TOF-SIMS and Principal Component Analysis Characterization of the

TOF-SIMS and Principal Component Analysis. Characterization of the Multilayer Surface Grafting of Small Molecules: Antibacterial Furanones. Sameer A...
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Anal. Chem. 2008, 80, 430-436

TOF-SIMS and Principal Component Analysis Characterization of the Multilayer Surface Grafting of Small Molecules: Antibacterial Furanones Sameer A. Al-Bataineh,*,†,‡,§ Marek Jasieniak,† Leanne G. Britcher,† and Hans J. Griesser†

Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia, and Vision CRC, University of New South Wales, Sydney, NSW 2052, Australia

Furanone compounds (fimbrolides) have attracted interest as antibacterial compounds for use in human health care, for instance, as an antibacterial coating for medical devices to combat device-centered infections. To ensure effectiveness for extended periods of time, they must be immobilized covalently onto a device surface; in this study, this was done via azide/nitrene chemistry and photochemical coupling. However, the detection and quantification of surface-immobilized small molecules such as furanones presents a considerable analytical challenge, yet is necessary for optimization of coatings and reliable interpretation of biological responses. We have utilized the surface sensitivity and chemical specificity of time-of-flight secondary ion mass spectrometry (TOFSIMS) to characterize each step of the grafting sequence. On account of the complexity of the data, principal component analysis (PCA) was used to interpret and compare spectra. The results demonstrate the utility of TOF-SIMS with PCA for the detection of the surfacegrafted small molecules azidoaniline and a brominated furanone; imaging of the bromine ion peaks also enabled assessment of grafting uniformity. Thus, successful multilayer coating and furanone grafting was observed, and substantial and uniform coverage of furanone molecules on the surface. Even multiple grafting steps involving, in the present case, two low molecular weight compounds can readily be disentangled by PCA. The utility of TOFSIMS analysis with PCA is particularly well illustrated in the present case by the grafting of the furanone molecules, which did not yield a singular unique peak in the positive ion mass spectra, whereas the collective spectral changes elucidated by PCA provided unambiguous verification of successful grafting of this low molecular weight compound. The Australian marine red algae Delisea pulchra produces fimbrolides, a class of compounds comprising a five-membered lactone ring and halogen substituents. They reside in vesicles on * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: +41 44 633 63 76. Fax: +41 44 633 10 27. † University of South Australia. ‡ University of New South Wales. § Current address: Laboratory for Surface Science and Technology, Department of Materials, ETH Zu ¨ rich, Ho ¨nggerberg, CH-8093, Switzerland.

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the surface of the algae and provide a defense against fouling of algal surfaces by marine organisms. Compounds of particular interest are the brominated furanones, as a number of naturally occurring halogenated furanones as well as their synthetic analogues have shown interesting antimicrobial properties such as interference with the quorum sensing process in biofilm formation of many bacteria including human pathogens.1-4 Given the often severe consequences of nosocomial medical device infections and the present lack of successful long-term preventative strategies, these compounds have attracted growing recent interest as promising antibacterial molecules for use in human health care. Potential applications include usage in solution or by slow release from a reservoir. Of particular interest to us is their potential use as an antibacterial surface coating for medical devices5-7 such as orthopedic implants, catheters, and contact lenses. Although it was thought initially that the mode of action of these compounds is via intracellular gene blocking,1,4 these compounds unexpectedly also showed activity after covalent grafting onto solid surfaces.5-7 This indicates that these compounds may interact with bacterial cells via another mechanism; a reasonable hypothesis appears to be a transmembrane signaling mechanism. However, before speculating on another, unprecedented mode of antibacterial action, it is important to ascertain that these compounds do not detach from the surface to interact via an intracellular mode of action. Ratner and Castner8,9 have emphasized the need for detailed characterization of biomaterial surfaces before attempting interpretation of observed biological responses in terms of properties (1) Manefield, M.; Rasmussen, T. B.; Henzter, M.; Andersen, J. B.; Steinberg, P.; Kjelleberg, S.; Givskov, M. Microbiology 2002, 148, 1119-1127. (2) Reichelt, J. L.; Borowitzka, M. A. Hydrobiologia 1984, 116/117, 158-167. (3) de Nys, R.; Steinberg, P. D.; Willemsen, P.; Dworjanyn, S. A.; Gabelish, C. L.; King, R. J. Biofouling 1995, 8, 259-271. (4) Rasmussen, T. B.; Manefield, M.; Andersen, J. B.; Eberl, L.; Anthoni, U.; Christophersen, C.; Steinberg, P.; Kjelleberg, S.; Givskov, M. Microbiology 2000, 146, 3237-3244. (5) Read, R.; Kumar, N.; Wilcox, M.; Zhu, H.; Griesser, H. J.; Thissen, H.; Muir, B.; Hughes, T. EP1296673, 2003. (6) Muir, B. W.; Thissen, H.; Read, R. W.; Wilcox, M. D.; Griesser, H. J. PolyMillennial, Proceeding of the 6th World Biomaterial Congress, Hawaii, May 2000. (7) Hume, E. B. H.; Baveja, J.; Muir, B.; Schubert, T. L.; Kumar, N.; Kjelleberg, S.; Griesser, H. J.; Thissen, H.; Read, R.; Poole-Warren, L. A. Biomaterials 2004, 25, 5023-5030. (8) Ratner, B. D. Biosens. Bioelectron. 1995, 10, 797-804. (9) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28-60. 10.1021/ac701720y CCC: $40.75

