Nanometer-Scale Roughness Having Little Effect on the Amount or

9868

Langmuir 2003, 19, 9868-9872

Nanometer-Scale Roughness Having Little Effect on the Amount or Structure of Adsorbed Protein Mina Han, Ananthakrishnan Sethuraman, Ravi S. Kane, and Georges Belfort* Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180-3590 Received March 31, 2003. In Final Form: August 26, 2003 Neither the adsorbed amount per unit actual surface area nor the structural stability of hen egg lysozyme is influenced by increasing the nanometer-scale roughness (5 < Rave < 60 nm) of a series of model substrates. Seven poly(ether sulfone) (PES) ultrafiltration membranes of increasing mean pore size with the same surface chemistry were chosen as model rough surface substrates. Topographical images, using atomic force microscopy, combined with attenuated total reflection Fourier transform infrared spectroscopy (ATR/ FTIR) and sessile captive bubble contact angle measurements were used to characterize the surface properties of the substrates. ATR/FTIR spectroscopy together with a newly developed optimization algorithm for predicting the content of secondary structure motifs is used to correlate the secondary structure and amount of adsorbed lysozyme with the substrate surface roughness. From the adsorption measurements, the net adsorbed amount (total minus nonspecific adsorbed amount) of lysozyme corresponded to approximately one monolayer of coverage for all the substrates independent of the roughness. Although lysozyme was structurally disturbed through adsorption to PES substrates, no significant changes in its secondary structure were observed with the increasing roughness.

Introduction Protein-substrate interactions are important because they can affect the efficacy of many applications, including the performance of medical implants, sensors, bioreactors, separation processes of biological molecules, and devices in the marine environment, to name a few.1 That the surface chemical composition of substrates has a strong influence on the adsorption process of proteins is welldocumented.1,2 Many researchers have addressed the underlying reasons for the extent of adsorption through experiment, theory, and simulation.3-15 To simplify these investigations, a propitious choice of experimental conditions and theoretical assumptions have been made. For example, heterogeneous polymeric surfaces have been replaced by well-defined surface chemistries at the molecular level using self-assembly,16-18 low-temperature * To whom correspondence should be addressed. Phone: 518276-6948. Fax: 518-276-4030. E-mail: [email protected]. (1) Malmsten, M., Ed. Biopolymers at Interfaces; Marcel Dekker: New York, 2003. (2) Horbett, T. A., Brash, J. L., Eds. Proteins at Interfaces II: Fundamentals and Applications; ACS Symposium Series No. 602; American Chemical Society: Washington, DC, 1995. (3) Norde, W. In Polymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 27-54. (4) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 17, 1. (5) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267. (6) Norde, W.; Lyklema, J. J. Biomater. Sci., Polym. Ed. 1991, 2, 183. (7) Malmsten, M.; Arnebrant, T.; Billsten, P. In Polymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 119-142. (8) Hannemaaijer, J. H.; Robbertsen, T.; Van den Boomgaard, Th.; Olieman, C.; Both, P.; Schmidt, D. G. Desalination 1988, 68, 93. (9) Palecek, S. P.; Mochizuki, S.; Zydney, A. L. Desalination 1993, 90, 147. (10) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464. (11) Roth, C. M.; Lenhoff, A. M. In Polymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 89-118. (12) Lee, S.-C.; Belfort, G. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8392. (13) Yoon, B. J.; Lenhoff, A. M. J. Phys. Chem. 1992, 96, 3130. (14) Roth, C. M.; Lenhoff, A. M. Langmuir 1993, 9, 962. (15) Roth, C. M.; Lenhoff, A. M. Langmuir 1995, 11, 3500. (16) Haeussling, L.; Ringsdorf, H.; Schmitt, F. J.; Knoll, W. Langmuir 1991, 7, 1837.

