Time-of-Flight-Secondary Ion Mass Spectrometry Study of the

Jul 17, 2009 - ... on plasma polymerized NIPAM coatings (ppNIPAM)(14) and protein adsorption on pNIPAM grafted to a plasma polymer support layer.(15) ...
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Anal. Chem. 2009, 81, 6905–6912

Time-of-Flight-Secondary Ion Mass Spectrometry Study of the Temperature Dependence of Protein Adsorption onto Poly(N-isopropylacrylamide) Graft Coatings Martin A. Cole,*,† Marek Jasieniak,† Helmut Thissen,‡ Nicolas H. Voelcker,§ and Hans J. Griesser† Ian Wark Research Institute, University of South Australia, Adelaide, SA 5095, Australia, CSIRO Molecular and Health Technologies, Melbourne, VIC 3169, Australia, and School of Chemistry, Physics and Earth Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5042, Australia Stimuli-responsive materials show considerable promise for applications that require control over biomolecule interactions at solid material interfaces. Graft coatings of poly(N-isopropylacrylamide) (pNIPAM) are of interest for biomedical and biotechnological applications due to their temperature-dependent switching of surface properties between adhesive and nonadhesive states for cells and proteins. The characterization of protein adsorption to these switchable coatings is a formidable task since switching not only influences the affinity for proteins but at the same time induces a significant change in the coating. Here, the highly sensitive analytical technique of time-of-flight-secondary ion mass spectrometry (TOFSIMS) combined with principal component analysis (PCA) was used for the characterization of protein adsorption onto pNIPAM coatings prepared by free radical polymerization onto surface-bound polymerizable groups. Adsorption of bovine serum albumin and lysozyme onto pNIPAM coatings from phosphate buffered solutions was investigated at temperatures above and below the polymer’s lower critical solution temperature (LCST). Below the LCST, no adsorbed proteins could be detected even with this ultrasensitive method. Whereas above the LCST, adsorbed protein was detected in amounts corresponding at less than the monolayer. PCA loadings plots showed that adventitious contaminants, which might lead to confounding or misleading spectral changes upon protein exposure, were not observed. Switchable surface coatings utilizing stimuli-responsive polymers afford the ability to transform surface properties of biomaterials, biodiagnostic devices, and biotechnological materials on demand. This can provide dynamic control over surface-biomolecule interactions such as protein adsorption and desorption1-4 * To whom correspondence should be addressed. E-mail: Martin.Cole@ unisa.edu.au. † University of South Australia. ‡ CSIRO Molecular and Health Technologies. § Flinders University. (1) Balamurugan, S.; Ista, L. K.; Yan, J.; Lopez, G. P.; Fick, J.; Himmelhaus, M.; Grunze, M. J. Am. Chem. Soc. 2005, 127, 14548–14549. 10.1021/ac9009337 CCC: $40.75  2009 American Chemical Society Published on Web 07/17/2009