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and chemistries of surfaces and coatings. The literature contains numerous examples of erroneous interpretations and inadequate surface characterization. Therefore, one of our main aims was to perform detailed spectroscopic characterization of the furanone coatings in order to collect data about the chemical composition, molecular structure, and uniformity of the grafted layers. Such information assists with the rational design and optimization of well-defined and stable antibacterial furanone coatings. The detection and quantification of surface-grafted molecules of low molecular weight, such as furanones, is a considerable analytical challenge. Whereas the characterization of surface immobilization of proteins and other macromolecules (e.g., poly(ethylene glycol)) is fairly straightforward using established analytical methods such as X-ray photoelectron spectroscopy (XPS),8,9 smaller molecules pose a problem with regard to insufficient sensitivity and detection limits of this technique. If a specific elemental signal is diagnostic of the grafted molecules, for instance, P for oligonucleotides or Br for brominated furanones, XPS can be used to detect such molecules,10-12 albeit at times with limited signal-to-noise (S/N) ratios. However, if no specific element exists, such as is the case for some nonhalogenated furanones of interest for biomedical applications, or the surface density is low, the range of applicable analytical techniques becomes quite limited. Static time-of-flight secondary ion mass spectrometry (TOFSIMS) is a powerful technique for the characterization of chemical species present on a surface and for the differentiation of surfaces of similar chemical compositions. The method provides extremely low detection limits, high surface sensitivity (sampling depth 1-2 nm), a rich information content inherent in spectra containing a large number of signals from many mass fragments, imaging capability, and high mass resolution, the latter enabling separation of different species with very close mass values. TOF-SIMS should thus be well suited to the detection of grafted small molecules. One of the difficulties associated with TOF-SIMS, however, is the large number of peaks present in each spectrum; thus, spectral analysis protocols, such as analysis of means and principal component analysis (PCA),13-15 have been used to simplify data sets and interpret static secondary ion mass spectra. PCA has been used in surface analysis applications including adsorbed protein films,15,16 polymers,17-20 self-assembled monolayers,21,22 nonfouling (10) Al-Bataineh, S. A. Ph.D. Thesis, University of South Australia, Adelaide, 2006. (11) Al-Bataineh, S. A.; Britcher, L. G.; Griesser, H. J. Sur. Sci. 2006, 600, 952962. (12) Al-Bataineh, S. A.; Britcher, L. G.; Griesser, H. J. Surf. Interface Anal. 2006, 38, 1512-1518. (13) Delcorte, A.; Bertrand, P.; Arys, X.; Jonas, A.; Wischerhoff, E.; Mayer, B.; Laschewsky, A. Surf. Sci. 1996, 366, 149-165. (14) Belu, A. M.; Graham, D. J.; Castner, D. G. Biomaterials 2003, 24, 36353653. (15) Wagner, M. S.; Tyler, B. J.; Castner, D. G. Anal. Chem. 2002, 74, 18241835. (16) Hartley, P. G.; McArthur, S. L.; McLean, K. M.; Griesser, H. J. Langmuir 2002, 18, 2483-2494. (17) Vanden Eynde, X.; Bertrand, P. Appl. Surf. Sci. 1999, 141, 1-20. (18) Vanden Eynde, X.; Bertrand, P. Surf. Interface Anal. 1997, 25, 878-888. (19) Wagner, M. S.; Pasche, S.; Castner, D. G.; Textor, M. Anal. Chem. 2004, 76, 1483-1492. (20) O ¨ hrlund, Å.; Hjertson, L.; Jacobsson, S. P. Surf. Interface Anal 1997, 25, 105-110. (21) Graham, D. J.; Price, D. D.; Ratner, B. D. Langmuir 2002, 18, 1518-1527. (22) Graham, D. J.; Ratner, B. D. Langmuir 2002, 18, 5861-5868.

