870
Langmuir 2005, 21, 870-881
Plasma Deposition and Surface Characterization of Oligoglyme, Dioxane, and Crown Ether Nonfouling Films Erika E. Johnston, James D. Bryers, and Buddy D. Ratner* University of Washington Engineered Biomaterials, Box 351720 Bagley Hall #484, Seattle, Washington 98195-1720 Received December 3, 2003. In Final Form: September 6, 2004 Plasma-deposited PEG-like films are emerging as promising materials for preventing protein and bacterial attachment to surfaces. To date, there has not been a detailed surface analysis to examine the chemistry and molecular structure of these films as a function of both precursor size and structure. In this paper, we describe radio-frequency plasma deposition of a series of short-chain oligoglymes, dioxane, and crown ethers onto glass cover slips to create poly(ethylene glycol)-like coatings. The resultant films were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), dynamic contact angle goniometry, and radiolabeled fibrinogen adsorption. Detailed analysis of the high-mass (120-300 m/z) TOF-SIMS oligoglyme film spectra revealed six classes of significant fragments. Two new models are proposed to describe how these fragments could be formed by distinct film-building processes: incorporation of intact and fragmented precursor molecules. The models also provide for the incorporation of hydrocarbonsa species that is not present in the precursors but is evidenced in XPS C1s spectra of these films. Two additional models describe the effects of incorporating intact and fragmented cyclic precursors.
Introduction There is a recognized need for chemical treatments that can render a surface resistant to random protein adsorption, bacterial attachment, and subsequent biofilm formation. A detrimental aspect of randomly adsorbed protein layers is their ability to promote bacterial colonization of implant surfaces and other surfaces exposed to aqueous biological environments. Depending on the species present and the growth conditions, bacteria can accumulate to form biofilms from a few micrometers to several centimeters thick. Once formed, bacterial biofilms are often resistant to antibiotics, disinfectants, and oxidants, even at concentrations several orders of magnitude greater than levels observed to be toxic for suspended microbial cultures. In medicine, it is estimated that over half of hospital-acquired bacterial infections are associated with implants or indwelling medical devices; with the caseto-fatality ratio of these infections ranging between 5 and 60%.1,2 In industrial systems, inefficiencies caused by biofilms contribute to significant maintenance and energy costs, costly system downtime, and environmentally damaging chemical control measures.3 Two approaches to preventing infection or biofilm formation are to (1) develop a nonadhesive surface by modifying the surface chemistry or (2) design a material to slowly release an agent that is lethal to the approaching bacterial cells. Here we aim to develop a surface treatment that can prevent nonspecific adsorption of proteins conditioning layers, and thereby reduce or eliminate subsequent bacterial adhesion and biofilm formation. Plasma deposition has been chosen because this method can form tightly adherent, conformal films on a wide variety of surfaces. (1) Dankert, J.; Hogt, A. H.; Feijen, J. Biomedical polymers: bacterial adhesion, colonization, and infection, In CRC Critical Reviews in Biocompatibility 1986, 2, 219-301. (2) Stamm, W. E. Infection related to medical devices. Ann. Intern. Med. 1978, 89, 764. (3) Bryers, J. D. Biofilms II: Process Analysis and Applications; J. Wiley Interscience: New York, 2000.
Nonfouling Aspects of PEG-Like Surface Modifications. Previous studies have indicated that poly(ethylene glycol)-like (PEG-like) surfaces can reduce protein adsorption.4-18 However, the relationship between surface properties and the prevention of protein adsorption is still unclear. The protein resistance of PEG-modified surfaces has been attributed to many factors, including excluded volume effects,19 loss of conformational entropy,13 and osmotic effects as water is driven from the polymer upon approach of the protein.20 There are also hypotheses based on the low PEG/aq interfacial free energy,14 the structuring of water at the PEG/water interface,21 the (4) Cornelius R. M.; Archambault, J. G.; Berry, L.; Chan, A. K.; Brash, J. L. JBMR 2002, 60, 622-632. (5) Price, M. E.; Cornelius, R. M.; Brash, J. L. Biochem. Biophys. Acta 2001, 1512, 191-205. (6) Brash, J. L. J. Biomater. Sci., Polym. Ed. 2000, 11, 1135-1136. (7) Bergstrom, K.; Holmberg, K.; Safranj, A.; Hoffman, A. S.; Edgell, M. J.; Kozlowski, A.; Hovanes, B. A.; Harris, J. M. J Biomed. Mater. Res. 1992, 26, 779-790. (8) Efremova, N. V.; Bondurant, B.; O’Brien, D. F.; Leckband, D. E. Biochem. 2000, 39, 3441-3451. (9) Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci., U.S.A. 1997, 94, 8399-8404. (10) Braatz, J. A.; Heifetz, A. H.; Kehr, C. L. J. Biomater. Sci., Polym. Ed. 1992, 3, 451-462. (11) Llanos, G. R.; Sefton, M. V. J. Biomater. Sci. Polym. Ed. 1993, 4, 381-400. (12) Andrade, J. D.; Herron, J.; Lin, J. N.; Yen, H.; Kopecek, J.; Kopeckova, P. Biomaterials 1988, 9, 76-80. (13) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; Gennes, P. G. D. J. Colloid Interface Sci. 1990, 142, 149-158. (14) Lee, J. H.; Kopecek, J.; Andrade, J. D. J. Biomed. Mater. Res. 1989, 23, 351-368. (15) Gombotz, W. R.; Guanghui, W.; Horbett, T. A.; Hoffman, A. S. J. Biomed. Mater. Res. 1991, 25, 1547-1562. (16) Mori, Y.; Nagaoka, S.; Takiuchi, H.; Kikuchi, T.; Noguchi, N.; Tanzawa, H.; Noishiki, Y. Trans. Am. Soc. Artif. Intern. Organs 1982, 28, 458-463. (17) Desai, N. P.; Hubbell, J. A. J. Biomed. Mater. Res. 1991, 25, 829-843. (18) Sheu, M.-S. Glow discharge immobilization of polyethylene-oxidecontaining surfactants for non-fouling surfaces. Ph.D. Dissertation, University of Washington, 1992. (19) Arakawa, T.; Timasheff, S. N. Biochemistry 1985, 24, 67566762. (20) Klein, J. Makromol. Chem., Macromol. Symp. 1986, 1, 125137.
