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Mar 24, 2010 - 601 West Main Street, Richmond, Virginia 23284, and ‡Department of Chemistry,. Virginia Commonwealth University, 1001 West Main Stree...
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Quantifying Surface-Accessible Quaternary Charge for Surface Modified Coatings via Streaming Potential Measurements Murari L. Gupta,† Kennard Brunson,† Asima Chakravorty,† Pinar Kurt,† Julio C. Alvarez,‡ Fernando Luna-Vera,‡ and Kenneth J. Wynne*,† †

Department of Chemical and Life Science Engineering, Virginia Commonwealth University, 601 West Main Street, Richmond, Virginia 23284, and ‡Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, Virginia 23284 Received December 23, 2009. Revised Manuscript Received March 10, 2010

Prior research established that P[AB]-copolyoxetane polyurethanes with soft blocks having A = trifluoroethoxy (CF3CH2-O-CH2-, 3FOx) and B=dodecylammonium-butoxy (C12) are highly effective as polymer surface modifiers (PSMs). These PSMs displayed high contact antimicrobial efficiency against spray challenge that was attributed to surface concentration of quaternary charge. Herein, using a novel cell design and polymer coating process, streaming potential (SP) measurements are reported for estimating accessible surface charge density. Fused-silica capillaries were embedded in flat polypropylene sheets, and the inner capillary walls were coated with neat HMDI-BD(30)-P[(3FOx)(C12)-87:13-5100] (PU-1) and 1 wt % PU-1 in HMDI-BD(50)-PTMO-1000 (base polyurethane 2). Effects of annealing (60 °C) and electrolyte flow cycles on near-surface quaternary charge concentration were determined. Neat PU-1 had a constant SP that was cycle-independent and actually increased on annealing. As-cast 1 wt % PU-1 showed initial SPs about half those for neat PU-1, with substantial attenuation over 16 measurement cycles. SPs for annealed 1 wt % PU-1 displayed lower initial values that attenuated rapidly over multiple cycles. Zeta potentials and surface charge densities were calculated from SPs and discussed relative to contact antimicrobial properties. Tapping mode atomic force microscopy (TM-AFM) imaging was employed for investigation of 1 wt % PU-1 surface morphology. Microscale phase separation occurs on annealing 1 wt % PU-1 for 24 h at 60 °C. Surprisingly, phase separation was also observed after short immersion of 1 wt % PU-1 coatings in water. The morphological changes are correlated with instability of near-surface charge found by SP measurements. A model is proposed for near-surface spinodal decomposition of metastable as-cast 1 wt % PU-1. The formation of a fluorous modifier rich phase apparently sequesters near-surface quaternary charge and accounts for temporal instability of antimicrobial properties. The results are important in providing a facile method for screening polycation-based, contact antimicrobial coatings for accessible charge density and in assessing durability.

Introduction Because the development of contact antimicrobial surfaces is important to human health, surface concentration of polycation charge for contact kill has been investigated broadly. It is important to distinguish contact kill that results from bacteria impinging on a surface, such as one containing covalently attached alkylammonium groups or other biocides, from biocide release, which occurs when biocides are leached from polymer-biocide mixtures. Over 30 years ago, the work of Isquith and McCollum focused on alkoxy(aminopropyl)silanes that form contact antimicrobial films on glass.1 More recently, the trialkoxy silane function has been used to form quaternized coatings on silicones.2,3 The trialkoxy group also facilitates attachment of well-defined polycations.4 Alternative methods for generating polycation nanofilms5-7 *To whom correspondence should be addressed. Telephone: þ1 804 828 9303. Fax: þ1 804 828 3846. E-mail: [email protected]. (1) Isquith, A. J.; McCollum, C. J. Appl. Environ. Microbiol. 1978, 36, 700–704. (2) Gottenbos, B.; van der Mei, H. C.; Klatter, F.; Nieuwenhuis, P.; Busscher, H. J. Biomaterials 2002, 23, 1417–1423. (3) Kugler, R.; Bouloussa, O.; Rondelez, F. Microbiology-SGM 2005, 151, 1341–1348. (4) Huang, J. Y.; Koepsel, R. R.; Murata, H.; Wu, W.; Lee, S. B.; Kowalewski, T.; Russell, A. J.; Matyjaszewski, K. Langmuir 2008, 24, 6785–6795. (5) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981–5985. (6) Lin, J.; Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol. Lett. 2002, 24, 801–805. (7) Lin, J.; Qiu, S. Y.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2003, 83, 168–172. (8) Lee, S. B.; Koepsel, R. R.; Morley, S. W.; Matyjaszewski, K.; Sun, Y. J.; Russell, A. J. Biomacromolecules 2004, 5, 877–882.

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include surface-initiated polymer brushes8,9 and polyelectrolyte layers.10 Development of coatings with micrometer scale thicknesses has utilized alkyammonium side chain polymers.11,12 However, quaternary function in the bulk does not contribute to contact bacterial kill and may jeopardize conventional coating durability. The development of coating surface modifiers offers the preservation of bulk properties and conventional processing while introducing target surface functionality. Sauvet et al. developed antimicrobial poly(dimethylsiloxane) elastomers by incorporating a copolymer telechelic having alkylammonium side chains into conventional resin cure.13 Second, Tiller et al. modified a UV-cured poly(methyl methacrylate) with a quat-containing macromer.14 The attractive features of “processing friendly” surface modifiers have motivated our research in polyurethanes.15-17 Prior (9) Murata, H.; Koepsel, R. R.; Matyjaszewski, K.; Russell, A. J. Biomaterials 2007, 28, 4870–4879. (10) Lichter, J. A.; Rubner, M. F. Langmuir 2009, 25, 7686–7694. (11) Haldar, J.; An, D.; Alvarez de Cienfuegos, L.; Chen, J.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17667–17671. (12) Park, D.; Wang, J.; Klibanov, A. M. Biotechnol. Prog. 2006, 22, 584–589. (13) Sauvet, G.; Dupond, S.; Kazmierski, K.; Chojnowski, J. J. Appl. Polym. Sci. 2000, 75, 1005–1012. (14) Waschinski, C. J.; Barnert, S.; Theobald, A.; Schubert, R.; Kleinschmidt, F.; Hoffmann, A.; Saalwachter, K.; Tiller, J. C. Biomacromolecules 2008, 9, 1764–1771. (15) Makal, U.; Wynne, K. J. Langmuir 2005, 21, 3742–3745. (16) Makal, U.; Wood, L.; Ohman, D. E.; Wynne, K. J. Biomaterials 2006, 27, 1316–1326. (17) Wynne, K. J.; Makal, U.; Kurt, P.; Gamble, L. Langmuir 2007, 23, 10573–10580.