polysaccharide coatings,23 and others.24,25 In this report, we describe the use of TOF-SIMS in combination with PCA for the detection of grafted small molecules, in this case an azide linker and a furanone, and show that TOF-SIMS combined with PCA can reliably distinguish sequential grafting steps involving these small molecules. EXPERIMENTAL SECTION Materials. The furanone compound used in this study, abbreviated as FU, was 3-butyl-4-bromo-5- (bromomethylene)2(5H)-furanone; its structure is shown in Figure 1. It was kindly provided by Dr. Naresh Kumar, School of Chemistry, University of New South Wales. Perfluorinated poly(ethylene-co-propylene) polymer (Teflon FEP 100 type A: 12.7 mm wide, 25 µm thick, Dupont) was used as the substrate for plasma coating. Prior to plasma modification, It was cleaned by consecutive ultrasonication in methanol (HPLC, Ajax Fine Chemicals) and acetone (Analytical Reagents, Asia Pacific Speciality Chemicals Ltd.) for 20 min each to remove surface contaminants and then rapidly dried with a jet of nitrogen. Heptylamine (n-HA; 99% purity, Sigma-Aldrich) was used as the precursor (“monomer”) for plasma polymerization. Poly(acrylic acid) (PAAC, MW 250 kDa, Aldrich), 4-azidoaniline hydrochloride (AZA, Fluka), and N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC, Sigma) were used as received. High-purity water (MilliQ) was used in all experiments. A UV lamp (6 W, 254 nm, 710 W/cm2, Cole-Parmer) was used as the UV source for photoactivation. Multilayer Coating onto FEP. On account of the absence of a chemical group capable of participating in a well-controllable interfacial bonding reaction and the lability of the furanone ring to acidic and alkaline reaction conditions, there are very few options for the covalent immobilization of the above furanone compound, and this applies to many others of the furanones with good antibacterial activity. Thus, as in previous work,11 we selected an approach utilizing azide/nitrene chemistry. Nitrenes are capable of reacting with the alkyl tail of furanones by C-H bond cleavage and insertion, though we also expect reaction with other parts of the furanone molecules, and hence, the immobilized furanones may be grafted at various positions. The grafting sequence is shown in Figure 1. Azide groups were attached onto a poly(acrylic acid) hydrogel interlayer, whose purpose was to make the composite coating sufficiently hydrophilic for biomedical applications, given the rather hydrophobic nature of the furanone molecule. Experimental details of the coating sequences have been reported previously.11 Briefly, a thin polymeric interlayer with surface amine groups was deposited by the plasma polymerization of n-heptylamine; this plasma polymer interlayer has been well characterized.26-29 Poly(acrylic acid) was (23) McArthur, S. L.; Wagner, M. S.; Hartley, P. G.; McLean, K. M.; Griesser, H. J.; Castner, D. G. Surf. Interface Anal. 2002, 33, 924-931. (24) Me´dard, N.; Aouinti, M.; Poncin-Epaillard, F.; Bertrand, P. Surf. Interface Anal.2001, 31, 1042-1047. (25) Lu, H. B.; Campbell, C. T.; Graham, D. J.; Ratner, B. D. Anal. Chem. 2000, 72, 2886-2894. (26) Griesser, H. J.; Chatelier, R. C. J. Appl. Polym. Sci. 1990, 46, 361-384. (27) Griesser, H. J.; Chatelier, R. C.; Gengenbach, T. R.; Johnson, G.; Steele, J. G. J. Biomater. Sci., Polym. Ed. 1994, 5, 531-554. (28) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal.1996, 24, 271-281. (29) Dai, L.; StJohn, H. A. W.; Bi, J.; Zientek, P.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 2000, 29, 46-55.