10.1021/la036274s CCC: $30.25 © 2005 American Chemical Society Published on Web 12/22/2004
Oligoglyme, Dioxane, and Crown Ether Nonfouling Films
Langmuir, Vol. 21, No. 3, 2005 871
absence of ionic groups,21 and optimal polar-nonpolar interactions with proteins.22,23 Most studies conclude that long PEG chains resist protein adsorption better than short chains, but studies of self-assembled monolayers indicate that surfaces that contain short-chain (n ) 2, 3) oligo(ethylene glycol) are sufficient to reduce protein adsorption as long as the packing density of chains exceeds a threshold value.24 Bacterial attachment studies have shown that PEGmodified surfaces can reduce the attachment of several strains of bacteria relative to untreated controls under a variety of culture conditions.25-30 One of these studies indicated that PEG chains as short as three repeat units were capable of reducing bacterial attachment and that long PEG chains may not be required to impart biofouling resistance.29 Lo´pez, et al. first showed that it was possible to impart surfaces with ether-carbon rich, PEG-like functionality by plasma deposition of short chain oligoglyme ((CH3O-(CH2-CH2-O)n-CH3), n ) 1-4) precursors and that tri- and tetraglyme surfaces were able to reduce protein adsorption31,32 Shen, et al., have shown an inverse relationship between deposition power and protein adsorption to tetraglyme films. Measures consistent with preservation of the precursor structure such as high ethercarbon content, high O:C ratio, and 59+, 71+, and 103+ m/z peaks were found to correlate well with low fibrinogen deposition.33 Johnston et al. have shown that both linear and cyclic oligo(ethylene glycol) precursors are capable of resisting protein adsorption and bacterial attachment,34,35 stimulating the current work to more thoroughly characterize PEG-like plasma-deposited films (PDFs) and to use multivariate methods to correlate the results to measures of biofouling resistance. Protein adsorption and bacterial attachment were found to be higher on the crown ether films than the glyme PDFs. Consistent with these results, Denes recently reported that a mixed culture of three bacterial strains attached less readily to triglymepulsed plasma films than to 12-crown-4-pulsed plasma and that both were significantly lower than stainless steel controls.36 Also, Wu et al. have shown that pulsed-plasma
12-crown-4 ether films have good stability in aqueous solution and that their protein resistance can be minimized by optimization of on/off time of the plasma.37 PEG-like plasma-deposited films are of increasing interest because of their protein- and cell-resistant properties. So far there has not been a detailed surface analysis to examine the chemistry and molecular structure of these films as a function of both precursor size and structure. Nor has there been an effort to correlate individual bacterial attachment, detachment, cell division, and cell erosion rates to PEG-like PDF surface properties. Therefore, the current two-part study was undertaken to clarify the relationship between surface properties of plasma-deposited PEG-like films and the ultimate resistance of the films to protein adsorption and initial bacterial attachment and growth rates. Here (Paper I), we report on surface chemical characterization and protein adsorption of oligoglyme, dioxane, and crown ether plasmadeposited thin films. In Paper II, correlations between surface spectral and wettability features, protein adsorption levels, and contributions to Pseudomonas aeruginosa accumulation rates will be detailed.
(21) Merrill, E. W.; Salzman, E. W. ASAIO J. 1983, 6, 60. (22) Coleman, D. L.; Gregonis, D. E.; Andrade, J. D. J. Biomed. Mater. Res. 1982, 16, 381-398. (23) Ratner, B. D.; Hoffman, A. S.; Hanson, S. R.; Harker, L. A.; Whiffen, J. D. J. Polym Sci. Polym. Symp. 1979, 66, 363-375. (24) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (25) Marsh, L. H.; Coke, M.; Dettmar, P. W.; Ewan, R. J.; Hovler, M.; Nevell, T. G.; Smart, J. R.; Smith, J. R.; Timmins, B.; Tsiboukis, J.; Alexander, C. J. Biomed. Mater. Res. 2002, 61, 641-652. (26) Vacheethasanee, K. and Marchart, R. E. J. Biomed. Mater. Res. 2000, 50, 302-312. (27) Humphries, M.; Nemcek, J.; Cantwell, J. B.; Gerrard, J. J. FEMS Microbiol. Ecol. 1987, 45, 297-304. (28) Dunkirk, S. G.; Gregg, S. L.; Duran, L. W.; Monfils, J. D.; Haapala, J. E.; Marcy, J. A.; Amos, D. L. C. R. A.; Guire, P. E. J. Biomater. Appl. 1991, 6, 131-156. (29) Bridgett, M. J.; Davies, M. C.; Denyer, S. P. Biomaterials 1992, 13, 411-416. (30) Desai, N. P.; Hossainy, S. F. A.; Hubbell, J. A. Biomaterials 1992, 13, 417-420. (31) Lo´pez, G. P. Molecular adsorption and the chemistry of plasmadeposited organic films. Ph.D. dissertation, University of Washington, 1991. (32) Lo´pez, G. P.; Ratner, B. D.; Tidwell, C. D.; Haycox, C. L.; Rapoza, R. J.; Horbett, T. A. J. Biomed. Mater. Res. 1991, 26, 415-436. (33) Shen, M.-C.; Wagner, M. S.; Castner, D.; Ratner, B. D.; Horbett, T. A. Langmuir 2003, 19, 1692-1699. (34) Johnston, E. E.; Ratner, B. D.; Breyers, J. D. Polym. Mater. Sci. Eng. 1997, 77, 577. (35) Johnston, E. E.; Ratner, B. D. Mater. Res. Soc. Abstracts, Fall Meeting, Boston, MA, 1998; 464. (36) Denes, A. R.; Somers, E. B.; Wong, A. C. L.; Denes, F. J Appl. Polym. Sci. 2001, 81, 3425-3438.