and subsequent events such as cell attachment, spreading, and also cell detachment.5-8 The best known temperature-dependent polymer of interest for biointerface applications is poly(N-isopropylacrylamide) (pNIPAM), which has attracted considerable attention since the pioneering work by Hoffman.9 Above the lower critical solution temperature (LCST), this polymer adopts a collapsed and partially dehydrated state to which cells can attach. When one lowers the temperature below the LCST, the polymer assumes a highly hydrated conformation which is resistant to cells and can induce their detachment.5,6 This temperature-dependent switching of bioadhesion has been utilized for the fabrication of surfaces that enable cell sheet culturing,8 and many other applications are also being studied (reviewed in ref 10). Although cycling of the phase transition of pNIPAM graft coatings across the LCST has been found to yield favorable surface and mechanical properties for reversible cell attachment, less attention has been paid to the question of whether protein adsorption is also fully reversible. Some extracellular matrix proteins may remain on the pNIPAM surface upon cooling, and we hypothesize that cell detachment may, therefore, not be due to detachment of the adhesive protein under layer but due to the mechanical properties of the highly hydrated pNIPAM below the LCST preventing cells from establishing and maintaining a wellspread functional cytoskeleton. Moreover, if one cycles temprature repeatedly, increasing amounts of proteins might become irreversibly adsorbed. It is also possible that the properties of pNIPAM coatings are sensitive to the coating architecture, such as chain (2) Huber, D. L.; Manginell, R. P.; Samara, M. A.; Kim, B. I.; Bunker, B. C. Science 2003, 301, 352–354. (3) Hyun, J.; Lee, W. K.; Nath, N.; Chilkoti, A.; Zauscher, S. J. Am. Chem. Soc. 2004, 126, 7330–7335. (4) Nath, N.; Hyun, J.; Ma, H.; Chilkoti, A. Surf. Sci. 2004, 570, 98–110. (5) Kushida, A.; Yamato, M.; Konno, C.; Kikuchi, A.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 1999, 45, 355–362. (6) Tsuda, Y.; Kikuchi, A.; Yamato, M.; Nakao, A.; Sakurai, Y.; Umezu, M.; Okano, T. Biomaterials 2005, 26, 1885–1893. (7) Canavan, H. E.; Cheng, X. H.; Graham, D. J.; Ratner, B. D.; Castner, D. G. J. Biomed. Mater. Res., Part A 2005, 75A, 1–13. (8) Yang, J.; Yamato, M.; Kohno, C.; Nishimoto, A.; Sekine, H.; Fukai, F.; Okano, T. Biomaterials 2005, 26, 6415–6422. (9) Hoffman, A. S. J. Controlled Release 1987, 6, 297–305. (10) Cole, M. A.; Voelcker, N. H.; Thissen, H.; Griesser, H. J. Biomaterials 2009, 30, 1827–1850.

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segment density, as is the case for PEO graft coatings.11-13 Thus, in the same way as one cannot generalize adsorption behavior of proteins, one may not be able to generalize observations from a specific pNIPAM graft coating to another. It is, therefore, important to investigate the interfacial interactions of pNIPAM surfaces, prepared in different ways, and proteins such as to elucidate the key factors that determine whether and how protein adsorption takes place. While some studies of protein adsorption have been reported,2,14-16 reported observations do not yet enable the derivation of general principles. One challenge in such studies is the probing of proteins adsorbed at low amounts. Adsorbed protein amounts as low as a few ng/cm2, which corresponds to ∼1/100th of a monolayer coverage or less, can induce biological consequences.17 Unfortunately, not all studies of protein adsorption have utilized methods capable of detecting such low amounts, and hence, some conclusions of nonfouling behavior are not well secured. Highly sensitive analytical techniques need to be employed to ascertain whether the pNIPAM surface is truly protein resistant or just low-fouling below the LCST. Acoustic and optical instruments such as optical waveguide lightmode spectroscopy (OWLS),18 surface plasmon resonance (SPR),19 and quartz crystal microbalance (QCM)20 have high sensitivity for protein adsorption but impose coating limitations and may have difficulty with simultaneous changes to pNIPAM during adsorption and variation of environmental conditions. Physico-chemical surface analysis methods can provide highly sensitive detection of protein adsorption events.21,22 However, for pNIPAM, the technique of X-ray photoelectron spectroscopy (XPS) is poorly suited due to the spectral similarities with protein.23,24 Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) is an extremely sensitive analytical technique capable of identifying proteins, small molecules, and chemically modified end groups on surface tethered coatings.25-28 TOF-SIMS has also been (11) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002, 23, 2043– 2056. (12) Pasche, S.; Textor, M.; Meagher, L.; Spencer, N. D.; Griesser, H. J. Langmuir 2005, 21, 6508–6520. (13) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Biomaterials 2005, 26, 5927– 5933. (14) Cheng, X.; Canavan, H. E.; Graham, D. J.; Castner, D. G.; Ratner, B. D. Biointerphases 2006, 1, 61–72. (15) Heinz, P.; Bretagnol, F.; Mannelli, I.; Sirghi, L.; Valsesia, A.; Ceccone, G.; Gilliland, D.; Landfester, K.; Rauscher, H.; Rossi, F. Langmuir 2008, 24, 6166–6175. (16) Janzen, J.; Le, Y.; Kizhakkedathu, J. N.; Brooks, D. E. J. Biomater. Sci., Polym. Ed. 2004, 15, 1121–1135. (17) Tsai, W. B.; Grunkemeier, J. M.; Horbett, T. A. J. Biomed. Mater. Res. 1999, 44, 130–139. (18) Ho ¨o ¨k, F.; Vo ¨ro ¨s, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloid Surf., B-Biointerfaces 2002, 24, 155–170. (19) Schasfoort, R. B. M.; Tudos, A. J. Handbook of Surface Plasmon Resonance; Royal Society of Chemistry: Cambridge, U.K., 2008. (20) Teichroeb, J. H.; Forrest, J. A.; Jones, L. W.; Chan, J.; Dalton, K. J. Colloid Interface Sci. 2008, 325, 157–164. (21) Griesser, H. J.; Kingshott, P.; McArthur, S. L.; McLean, K. M.; Kinsel, G. R.; Timmons, R. B. Biomaterials 2004, 25, 4861–4875. (22) Malmsten, M. Biopolymers at Interfaces, 2nd ed.; Marcel Dekker, Inc.: New York, 2003. (23) Canavan, H. E.; Cheng, X. H.; Graham, D. J.; Ratner, B. D.; Castner, D. G. Langmuir 2005, 21, 1949–1955. (24) Cole, M. A.; Jasieniak, M.; Voelcker, N. H.; Thissen, H.; Horn, R.; Griesser, H. J. Proc. SPIE Int. Soc. Opt. Eng. 2006, 6416, 641606–641610.