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Figure 1. Schematic of P[AB]-copolyoxetane polyurethane surface modification, soft block structure, and notation.

work has shown that polyurethane soft blocks are surfaceconcentrated.18-20 Thus, we have leveraged this tendency and employed functional soft blocks, namely, P[AB]-copolyoxetanes, in a new approach to surface modification. A model for this concept applied to introducing quaternary surface function into polyurethane blends is shown in Figure 1. Here, the surface concentrated functional soft block of the polymer surface modifier (PSM) is shown in red along with side chains A and B. In addition to surface modification, P[AB]-copolyoxetane polyurethanes themselves have proved interesting by virtue of unexpected A-B side chain interactions that have led to new surface phenomena such as contraphilic wetting.21 The model shown in Figure 1 has been used to guide the development of hydantoin and quaternary alkylammonium antimicrobial PSMs.16,22,23 Herein, we focus on the surface dynamics of a specific PSM, namely, the P[(3FOx)(C12)]-copolyoxetane polyurethane PU-1, whose soft block structure 1 is shown in Figure 1. This modifier results in contact kill due to the presence of quaternary surface functionality.22,23 At present, the surface gradient of modifier/bulk composition is unknown, but assuming efficient modifier surface concentration the outer PSM layer for a conventional 50 μm coating at 1 wt % PSM is ∼0.5 μm or 500 nm. Compositional economy is thus ensured by using the P[AB]-soft block polyurethane as a minor constituent in a blend (Figure 1). Such economy via a blend approach was discussed by Mayes et al., who modified poly(vinylidene fluoride) membranes for resistance to protein adsorption.24 Quantification of accessible quaternary ammonium charge is of critical importance in understanding biocidal efficacy. PSM structure/near-surface charge correlations are essential in guiding the choice of P[AB]-copolyoxetane polyurethane surface modifiers so as to optimize efficacy and durability. Measurement of quaternary charge density has most often employed fluorescein dye binding and subsequent release of bound dye by an ion exchange surfactant such as dodecyl trimethyl ammonium (18) Andrade, J. D.; Tingey, K. G. Langmuir 1991, 7, 2471–2478. (19) Garrett, J. T.; Lin, J. S.; Runt, J. Macromolecules 2002, 35, 161–168. (20) Ratner, B. D.; Yoon, S. C.; Kaul, A.; Rahman, R. In Polyurethanes in biomedical engineering II; Planck, H., Syre, I., Dauner, M., Egbers, G., Eds.; Elsevier: New York, 1986; Vol. 3, pp 213-229. (21) Makal, U.; Uilk, J.; Kurt, P.; Cooke, R. S.; Wynne, K. J. Polymer 2005, 46, 2522–2530. (22) Kurt, P.; Gamble, L.; Wynne, K. J. Langmuir 2008, 24, 5816–5824. (23) Kurt, P.; Wood, L.; Ohman, D. E.; Wynne, K. J. Langmuir 2007, 23, 4719–4723. (24) Hester, J. F.; Banerjee, P.; Mayes, A. M. Macromolecules 1999, 32, 1643–1650.

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chloride.3,5,9,25,26 We have been unsuccessful in adapting this method to quantify surface charge for polyurethanes containing PSMs such PU-1 (Figure 1). We attribute this failure to nonspecific dye adsorption and slow desorption from coatings that have thicknesses of tens of micrometers. Streaming potential measurements have long been known for quantifying surface charge.27 Assessing surface accessible charge by the streaming potential method was elegantly demonstrated for thin films formed by alternating polyelectrolyte deposition.28 Here, the alternating positive and negative potentials attest to the charge of the last polyeletrolyte deposited. Rubner and Lichter have also employed measurement of streaming potentials for studying antimicrobial function of polyelectrolyte multilayers comprising poly(allyl amine) hydrochloride and poly(sodium 4-styrene sulfonate).10 Streaming potential measurements have been carried out by Alvarez et al. for label-free detection of heparin and streptavidin in microfluidic channels made of cyclic olefin copolymer (COC).29 Below, we describe an adaptation of this method employing a novel microfluidic channel design and polymer coating process that provides reproducible streaming potentials on neat and modified polyurethanes containing soft block 1. The kinetic and thermal stability of accessible surface charge density is correlated with tapping mode atomic force microscopy (TM-AFM) imaging of surface morphology and phase separation. Microfluidic capillary streaming potential (MCSP) measurements are very promising for linking alkylammonium PSM composition and structure to surface concentration of quaternary charge in conventional coatings. Acronyms/Designations. The formula for the polyurethane PSM containing 1 is H12MDI-BD(30)/P[(3FOx)(C12)-87:135100]; this is designated PU-1, where H12MDI-BD represents the hard block derived from 4,40 -(methylene bis(p-cyclohexyl isocyanate) (H12MDI) and butanediol (BD) followed by wt % (30); “P” indicates polymerized monomer-in-telechelic followed by ratios of repeat units (m:n); the repeat unit designation “C12” stands for the 2-decylammonium butoxymethyl-2-methyl 1,3propylene oxide segment; 5100 is the Mn for soft block 1. Complete structural representations for 1 and PU-1 are in Figure 1; 2 is provided in the Supporting Information.

