis. The QMS has unit mass resolution, and this served as an accurate calibration. We report data taken with the TOF because the overall signal intensities are generally higher. Roles of Water, Silanol Groups, and Surface Acidity. To quantitatively analyze the amounts and role of water on the different substrates, pure water was deposited onto the clean substrates at low temperature (120 K) via gas-phase dosing. The controlled leak rate of water vapor was 0.01 ML/s. The integrated TPD peak areas of water were calibrated by the known water coverages (1 ML, 2 ML, 5 ML, 10 ML, 50 ML, etc.) to obtain a normalized peak area constant per ML. Similar TPD experiments were then carried out with samples containing amino acid analytes deposited from the aqueous solution at room temperature. The integrated TPD peak areas of water can then be converted to water coverage using the normalized peak area constant per ML. The number of water molecules on each substrate containing analytes at room temperature were estimated as 1018, 1019 ∼ 1020, and 1016 on graphite, porous silicon, and Si(100), respectively. Compared to the amount of analyte (∼1015 molecules), the number of solvent molecules were between three and five orders of magnitude higher on the porous surfaces. To understand the role of physisorbed and chemisorbed water, a series of SALDI experiments were performed at different temperatures and compared to the TPD of arginine and water from porous Si. Specifically, Figure 3a shows the SALDI yield of protonated arginine at different temperatures, in which each data point represents a separate experiment at a specific temperature on a clean and newly deposited analyte film. Note

Chen et al. that initial analyte deposition used an aqueous solution at room temperature; these films were then cooled or heated to a stable temperature for SALDI measurments. The substrate temperature was not changed during the SALDI experiments. Figure 3b shows the TPD of neutral arginine, and Figure 3c presents the TPD of water. For the data shown in Figure 3b and c, the analyte was initially deposited at room temperature and then cooled to 100 K. Because the main chamber pressure was 10-9 Torr, a very limited amount of background gas could physisorb to the cold (100 K) surface during data acquisition. The protonated arginine SALDI signal (Figure 3a) is present at the lowest temperature studied (∼110 K) and rises slowly until it peaks near 150 K. The SALDI signal increase follows the temperature dependence of proton production and desorption from amorphous and crystalline ice.34-36 The SALDI signal then decreases dramatically when the substrate temperature exceeds 150 K. This decrease can be correlated with the onset of thermal induced desorption of both arginine (Figure 3b) and physisorbed water (Figure 3c) from the surface. Though the SALDI yield dropped to a relatively low level at the TPD maximum for arginine and multilayer water, it did not disappear. Rather, it remained at a fixed level during the thermal desorption of chemisorbed and trapped water between 200 and 350 K (see Figure 3c). These results indicate that (i) physisorbed or weakly trapped water is necessary to maximize the protonated amino acid desorption products and (ii) the SALDI signal may arise primarily from analyte molecules at the vacuum-surface interface rather than from molecules trapped within the small pores. This is consistent with the fact that the SALDI yields showed a strong dependence on the surface terminal Si-OH groups rather than just the presence of water. These Si-OH groups undergo recombinative desorption at temperatures >560 K and release primarily H2O and H2.37 This is shown in Figure 3c as an increase in the H2O TPD signal at 560 K. A weak TPD signal from a small amount of chemisorbed arginine is also observed at 560 K in Figure 3b. The SALDI signal is completely quenched once the Si-OH groups and arginine are removed as expected. To summarize Figure 3, there is an increase in SALDI signals from 110 to 150 K, continued SALDI yield after the removal of physisorbed and chemisorbed water, a clear reduction of SALDI signals by surface interactions, and the complete quenching of SALDI after the removal of silanol groups. Acidity effects in SALDI were studied by adding HCl to adjust the initial sample solution pH from 6 to 1. The protonated molecular ion yields at room temperature were all enhanced by factors of 20-30 when acid was added (not shown), and the results show similar trends for SALDI from porous graphite and silicon. This large increase is consistent with previous studies using acetic acid addition26 and demonstrates that protonation is critical to the SALDI process. In Figure 4, the relative SALDI yields of each amino acid from both graphite and Si substrates are compared to the known gas-phase proton affinities.38 There is a general increase with higher proton affinity, and this trend is similar for initial solution pH levels of 6 and 1. Trytophan and arginine are hydrophobic, and their yields are the highest relative to the other amino acids studied. The general hydrophobicity of these amino acids and the aromatic character of tryptophan may also lead to repulsive interactions with the water covered Si and graphite. This could actually minimize their interactions with the wet surfaces, implying that the charge states of these amino acids in solution are preserved. If the SALDI mechanism involves solely preformed ions, then a similar trend and correlation is expected