plasma,19-21 ultraviolet irradiation methods22,23 and the grafting of polymers such as poly(ethylene glycol) and poly(vinyl pyrrolidone).24,25 Also, well-defined adsorbates (building blocks) such as amino acids and small peptides with known secondary structure have been used to simulate larger and more complex proteins.26,27 In several recent studies, surface chemistry and surface topology were considered and thought to affect protein adsorption as well as cell adhesion.28-35 Unfortunately, in these and other studies, the two effects were not convincingly decoupled because most topographical modifications were accompanied by chemical heterogeneities.33,36-39 (17) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (18) Cheng, S.-S.; Chittur, K. K.; Sukenik, C. N.; Culp, L. A.; Lewandowski, K. J. Colloid Interface Sci. 1994, 162, 135. (19) Kiaei, D.; Hoffman, A. S.; Horbett, T. A.; Lew, K. R. J. Biomed. Mater. Res. 1995, 29, 729. (20) Lopez, G. P.; Ratner, B. D.; Rapoza, R. J.; Horbett, T. Macromolecules 1993, 26, 3247. (21) Ulbricht, M.; Belfort, G. J. Membr. Sci. 1996, 111, 193. (22) Yamagishi, H.; Crivello, J. V.; Belfort, G. J. Membr. Sci. 1995, 105, 249. (23) Pieracci, J.; Crivello, J. V.; Belfort, G. J. Membr. Sci. 1999, 156, 223. (24) Sheu, M. S.; Hoffman, A. S.; Ratner, B. D.; Feijen, J.; Harris, J. M. J. Adhes. Soc. Technol. 1993, 7, 1065. (25) Chen, C.; Belfort, G. J. Appl. Polym. Sci. 1999, 72, 1699. (26) Molnar, I.; Horvath, C. J. Chromatogr. 1977, 142, 623. (27) Basiuk, V. A. In Polymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 55-87. (28) Lestelius, M.; Liedberg, B.; Tengvall, P. Langmuir 1997, 13, 5900. (29) Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639. (30) Buijs, J.; Hlady, V. J. Colloid Interface Sci. 1997, 190, 171. (31) Kondo, A.; Urabe, T.; Yoshinaga, K. Colloids Surf., A 1996, 109, 129. (32) McClary, K. B.; Ugarova, T.; Grainger, D. W. J. Biomed. Mater. Res. 2000, 50, 428. (33) Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573. (b) Curtis, A.; Wilkinson, C. Biochem. Soc. Symp. 1999, 65, 15. (34) Boyan, B. D.; Hummert, T. W.; Dean, D. D.; Schwartz, Z. Biomaterials 1996, 17, 137. (35) Schwartz, Z.; Kieswetter, K.; Dean, D. D.; Boyan, B. D. J. Periodontal. Res. 1997, 32, 166. (36) Mu¨ller, B.; Riedel, M.; Michel, R.; De Paul, S. M.; Hofer, R.; Heger, D.; Gru¨tzmacher, D. J. Vac. Sci. Technol., B 2001, 19, 1715.

10.1021/la030132g CCC: $25.00 © 2003 American Chemical Society Published on Web 10/03/2003