Experimental Methods Films were deposited using a two-stage (high power, low power) plasma-deposition process to create tightly adherent films that retain high precursor functionality. The films were analyzed by X-ray photoelectron spectroscopy (XPS),38,39 time-of-flight SIMS (TOF-SIMS),40-43 and dynamic contact angle goniometry (DCA).44 1. Plasma-Deposition of PEG-Like Thin Films. A previously described radio frequency plasma reactor was used to deposit PEG-like films onto glass coverslips.45,46 The main reaction chamber consisted of a glass tube 10 cm in diameter and approximately 60 cm in length. The reactor was separated from the vacuum pump by a liquid nitrogen cold trap and a filter to prevent pump oil from backstreaming into the reactor. Plasma reactor walls, inlet, and outlet lines were warmed with heating tapes to diminish condensation and accumulation of plasma byproducts. Flow of argon and oxygen into the reaction vessel was controlled by mass flow controllers. Flow of the precursor vapor was controlled by a mass flow controller with customized internal heating to 100 °C. A thermal pressure transducer was located in the gas feed line and a capacitance pressure manometer, mounted 3 in. downstream of the reactor inlet, measured pressure within the reaction vessel. Feedback control of the reactor pressure was achieved by control of a motorized valve on the exhaust line. Capacitively coupled, external ring electrodes consisted of 1-in. wide copper strips wrapped around the reaction chamber. The active ring electrode was fixed 3 in. downstream from the pressure transducer port and the grounded ring electrode was positioned 6 in. further downstream. A radio frequency generator powered the oscillating electric field to the active electrode, and a variable (37) Wu, Y. J.; Griggs, A. J.; Jen, J. S.; Manolache, S.; Denes, F. S.; Timmons, R. B. Plasmas Polym. 2001, 6, 123-144. (38) Briggs, D.; Seah, M. P., Eds. Practical Surface Analysis, 2nd ed.; John Wiley and Sons: Chichester, 1990; Vol. 1: Auger and X-ray Photoelectron Spectroscopy. (39) Beamson, G.; Briggs, D. High-Resolution XPS of Organic Polymers; J. Wiley and Sons: Chichester, 1992. (40) Vickerman, J. C., Brown, A., Reed, N. M., Eds. Secondary Ion Mass Spectrometry: Principles and Applications; Clarendon Press: Oxford, 1989. (41) Niehuis, E. Time-of-flight SIMS in Second. Ion Mass Spectrom., SIMS 8, Proc. Int. Conf.; Benninghoven, A., Ed.; Wiley: Chichester, 1992; 269-76. (42) Hercules, D. M. J. Mol. Struct. 1993, 292, 49-64. (43) Benninghoven, A. J. Surf. Anal. 1997, 3, 248-251. (44) Berg, J. C. Wettability; Marcel Dekker: New York, 1993; Vol. 49, 531. (45) Lo´pez, G. P.; Ratner, B. D. Plasmas Polym. 1996, 1, 127-151. (46) Johnston, E. E. Surface and biological properties of biofoulingresistant, poly(ethylene oxide)-like plasma deposited films. Ph.D. dissertation, University of Washington, 1997.