Roles of Water, Acidity, and Surface Morphology

Figure 4. Comparison of amino acid proton affinities with their SALDI yields at an initial solution pH of 6 on (a) graphite and (b) porous silicon.

when comparing the SALDI yields to the aqueous-phase pKb values for the amino acids at pH ) 6. However, as shown in Figure 5, the correlation with these pKb values is very poor. Because glycine, glutamine, methionine, and tryptophan have similar pKb values, their SALDI yields should be similar. However, the yields of protonated glycine and glutamine are less than those of protonanted trypotophan and methionine. These low yields indicate strong specific adsorption on silica and graphite.39 Indeed, glycine can bind to two or three silanol groups on silica with a binding energy as high as 70 and 80 kJ mol-1, respectively.40 It can also bind in the zwitterionic form with the amine group pointing into graphite.41 Though it is reasonable to implicate desorption of only preformed solvated ions,42 the process seems more complicated. Because photoexcitation (355 nm) of porous graphite, porous silicon, and Si(100) containing water yield essentially the same SALDI results, a consistent model must be developed. Although laser-induced thermal desorption followed by plume interactions and/or some laser-induced desorption of pre-existing ions are not ruled out, we focus on the roles of hole transfer and ionization of interfacial water in SALDI when using samples from an aqueous solution. Mechanism of SALDI on Graphite and Porous Silicon. Electronic Structure of Interfacial and Condensed Water. Water has been found to affect the ion yield in MALDI43-45 and SALDI26 measurements. Although UV laser irradiation could not produce satisfactory SALDI results on silicon single crystals, infrared laser irradiation has been reported to successfully ionize target molecules (up to 6 kDa) on Si(100) wafers.46 It has been

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Figure 5. Comparison of the aqueous-phase pKb values of amino acids with their SALDI yields at an initial solution pH of 6 on (a) graphite and (b) porous silicon.

postulated that photons were absorbed by the solvent and lead to desorption and ionization of analyte molecules. In order to understand this, we briefly review the electronic structure of water; the solvent used in the present studies. The O-H bond of water is composed of mainly the 1b2 and 2a1 orbitals, the 1b1 and 3a1 contribute to the oxygen lone pair orbitals, and the lowest unoccupied molecular orbitals are the 4a1 and 2b2. These unoccupied levels, particularly the 4a1, are strongly antibonding and lead to O-H breakage if occupied. Photoemission studies of thin films of water36 indicate that these molecular orbitals retain much of their character in the condensed phase with some broadening and minor shifts in energy. The unoccupied states lead to the formation of an illdefined conduction band, which is essentially an exponentially decaying density of states relative to the vacuum level. The most prominent state in the band gap of pure water has a threshold >6.5 eV and a defined peak at 8.4 eV. This feature is present for crystalline ice, amorphous ice, liquid water, and physisorbed and weakly adsorbed water. It is has been well-described theoretically as a weakly localized exciton using a many-body Green’s function approach.47 Briefly, occupation of the lowest unoccupied level (i.e., the mixed 4a1/2b2 level of condensed water) shows a significant electron probability density in the proximity of the O atom, as well as two lobes that extend along the hydrogen atom directions. These lobes are extended considerably relative to the same excitation in the gas phase, and the exciton radius, R, is calculated to be 4.02 Å relative to a gas-phase value of 2.27 Å.47 Because the nearby accepting hydrogens have partial positive charges, this excitation provides

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Figure 6. SEM images resulting from the room-temperature deposition of 10 µL aqueous solution containing six amino acids (5 µg/mL for each) on (a) graphite (b) porous silicon, and (c) Si(100). These samples were treated and prepared as described in the text.