Nanometer-Scale Roughness Effect on Adsorbed Protein

The goal of this research was to measure the adsorbed amount and secondary structural changes resulting from the adsorption of lysozyme on a series of poly(ether sulfone) (PES) substrates (commercial ultrafiltration membranes) that are compositionally identical but have different surface roughness characteristics. Thus, the surface chemical composition was uncoupled from the surface roughness and conformational changes of the protein were monitored for different roughnesses. Experimental Section Materials. PES membranes with 10, 30, 70, 100, 300, 500, and 1000 kDa molecular weight cutoffs (MWCOs) from lots 7099D, 8220B, 7309A, 7265G, 9336J, 0073F, and 0083B, respectively, were obtained from Pall Filtron Corp. (East Hills, NY) and were used as model rough surfaces. These Omega series membranes have been slightly hydrophilized by the manufacturer by an undisclosed process (most membrane manufacturers add poly(vinyl pyrrolidone) to the casting solution to impart increased hydrophilicity.) Spectra, using attenuated total reflection Fourier transform infrared spectroscopy (ATR/FTIR: Magna-IR 550 Series II, Nicolet Instruments, Madison, WI), were taken for each membrane. Acetic acid, hydrochloric acid, sodium hydroxide, a solution of Ponceau S (2% w/v, in water with trichloroacetic acid, 30% w/v, and sulfosalicyclic acid, 30% w/v), phosphate buffered saline (PBS, 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride), and chicken egg white lysozyme (lyophilized powder) were purchased from Sigma-Aldrich. Lysozyme was chosen as the model protein adsorbate because its adsorption characteristics have been extensively studied in our and other groups.10,40-45 Lysozyme (14.6 kDa, pI 11.0, 3.0 × 3.0 × 4.5 nm3) is fairly hard and ellipsoidal and is known to be structurally robust in solution.43,46 Atomic Force Microscopy (AFM). Topographical images of 5 × 5 µm2 sections of the membrane surfaces were obtained in contact mode using silicon nitride cantilevers (Park Scientific Instruments, Sunnyvale, CA) with an atomic force microscope (Auto Probe PC, Park Scientific Instruments) and surface analysis and data acquisition software (PSI ProScan Version 1.5, Park Scientific Instruments). The mean vertical, δV, and horizontal, δH, length scales were obtained from more than 300 measurements of the depth (mean vertical distance from the top of the peak to the bottom of the groove) and the width (mean horizontal from peak to peak) for each membrane surface, respectively.47 These length scales were used to correct for the roughness in estimating the intrinsic contact angle of the membrane surfaces according to Taniguchi and Belfort’s protocol.47 Contact Angle. A captive air bubble was placed under a membrane substrate, both of which were submerged in deionized water, and sessile contact angle values were measured using an optical system (SIT camera, SIT66, Dage-MTI, Michigan, IN) converted to a video display. The average value of each contact angle was obtained for at least five different bubbles at different locations on the substrate surface.44,45 Protein Adsorption and Desorption. Static adsorption measurements were achieved by immersing substrate swatches (37) Cacciafesta, P.; Hallam, K.; Watkinson, A. C.; Allen, G. C.; Miles, M. J.; Jandt, K. D. Surf. Sci. 2001, 491, 405. (38) Denis, F. A.; Hanarp, P.; Sutherland, D. S.; Gold, J.; Mustin, C.; Rouxhet, P. G.; Dufreˆne, Y. F. Langmuir 2002, 18, 819. (39) Dufreˆne, Y. F.; Marchal, T. G.; Rouxhet, P. G. Langmuir 1999, 15, 2871. (40) Koehler, J. A.; Ulbricht, M.; Belfort, G. Langmuir 1997, 13, 4162. (41) Koehler, J. A.; Ulbricht, M.; Belfort, G. Langmuir 2000, 16, 10419. (42) Sethuraman, A.; Vedantham, G.; Przybycien, T.; Imoto, T.; Belfort, G. Manuscript submitted for publication, 2003. (43) Imoto, T.; Johnson, L. N.; North, A. C. T.; Phillips, D. C.; Rupley, J. A. Enzymes 1970, 7, 665. (b) Sundaram, S.; Ferri, J. K.; Vollhardt, D.; Stebe, K. J. Langmuir 1998, 14, 1208. (44) Pieracci, J.; Crivello, J. V.; Belfort, G. J. Membr. Sci. 1999, 156, 233. (45) Pieracci, J.; Wood, D. W.; Crivello, J. V.; Belfort, G. Chem. Mater. 2000, 12, 2123. (46) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (47) Taniguchi, M.; Belfort, G. Langmuir 2002, 18, 6465.