872
Langmuir, Vol. 21, No. 3, 2005
Table 1. Plasma Precursors and Deposition Conditions
transformer was tuned to match the impedance of the discharge and reactor to that of the RF generator, thus optimizing power delivery. Plasma films were deposited onto glass coverslips. All coverslips were rubbed with cotton cloth to remove glass fines, then sonicated sequentially in H2SO4/Nochromix solution and distilled, deionized water, then rinsed copiously with more distilled, deionized water. We previously observed that the molecular-ion peak was missing in the TOF-SIMS spectra of crown ether PDFs that were deposited between the electrodes. To better preserve functionality of the cyclic precursors, all samples were placed upstream of the active electrode for these experiments. Plasma precursors, molecular weights, and vapor flow rates are listed in Table 1. Flow rates were chosen to achieve conditions of constant power delivery per unit mass of precursor, W/FMw, where W is the plasma power, F is the precursor flow rate and Mw is the precursor molecular weight. Dioxane and the oligoglymes were acquired from Aldrich (Milwaukee, WI), and the crown ethers were purchased from Sigma (St. Louis, MO). All precursors were of the highest available purity and used as received. To assist interpretation of TOFSIMS spectra (see below), a film of 18-crown-6 was spin cast onto a silicon wafer; 18-crown-6 was chosen because it was a solid at room temperature and could be analyzed under ultrahigh vacuum conditions without requiring cross-linking. 18-Crown-6 was also purchased from Sigma and used as received. Prior to each series of depositions, the glass reactor, glass end cap, and a round-bottom flask modified with a thermocouple well were baked out by heating to 400 °C and cooling overnight. After the reactor was assembled and leak tested, the flask was loaded with ∼10 mL of precursor and sealed to the inlet of the mass flow controller. The reactor was evacuated to a base pressure of 10-20 mTorr and the precursor was degassed by freezing with liquid nitrogen and thawing under vacuum three times or until no bubbles could be seen escaping the liquid. After being degassed, the monomer was either warmed by heat tapes (diglyme, triglyme, tetraglyme, and the crown ethers) or cooled by an ice bath (monoglyme and dioxane) to achieve suitable vapor pressures for the flow controller. After the vapor flow rate was tested, the reactor was evacuated and etched with argon (4.0 sccm, 30 min, 80 W). The samples were then loaded, evacuated to base pressure, etched with argon (10 min, 80 W, 4.0 sccm, ∼130 mTorr), and evacuated again. To deposit films, vapor was introduced to the reactor at a controlled rate. When the reactor pressure reached 200 mTorr,
Johnston et al. the RF generator was activated and the plasma tuned to 80 W power at 250 mTorr to form a tightly adhesive film. Under these conditions, a faint purple glow was visible between the electrodes. After 2 min, the plasma power was reduced to 5 W for 5 min more to form a film that better retained the precursor functionality; the glow was then barely visible. After extinguishing the plasma, the reactor was flooded with argon for 10 min (4.0 sccm, ∼130 mTorr) then evacuated to base pressure to evaporate any condensate. Samples were removed and stored in air in lidded polystyrene dishes. Prior to surface analysis or use in biological studies, samples were soaked for 10 h in either singly or doubly distilled, reverse-osmosis-purified water to remove any extractable material. 2. Surface Analysis. 2.1. X-ray Photoelectron Spectroscopy (XPS). XPS spectra were collected on a Surface Science (Mountain View, CA) X-Probe instrument with a monochromatized Al KR X-ray source and a hemispherical analyzer. The take-off angle was 55° from the surface normal, resulting in a sampling depth of 50 Å. Samples were mounted on an aluminum sample platform with double-sided tape. Charge compensation was provided by a 5-eV flood gun and a fine nickel screen mounted 1-2 mm above the samples. Survey scans for elemental composition and highresolution scans for carbon were collected at two 1000-µm spots per film. XPS data analysis was performed on Surface Science M-Probe software, version 1.31. High-resolution XPS C1s spectra were referenced to the dominant peak in the spectraseither the hydrocarbon at 285 eV or the ether carbon at 286.5 eV. In initial fits, minor peaks were constrained to 288 eV for carbons with two oxygen bonds and to 289.5 eV for carbons with three oxygen bonds. These constraints were removed for final fits. 2.2. Time-of-Flight Secondary Ion Mass Spectroscopy (TOFSIMS). TOF-SIMS measurements were made on a Charles Evans & Associates T-2000 TRIFT instrument. An 11-keV cesium ion source with an average ion current of 2 pA, a 156-ps pulse width, and a 10-kHz repetition rate was used to raster a 65 µm × 65 µm area. Charge compensation was provided by a 24-V, 4-nA pulsed electron beam. Sampling times varied, but the integrated ion dose for both quadrupole and TOF-SIMS was kept below 1013 ions/cm2 in order to maintain static sampling conditions. TOF-SIMS spectra were calibrated to CH3 at 15.023551 amu, C2H3 at 27.023351 amu, and cesium at 132.905476 amu. The spectra were analyzed using the templating feature of PHI’s TOFPAK software version 2.0. In the high-mass region (120-300 m/z) of the template, peaks were defined in 1-amu increments. In the low-mass (1-120 m/z) range, sub-amu peaks were defined corresponding to stoichiometric combinations of 12C, 1H, and 16O. Additionally, peaks corresponding to other observed trace elements, such as Na+, K+, and Si+, were identified and included in the template. The template was applied to each spectrum to integrate the counts at each defined peak. Relative abundances (RA) were calculated by normalizing the total ion count of each 0-300 m/z spectrum to 106. Analyses of the TOF-SIMS spectra are based on a single spectrum from each material. 2.2.1. Filtering of High-Mass (120-300 m/z) Regions. A common challenge in interpreting high-mass regions of SIMS spectra is selecting significant peaks for analysis. In this study a moving average filter was applied to identify significantly strong peaks for interpretation. Odd and even mass peaks were filtered separately. The filter calculated the average and standard deviation of the five odd (even) peaks directly preceding and following an odd (even) peak in question. Each peak in the 120300-amu region was tested in succession. Only peaks greater than two standard deviations above the calculated local average were selected for interpretation. 2.3. Wilhelmy Plate Dynamic Contact Angle Goniometry. Advancing and receding contact angles were measured by the Wilhelmy plate technique using a commercial dynamic contact angle analyzer (Model DCA-312 Cahn, Cerritos, CA). Measurements were performed using HPLC grade water (JT Baker) and acid-cleaned glass beakers. Cleanness of the system was ensured by determining that the advancing and receding contact angle of an acid-cleaned and flamed glass coverslip was lower than 5°. Samples consisted of thin films that had been plasma-deposited simultaneously onto both sides of inch-square glass coverslips. Each sample was dipped in several changes of water and then soaked overnight for 10 h. Then, using a fresh change of water,
Oligoglyme, Dioxane, and Crown Ether Nonfouling Films
Langmuir, Vol. 21, No. 3, 2005 873
Table 2. Summary of XPS Atomic Content and Contact Angle Measurements for Various PDF Coating O:C ratio atomic percent precursor
%C
%O
%Si
monoditritetra, 5 W tetra, 20 W dioxane 12C4 15C5, 5 W 15C5, 20 W
61.1 67.3 68.9 67.2 68.4 74.3 68.3 66.6 66.6
33.5 32.3 31.1 32.8 31.7 23.8 31.8 32.3 32.4
5.4 0.5
a
%Cl
Meas.