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utilized for the detection of minute amounts of adsorbed proteins on PEG graft surfaces, undetectable by other methods.29 Accordingly, in this study we have utilized TOF-SIMS to probe for protein adsorption onto free radical polymerized pNIPAM graft coatings. We have also investigated by TOF-SIMS whether the coating itself changes, apart from the extent of hydration and thus microstructure, when cycled past the LCST, because any such changes might interfere with interpretation of protein adsorption and also because such changes might cause increasing protein adsorption upon repeated cycling. Principal component analysis (PCA)27,29,30 was utilized to enable an objective study of differences in peak intensities in these complex spectra with few unique mass signals. EXPERIMENTAL SECTION Substrate Preparation and Functionalization. Silicon wafers 〈100〉 (MMRC, Melbourne, Australia) cut to 1 × 1 cm were ultrasonically cleaned in ultrapure Milli-Q (MQ) grade water (18.2 MΩ.cm). Wafers were then immersed in 7:3 (v/v) 97% H2SO4/ 30% H2O2 (Piranha) solution for a minimum of 30 min, washed with copious amounts of MQ water, and dried under a stream of nitrogen. Caution, Piranha solution is a strong oxidant and reacts violently with organic substances! Diced wafers were immersed in a 5% v/v solution of 3-trimethoxysilylpropyl methacrylate (γMPS, 98%, Aldrich, Sydney, Australia) in distilled toluene for 2 h at room temperature. The resulting Si-γMPS samples were washed with toluene, acetone, and MQ water, followed by drying under a gentle stream of nitrogen. Grafting of Poly(N-isopropylacrylamide). N-isopropylacrylamide (NIPAM, 97%, Sigma, Sydney, Australia) purified by recrystallization from distilled n-hexane was made to 7% w/v solutions in MQ water along with initiator 4,4′-azobis(4-cyanopentanoic acid) (ACPA, 98%, Fluka) 0.1% w/v. The methacrylate functionalized Si-γMPS wafers were immersed in the monomer/ initiator solution and purged with nitrogen for a minimum of 20 min. The stirring reaction was heated to 55 °C under inert conditions, and polymerization was conducted for 40 min. Polymerized (Si-γMPS-pNIPAM) samples were washed and soaked in MQ water overnight at 20 °C and finally dried under a stream of nitrogen. Protein Adsorption. The adsorption of proteins at temperatures above and below the LCST was investigated using bovine serum albumin (BSA, MW 66 kDa, >96%, Sigma, Sydney, Australia) and lysozyme (Lys, MW 14.7 kDa, >90% Sigma, Sydney, Australia). The tertiary structure of protein is prone to unfolding in response to physical interactions such as those encountered when adsorbed at an interface. Proteins such as BSA that are more susceptible to unfolding are often termed “soft” and unstable, while (25) Al-Bataineh, S. A.; Jasieniak, M.; Britcher, L. G.; Griesser, H. J. Anal. Chem. 2008, 80, 430–436. (26) Takahashi, H.; Emoto, K.; Dubey, M.; Castner, D. G.; Grainger, D. W. Adv. Funct. Mater. 2008, 18, 2079–2088. (27) Wagner, M. S.; McArthur, S. L.; Shen, M. C.; Horbett, T. A.; Castner, D. G. J. Biomater. Sci., Polym. Ed. 2002, 13, 407–428. (28) Cheng, F.; Gamble, L. J.; Castner, D. G. Anal. Chem. 2008, 80, 2564– 2573. (29) Kingshott, P.; McArthur, S.; Thissen, H.; Castner, D. G.; Griesser, H. J. Biomaterials 2002, 23, 4775–4785. (30) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28–60.