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Table 1. Four Groups of Positive Fragments Used in the Evaluation of the Modification Stages by Analysis of Means and Principal Component Analysis

Figure 1. Schematic diagram of the grafting sequence and the molecular structure of the furanone used.

then attached from aqueous solution via carbodiimide chemistry, followed by attachment of AZA, again via carbodiimide chemistry. Due to the light sensitivity of the azide group, the latter reaction needed to be performed in a dark room, as did the subsequent immobilization of FU molecules. After attaching AZA onto the PAAC layer, the furanone compound (10 mg/mL in acetone) was applied onto the surface, still under dark room conditions. After complete evaporation of the acetone solvent, the sample was illuminated with UV light for 2 min. Finally, the sample was extensively washed with ethanol and then water and air-dried. Static TOF-SIMS Analyses. TOF-SIMS analyses were performed with a PHI TRIFT II model 2100 spectrometer (PHI Electronics Ltd.) equipped with a 69Ga liquid metal ion gun. A pulsed primary ion beam was used to desorb and ionize species 432 Analytical Chemistry, Vol. 80, No. 2, January 15, 2008

[CmHn]+ (m/z)

[CmHnO]+ (m/z)

[CmHnN]+ (m/z)

[CmHnNO]+ (m/z)

[CH2]+ (14) [CH3]+ (15) [C2H2]+ (26) [C2H3]+ (27) [C2H4]+ (28) [C2H5]+ (29) [C3H]+ (37) [C3H2]+ (38) [C3H3]+ (39) [C3H4]+ (40) [C3H5]+ (41) [C3H6]+ (42) [C3H7]+ (43) [C4H3]+ (51) [C4H4]+ (52) [C4H5]+ (53) [C4H7]+ (55) [C4H8]+ (56) [C4H9]+ (57) [C5H5]+ (65) [C5H6]+ (66) [C6H9]+ (81)

[CHO]+ (29) [CH3O]+ (31) [C2H2O]+ (42) [C2H3O]+ (43) [COOH]+ (45) [C2H5O]+ (45) [C2H6O]+ (46) [C3HO]+ (53) [C3H2O]+ (54) [C3H3O]+ (55) [C3H7O]+ (59) [C4H4O]+ (68) [C5H5O]+ (81)

[NH4]+ (18) [CH2N]+ (28) [CH3N]+ (29) [CH4N]+ (30) [CH5N]+ (31) [C2H4N]+ (42) [C2H5N]+ (43) [C2H6N]+ (44) [C2H7N]+ (45) [C3H4N]+ (54) [C3H6N]+ (56) [C3H8N]+ (58) [C3H9N]+ (59) [C4H6N]+ (68) [C4H8N]+ (70) [C4H10N]+ (72) [C5H4N]+ (78) [C5H6N]+ (80) [C5H8N]+ (82) [C5H10N]+ (84) [C5H12N]+ (86) [C6H4N]+ (90) [C6H8N]+ (94)

[CH2NO]+ (44) [CH4NO]+ (46) [C2H2NO]+ (56) [C3H4NO]+ (70) [C3H6NO]+ (72) [C4H2NO]+ (80) [C4H4NO]+ (82) [C4H6NO]+ (84) [C4H8NO]+ (86)

from sample surfaces. Pulsed low-energy electrons were used for charge compensation. Mass axis calibration was done with CH3+, C2H5+, and C3H7+ in positive mode and with CH-, C2H- and Clin negative mode of operation. A mass resolution m/∆m of 4500 at nominal m/z ) 27 amu (C2H3+) was typically achieved. The TOF-SIMS technique is “destructive” by its nature; however, by applying an ion beam of low current, it is possible to derive data from a virtually intact surface. The primary ion fluxes used in this study were between 3 × 1011 and 6 × 1011 ions cm-2, meeting the static conditions regime.30 Each sample was characterized by 10 positive and 1 negative mass spectra collected from different, non-overlapping areas. The spectra were initially subjected to processing by analysis of means,31,32 but the major focus was on PCA, with four series of positive fragments, listed in Table 1, selected from the 0-100 amu spectral regions. The series are abbreviated by the formulas [CnHm]+, [CnHmO]+, [CnHmN]+, and [CnHmNO]+. The analysis range was restricted to 100 amu, as for the monatomic gallium projectile used in this work, the yield of secondary molecular ions drops dramatically with the increase of the molecular weight. After a 60-s acquisition, assuring the static regime, the fragment ions of m/z up to 100 amu were of satisfactory intensity and definition for further processing. Multivariate Analysis. The positive secondary ion mass spectra recorded for the substrate and each sequential modification step were complex. As they did not contain any obvious, sample-unique molecular ions, they were classified into groups by detecting differences in the fragmentation patterns. The large (30) Briggs, D. Surface Analysis of Polymers by XPS and Static SIMS; Cambridge University Press: Cambridge, UK, 1998. (31) Zuwaylif, F. H. General Applied Statistics; Addison-Wesley Publishing Co., Inc.: Reading, MA, 1980. (32) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry; Ellis Horwood Ltd.: Chichester, UK, 1993.