assume C-OR
1 1
0.37 0.47 0.45 0.49 0.46 0.26 0.47 0.48 0.49
0.41 0.47 0.43 0.48 0.48 0.27 0.48 0.49 0.48
2.4
contact angles
hysteresis
assume C-OH
Adv high speeda
Adv low speeda
Rec high speeda
Rec low speeda
high speed, low speeda
0.69 0.84 0.77 0.90 0.91 0.43 0.87 0.89 0.91
68 65 60 70 69 64 52 69 69
66 62 56 61 59 61 44 60 56
42 44 37 29 31 21 30 29 25
43 45 36 23 29 20 29 19 24
26, 23 21, 17 23, 20 41, 38 38, 30 43, 41 22, 15 40, 41 44, 32
high speed ) 264 µm/sec; low speed ) 20 µm/sec.
advancing (ΘAdv) and receding (ΘRec) contact angles were measured, first at high (264 µm/s) and then at low (20 µm/s) immersion speeds. Travel distance was approximately 1 cm. All measurements were performed at 24 °C. Humidity was not controlled. 2.4. 125I-Radiolabeled Protein Adsorption and Retention. Films of monoglyme, diglyme, triglyme, tetraglyme, dioxane, 12-crown4, and 15-crown-5 were deposited as described in the preceding section onto both sides of acid-cleaned 9-mm glass disks. Poly(tetrafluoroethylene) (PTFE) controls consisted of disks stamped from skived PTFE films (Berghof America, Concord, CA). Glass disk controls were identical to those used as substrates for plasmadeposited films. Both glass and PTFE controls were cleaned in H2SO4/Nochromix solution, rinsed with reverse-osmosis-purified water, and dried immediately prior to use in protein adsorption studies. Human fibrinogen (Fb, 95% clottable, KABI, Franklin, OH) was radiolabeled with Na125I (Amersham Corp., Arlington Heights, IL) according to an iodine monochloride method previously described.47 All protein adsorption and rinsing was performed with degassed citrate phosphate buffered saline (dCPBS: 3.5 g NaCl, 1.05 g citric acid, 0.69 g NaH2PO4‚H2O, 0.79 g NaOH, pH adjusted to 7.4 by addition of NaOH). Freshly radiolabeled protein was purified from free 125I by two passes through gel permeation chromatography columns (BioRad Econopac 10DG) and stored at -70 °C overnight prior to use. A 0.1-mg/mL fibrinogen solution at 37 °C was spiked with radiolabeled protein to achieve a solution of 16 cts/ng Fb‚min. RF plasma-deposited samples were soaked 10 h in reverseosmosis-purified water to remove extractable components from the plasma polymer. Samples and controls were then placed in 2-mL conical polystyrene cups and equilibrated in dCPBS for 4 h at 37 °C. The buffer was aspirated and replaced by 1 mL of 0.1 mg/mL 125I-radiolabeled fibrinogen solution. After a 2-h incubation, the samples were rinsed by displacement with 12 pulses of dCPBS buffer and transferred to polystyrene counting tubes containing 2 mL of sodium dodecyl sulfate (SDS) surfactant solution (3% w/v SDS in 0.003 M H3PO4, 0.01 M Tris, pH 7). Sample radioactivity was determined by counting γ emissions for 10 min with a Gamma Trac gamma counter (Model 1185, TM analytical, Inc.). Protein adsorption levels were calculated by dividing the sample’s radioactivity (after subtracting the background signal) by the specific γ activity of the fibrinogen and the planar area of the sample. After 24 h ,the samples were removed from the surfactant solution, rinsed by dipping 12 times in reverse-osmosis-purified water, and the retained protein levels determined from γ emissions.
Results and Discussion 1. XPS Surface Analysis. Elemental compositions were averaged from two replicates of each PDF sample. Results are presented in Table 2. As expected, all films were composed primarily of carbon and oxygen. Traces of chlorine (1%) were detected in the 15-crown-5 films and may have originated in the precursor liquid. Silicon from the glass substrate was detected in the monoglyme (5.4%), (47) Kiaei, D.; Hoffman, A. S.; Horbett, T. A.; Lew, K. R. J. Biomed. Mater. Res. 1995, 29, 729-739.