an effective path for producing hydrogen atoms and protons (or separated charge) at energies as low as 6.5 eV.48 Multiphoton ionization of condensed water is well known at wavelengths ranging from the deep UV to far IR46,49,50 and can occur easily at the laser fluences used in this work. In fact, the 7.0 eV two-photon excitation energy using 355 nm photons is above the deep UV threshold. Therefore, a primary and efficient source of protons is hot hole formation by direct two-photon ionization or autoionization of excited interfacial water. Once formed, these holes or protons will migrate quickly through the hydrogen-bonded water network and trap at the interface or at the “defect” configurations associated with the solvated amino acid. If the holes or protons are localized at a shorter distance than the typical screening length, then Coloumb ejection of cations and protonated species will occur.36 The efficacy of this Coloumb ejection process can be enhanced by the existence of pre-existing ions at interfaces,51 and this could be associated with the strong effect observed with increasing acidity. Substrate Electronic Structure and Energy Exchange. It is well established that laser desorption and ionization is promoted by absorption of energy by the substrate. For graphite, there is a maximum in the density of states around 3.6 eV, which is attributed to the flat π band near the Q2g-(π*) point in the Brillion zone.52,53 In addition, there are unoccupied interlayer surface states around 3.2 eV above the graphite Fermi level.54 Though water does not wet graphite, these surface states can be perturbed by hydrogen and water. The presence of adsorbed hydrogen atoms can also lead to weak interaction of the first monolayer of water. This may lead to ideal oxygen atom down configurations for substrate mediated hot hole or proton transfer. Photoexcitation of graphite using 355 nm (3.5 eV) is very efficient, and some positive surface charging (i.e., hole production) can proceed via excitation of these interfaces or surface states. Because graphite is a quasi-2D electron gas with large lateral dispersion, hole localization would only be prominent at the oxidized or water-covered edges of graphite sheets that make up the ledges and pores.55 Bulk Si also has a strong absorption in the 3.5 eV range and surface states at slightly lower energy.56 Photodesorption of H+ from H-terminated Si(100) has been observed during UV 193 nm (6.4 eV) excitation, but this required at least three photons and the formation of screened Si-H two-hole final states.57 The laser fluence used in these studies was very high (200-1000 mJ/cm2), and the H+ photodesorption cross section was extremely low (10-73 /cm6). The porous Si and water-treated Si(100) have terminal hydride and silanol groups, which lead to the adsorption and trapping of water. Holes from Si-H or Si-OH sites will end up trapping where strain and bond elongation causes reduced hole-hopping probabilities. This could cause a propensity for weakly screened surface charge buildup near the protrusions, edge sites, and internal pore surfaces. Though the cross section for VUV (>7 eV) excitation of adsorbed or interstitial water is 2 orders of magnitude higher than direct excitation of silanol groups, our observation that

SALDI signals persist until silanol groups are removed via recombinative desorption indicates that these surface states are active. The propensity for hole or energy localization near defects, protrusions, and edge sites also makes these areas more active in SALDI. As noted above, glycine, glutamine, and methionine may be initially adsorbed on the Si(100) surface. Because protonated forms of these amino acids can be removed only after a critical amount of water is present, water-induced solvation or waterderived surface/interface states may also be important on the Si(100) surface. In addition, GlyNa+, GlyK+, GlnNa+, and MetNa+ are only observed from the low-temperature hydrated Si(100) surface. This further indicates the important role of direct excitation of the surface states associated with the hydrated surface-adsorbed species in SALDI. Pore Size and Surface Morphology. To better understand the desorption/ionization of analyte molecules on SALDI substrates, the surface morphology and distribution of analyte molecules on graphite, porous silicon, and Si(100) were characterized by scanning electron microscopy (SEM). Though SEM is not chemically specific, as seen in Figure 6a, on rough graphite substrates, analyte molecules can localize in 1-10 µm pores or at the edges and tips. On porous silicon, many small clusters (20 µm) spherical clusters on Si(100) surfaces (Figure 6c). Comparing the SEM results with SALDI MS signals in Figures 2 and 3, an apparent link between surface morphology and SALDI yields is found. On rough and high-surface-area substrates such as graphite and porous silicon, analyte and solvent molecules can be coadsorbed/trapped and protonated analyte molecules or zwitterions can be deposited and preserved. On a single crystal, water does not wet the surface at room temperature and large clusters of nearly solvent-free analytes form. Under these conditions, there is little probability of proton transfer from coadsorbed water and there is less interaction between the sample and the substrate relative to the intra-analyte coupling. High pore density is helpful because this serves to increase the overall surface area and analyte/matrix coverage. As pointed out already, the aspect ratio and narrowness of the pore must also be considered.23 If one invokes electronemission, plume expansion, and plasma interactions, then selffocusing and dimensional collapse of the expanding plasma occurs due to wall effects and space charge interactions.27 Protonated water cluster ions are always observed in plasmas that contain trace amounts of water. However, wall interactions and water-induced suppression of thermionic emission reduces the potential importance of electron-impact mediated gas-phase ionization, ion-molecule reactions, and effects of space charge.