those that are least inclined to denature (e.g., Lys) are known as hard or stable proteins. These two proteins were chosen as model “soft” and “hard” proteins, respectively. Additionally, BSA and Lys represent different sizes and net charges and may be expected to have different adsorption characteristics. Solutions of each protein were made in phosphate buffered saline (PBS, 137 mM NaCl, 27 mM KCl, 6.64 mM Na2HPO4 · H2O, 1.76 mM KH2PO4) at pH 7.4. Samples of Si-γMPS-pNIPAM were first incubated in buffer solutions at a controlled temperature for 30 min prior to addition of protein solutions, equilibrated to the same temperature, such that the final concentration of BSA or Lys was 1 mg/mL. Samples were incubated in a protein solution at 20 ± 2 °C with gentle shaking for a period of 4 h or at 37 ± 2 °C for 15 min. Following incubation with protein at either temperature, Si-γMPS-pNIPAM samples were carefully washed twice with PBS and thrice with MQ H2O equilibrated to the same temperature as the respective protein incubation step. Due to the sensitivity of TOF-SIMS to small amounts of metal ions, extensive washing of the samples with MQ H2O was important. Samples were allowed to dry in air at room temperature before being stored under vacuum in a desiccator for analysis by TOF-SIMS. Parallel samples incubated in PBS in the absence of protein served as controls for the interpretation of spectra recorded on protein incubated samples. TOF-SIMS Analysis. TOF-SIMS analyses were performed using a PHI TRIFT II (model 2100) spectrometer (PHI Electronics Ltd., USA) with a 69Ga liquid metal ion gun (LMIG). A 15 keV pulsed primary ion beam was used to desorb and ionize species 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 Cl- in 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 nature; however, by applying an ion beam of low current and limited acquisition time, it is possible to limit the probability of multiple ion impact and, thus, derive data from a virtually intact surface. Primary ion fluxes employed here were between 3 × 1011 and 6 × 1011 ions cm-2, which meets the static conditions criteria.31 This ensures that less than 0.1% of the surface atomic sites (1 in 1000) were struck by the primary ion beam within the time of the measurement. Thus, the spectra acquired under such “static SIMS” conditions allowed collection of detailed information of the upper 10-15 Å without significantly affecting the overall chemical integrity of the coatings. Samples of Si-γMPS-pNIPAM were characterized by ten positive and one or two negative mass spectra collected from different, nonoverlapping areas. Normalization of peak intensity to the total intensity of selected peaks was performed prior to data analysis. Spectra were subjected to further processing by Analysis of Means.32 This method yielded statistical variability within and between the samples based on a single variable (31) Briggs, D. Surface Analysis of Polymers by XPS and Static SIMS; Cambridge University Press: Cambridge, U.K., 1998. (32) Zuwaylif, F. H. General Applied Statistics; Addison-Wesley Publishing Company, Inc.: Reading, MA, 1980.