diglyme (0.5%), and dioxane (2.4%) films, suggesting that these films were thinner than the ∼50-Å sampling depth. Subsequent angle-dependent XPS analysis of a monoglyme film revealed that the silicon signal decreased exponentially with the cosine of the takeoff angle, indicating an intact PDF overlayer. A useful indicator of the retention of precursor stoichiometry in PEG-like plasma-deposited films is the O:C ratio, presented in Table 2. Silicious oxygen (SiO2) in the monoglyme, diglyme, and dioxane PDF spectra was accounted for by subtracting twice the % silicon content from the measured oxygen content of each film. The expected O:C ratio based on precursor stoichiometries is 0.5. O:C ratios of 0.45-0.49 were observed for all but the monoglyme and dioxane films, which had O:C ratios ) 0.37 and 0.26, respectively. The fact that the glass substrate was observed in the monoglyme and dioxane spectra implies that the interfacial film deposited at 80 W was being sampled. The interfacial layer is, by design, more tightly cross-linked and probably enriched in carbon, therefore contributing to the attenuation of O:C. The O:C ratio of the dioxane film is markedly lower than that of the monoglyme film. Considering that dioxane is cyclic and has fewer vibrational degrees of freedom available to dissipate energetic impacts sustained in the plasma glow, dioxane is probably more susceptible to damage than monoglyme. Relaxation by bond scission and subsequent oxygen loss also likely contributed to lower O:C ratios in the dioxane PDFs. Retention of PEG-like functionality in the oligoglyme and crown ether films is evidenced in the XPS C1s envelopes shown in Figures 1 and 2. The C1s spectra were resolved into four Gaussian peaks at 285.0, 286.5, 288.0, and 289.4 eV corresponding, respectively, to carbon sharing zero (i.e., hydrocarbon), one (C-OH or C-OR), two (CdO or O-C-O), and three (O-CdO) bonds with oxygen. The prominent peak for all PDFs, except dioxane, occurs at 286.5 eV, which is indicative of carbon singly bound to oxygen and existing as either hydroxyl carbon (C-OH) or ether carbon (C-OR). To correctly assign the functionality of 286.5 eV, the measured O:C ratio was compared to an O:C ratio calculated from the deconvoluted C1s peaks. To calculate the O:C ratio from the C1s envelope, it was assumed that each carbon in the 285.0-eV peak was bound to no oxygen, that the carbon in the 286.5-eV peak was bound to either one oxygen (assuming C-O-H) or 0.5 oxygen (assuming C-O-R), that the carbon in the 288.0-eV peak was bound to one oxygen (assuming CdO), and that the carbon in the 289.4-eV peak was bound to two oxygen atoms (assuming O-CdO). The O:C ratios were then calculated by dividing the sum of estimated oxygens by the sum of C1s counts. In Table 2, close agreement between measured O:C values and those calculated using the ether carbon assumption indicates
874
Langmuir, Vol. 21, No. 3, 2005
Figure 1. XPS C1s environment of oligoglyme plasma deposited films. The highest peak in each spectrum is normalized to 10 000 counts.
Figure 2. XPS C1s environment of dioxane and crown ether plasma-deposited films. The highest peak in each spectrum is normalized to 10 000 counts.
that the 286.5-eV peak can be attributed to ether carbon for all PDFs. Therefore, the dominance of the 286.5-eV peak reflects retention of the ether carbon functionality that is present in all of the precursors. The C1s spectrum of PEG consists of a single peak at 286.5 eV;39 hence, retention of this peak is considered a minimum requirement for achieving PEG or PEG-like materials. Figure 3 shows the separate contribution of the fitted peaks to the C1s envelope. The C1s environments of tetraglyme and 15-crown-5 films are virtually identical and contain the highest ether carbon contributions of 8085%. The hydrocarbon (10.6-12.5%), carbonyl carbon (3.2-5.4%) and carboxyl or ester carbon (0.9-1.4%) contents for the tetraglyme and 15-crown-5 films are also similar. Elevated hydrocarbon contents among the monoglyme and dioxane films occur largely at the expense of ether carbon content. Models in Figures 4-7 illustrate how incorporation of precursor molecules and fragments can introduce hydrocarbon and carbon doubly bound to oxygen through film formation processes. Figures 4 and 5 show that incorporation of intact precursor preserves ether carbon content. Figures 6 and 7 show that incorporation of precursor fragments can either incorporate ether carbon, or incorporate carbon with zero or two oxygen bonds with simultaneous loss of ether carbon functionality. Therefore, the source of hydrocarbon and carbon with two oxygen bonds in the C1s spectra of plasma-deposited PEG-like films could be due to the incorporation of simple precursor fragments into the growing film.
Johnston et al.
2. TOF-SIMS. 2.1 Low-Mass Region 1-120 m/z. 2.1.1. Retention of Plasma Precursor Structure in Oligoglyme and Crown Ether Films. The primary difference between the 1-120 m/z regions of the positive ion SIMS spectra of oligoglyme and cyclic precursor PDF coatings (Supporting Information, Supplements 2 and 3, respectively) is the dominance of the 59+ peak in the oligoglyme PDF spectra. Dominant 59+ peaks have been previously observed in quadrupole SIMS spectra of plasma-deposited oligoglyme films and attributed to [C3H7O]+ and/or [C2H3O2]+.45 Here we confirm that the observed 59+ peak is centered at 59.050+ m/z, corresponding to [C3H7O]+. This stoichiometry is consistent with methyl-terminated chain ends of the oligoglyme precursor, and the assignment is supported by the presence of repeat peaks at 59 + n44 corresponding to 103+ m/z, 147+ m/z, etc. The abundance of the 59.050+ counts is attributed to fragment formation by inductive cleavage of a carbon-oxygen bond with preferential cationization and detection of the carbonterminated fragment.48 A decrease in the 59.050+ contribution to the tetraglyme 20-W film indicates a slight reduction of the methyl-terminated chain ends deposited under more-energetic plasma conditions. In contrast with the oligoglyme films, the low-mass region of crown-ether PDF coatings is dominated by peaks at 45.034+ and 43.018+ amu, corresponding to [C2H5O]+ and [C2H3O]+. Figure 8 shows the individual contributions of 43.018+, 45.034+, and 59.050+ to the PDF coatings and the 18-crown-6 spin-cast film spectra. The similarity between contributions from 43.018+, 45.034+, and 59.050+ in the plasma-deposited crown ether films and the spincast crown ether films suggests that the structural environment of the -[CH2CH2O]- repeat unit is also similar, i.e., that the ring structure remains intact. 2.1.2. Shard Analysis of PEG Backbone-Like Character. The dominant 45+ peak in the crown ether C3 cluster is a property shared with spectra of hydroxyl-terminated PEG (HO-PEG-OH).49 This observation raises the question of whether the shared structural characteristic is due to the hydroxyl-terminated chain ends, indicative of ring cleavage in the crown ether film, or the influence of the PEG backbone, indicative of retention of the ring structure. In a study of hydroxyl-terminated PEGs varying in molecular weight, Shard, et al.49 found that as the polymer molecular weight increased from 1000 to 100 000 Da, the positive ion quadrupole SIMS ratio 45/(43 + 45) decreased from 0.85 to a plateau level of 0.7. These results suggest that hydroxyl-terminated chain ends increase the 45+ ion intensity relative to the 43+ ion. To compare our TOFSIMS results with the quadrupole-SIMS data of Shard, both 43.018 [C2H3O+] and 43.055 [C3H7+] were combined and used in the 45/(43 + 45) denominator. Results are shown in Figure 9. Oligoglyme PDF coating results appear to extend a trend observed for low-molecular-weight methoxy-terminated PEG-amines. The crown ether films and the 18-crown-6 spin-cast film all have similar 45/ (43 + 45) ratiossslightly less than the 0.7 “backbonelike” value observed for high-molecular-weight HO-PEGOH. These results suggest that the plasma deposition process does not fragment either oligoglyme or crown ether molecules so extensively as to introduce significant numbers of hydroxyl-terminated chain ends. This is consistent with the XPS C1s analysis, indicating that most carbon singly bound to oxygen is in the form of ether carbon. The comparison with Shard’s results must be (48) McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra, 4th ed.; University Science Books: Sausalito, CA, 1993. (49) Shard, A. G.; Davies, M. C.; Schacht, E. SIA 1996, 24, 787-93.