quantities of collected data required proper assessment and interpretation. For interpretation, the primary task was to compress the original data sets such that the most essential and useful information was retained. Accordingly, data compression and information extraction were performed with the aid of PCA. The aim of PCA is to find a new coordinate system that can be expressed as a linear combination of the original variables. Principal component analysis projects the original data onto new axes, called principal components (PCs). These new axes are such that the greatest amount of the original spectral variance is retained.13,15 Principal components are graded in accordance with the amount of the original data variance. Principal component 1 (PC1) describes the direction of the greatest variation in the data set and consecutive PCs account for less and less variability. In the case of static secondary ion mass spectra, the original data set, consisting of hundreds of variables, can be adequately described by a few uncorrelated PCs. Principal component analysis requires that the spectra be represented in a matrix format. The process of data decomposition by PCA results in the formation of two new matrices: a score matrix and a loading matrix. The scores show the relationship between the samples in the new coordinate (PC defined) space, while the loadings illustrate the relationship between the original variables and the principal components. If two or three PCs form a satisfactory model, then the data processed by PCA can be visualized (2- or 3D scores plots).15,33 Principal component analysis was performed using PLS_Toolbox (Version 3.0, Eigenvector Research, Inc., Manson, WA) working within MATLAB software (Version 6.5, MathWorks Inc., Natick, MA). The original data matrix was built of “n” columns, comprising the intensities of peaks selected from the mass spectra, and “m” rows corresponding to m spectra subjected to multivariate analysis. The peaks intensities were normalized to the total intensity of the selected peaks and mean-centered prior to PCA. RESULTS AND DISCUSSION While XPS can be utilized for monitoring the reaction sequence of Figure 1, the limited chemical bonding information available by XPS analysis and the close similarity of the C 1s spectra for these samples allow rather limited analysis.10 Moreover, it was not possible to adequately assess coating uniformity by XPS imaging. The enhanced chemical information content in secondary ion mass spectra from TOF-SIMS renders this technique suitable for more detailed analysis and in particular for the detection of the addition of small molecules, although the issue of quantification remains. Thus, our study focused on the question of whether TOF-SIMS analysis would enable direct detection of the immobilization of AZA and the furanone compound via characteristic fragments and whether any mass fragments indicative of side reactions might be detectable. Replicate positive static secondary ion mass spectra were recorded on the fluoropolymer substrate (FEP), n-HA plasma polymer-coated FEP substrate (FEP-HApp), and the sequential modifications denoted FEP-HApp-PAAC, FEP-HApp-PAAC-AZA, and FEP-HApp-PAAC-AZA-FU. While the spectrum of the FEPHApp sample differed considerably from that recorded on un(33) Jurs, P. C. Science 1986, 232, 1219-1224.

Figure 2. Positive static secondary ion mass spectra in the range m/z ) 44.9-45.2 before and after grafting PAAC onto the HApp film. Note the different intensity scales.