Experimental Section Materials. 3-(2,2,2-Trifluoroethoxymethyl)-3-methyloxetane (3FOx) and 3-bromomethyl-3-methyloxetane (BrOx) were gifts from OMNOVA Solutions (Akron, OH). The synthesis and characterization of monomers, the P[AB]-copolyoxetane telechelic 1 and P[AB]-copolyoxetane polyurethane 2, have been described in detail.30 The Mn for 1 was determined by end group analysis using 1H NMR spectroscopy.30 The P[AB]-telechelic was incorporated into a polyurethane by the soft block first method. The base polyurethane H12MDI-BD(50)/PTMO(1000), 2, was synthesized via a two-step solution polymerization31 using PTMO (1000 Da) as soft block and H12MDI-BD as hard block (50 wt %).32 Coating Process. Neat PU-1 was dissolved in tetrahydrofuran (THF) to give 10 wt % solution. For the modified blend, (25) Goddard, J. M.; Hotchkiss, J. H. Prog. Polym. Sci. 2007, 32, 698–725. (26) Ledbetter, J. W., Jr.; Bowen, J. R. Anal. Chem. 1969, 41, 1345–1347. (27) Stanley, J. S. J. Phys. Chem. 1954, 58, 533. (28) Adamczyk, Z.; Zembala, M.; Warszynski, P.; Jachimska, B. Langmuir 2004, 20, 10517. (29) Pu, Q. S.; Elazazy, M. S.; Alvarez, J. C. Anal. Chem. 2008, 80, 6532–6536. (30) Kurt, P.; Wynne, K. J. Macromolecules 2007, 40, 9537–9543. (31) Grasel, T. G.; Cooper, S. L. Biomaterials 1986, 7, 315–328. (32) Makal, U.; Fujiwara, T.; Cooke, R. S.; Wynne, K. J. Langmuir 2005, 21, 10749–10755.

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Figure 2. Imbedding fused-silica capillary between flat and gated PP sheets to obtain an airtight microfluidic channel for measuring streaming potential. (1) PP sheet with drilled holes, (2) fused silica capillary with i.d. 100 μm, (3) glass microscope slide, (4) binder clip, (5) clamped glass slide PP sheets for thermal treatment, (6) complete assembly (cell) having imbedded silica capillary (red) between PP sheets. Blue fill in (5) and (6) indicates fluid flow path. 1 wt % PU-1 was codissolved with 99 wt % 2 in THF to give a solution containing 10 wt % solids. Microfluidic Cell Fabrication. Figure 2 shows a schematic of the microfluidic capillary cell for streaming potential measurements. Rectangular pieces (5  2 cm2) were cut from polypropylene (PP) flat sheets (0.0125 in., McMaster and Carr Company). Fused silica capillaries (i.d. 100 μm) having a UV-transparent fluoropolymer outer coating were obtained from Polymicro Technologies, Phoenix, AZ. Sigma Aldrich NaBr (99%) was used to generate the aqueous electrolyte. Fused-silica capillaries were cut into 3 cm long sections with a capillary cutter (Agilent Technologies), as this tool provided a clean end cut that was perpendicular to the capillary tube length. Holes 3 cm apart were drilled in one 5  2 cm2 PP piece so as to provide an inlet and outlet for the NaBr electrolyte. The capillary was placed on the PP sheet so that the ends were adjacent to the holes. Another PP sheet of the same dimensions was placed so as to sandwich the capillary. The temporary assembly was rinsed with ethanol, positioned between two glass microscope slides, and clamped with four binder clips to create a press for subsequent processing. The assembly was placed in an oven at 155 °C for 15 min. During this time, PP softened sufficiently so as to embed and seal the capillary tube. The assembly was removed from the oven and cooled, and the binder clips and glass slides were removed to provide a sealed microfluidic channel (Figure 2). Channel Coating. Stock polymer solutions (10 wt %) of neat PU-1, 2, and 1 wt % PU-1/base PU 2 were prepared. The coating process involved the use of a syringe pump (Harvard Apparatus, PHD 2000 Infusion) to infuse the viscous polymer solution into the fused-silica capillary at a rate of 50 μL/min (Figure 3). The syringe pump was removed, and air was slowly pumped through the capillary using a 10 mm diameter Tygon tube fitted with a conical polypropylene adapter. Air flow simultaneously removed solvent and left a polymer coating on the inner walls (Figure 3). Residual solvent was removed from the coated capillary channel by vacuum drying overnight at ambient temperature. Measurement of Streaming Potential. Six capillary cells were fabricated/coated for each material. Streaming potentials (SPs) were measured using Labview software as described previously.29 Four cycles were obtained per run with four runs per coated capillary. As controls, streaming potentials were obtained for the fused silica capillary and a capillary coated with base polyurethane 2. SP values in Figure 5 and Table 1 are average values (standard error noted) from six cells for the respective materials. Determination of Charge Density. In previous studies, minimal water uptake was observed for a close analogue of surface 9034 DOI: 10.1021/la904849u