Oligoglyme, Dioxane, and Crown Ether Nonfouling Films
Langmuir, Vol. 21, No. 3, 2005 875
Figure 3. Results of XPS C1s peak fitting.
Figure 4. Models illustrating incorporation of oligoglyme precursor molecules into previously deposited film.
Figure 5. Model illustrating incorporation of intact cyclic molecule into a previously deposited film; ether carbon content is preserved.
viewed with some caution, as their data were taken on different instruments with different ionization environments and detection characteristics. Nevertheless, the similarity between the results is worth noting. 2.1.3. Distinct Dioxane PDF Character. In Figure 9, the dioxane 45/(43 + 45) ratio is distinct from the other PDF coatings. The abundance of [C3H7]+ ions at 43.055 amu decreases the 45/(43 + 45) ratio to 0.27. To find an analogue to dioxane, the spectrum was compared with published quadrupole SIMS spectra of oxygen-containing model polymers: polyacrolien, poly(ethylene glycol), poly(propylene glycol), poly(methacrylic acid), poly(oxy-methylene), poly(methyl vinyl ether), and poly(vinyl alcohol).50 Of these, only poly(vinyl alcohol) (PVA) shared the dioxane PDF trend in C3 cluster peak heights: 43+ > 41+ > 45+. The major peaks in PVA’s C2, C4, and C5 clusters also
occurred at the same masses as those of the dioxane film. Whether the major peaks in PVA and dioxane PDF correspond to identical stoichiometries could not be determined due to the lower resolution of the quadrupole SIMS spectra. Comparisons between PVA and dioxane PDF cannot be used too literally to assign surface structure as it is unlikely that dioxane is polymerizing to PVA within the plasma glow. Rather, the similarity suggests that the outermost 15-20 Å of dioxane films and PVA share structural featuressin this case, hydroxyl groups pendant to a hydrocarbon backbone or matrix rather than terminating chains. The obvious difference between PVA and dioxane PDF spectra is the presence of the dioxane molecular ion [M - 1]+ at 87+ amu. Whether the molecular fragment is intact in the cyclic form or incorporated as a linear species after ring opening, the group undoubtedly provides much of the ether carbon content detected in the XPS spectra. 2.1.4. Sources of Hydrocarbon Fragments. The contributions of low-mass hydrocarbon fragments are also distinct among the different films. Figure 10 shows contributions of selected hydrocarbon fragments to the positive ion TOF-SIMS spectra. The percent of total ion (50) Newman, J. G., Carlson, B. A., Michael, R. S., Moulder, J. F., Hohlt, T. A., Eds. Static SIMS Handbook of Polymer Analysis; PerkinElmer Corp.: Eden Prairie, MN, 1991.
876
Langmuir, Vol. 21, No. 3, 2005
Johnston et al.
Figure 6. Models illustrating incorporation of oligoglyme fragments into previously deposited films.
Figure 7. Incorporation of cyclic precursor after ring opening between either C-C or C-O bonds.
count attributable to CH3+ and the monounsaturated fragments C2H3+, C3H5+, C4H7+, and C5H9+ are shown. Monounsaturated fragments are more abundant than either saturated or multiply unsaturated fragment ions. The 18-crown-6 spin-cast film has strong peaks attributable to CH3+ and C2H3+ fragments, both of which could be abstracted from CH2CH2O repeat units in the crown ether backbone. The strong C3H5+ peak in the 18-crown-6 spectra indicates that rearrangement of carbon bonds occurs during selvedge processes. Very low counts from C4H7+ and C5H9+ suggest that the extent of rearrangement during selvedge processes is limited. In the dioxane and crown ether PDF coatings, C3H5+ peak counts outnumber CH3+ and C2H3+, reflecting the cross-linking occurring during plasma processing. C3 carbon clusters can be created by incorporation of either fragmented cyclic molecules, as seen in Figure 7, or by incorporation of intact cyclic precursors, as seen in Figure 5. C4H7+ and C5H9+ structures are strongest in dioxane films reflecting the more intense rearrangement of carbon bonds and extraction of oxygen from the intact precursor. This result is consistent with the high hydrocarbon content observed in the dioxane films.