(univariate) assessment. Spectra were also processed by PCA as described in the following manner. Principal Component Analysis. The complex multiple mass static SIMS spectra were evaluated with the aid of PCA. This enabled statistically secured, objective simplification of spectral data and, consequently, aided in better understanding and meaningful interpretation. The idea of PCA is to transform a set of intensities of fragment ions (correlated variables) into a smaller number of new uncorrelated variables, called principal components (PCs); which can be expressed as linear combinations of the original variables. The PCs are arranged in decreasing order of the original data variance content such that the first PC accounts for the highest variance and consecutive PCs account for increasingly less variability. If two or three PCs form a satisfactory model (i.e., account for a large proportion of the total data variance), then the PCA scores and loadings can be interpreted with confidence. This is a practical way to determine clusters in data. PCA also allows assessment of the importance of specific original variables (mass fragments) and elimination of those that contribute little information or are subject to signal/noise ratios that would make interpretation unreliable. The application of PCA for TOF-SIMS data analysis has been described extensively elsewhere.33-38 Briefly, a sufficient number of apparently significant fragments are selected from the TOFSIMS spectra. The peaks are normalized to the sum of the intensities of the selected peaks and mean-centered prior to analysis. Data transformation by PCA results in the formation of two new matrices: a scores matrix and a loading matrix. The scores show the relationship between the samples in the new coordinate space (PC defined), while the loadings illustrate the relationship between the original variables and the principal components and, thereby, reveal which mass peaks contribute most to the differences between samples. PCA was carried out using PLS_Toolbox software version 3.0 (Eigenvector Research, Inc., Manson, WA) along with MATLAB software v. 6.5 (MathWorks Inc., Natick, MA). RESULTS AND DISCUSSION The successful execution of each step in the coating sequence was monitored using XPS as described earlier.24 XPS, however, is unsuitable for the purpose of studying protein adsorption onto pNIPAM graft coatings due to close XPS spectral similarity between pNIPAM and proteins, which prevents detection of adsorbed proteins unless there is very high coverage. This situation arises because XPS essentially performs elemental analysis, with some information also gained on next-neighbor effects; the similar elemental composition of pNIPAM and proteins, containing C, N, and O (H is not detectable by XPS) at similar proportions, causes spectral differences to be relatively small. TOFSIMS, on the other hand, relies on the detection of mass fragments, which are expected to differ for pNIPAM and proteins. Eynde, X. V.; Bertrand, P. Surf. Interface Anal. 1997, 25, 878–888. Graham, D. J.; Ratner, B. D. Langmuir 2002, 18, 5861–5868. Pacholski, M. L. Appl. Surf. Sci. 2004, 231-2, 235–239. Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649–4660. Wagner, M. S.; Graham, D. J.; Ratner, B. D.; Castner, D. G. Surf. Sci. 2004, 570, 78–97. (38) Wagner, M. S.; Graham, D. J.; Castner, D. G. Appl. Surf. Sci. 2006, 252, 6575–6581. (33) (34) (35) (36) (37)

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Moreover, TOF-SIMS is exceptionally sensitive, capable of detecting adsorption of extremely small amounts of proteins.27,29 For these reasons, we used TOF-SIMS to study the temperature dependence of protein adsorption onto pNIPAM coatings. Due to the sensitivity of TOF-SIMS to small changes in sample chemistry, we also used this technique to study whether the chemical composition of the pNIPAM surface might change upon temperature cycling and immersion in PBS. Such control experiments were considered important as any such changes to the samples themselves would complicate interpretation of changes upon exposure to protein solutions. Figure 1 shows positive ion survey spectra of lysozyme (Lys) and bovine serum albumin (BSA) layers adsorbed on Si wafer surfaces as well as spectra of pNIPAM graft coatings recorded before and after immersion in BSA solutions (results for Lys were analogous and are not shown). Figure 1a-c shows that in the reference spectra many common peaks occur, yet the three samples can be readily distinguished by the different mass fragment intensity profiles, which are characteristic of their structures (Figure 1a-c). In contrast, after immersion of pNIPAM samples in BSA solution, the spectra appear visually to be quite similar (Figure 1c-e), even with magnification of lower intensity peaks. Clearly, interpretation requires more detailed information than offered by survey spectra, and it is also essential to ensure that the relatively small spectral differences are statistically significant (as opposed to resulting from finite signal/noise). This can be done in two ways: by univariate analysis of specific peaks that can be interpreted reliably or by multivariate analysis. High resolution analysis of peaks characteristic of specific elements such as sulfur or structures such as amino acid fragments can be used to probe for the presence of small amounts of adsorbed proteins. Figure 2 shows negative mass fragment signals recorded in high resolution in the region around 32 amu where sulfur (S-, 31.97 amu) and oxygen (O2-, 31.99 amu) can be observed. Lysozyme yields a particularly strong sulfur peak at 31.97 amu as shown in Figure 2a. The presence of sulfur arises from cysteine and methionine amino acids in BSA (not shown) and Lys, whereas the pNIPAM coating lacks sulfur in its structure and only minute trace amounts are observed (Figure 2b). The 31.97 amu signal shows that for pNIPAM coatings exposed to protein solutions at 20 and 37 °C, lysozyme adsorption did not occur on the well-hydrated pNIPAM surface at 20 °C whereas some adsorption did occur on the collapsed coating at 37 °C (Figure 2c,d). Analysis of the 58 amu region in the positive spectra leads to the same conclusion. The protein spectra and the pNIPAM spectrum all show a C3H8N+ peak at 58.0656 amu due to the presence of isopropyl groups in their structures, but the C2H4NO+ peak at 58.0293 amu (arising from the amino acid serine) exists only in the protein structure. This latter peak was observed only with protein control samples (on Si wafer surfaces) and for pNIPAM immersed in protein solution above the LCST but not on pNIPAM or on pNIPAM samples exposed to proteins at 20 °C. Spectra recorded upon adsorption of BSA are provided in the Supporting Information (Figure 1S); results were similar for Lys (not shown). Thus, these analyses suggest that even within the detection limit of TOF-SIMS there is no 6908