Langmuir, Vol. 19, No. 23, 2003 9869 (2 cm in diameter) into 9 mL of protein solution (10 mg/mL in a 50 mM PBS solution at pH 7.4) at 25 ( 1°. Analysis of the amount of adsorbed lysozyme was performed by rinsing a membrane swatch with the starting buffer solution three times and then staining with Ponceau S.48 For the AFM image measurements, after immersion into the protein solution at 25 ( 1 °C for 300 min, the samples were immersed in deionized water for 10 s, dried with a gentle nitrogen stream for about 10 s, and immediately analyzed with the atomic force microscope. For analysis of the desorbed amount of lysozyme, after immersion in a 10 mg/mL protein solution for 960 min (g300 min, that is, sufficient time to reach the saturated adsorption level), each sample was dipped into a solution of Ponceau S for 1 h.48 After washing with deionized water five times, the samples were immersed in deionized water and the desorbed amount of protein was measured by following the solution absorbance at 515 nm. Protein Conformation in Solution and on Adsorption. ATR/FTIR spectroscopy. Because the usual method for assessing the secondary structure in aqueous solution, circular dichroism, is not easily adaptable to adsorbed proteins on solid substrates in aqueous solution, a quantitative approach based on amide I band ATR/FTIR spectroscopy was used.42,49 Analysis of irreversibly adsorbed lysozyme was performed by rinsing membranes with starting buffer. The membranes analyzed were cut into strips of dimension 7 × 0.7 cm2 such that they fitted neatly onto the germanium element of the internal reflection accessory. The background, buffer, virgin membrane, and protein-adsorbed membrane spectra were collected in the 1000-4000 cm-1 range as sets of 1024 time-averaged, double-sided interferograms with Happ-Genzel apodization. The spectral resolution was set at 2 cm-1, the gain was set at 8, and an aperture of 38 was used. After each experiment, the exposed surface of the germanium crystal was cleaned with 1% (w/w) sodium dodecyl sulfate solution for 10 min. Spectra of protein adsorbed onto wet membrane sections were obtained. Spectral Analysis Algorithm. To obtain the secondary structure content, a holistic spectral analysis used amide I band infrared spectra (ATR/FTIR spectroscopy) for estimating the protein secondary structure.49 It combines the superposition of reference spectra of pure secondary structural elements with the simultaneous aromatic side chain, water vapor, and solvent background subtraction. When a single optimization algorithm was used, a single mathematical function was defined for the protein spectra, permitting all subtractions, normalizations, and amide band deconvolution steps to be performed simultaneously. A key element of the technique was the calculation of the reference spectra for the ordered helix (called R helix), sheet (called β sheet), unordered and unordered helix, and turn structures from a basis set of well-characterized proteins. a-chymotrypsin (bovine pancreas; C7762), concanavalin-A (canavalia ensiformis; C7275), myoglobin (sperm whale; M7527), papain (papaya latex; P4762), subtilisin (bacillus licheniformis; 85968), and triosephosphate isomerase (rabbit muscle; T6258) were purchased from SigmaAldrich. Lysozyme (chicken egg white; LS002933) and ribonuclease A (bovine pancreas; LS003433) were purchased from Worthington Biochemical Corp., Lakewood, NJ). Structural reference spectra were generated in the amide I (1600-1700 cm-1) and amide III (1200-1300 cm-1) bands, both of which were sensitive to protein secondary structural content. The amide I band estimates were significantly better than the amide III band estimates when comparing the secondary structure predictions (48) For dye binding analysis, the membrane swatches (2 cm in diameter) with adsorbed lysozyme were immersed for 1 h into a solution of Ponceau S, then washed five times with deionized water, immersed for 1 h into 5% (v/v) acetic acid, and again washed three times. The protein-dye complex was quantitatively eluted (desorbed from the membrane) with 3 mL of 10 mM NaOH solution for 1 h. The membranes were removed, the solutions were neutralized by addition of 50 µL of 6 M HCl, and the 515-nm absorbance of the red solutions was measured (model U-2000, Hitachi UV/visible spectrophotometer, Japan). A calibration curve was prepared from a known amount of lysozyme in solution. For further details, see Ulbricht, M.; Matuschewski, H.; Oechel, A.; Hicke, H.-G. J. Membr. Sci. 1996, 115, 31. (49) Vedantham, G.; Sparks, H. G.; Sane, S. U.; Tzannis, S.; Przybycien, T. M. Anal. Biochem. 2000, 285, 33.

9870

Langmuir, Vol. 19, No. 23, 2003

Han et al.

Figure 1. AFM topographical images (5 × 5 µm2) recorded in air for different MWCO PES membranes before (a-d) and after (e-h) lysozyme adsorption at 10 mg/mL for 300 min in PBS solution at pH 7.4. The 5-µm line scans below each image depict a roughness profile with different scales. Insets in parts a and e: 300 × 300 nm2. with known secondary structure components from X-ray data. The mean peak positions for Gaussian-Lorentzian band shapes corresponding to these reference spectra and structural motifs were R helix (1650 cm-1), β sheet (1693 and 1633 cm-1), unordered and unordered helix (1681, 1657, and 1634 cm-1), and turn structures (1723, 1666, 1633, and 1617 cm-1).49 For obtaining the adsorbed amounts of lysozyme, the Ponceau S dye technique correlated well with the peak areas in the amide II band region (the calibration between the protein surface concentration determined by the Ponceau S dye binding technique and the area under the amide II band gave a straight line: y ) 1.3963x, R2 ) 0.98).

Results and Discussion Model Substrates. A series of seven model substrates of increasing mean pore size (PES ultrafiltration membranes with 10-1000 kDa MWCOs) was chosen for this study. ATR/FTIR spectra (data not shown) clearly demonstrate that all the substrates have essentially the same surface chemistry because the spectra in the region of 800-1900 cm-1 are similar.47 In addition, they all have a peak around 1670 cm-1 not seen for pure PES42 (data not shown) due to an additive to the casting solution to impart increased hydrophilicity. The functional group exhibits a peak at this frequency.50,51 Topographical AFM images of four of the substrates before (a-d) and after (e-h) adsorption of lysozyme for 300 min from a 10 mg/ mL solution of protein in PBS at pH 7.4 are depicted in Figure 1. A line scan of the roughness for each substrate is shown below each image. First, it is clear that the roughness increases with the MWCO and that the images are quite different for the different substrates. Individual pores can be identified for the 300 and the 1000 kDa MWCO membranes. Changes in the surface topology (50) Pieracci, J.; Crivello, J. V.; Belfort, G. Chem. Mater. 2002, 14, 256. (51) Pieracci, J.; Crivello, J. V.; Belfort, G. J. Membr. Sci. 2002, 202, 1.