modified FEP (spectra not shown), in accord with XPS data showing complete disappearance of F upon HApp coating,10,11 all the other spectra were superficially quite similar. Apart from the HApp layer, which is ∼20 nm thick, each subsequent grafting step produces only an ultrathin additional “layer”, and it is thus not surprising that the secondary ion mass spectra contain superimposed contributions from each grafting step. For the sake of brevity, these full spectra are not shown here, but they are published elsewhere.10 The most intense peaks in the spectra are at m/z values corresponding to fragment ions assignable to hydrocarbon structures. There are, however, significant differences between the spectra, which emerge when high mass resolution is employed. This is illustrated in Figure 2, which shows the changes in the intensities of the [COOH]+, [C2H5O]+, and [C2H7N]+ fragment ions upon grafting of the HApp layer with poly(acrylic acid). The high mass resolution achievable in TOF-SIMS allows discrimination between chemical species that have very close m/z values and thereby enable the elucidation of chemical information that identifies fragment ions diagnostic of particular grafted molecules. The sequential grafting steps in the furanone immobilization sequence were monitored and compared with the previous surfaces using a number of positive fragment ions, which are listed in Table 1. They were grouped into hydrocarbon, oxygencontaining, nitrogen-containing, and nitrogen/oxygen-containing hydrocarbon fragments. Inspection of high-resolution spectra revealed many differences such as those shown in Figure 2, but the analysis and interpretation of such single peak changes is not efficient, and we will in the following take the path of global analysis of the spectra. Analytical Chemistry, Vol. 80, No. 2, January 15, 2008

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Figure 3. Scores plot on PC1 and PC2 of the positive secondary ion mass spectra of the different modification steps in the FU immobilization sequence.

The collected data were initially subjected to further processing by analysis of means. This methodology yielded statistical differences within and between the samples based on a single variable (univariate) assessment. The interpretation of the collected data, including figures, from univariate analysis is given in the Supporting Information. The intensity changes for the sequential grafting steps in the furanone immobilization sequence are irregular across the selected fragments, especially for the AZA and FU modification stages, hindering interpretation of these spectra by univariate analysis; although consideration of individual peaks can be done and yields information on specific chemical structures, we will not elaborate on those interpretations here and will, instead, move on to multivariate analysis, which is a much faster way of elucidating and interpreting differences between the samples. Principal Component Analysis. Principal component analysis was performed on the positive secondary ion mass spectra using all the peaks listed in Table 1; this led to the scores plot shown in Figure 3. The plot shows four distinct clusters; each cluster contains multiple spectra representing HApp, HApp-PAAC, HApp-PAAC-AZA, and HApp-PAAC-AZA-FU. The first two components (PC1 and PC2) contain around 70 and 21% of the original data variance, respectively. Since the total variance captured is ∼91%, this indicates that most of the original spectral information is retained in the first two components. The scores plot in Figure 3 shows that the resolving power of the PCA model with two principal components is sufficient for unambiguous sample discrimination. All stages of the multistep grafting sequence are well separated from each other. The PAAC and AZA surfaces have positive scores on PC1, while PC1 scores are negative for the HApp and FU surfaces. The first principal component discriminates these two groups with a high degree of confidence. Use of both PC1 and PC2 is sufficient to clearly separate all four spectral data sets. While three data sets cluster within the usual degree of variance, which arises from the S/N ratios in the peak intensities as well as possibly from small local variations in the surface composition, the experimental points for the AZAmodification step show a higher degree of scatter, which could be associated with its instability upon exposure to light. Whereas the coupling of the small AZA molecule onto the PAAC layer does not incur marked, easily identifiable spectral changes in univariate analysis, the difference between PAAC and PAAC-AZA samples is clearly evident by PCA. The PC1 score is 434