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Figure 3. Schematic for coating an embedded fused-silica capillary with 1 wt % HMDI-BD(30)-[P(3FOx)(C12)-87:13-5100], (1 wt % PU-1). modifier PU-1.30 In the later case, the soft block Mn was 6.5 kDa compared to 5.1 kDa for 1. Minimal water uptake was also observed for a 2 wt % analogue and base PU 2.22 After 24 h immersion, a typical weight gain was ∼5 wt %. The high mole fraction of fluorous repeats contributes to hydrophobic character. This stands in contrast to the results of Cooper and Grapski,33 who functionalized polyurethane hard blocks with quaternary side chains. Hard block functionalization disrupts hydrogen bonding, introduces high weight fractions of quaternary function, and leads to high degrees of swelling by water. In view of the hydrophobic nature of the polyurethane coatings, the Helmholtz-Smoluchowski approach34 (eq 1) was chosen for calculating zeta potentials. The models developed by Duval and co-workers35,36 and Ohshima37 for soft surfaces such as particles or hydrogel-like polyelectrolytes were deemed inappropriate. ΔE ηK ζ ¼ ΔP εε0

ð1Þ

Here, ΔE=measured streaming potential across the channel (V), ΔP = applied pressure, κ = conductivity of electrolyte solution (S/m), η=viscosity (kg/mS)=10-3 Pa 3 s at 20 °C, ε=permittivity of the solution = 80 at 20 °C, ε0 = vacuum permittivity = 8.8  10-12 C/mV, and ζ = zeta potential (V). Surface charge density was determined using the Gouy equation (eq 2).34   pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi zFζ σ ¼ 2εε0 RTc sinh ð2Þ 2RT Here, σ=charge density (C/m2), R=gas constant (8.31 J/K mol), T=293 K, F=Faraday constant (9.6104 C/mol), z=valence of counterion charge (1), and c = bulk electrolyte concentration (mol/L). With 10-3 M NaBr, the Fairbrother-Mastin approach was followed to calculate zeta potentials.38 Yaroshchuk and Ribitsch39 noted that a surface conductance correction is needed for porous membranes because the conduction current does not necessarily take the same path as streaming current and may circulate through the body of the porous membrane. The polyurethane coatings reported herein are not porous, and we believe that measuring surface conductivity is not essential. The charge density in eq 2 was normalized by inner capillary surface area. The lower (33) Grapski, J. A.; Cooper, S. L. Biomaterials 2001, 22, 2239–2246. (34) Berezkin, V. V.; Volkov, V. I.; Kiseleva, O. A.; Mitrofanova, N. V.; Sobolev, V. D. Colloid J. 2003, 65, 119–121. (35) Langlet, J.; Gaboriaud, F.; Gantzer, C.; Duval, J. F. L. Biophys. J. 2008, 94, 3293–3312. (36) Duval, J. F. L.; Huijs, G. K.; Threels, W. F.; Lyklema, J.; van Leeuwen, H. P. J. Colloid Interface Sci. 2003, 260, 95–106. (37) Ohshima, H. Colloids Surf., A 2005, 267, 50–55. (38) Fairbrother, F.; Mastin, H. J. Chem. Soc. 1924, 125, 2319–2330. (39) Yaroshchuk, A.; Ribitsch, V. Langmuir 2002, 18, 2036–2038.

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Table 1. Streaming Potentials (SPs) and Accessible Quaternary Charge Density (CD) for Runs on Neat and 1 wt % HMDI-BD(30)-[P(3FOx)(C12)-87:13-5100] as-cast PU-1a,b run

SP

CD

annealed PU-1a,b SP

CD

as-cast 1 wt % PU-1a,c

annealed 1 wt % PU-1a,c

SP

SP

CD

CD

1 2 3 4

174 (31) 11.1 (1.8) 223 (26) 15.2 (1.5) 93 (13) 5.5 (0.8) 55 (13) 3.2 (0.8) 180 (31) 11.6 (1.9) 226 (24) 15.5 (1.4) 83 (15) 4.9 (0.8) 23 (11) 1.3 (0.6) 179 (31) 11.5 (1.9) 225 (22) 15.4 (1.3) 72 (14) 4.2 (0.8) 5 (11) 0.3 (0.6) 178 (31) 11.5 (1.9) 225 (20) 15.4 (1.2) 62 (14) 3.6 (0.8) -7 (8) -0.4 (0.5) a Units: SP is mV with standard error in parentheses. CD is charge/cm2  1016 with standard error in parentheses. CD values are derived from SP values using eq 2. b For each run, SP values are the average of measurements on six cells and four cycles for a total of 24 data points. c SP for 1 wt % PU-1 changes with cycle. Reported values are the average of measurements on six cells using only cycle 2 for each run. Each SP is the average of six data points from the separate cells.

limit for the inner surface area was calculated assuming a coating thickness based on a 10 wt % solution filling the capillary prior to solvent removal. Sample calculations for coating thickness, surface area, zeta potential, and charge density are provided in the Supporting Information. Atomic Force Microscopy (AFM). Morphological analyses of polyurethane surfaces were carried out using a Dimension-3100 (Digital Instruments, CA) atomic force microscope with a NanoScope V controller. Imaging was performed in tapping mode using a microfabricated silicon cantilever (40 N/m, Veeco, Santa Barbara, CA) in air. The tapping force was increased from soft to hard by decreasing the set point ratio rsp or Aexp/Ao, where Ao is free oscillation amplitude and Aexp is the experimental oscillation amplitude. Images were analyzed by using NanoScope v710r1 software.