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Figure 1. SIMS survey spectra (positive fragments) for lysozyme (a); BSA (b); pNIPAM (c); pNIPAM + BSA 20 °C (d); pNIPAM + BSA 37 °C (e). Note 10× magnification of m/z 55-150 in c-e.

adsorption of BSA and Lys onto the pNIPAM surface below the LCST while there is measurable adsorption of both proteins above the LCST. However, it is well-known in the TOF-SIMS field that one needs to be careful with such univariate analysis, and better reliability can be obtained with PCA. PCA is highly useful to study subtle differences in surface chemistry and can be employed to investigate possible changes between coatings that are technically

Figure 2. High resolution negative SIMS spectra for (a) Lys; (b) pNIPAM; (c) pNIPAM + Lys (20 °C); (d) pNIPAM + Lys (37 °C).

identical but may have, for instance, been prepared from different batches. It is also an effective method to probe control samples for evidence of changes occurring from exposure to buffer solution, oxidation, delamination, and contamination. For this study, a particular point of interest was whether there is indeed no measurable adsorption of proteins below the LCST or whether the adsorbed amount, though clearly less than above the LCST, was not detected by the above univariate analyses due to finite signal/noise. By the simultaneous analysis of many peaks, PCA offers the power to enhance detection, as the signal/noise ratio of any one single peak becomes less critical. Equally important is the fact that interpretation of single peaks can be misled by the presence of even small amounts of adventitious surface contaminants such as fatty acid esters, hydrocarbons, or silicones. The simultaneous analysis of multiple peaks

by PCA is a most useful way of probing for any such adventitious effects, as the loadings plots will reveal either expected mass signals such as immonium ions from amino acids or signals indicative of contaminants. It is unfortunate that in many surface analysis studies the possibility of unintended effects from contaminants is not checked. Prior to PCA of protein adsorption, we considered it essential to probe for any possible effects of immersion of pNIPAM samples into the aqueous environment at the specific temperatures employed. Thus, we tested the stability of pNIPAM samples under the conditions used for protein adsorption studies using PBS (without added proteins). Nominally identical samples prepared in parallel but not exposed to PBS were used to record reference spectra. The positive survey spectra of the pNIPAM coating after stability testing in buffer (Figure 2S of the Supporting Information) is identical to that of freshly prepared pNIPAM shown in Figure 1. Thus, PCA and analysis of means were employed to investigate possible changes that may not be apparent from visual inspection of survey spectra. The scores plots of fresh and stability tested pNIPAM coatings overlap within experimental uncertainty, indicating negligible differences between samples (Figure 3a) and, hence, no observable effects upon PBS exposure. Analysis of means (Figure 3b,c) of selected structural peaks also shows close agreement between spectra from the two samples in agreement with the PCA. These results confirm that the pNIPAM coatings are stable under experimental conditions used here. Since the contributions from exposure to aqueous conditions were excluded, any observable spectral differences can be assigned to adsorbed proteins (or adventitious contaminants) and should enable detection of very low adsorbed amounts. Spectra from fresh pNIPAM samples and pNIPAM samples exposed to solutions of BSA and Lys were analyzed by PCA. The overlap of data clusters for pNIPAM by itself and pNIPAM incubated in a 1 mg/mL BSA solution at 20 °C in the scores plot of Figure 4a indicates that the samples are not significantly different and, thus, indicates an absence of detectable amounts of adsorbed BSA on the pNIPAM coated sample incubated with this protein. On the other hand, the data cluster for pNIPAM incubated with BSA at 37 °C has shifted significantly, indicating a chemical difference between the samples, that is, presumably adsorbed protein. The fragment ions contributing most strongly to the observed overall differences and the PC scores can be identified via loadings plots. From inspection of the loadings (Figure 4b), it is apparent that pNIPAM structural elements such as C3H8N+ (N-isopropyl) and C6H12NO+ (monomer) fragments load positively as does the isopropyl hydrocarbon fragment C3H7+ (not shown). Immonium ion (HC-N) signals such as C3H6N+, C4H8N+, and C5H10N+ and HC-NO fragments C2H4NO+ and C3H8NO+ fragments, which originate from amino acids (Table 1), load negatively and are indicative of adsorbed protein. There is no evidence in the loadings plots of any contribution from contaminants. Note that the characteristic pNIPAM fragment ions (Table 2) load positively and the protein fragment ions load negatively in the loadings plot because the scores plot shows that the proteincontaining surface is at a more negative location in the scores plot. The scores plot in Figure 5a shows the outcome of PCA for lysozyme adsorption; similar to the result with BSA, Lys is found Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 4. (a) PCA scores plot of the adsorption of BSA on pNIPAM coatings. (b) Loadings of HC-N (red) and HC-NO (black) fragments for PC1 from positive spectra.