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similar for the two surfaces, but the second principal component clearly distinguishes them. The HApp and the AZA surfaces form well-separated clusters, which indicates significant compositional differences between these two samples although both are nitrogenrich and much less clearly separated by XPS.11 Likewise, the grafting of FU onto AZA is clearly evident in the scores plot although the added mass is very small. It is noteworthy how well the clusters are separated; TOF-SIMS with PCA has no difficulty revealing the differences in surface chemistry upon grafting of the small molecules AZA and FU. The analysis of surface grafting of such small molecules is often challenging by other surface analytical methods such as XPS, unless a unique label such as the Br in FU is available (but even then the S/N ratio is low).11 The very high sensitivity and the shallow depth of analysis of TOFSIMS are the main causes for the suitability of TOF-SIMS for such analyses; within its very shallow probe depth, even small grafted molecules matter. For reasons of convenience and availability, we have used a brominated furanone. Clearly, TOF-SIMS combined with PCA analysis is equally applicable to nonhalogenated furanones, some of which are also of antibacterial interest, and other antibacterial molecules, as the peaks used for PCA (Table 1) do not contain Br and thus the differentiation by the scores plot in Figure 3 is based on spectral information derived from the furanone molecular skeleton rather than from the bromine substituents of the presently grafted molecule. We note that it is difficult to identify individual peaks that change strongly after furanone grafting, but the collective spectral changes revealed by PCA provide unambiguous proof of FU grafting. Data processing by PCA produces a second matrix, the matrix of loadings. Each variable has a loading on each PC, which reflects how much the variable contributes to the particular PC. Thus, analysis of the loadings reveals which individual peaks contribute most strongly to a PC and hence yields information on the most important chemical differences between samples. Loadings plots can also be used to check that the differences in scores plots are consistent with expectations; for instance, two samples could show different scores if one sample was contaminated with adventitious silicone or hydrocarbon contaminants, which would be revealed by peaks that are diagnostic of those materials rather than the intended grafted surface chemistries. Figure 4 shows loading of positive fragment ions on PC1 (a) and PC2 (b). Most of the hydrocarbon peaks are loaded negatively on PC1. Clearly, these intensities are more prevalent on HApp and FU surfaces, and this is consistent with the higher content of alkyl structures in the expected chemical compositions of these surfaces. Their contribution to the discrimination between the samples by PC1 is significant. A number of peaks have substantial loadings; it is not surprising that both HApp and FU can produce various alkyl fragments. The hydrocarbon fragments are not informative with regard to more detailed characterization of the surface chemistries. In contrast, the [CmHnN]+, [CmHnO]+, and [CmHnNO]+ fragment ions have positive loadings on PC1. They are assignable to structures in the PAAC and AZA surface chemistries. The [CnHmNO]+ fragment ions can be assigned to originate from the amide linkages used for the grafting of both PAAC and AZA. The loading of the [C3H8N]+ fragment ion, the highest positive among the

Figure 4. Loadings plots on PC1 (a) and PC2 (b) of the positive secondary ion mass spectra of the different modification steps in the FU immobilization sequence.

nitrogen-containing fragments, correlates strongly with attachment of the nitrogen-rich AZA molecules. No ions with multiple N are observed; the unavoidable exposure to light between grafting of AZA in the darkroom and the TOF-SIMS analysis leads to photoactivated reaction of azide to nitrene, thus eliminating the azide groups as a possible source of fragments containing N-N structures. The nitrogen-containing fragments, even those with high positive loadings on PC1, such as [C3H8N]+, [C2H6N]+, [C2H4N]+, or [CH4N]+, cannot be directly correlated with structural elements expected on the AZA sample. On exposure to light, the nitrene thus produced will react with various other molecular structures; nitrenes are rather indiscriminate in their reactivity and can in principle insert into any C-H, N-H, and O-H bonds, as well as into ring structures. When a furanone is present on the AZA surface, it can act as a grafting partner, but when we remove an AZA sample from the darkroom for TOF-SIMS analysis, the nitrenes will undergo various other reactions. Thus, a diversity of surface chemistries, and a diversity of fragment ions, can be expected. In addition, the process of sputtering gives rise to various complex fragmentations and transformations. Nevertheless, even though structural assignments are difficult, the ions observed and the scores and loadings plots clearly show that a high surface density of AZA had been grafted. Figure 4a shows that virtually all the [CmHnO]+ and [CmHnNO]+ fragments load positively on PC1; thus, they are characteristic of PAAC and AZA surfaces. In contrast to the hydrocarbon and nitrogen-containing fragments, some of the [CmHnO]+ and [CmHnNO]+ fragments can be related to the structure of the grafted molecules. For instance, modification of the HApp surface with PAAC can be easily detected by the presence of [C3H3O]+,