Results and Discussion Knowledge of accessible surface charge density for P[AB]copolyoxetane polyurethane (PU) polymer surface modifiers (PSMs) is essential for correlating biocidal efficacy with PSM weight percent, coating processing conditions, surface morphology, and time dependence of quaternary charge concentration. As noted earlier, efforts to quantify surface concentration of charge by dye release were unsuccessful. The elegant work of Adamczyk et al.,28 who followed alternating polyelectrolyte deposition by streaming potential or apparent zeta potential measurements, inspired an investigation of this method for quantifying accessible surface charge in coatings modified with PU-1. In adapting this method, a sampling process was sought that would mimic dip coating.22,23 The method and cell fabrication is summarized below followed by a discussion of results and correlation with near-surface morphology by TMAFM. Method/Cell. Kirby and Hasselbrink have reviewed streaming potential (SP) measurements in microfluidic devices fabricated from nylon, poly(ethylene terephthalate), poly(tetrafluoroethylene), poly(methyl methacrylate), poly(dimethylsiloxane), and polycarbonate.40 Kirby and Hasselbrink pointed out that while polymers are ubiquitous in microchip analytical systems, the surface science of most polymeric substrates is not well-known. The measurements described herein had a specific priority, namely, mimicking dip coating for PSM-polyurethanes. Therefore, we sought to minimize the device complexity and to focus on a single coated capillary. Streaming potential measurements in embossed polycarbonate (PC)10 or cyclic olefin copolymer (COC) cells29,41 were described by Alvarez and co-workers. The straightforward fabrication of cells described by the latter work was attractive. To avoid swelling by THF, the solvent routinely used for polyurethane coatings, and to parallel a dip coating procedure, polypropylene (PP) was selected. Initially, microchannels were embossed in PP using a (40) Kirby, B. J.; Hasselbrink, J. E. F. Electrophoresis 2004, 25, 203–213. (41) Luna-Vera, F.; Alvarez, J. C. Biosens. Bioelectron. 2010, 25, 1539–1543.

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125 μm steel wire at 155 °C. The embossed channel was sealed with another flat PP sheet at 150 °C. The microfluidic channel was coated, but streaming potentials for different channels were not reproducible or not measurable. Inability to measure SPs or sudden failure of SP measurements was thought to be due to channel clogging by poorly adherent coatings. These problems were circumvented by using 100 μm i.d. fusedsilica capillaries embedded in PP coupons. As described in the Experimental Section, microscope slides with binder clips were used to hold a sandwich comprising two PP plates and a 3 cm capillary (Figure 2). The device was heat-sealed at the softening temperature of PP (155 °C). In use, clogging was uncommon, suggesting good adhesion of PU-1 and base polyurethane 2 to glass. NaBr was selected as the polyelectrolyte to provide a common anion for PU-1. A test of a fused-silica capillary with 1 mM NaBr gave a steady streaming potential (SP) of ∼ -300 mV. The negative SP is a result of the weakly acidic silica surface (pKa ∼ 5.3).42-44 Anion adsorption may also contribute to the negative SP as reported by Beattie45 and Kirby et al.46 Polymer Coatings. After exploring a number of options, a satisfactory result was obtained by infusing a 10 wt % polymer solution (THF) into the capillary by means of a syringe pump (50 μL/min). This was followed by the slow passage of air, which displaced some of the polymer solution and left a coating due to high solution viscosity. Continued air flow then removed solvent from the residual coating (Figure 3). The microcapillary cell was then placed in a vacuum oven overnight at 25 °C to remove solvent completely. In previous studies, thermal treatment at 60 °C was used for removal of residual solvent from coatings.22,23 Because of morphological changes (vida infra), the term “annealing” is now used, and “as-cast” refers to coatings from which solvent is removed in vacuum at 25 °C. As a control, microfluidic capillary streaming potential (MCSP) measurements were obtained for a capillary coated with base polyurethane 2 (Figure S2 in the Supporting Information). The streaming potential of the as-cast coating was -100 mV; annealing the same coating after SP measurements gave -120 mV. Beattie reported that negative streaming potentials for nominally neutral polymer surfaces are due to enhanced autolysis of water, which causes aqueous hydroxide ion adsorption.45 However, bromide ion adsorption is another equally plausible explanation. Kirby et al. summarized possible causes such as impurities, salt ion adsorption, and hydroxide ion adsorption but stated that conclusive evidence is not yet available to ascertain the origin of (42) Schwer, C.; Kenndler, E. Anal. Chem. 1991, 63, 1801–1807. (43) Melanson, J. E.; Baryla, N. E.; Lucy, C. A. TrAC, Trends Anal. Chem. 2001, 20, 365–374. (44) Horvath, J.; Dolnik, V. Electrophoresis 2001, 22, 644–655. (45) Beattie, J. K. Lab Chip 2006, 6, 1409–1411. (46) Tandon, V.; Bhagavatula, S. K.; Nelson, W. C.; Kirby, B. J. Electrophoresis 2008, 29, 1092–1101.

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Figure 4. Four cycles of SP measurements from one run for PU-1: (A) neat, as-cast and (B) neat, annealed. For 1 wt% PU-1: (C) as-cast and (D) annealed. SP values from cycle 2 (arrow) are used for Figure 5.

negative streaming potentials. This uncertainty is due to “complexities of slip phenomena, uncertainties in interfacial structure, and unknown surface chemistry.”46 Like Beattie,45 Kirby et al. concluded that water hydroxide ion adsorption is the likely reason for negative streaming potentials on nominally neutral surfaces such as polyurethane, but this needs experimental verification in light of the possibility of bromide ion adsorption. MCSP measurements were obtained for neat PU-1 and for coatings with 1 wt % PU-1 and 99 wt % base polyurethane 2. Six microcapillary cells were coated in order to test reproducibility of MCSP measurements. For each SP run, four cycles of SP measurements were recorded. A total of four runs were carried out for each cell. The time for recording four cycles was 20 s (Figure 4). H12MDI-BD(30)/P[(3FOx)(C12)-87:13-5100] (PU-1). Representative MCSP measurements for one SP run (one capillary, four cycles) are shown in Figure 4. The SP is the difference between the peak and base (mV) for each cycle. Inspection of Figure 4A shows that the SP for as-cast PU-1 is constant for four cycles. A total of 16 cycles were run for this capillary, yielding an average SP of 184 ( 2 mV. As this example illustrates, the 9036 DOI: 10.1021/la904849u