Figure 3. PCA of fresh pNIPAM (squares) and stability tested pNIPAM (circles) samples, and analysis of means of positive HC-N (b) and HC-NO (c) fragments for fresh and stability tested pNIPAM.

to adsorb at 37 °C and not at 20 °C, although a very slight shift in data points for pNIPAM + Lys (20 °C) may indicate that very low levels of this protein can adsorb below the LCST. The small size of Lys might allow some protein to become entangled in the coating or small coating defects may provide sites for small, localized protein adsorption. However, results are too close to the confidence limit for us to draw a firm conclusion. Significant peaks at 56, 70, and 84 amu (Figure 5b) are readily attributed to amino acids (Table 1) and indicate that Lys adsorption to pNIPAM does occur at 37 °C. Inspection of the loadings (Figure 5b) shows that in addition to the negatively loading immonium ion peaks identical to those found upon BSA adsorption, a number of others such as C3H4N+ (54.0321 amu), C5H8N+ (82.0735 amu), and C6H8N+ (94.0746 amu) are also apparent. The peak at 82.0735 amu is assigned to C5H8N+ rather than C4H6N2+ which is expected at 82.05 amu and is characteristic of histidine. Lys possesses only 6910

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one histidine residue, and thus, it is not surprising that its characteristic fragments at approximately 81, 82, and 83 amu39 do not feature in our Lys loadings (Figure 3S in the Supporting Information). In addition, two peaks at approximately 54 and 94 amu are not immediately identifiable. Further PCA investigation of lysozyme (adsorbed on Si) vs a pNIPAM coating (Figure S3 in the Supporting Information) showed an absence of these peaks. However, we can rule out that these two peaks originate from small amounts of adventitious contaminants as they are not part of well-documented reference spectra recorded in our laboratory of common contaminants such as silicones, hydrocarbons, and fatty acids, which would be expected to give rise to other, more prominent peaks (unpublished data). One possibility is that these peaks arise from amino acids that are more exposed upon surface contact; the mass spectra of adsorbed proteins are complex and may vary somewhat with the adsorbent surface; further study of protein and amino acid fragmentation on a number of surfaces is required to assess the effects of the adsorbent surface chemistry upon protein fragmentation in TOF-SIMS and identify the origin of some peaks. Our findings are consistent with, and extend, earlier work using TOF-SIMS to investigate proteins on plasma polymerized NIPAM coatings (ppNIPAM)14 and protein adsorption on pNIPAM grafted to a plasma polymer support layer.15 In the cases above, it has been shown that proteins do adsorb above the LCST and do not adsorb (or adsorb far less) protein below the LCST. For PEO, the grafting method and density can have a substantial influence on protein adsorption.11 Not surprisingly, (39) Suzuki, N.; Gamble, L.; Tamerler, C.; Sarikaya, M.; Castner, D. G.; Ohuchi, F. S. Surf. Interface Anal. 2007, 39, 419–426.