The oligoglyme films all exhibit similar hydrocarbon functionality with CH3+ ≈ C3H5+ > C2H3+ > C4H7+ > C5H9+. Greater CH3+ intensity in the oligoglyme films is consistent with abundant methyl-terminated chains. High C3H5+ counts probably reflect contributions from incorporation of precursor fragments, as illustrated in Figure 6, and selvedge rearrangements, as observed in the spincast 18-crown-6 spectrum. 2.1.5. Film Thickness. Absence of a 27.977+ silicon peak in the dioxane spectra indicates that although the films are thin, as noted in the XPS analysis, they are still thicker than the ∼15-20-Å SIMS sampling depth. A small peak (0.2%) at 27.977+ in the monoglyme PDF spectra indicated that the films were thinner than the ∼15-Å sampling depth and that silicon from the glass substratum was detectable. This observation corroborates XPS evidence that the monoglyme films were thin. 2.2. High-Mass Region 120-300 m/z. As previously mentioned, a moving average filter was applied to select peaks from the 120-300 m/z region (Supporting Information, Supplements 4-7) to ease interpretation. Peaks that were two standard deviations above the local baseline are listed in Supplement 8 for the oligoglyme films and
Oligoglyme, Dioxane, and Crown Ether Nonfouling Films
Langmuir, Vol. 21, No. 3, 2005 877
Figure 8. Contributions to positive ion TOF-SIMS spectra from peaks occurring at 43.018, 45.034, and 59.050 m/z.
Figure 9. 45/(43 + 45) versus molecular weight of plasma-deposited film precursors and spin-cast 18-crown-6 (TOF-SIMS, this work) and PEG polymers (quadrupole SIMS; Shard, et al., ref 55).
Supplement 9 for the cyclic films and the 18-crown-6 spincast control. Accompanying each peak is a note that describes how that fragment differs in mass from the molecular ion. Directly below this note are tentative assignments based upon combinations of the molecular ion (M), the (CH2CH2-O) repeat unit (R), the incorporation of CH2 methylene groups, and degrees of unsaturation by loss of hydrogen. Stabilized carbocations are suggested for some even mass peak assignments. 2.2.1. Cyclic Precursors. 2.2.1.1. Dioxane. Dioxane PDFs exhibit a fragmentation pattern distinct from both the oligoglyme and crown ether films. Above 120 m/z, significant counts are detected at 128+, 152+, 165+, 166+, 178+, 202+, and 252+ m/z. These masses do not correspond to simple combinations of dioxane molecules, CH2CH2O repeat units, or to simple saturated hydrocarbon species. Rather, the 128+, 152+, 165+, and 178+ mass fragments probably correspond to aromatically stabilized carbocations and radical cations, as have been observed in the 100-200 m/z region of polystyrene50 and styrene PDFs.51 Leggett attributed the fragments observed at these masses in styrene PDFs to naphthalene radical, biphenylene
radical, fluorene carbocation, and anthracene radical cations, respectively.51 Likely building blocks for the naphthalene fragment include methylene radicals and other small organic ions and radicals commonly created during fragmentation of organic precursors in the plasma phase. Assignment of the 202+ fragment is slightly ambiguous since the mass also corresponds to that of a pair of dioxane molecules modified by a pair of methylene groups. However, by analogy to the naphthalene and anthracene radical ions at 128+ and 178+ m/z, the 202+ and 252+ m/z peaks may represent quinoid additions to the 152+ fragment. Of the high-mass fragments occurring in polystyrene and styrene PDFs, only the two fragments attributed to seven-membered rings (91+ and 141+)47 and the three fragments corresponding to nominally intact styrene repeat units (103+, 117+, 193+)46 are not detected in significant amounts in the dioxane PDFs. The peak at (51) Leggett, G. J.; Ratner, B. D.; Vickerman, J. C. Surf. Interface Anal. 1995, 23, 22-28.
878
Langmuir, Vol. 21, No. 3, 2005
Johnston et al.
Figure 10. Contribution of representative hydrocarbon fragments to the positive ion TOF-SIMS spectra.
91+ corresponds to the highly stable C7H7+ tropylium ion that is prominent in mass spectra of aromatic compounds. Its absence suggests that little aromaticity exists in the dioxane PDF. This conclusion is supported by the absence of a π-π* shake-up satellite in the XPS C1s spectra. These aromatic fragments must be present in very small amounts, but due to the inherent stability of these carbocations, their detection may have been artificially enhanced. 2.2.1.2. Crown Ethers. Significant peaks in the 120300 m/z region of the crown ether PDFs are listed in Supplement 9. [M - 1]+ and [M - 2]+ are observed in all crown ether PDFs. [M - 1]+ peaks are conspicuously absent from the spectra of 18-crown-6 spin-cast films, leading to the conclusion that the loss of a hydrogen is part of the process of incorporating molecular species into the plasma-deposited layer. As with the oligoglyme films, loss of hydrogen from the precursor molecule would free a site on a carbon atom for covalent bonding of the molecule to the surface. It is difficult to discern whether the fragment incorporates primarily in its native cyclic configuration or as a chain via backbone scission since the fragment ions would be of equivalent molecular weight. The ability of the films to bind ions would suggest preservation of the crown ether cyclic conformation since ion binding by crown ethers is predicated on the arrangement of oxygen atoms to form a circular cavity into which cations of a corresponding radii easily fit. XPS results50 shows that sodium and potassium cations are observed in much higher levels in the crown ether spectra (0.1-4.5%) than in the oligoglyme (0.1-0.2%) or dioxane PDFs (