measured SP for a particular coated capillary has very good precision. However, due to considerations described below, there were significant variations from cell to cell. Because the SP for PU-1 is constant, the values for the four runs on all six capillaries (Figure 5, Table 1) is averaged to give a value of 178 ( 31 mV. The standard error for six capillaries of as-cast PU-1 is relatively high ((31 mV). A contributor to SP inconsistency may be sample to sample surface variability. The surface area of the polymer coating is ∼8.9 mm2, while coverslips used for XPS and wetting behavior22 have a surface area of 1760 mm2. Uncoated areas are readily visualized for coverslips but not for capillaries. The small capillary surface area may amplify differences in sample surface compositions. The coating thickness may vary depending on the amount of polymer solution that is removed by air flow in the formation of the channel. Thus, in its present form, while the measurement of SP for a given capillary is precise ((2 mV), the coating process apparently results in surface composition variations. The SP variability led to averaging results from six capillaries, a compromise between time and benefit. Langmuir 2010, 26(11), 9032–9039

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Figure 5. Streaming potentials for representative samples of neat PU-1 (blue triangle) and 1 wt % PU-1 (red triangle): as-cast (solid lines) and annealed (dashed lines). Each data point (average of six cells) is taken from the second cycle of the respective run. Table 1 lists SPs.

Neat PU-1 was annealed at 60 °C for 24 h under vacuum. One set of SP data for four cycles is shown in Figure 4B. Like as-cast PU-1, SPs for annealed PU-1 remain constant and are independent of electrolyte-flow time (Figure 5, Table 1). The average value for six capillaries is 225 ( 23 mV. Interestingly, annealing results in a 26% increase in SP compared to as-cast PU-1 (178 ( 31 mV). In addition, the accuracy of SP measurements for annealed PU-1 is improved by 8% compared to as-cast. Thus, variations in processing as-cast PU-1 (vida supra) are to some degree “annealed out”. Pioneering work in polyurethane surface morphology established that soft blocks are surface concentrated.47-49 For neat PU-1, the increase in SP after annealing is correlated with increased hard block-soft block phase separation and concomitant increase in quaternary C12 surface concentration. This point is explored further in TM-AFM imaging described below. Limited prior SP results are available on comparable systems. SPs have been measured for thin films formed by alternating polyelectrolyte deposition (APD). After deposition of poly(allylamine hydrochloride) (PAH), a typical SP was 63-75 mV.28 A lower range (33-38 mV) was observed for polyelectrolyte multilayers topped with PAH.10 A comparison of SPs with those reported herein cannot be made without knowing applied pressure and electrolyte concentration for the different measurements. However, it is clear that the results for PU-1 and the polyelectrolyte multilayers are in qualitative agreement. A more thorough treatment of surface charge demonstrates that actual surface charge (viz., measured by titration) may be different from that determined by streaming potential measurements.50 However, the electrokinetic measurements, that is, streaming potentials, reported herein provide a self-consistent measure of accessible surface charge relevant to near-surface phase separation and contact antimicrobial kill. TM-AFM on dip-coated samples was employed to evaluate surface morphology for evidence of increased PU-1 soft block surface phase separation. This trend might be expected considering the increased SPs for annealed PU-1. Figure 6 shows phase images for as-cast and annealed PU-1 at two different set point ratios. Paralleling the findings of Runt et al.,47 the phase images (47) Garrett, J. T.; Siedlecki, C. A.; Runt, J. Macromolecules 2001, 34, 7066–7070. (48) Garrett, J. T.; Xu, R.; Cho, J.; Runt, J. Polymer 2003, 44, 2711–2719. (49) Ratner, B. D.; Cooper, S. L.; Castner, D. G.; Grasel, T. G. J. Biomed. Mater. Res. 1990, 24, 605–620. (50) Lyklema, J. Fundamentals of Interface and Colloid Science Vol. II: SolidLiquid Interfaces; Academic Press: San Diego, CA, 1995; Chapter 4.

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Figure 6. Phase images (1  1 μm2, Rq =1.6 nm) for neat HMDIBD(30)-[P(3FOx)(C12)-87:13-5100] (A) as-cast and (B) annealed at 60 °C/24 h. (A-1, B-1) rsp = 0.95; (A-2, B-2) rsp = 0.90.