Roles of Water, Acidity, and Surface Morphology Recall that protonated water cluster ions are not observed during SALDI. A plume-dominated gas-phase clustering process should require a four-photon ionization event at 355 nm. This is less likely than the two-photon excitation required for ionization of surface states or interfacial water. A recent model for cluster ion production and release from ice has a temperaturedependent hole mobility term.34-36 At temperatures above 120 K, the holes move, cluster ion production stops, and proton release increases. Indeed, the temperature-dependent increase in the SALDI yield from frozen aqueous solution (Figure 2a) between 110 and 150 K follows closely the hole or proton mobility in ice.34-36 Assuming that SALDI involves mobile holes and protons, SALDI yields may be maximized at trapping sites such as edges or pore surfaces containing dispersed analytes within a 150 K overlayer. In addition to maintaining the temperature at 150 K, the use of a large number of pores with diameters greater than the depth is suggested for maximum sensitivity. Conclusions A detailed investigation of the roles of adsorbed water, terminal OH groups, interface acidity, and surface morphology in SALDI of amino acids from porous graphite and silicon substrates is reported. The SALDI yields and relative intensity ratios of protonated arginine, tryptophan, histidine, methionine, glutamine, and glycine are found to be very similar using porous graphite and silicon despite the very large differences in substrate electronic structure and surface chemistry. The UHV studies demonstrated that SALDI does not occur using initially pristine boron-doped Si(100) substrates; however, adsorption of water at 130 K produces a SALDI signal similar to that observed from porous graphite and silicon surfaces at 300 K. The SALDI yields are greatly reduced after removal of physisorbed and chemisorbed water and are completely quenched from Si(100) and porous Si once the surface terminal hydroxyls are removed via recombinative desorption. The yields of all amino acids from all three substrates increased greatly with a reduction of pH, indicating important roles of surface/interface layer acidity and proton affinity of the desorbing amino acid. Anomalies in the SALDI yield comparison to aqueous phase pKb data can be explained by the strong interaction of molecules such as glycine and glutamine with graphite and silica. These interactions serve to reduce or quench the SALDI signal relative to that expected for solution-phase ionization probabilities and illustrates that the use of the pKb concept to describe interactions at a surface/interface is perhaps improper. The data collectively indicates that SALDI depends more on the presence of water and the intrinsic proton or hydronium ion content than the substrate bulk electronic structure. Desorption of pre-existing ions can occur, but in view of the energetics, required laser fluence, and yields versus temperature, multiphoton-induced ionization of water and surface terminal groups may be primary ionization steps in SALDI. These ionization channels can be facilitated by perturbed states of interfacial water and surface states derived from OH terminal sites. SALDI and other laserinduced desorption ionization techniques are not “matrix-free” because they rely upon the proton yielding solvent inherent in the analyte deposition step. Acknowledgment. This work was supported by the U.S. Dept. of Energy, Contract DE-FG02-02ER15337. We thank Christopher Lane, Irene Anestis-Richard, and Gregory Grieves for useful discussions and technical support.

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