[C2H2NO]+, and [COOH]+ fragments in the spectrum (the structural formulas for [C3H3O]+ and [C2H2NO]+ are [CH2CHCO]+ and [CHCONH]+, respectively), and these fragments are consistent with expected mass spectrometric fragmentation pathways. Loadings of the hydrocarbon and nitrogen-containing fragments on PC2 shown in Figure 4b reveal interesting information about ion fragmentation. The low m/z hydrocarbon fragments such as [CH2]+, CH3+, [C2H2]+, and [C2H3]+ up to m/z ) 40 are negatively loaded on PC2 and they correlate with PAAC and FU surfaces. Fragments such as [C3H5]+, [C3H7]+, [C4H7]+, [C4H9]+, [C3H5]+, or [C6H9]+ with m/z > 40 load positively on PC2 and are associated with HApp and AZA surfaces. This observation is consistent with expectations based on mass spectrometric fragmentation pathways; the chemical structures of PAAC and FU do not lend themselves to the ejection of large hydrocarbon ions, whereas for HApp and AZA surfaces, a larger proportion of heavier molecular ions are contained in the spectra. Loadings of the nitrogen-containing fragments on PC2 indicate an analogous effect. Higher molecular weight fragments such as [C3H8N]+, [C5H10N]+, or [C5H12N]+ are loaded positively on PC2 and correlate with HApp and AZA surfaces. The [CmHnO]+ and [CmHnNO]+ fragments are loaded negatively on PC2. This indicates that PAAC and FU surfaces produce, as expected, more oxygen-containing fragments compared to HApp and AZA. Both PC1 and PC2 point to the PAAC surface as the richest in oxygen in the modification sequence, which is a logical consequence of its structure. The marked changes upon immobilizing AZA onto the PAAC layer indicates that there is a high efficiency of attaching AZA; if AZA molecules were at low density of the PAAC layer, we would expect the spectra to be dominated still by PAAC fragment ions. The presence of the brominated furanone was readily ascertained by the presence of two peaks at m/z ) 79 and 81 in the negative ion mass spectra (Figure 5a). These signals correspond to the two stable isotopes of bromine, 79Br and 81Br, and they are present only in the spectra of the FEP/HApp/PAAC/AZA/FU surface as expected. These singular, specific ion signals facilitate the verification of successful grafting of furanone molecules and enable easier imaging. However, as discussed above, PCA analysis of the positive ion spectra recorded on the FU surface also reveals the presence of furanone from mass fragments that do not contain Br, and hence, there is no requirement for a unique element in TOF-SIMS analysis, whereas by XPS it would not be possible to detect a nonhalogenated grafted furanone. Antibacterial Furanone Coating Uniformity. A great advantage of the presence of Br in the grafted furanone molecules, however, is the ease with which imaging can be performed by use of the Br ion peaks. Coating uniformity was examined by imaging the bromine isotope signal (m/z ) 79) over a 100 × 100 µm area using the TOF-SIMS imaging mode. As shown in Figure 5b, the FU molecules were distributed fairly evenly across the surface; the main effect in these images is pixel noise due to the low intensity of these peaks, as shown by comparison with other, known uniform reference surfaces (not shown). Clearly, there were no major gaps in the furanone coating within the selected area. Given the size of bacteria, there is no need for higher magnification imaging and it appears reasonable to assume that Analytical Chemistry, Vol. 80, No. 2, January 15, 2008

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CONCLUSIONS The results of this study highlight the utility of TOF-SIMS and PCA for the sensitive analysis of the surface grafting of small molecules, which is difficult to analyze by other techniques. Even multiple grafting steps involving, in the present case, two low molecular weight compounds can readily be disentangled by PCA, whereas analysis of individual peaks is time-consuming and does not necessarily produce identification of uniquely assignable peaks or intensity changes. This is particularly well illustrated in the present case by the grafting of FU molecules, which did not yield a singular unique peak in the positive ion spectra, whereas the collective spectral changes elucidated by PCA provided unambiguous verification of the successful grafting of this low molecular weight compound. The analyses provided information on each grafting step, and imaging enabled assessment of coating uniformity. ACKNOWLEDGMENT This research was supported in part by a University of South Australia President’s Ph.D. scholarship to S.A.A.-B., by the Commonwealth Government under the Cooperative Research Centre Scheme (Vision CRC), and by the Australian Research Council (Special Research Centre for Particle and Material Interfaces). The authors thank Prof. David G. Castner and Dr. Daniel J. Graham for making available copies of custom software that greatly facilitated PCA. Figure 5. Negative secondary ion mass spectrum of furanonecoated surface (a) and corresponding image of 79Br distribution across a 100 × 100 µm area of furanone coating (b) (beam diameter was120 nm).

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

the furanones do not need to be molecularly confluent. The uniformity of this coating appears sufficient to present a surface on which bacteria cannot find gaps large enough to settle.

Received for review August 15, 2007. Accepted October 19, 2007.

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