for light tapping (rsp =0.95) are largely featureless for both as-cast and annealed samples. Decreasing the set point ratio reveals a near-surface hard block (lighter colored regions) as expected for a well phase separated polyurethane (Figure 1). Near-surface hard block domains seem somewhat less prominent for annealed PU-1 (Figure 6B-2), but there is no dramatic change in morphology. The increased SP for the annealed sample is therefore attributed to subtle, near-surface morphological changes resulting in an increased area fraction of accessible quaternary charge. The latter reflects the minimization of surface free energy for the surface concentration of the P[(3FOx)(C12)-87:13-5100]-copolyoxetane soft block. 1 wt % H12MDI-BD(30)/P[(3FOx)(C12)-87:13-5100]. Newly Cast 1 wt % PU-1 Coatings. Figure 4C shows four cycles for a representative capillary coated with 1 wt % PU-1. The initial SP is 112 mV. It is noteworthy that 1 wt % PU-1 causes an increase in streaming potential from -100 mV to an average initial value of þ93 mV (Table 1). The positive steaming potential for the surface modified PU is an indication of the surface concentration of C12 quaternary charge. This result supports the model for P[(3FOx)(C12)]-copolyoxetane surface concentration after the ambient temperature, dip-coating/solvent evaporation process (Figure 1). However, the average SP for 1 wt % PU-1 is about 50% less than that for neat, as-cast PSM PU-1 (þ178 mV). The positive SP must account for the antimicrobial action of base polyurethane coatings modified with a PSM of similar composition to PU-1 (but 2 wt %).22,23 Figure 7 A shows TM-AFM phase images for 1 wt % PU-1 ascast coatings. The presence of PSM PU-1 clearly disrupts the base PU 2 morphology that is seen in the phase image at the same set point ratio (0.9) in Figure 6. The AFM images in Figure 7A thus support the notion of surface concentration for PSM PU-1 in the as-cast coating. From the phase images shown in Figure 7A, there is little evidence for near-surface phase separation of PSM PU-1. From the microscale (Figure 7A-1, 5050 μm2) to the nanoscale (Figure 7A-3, 1  1 μm2), the phase images are relatively featureless. Only Figure 7A-3 shows some faint evidence for near-surface nanoscale phase separation. Effects of Immersion and Temperature on 1 wt % PU-1 Coatings. Figure 4C shows that the SP for as-cast 1 wt % PU-1 decreases from 112 to 95 mV over four cycles. The downward DOI: 10.1021/la904849u

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Figure 8. Streaming potentials for 1 wt % PU-1 as a function of cycle: as-cast (solid line) and annealed (dashed line).

Figure 7. Phase images (rsp =0.90) for 1 wt % PU-1 modified PU 2: (A) as-cast, (B) as-cast after 2 min exposure to water, and (C) ascast, annealed at 60 °C/24 h; Rq for A-1, -2, -3 = 6.9, 2.0, 0.6 nm; Rq for B-1, -2, -3 = 21.1, 6.3, 1.6 nm; Rq for C-1, -2, -3 = 8, 1.5, 0.5 nm.

trend in SP measurements shown in Figure 4C prompted a closer evaluation. The SP was determined for four consecutive runs (16 cycles) on six capillaries. Representative values for one cell are shown in Figure 8. From an initial 112 mV, the SP for this cell decreases linearly (R2 = 0.99) to 34 mV. This change in SP from 112 to 34 mV occurs in less than 2 min cumulative flow time, as each run, consisting of four cycles, takes about 20 s and a total of four runs were obtained for each cell. To further investigate durability of surface charge, as-cast 1 wt % PU-1 coatings were annealed at 60 °C for 20 h. Four cycles for a representative coating shown in Figure 4D reflect a rapid decrease in SP. On average, annealing results in a decrease of SP from an initial value of þ67 mV to -7 mV over the course of 16 cycles (Figure 5, Table 1). Figure 8, which shows SPs over 16 cycles for a representative coating, reveals that, compared to as-cast coatings, the SP trend line for annealed coatings is nonlinear with a faster initial rate of decrease. TM-AFM images described below provide morphological evidence concerning the rapid drop in near-surface quaternary charge density. Surface Charge Density. Neat PU-1. Zeta potentials were calculated from streaming potentials using eq 1. Charge density was calculated using eq 2 which was normalized to the inner surface area of the capillary coating. Example calculations are provided in the Supporting Information. Accessible quaternary charge density for neat PU-1 was stable for 16 cycles on six different cells (1.1 ( 0.21017 charge/cm2) (Supporting Information Figure S3, Table 1). Paralleling the increased SP, the accessible quaternary charge density for neat PU-1 increased to 1.5 ( 0.1  1017 charge/cm2. These values of charge density exceed the threshold values for contact antimicrobial kill (g5  1015 charge/cm2) reported by Matyjaszewski et al.9 and a threshold of 1013-1014 charge/cm2 determined by Kugler et al.3 Both of the substrates in these studies were polycation nanofilms where charge density was determined by dye adsorption/desorption.3,9 Without a cross-comparison of methods, only qualitative com9038 DOI: 10.1021/la904849u

parisons can be made concerning results from SP measurements. However, the high accessible charge density for neat PU-1 must account for the consistent 100% contact kill against challenges of Gram postive/negative bacteria.22,23 1 wt % PU-1. Paralleling the streaming potential, the initial charge density for 1 wt % PU-1 (5.5 ( 0.8  1016 charge/cm2) is about 50% that of the neat modifier (1.1 ( 0.21017 charge/cm2). The calculated charge density for 1 wt % PU-1 decreases from an initial value of 5.5 ( 0.8  1016 to 3.6 ( 0.8  1016 charge/cm2. Because of the rapid decrease, the calculated charge density (Table 1) is arbitrarily based on the second cycle of SP measurements for each of four successive runs. Accessible quaternary charge is already negligible at the end of run 3 (Supporting Information Figure S3) and becomes “negative” at the end of run 4. While charge density cannot be negative, the value obtained (-0.4 ( 0.5  1016 charge/cm2 after 16 cycles) emphasizes the complete depletion of quaternary charge. SP measurements are rapid so that the elapsed time for this transformation is less than 2 min (Supporting Information Figure S3, Table 1). TM-AFM was used to provide information on near-surface morphological changes that might account for the drop in streaming potentials on annealing and after exposure to electrolyte. The investigation focused on the effect of NaBr solution or water on as-cast 1 wt % PU-1. Residual solvent was removed under vacuum overnight for a dip-coated slide (from 10% solution of 1 wt % PU-1). The coating was then immersed in 1 mM NaBr solution for 2 min followed by drying in vacuo at ambient temperature. TM-AFM images are shown in Figure 7B-1, -2, and -3. The 10  10 μm2 image (Figure 7B-2) provides a subtle but distinctly different morphology compared to the case of as-cast without aqueous exposure (Figure 7A-2). Careful inspection reveals irregular 1 μm circular domains characteristic of the onset of PU-1 phase separation (vida infra). These features are clearer in the large-scale image provided in the Supporting Information (Figure S4). A control experiment demonstrated that the same morphological change occurred for water (sans electrolyte). To further confirm phase separation, a 1 wt % PU-1 coating was immersed in water for 1 h. Further phase separation is particularly clear in the Supporting Information Figure S4-D2 image (1010 μm2), where phase separated features are seen with dimensions on the order of ∼1 μm. In contrast to newly as-cast coatings, AFM phase images for annealed coatings provide evidence of phase separation on the microscale and nanoscale (Figure 7C-1, -2, -3). In the 5050 μm2 phase image (Figure 7C-1), numerous irregular circular features are observed. Figure 7C-1,-2 shows that the size distribution of these features is roughly bimodal, with one set at 2-3 μm and Langmuir 2010, 26(11), 9032–9039