Table 1. Key Negatively Loading Fragments (Associated with Protein) from Figures 4b and 5b and Their Associated Parent Amino Acids experimental mass (amu)

characteristic fragment(s)

54.0321 56.0598 58.0199 59.0310 70.0678 74.0609 82.0735 84.0825 94.0746

C3H4N+ C3H6N+ C2H4NO+ C2H5NO+ or CH5N3+ C4H8N+ C3H8NO+ C5H8N+ C5H10N+ C6H8N+

associated amino acid(s)

amino acid composition in BSA (%)

amino acid composition in Lys (%)

Q, M, T, I, K S R K, I, P T

Q(3.3), M(0.8), T(5.8), I(2.5), K(9.9) S(5.3) R(4.3) K(9.9), I(2.5), P(4.6) T(5.8)

Q(2.0), M(2.0), T(4.8), I(4.8), K(4.1) S(8.2) R(8.2) K(4.1), I(4.8), P(2.0) T(4.8)

K, I

K(9.9), I(2.5)

K(4.1), I(4.8)

Table 2. Proposed Molecular Structures and Origins of Key Positively Loading Fragments (Associated with Polymer) from Figures 4b and 5ba

a

Proposed structures arise due to proton addition.

it has been reported that some pNIPAM grafting architectures do not adsorb proteins40 or allow cell attachment41 even at temperatures above the LCST. Our pNIPAM coatings were prepared in quite different ways to those of the two earlier studies,14,15 yet the results were analogous. Use of PCA and investigation of the possibility of changes to the pNIPAM itself from immersion in buffer enable our interpretations to

be more reliably secured than those obtained by univariate analyses. Even when we use highly sensitive detection and analysis protocols, protein adsorption cannot be detected on pNIPAM below the LCST. Thus, in this case, there is full modulation of the interfacial interactions of two model proteins by the surface tethered, stimuli-responsive pNIPAM chains. Switching of the Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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CONCLUSIONS The investigation of submonolayer protein adsorption onto biomaterial coatings is a challenge for surface analytical techniques and is further complicated when the coating of interest shares structural and chemical similarities with proteins. This is particularly relevant for acrylamide based coatings. In this study, we have used TOF-SIMS in combination with principal component analysis to establish the presence or absence of adsorbed proteins on poly(N-isopropylacrylamide) graft coatings. Clear differences were detected in protein adsorption onto pNIPAM coatings incubated at low (20 °C) and high (37 °C) temperature, and peaks responsible for the spectral differences between samples were identified. It was also shown that the pNIPAM samples were not affected by immersion in aqueous solutions, and when PCA loadings plots were used, it was demonstrated that the observed differences after protein exposure at 37 °C were indeed due to adsorbed proteins. ACKNOWLEDGMENT This work was supported by the Australian Commonwealth Government under the Australian Research Councils Special Research Centers Scheme (Special Research Center for Particle and Materials Interfaces).

Figure 5. (a) PCA scores plot of the adsorption of Lys on pNIPAM coatings. (b) Loadings of HC-N (red) and HC-NO (black) fragments for PC1 from positive spectra.

pNIPAM coating provides control such as to either prevent or induce adsorption. Our analysis by TOF-SIMS and PCA provides direct evidence for adsorbed proteins on pNIPAM coatings above the LCST, in agreement with the findings of Heinz et al.15 The methods utilized in this study are now being employed in further work to study the effects of other proteins as well as pNIPAM chain length and density. This will provide information on whether the switching of protein adsorption observed in this study and earlier reports14,15 is invariably maintained for various pNIPAM graft coatings and different proteins.

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NOTE ADDED AFTER ASAP PUBLICATION This manuscript originally posted ASAP on July 17, 2009. Corrections were made to Table 2, and the corrected manuscript posted ASAP on July 21, 2009. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 1, 2009. Accepted July 7, 2009. AC9009337 (40) Mizutani, A.; Kikuchi, A.; Yamato, M.; Kanazawa, H.; Okano, T. Biomaterials 2008, 29, 2073–2081. (41) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506– 5511.