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Figure 9. Surface phase separation model for 1 wt % PU-1. Red

A, 3FOx; blue Bþ, quaternary side chain. The dashed open circle represents a metastable, one-phase, near-surface composition. The open circle is postphase separation and represents a base polyurethane rich phase (PU-1 depleted), while the filled (colored) circle corresponds to the PU-1 rich modifier phase.

another set with nanoscale dimensions (500 nm). The 1  1 μm72 image (Figure 7C-3) provides additional evidence of near-surface phase separation at the nanoscale. The surface morphology for 1 wt % PU-1 after annealing or exposure to water provides clear evidence of near-surface phase separation. Such phase separation is characteristic spinodal decomposition/lower critical solution temperature (LCST) behavior for binary polymer blends.51 Figure 9 illustrates phase separation that appears to be driven by the insolubility of the 3FOx component of the PSM. Our first publication on quaternary surface modifiers made note of AFM images showing nanoscale phase separation for a 2 wt % PU-1 composition.23 The P[(3FOx)(C12)]-copolyoxetane soft block was similar to 1, but the Mn was slightly higher (6.5 kDa). At that time, favorable biotesting results did not raise concerns that surface phase separation had an adverse effect on antimicrobial activity. However, it is clear from the rapid decrease in charge density for 1 wt % PU-1 that phase separation sequesters quaternary C12, resulting in diminished streaming potentials. Thus, a reinvestigation of time dependent changes in contact antimicrobial activity for modified coatings is underway.

Conclusion Streaming potential measurements showed diminution of nearsurface charge in less than 2 min for as-cast 1 wt % PU-1 and even more rapid attenuation after annealing. As noted above, prior to previously reported antimicrobial testing, solvent was removed at 60 °C and antimicrobial testing was done typically 2-3 days after coating preparation. The instability of surface charge was not apparent as the bacterial spray wetted the surrface for only 10-15 s. (51) Rubinstein, M.; Colby, R. H. Polymer Physics; Oxford University Press: New York, 2003.

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During this time, the reduction of surface charge due the combined effects of “60 °C annealing” coupled with rapid phase separation on contact with water was not evident as 100% kill of Gram positive/negative bacteria was observed. The SP results indicate that water plasticizes the near-surface domain and increases the rate of thermodynamically favored phase separation illustrated in Figure 9. This figure depicts the formation of a metastable, one-phase, near-surface, 1 wt % PU-1 composition (dashed circle). Spinodal decomposition to modifier rich and poor domains is represented by the colored circle and open circles, respectively. From SP measurements and charge density calculations, the impact of this phase separation, clearly imaged by AFM, is the sequestration of surface accessible quaternary charge into the phase separated fluorous domain. The bulk Tg of the P[(3FOx)(C12)-87:13-5100] soft block is 60 °C below ambient.22,23 The softening temperature of PSM PU-1 and the linear base polyurethane 2 is 70-80 °C.52 This softening is principally due to hard block disordering. Considering the acceleration of surface charge diminution by water, plasticization of near-surface hard block likely facilitates chain mobility and phase separation along with sequestration of charge in the largely fluorous domain. This is illustrated in the upper portion of the phase diagram in Figure 9. In view of the streaming potential and AFM results, the durability of contact antimicrobial effectiveness for polyurethane coatings modified by PU-1 is being reinvestigated. Preliminary results suggest that, even at ambient temperature and humidity, phase separation for “as-cast” PSM PU-1 base polyurethane coatings occurs slowly. A key requirement for antimicrobial coatings is durability. Polymeric electrophoresis microfluidic substrates that are used extensively for microchip separations40 also require surface charge stability. Balancing near-surface charge stability along with mobility required for biocidal effectiveness10 is a continuing challenge for P[AB]-copolyoxetane based surface modifiers. The importance of SP and AFM results described above lies in providing quantitative guidance for achieving charge stability as new quaternary antimicrobial surface modifiers are developed. Acknowledgment. The authors thank the National Science Foundation (DMR- grants DMR-0207560 and DMR-0802452) for support of this research. JCA thanks NSF, Chemistry Division (grant CHE-0645494) for support of this research. The authors thank Ms. ThuTrang T. Nguyen for assistance with some of the SP measurements. Supporting Information Available: Calculation of capillary coating thickness, capillary surface area, zeta potential, and charge density; structural representation for 2, base polyurethane; streaming potentials for base polyurethane 2; accessible quarternary charge density for neat PU-1 and 1 wt % PU-1; enlargement of phase images shown in Figure 7 and phase images for as-cast 1 wt % PU-1. This material is available free of charge via the Internet at http:// pubs.acs.org. (52) Brunson, K. M. M.S. Thesis, Virginia Commonwealth University, 2006.

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