between clean (a-d) and protein-exposed (e-h) membranes are difficult to discern from the images, except perhaps for the 10 kDa MWCO membranes. Small roundshaped structures can be seen on the 10 kDa membrane after protein adsorption (inset in Figure 1e). As can be seen from the AFM scans (see scans below each image), in very few cases did the probe hit zero height. This suggests that the pores, which were contiguous (mostly connected to other pores), had very little linear depth, that is, less than the diameter of the pores. This means that the cantilever tip, which had a radius of about 20 nm, easily bottomed-out in the typical mean size pore. Quantitative roughness data are presented in Table 1 using the standard analysis of the surface roughness [roughness factor, γ (ratio of actual to projected surface area); root-mean-square roughness, Rrms; and average roughness, Rave] and a new measure of the roughness [R ) tan-1(δV/δH), where δV and δH are the mean horizontal and mean vertical length scales obtained from >300 AFM line scans of the surface, respectively].47,52 All four roughness parameters increase with the MWCO up to the 500 kDa MWCO membrane. The presence of adsorbed protein on the surface had little effect on the roughness parameters. Measured sessile captive bubble contact angles decreased from 49 to 24° for the clean original membrane. Adding R to the measured contact angle, θM, provides an estimate of the intrinsic (or corrected) contact angle of the surface, θ.47,53 Rave, θM, and θ are plotted in Figure 2 against the MWCO. As expected from the images and scans in Figure 1, as the roughness (Rave) increases with the MWCO, so θM decreases. The average corrected θ h and standard deviation was 47.4 ( 3.5° for the clean substrates. This is, within error, the same value as 46.8 ( 1.2° for a nonporous PES film.47 (52) Taniguchi, M.; Pieracci, J.; Belfort, G. Langmuir 2001, 17, 4312. (53) Wu, S. In Polymer Interface and Adhesion; Marcel Dekker: New York, 1982.

Nanometer-Scale Roughness Effect on Adsorbed Protein

Langmuir, Vol. 19, No. 23, 2003 9871

Table 1. Surface Parameters of Substrates (Synthetic PES Membranes) before and after Protein Adsorption clean substrate (no adsorbed protein)a MWCO (kDa) 10 30 70 100 300 500 1000 b

γb

c

Rrms (nm)

Raved,e

1.02 ( 0 1.02 ( 0 1.07 ( 0.01 1.08 ( 0.01 1.51 ( 0.26 2.49 ( 0.35 1.80 ( 0.23

8.1 ( 0.2 5.3 ( 0.6 8.4 ( 0.1 8.2 ( 0.5 25.4 ( 5.8 50.3 ( 6.9 52.9 ( 6.0

4.9 ( 0.2 3.9 ( 0.1 4.6 ( 0.2 7.2 ( 0.3 21.7 ( 0.9 37.4 ( 1.6 59.0 ( 3.3

(nm)

Re

protein-coated substrate (after adsorption)a θΜ(θ)f

(deg)

4.0 ( 0.3 5.2 ( 0.3 10.3 ( 0.5 13.0 ( 0.7 16.2 ( 1.7 22.8 ( 2.4 14.5 ( 2.5

49 (53) 45 (50) 37 (48) 30 (43) 31 (47) 24 (47) 29 (44)

γb 1.02 ( 0 1.09 ( 0 1.10 ( 0.02 1.60 ( 0.16 2.68 ( 0.25 1.97 ( 0.20

Rrmsc (nm)

Raved,e (nm)

Re (deg)

7.4 ( 0.3

6.3 ( 0.3

3.9 ( 0.2

5.5 ( 0.3 8.4 ( 0.6 29.1 ( 2.2 49.5 ( 6.7 50.1 ( 10.6

4.9 ( 0.2 7.8 ( 0.4 27.7 ( 1.2 42.1 ( 1.7 51.2 ( 1.5

7.6 ( 0.5 10.6 ( 0.6 17.7 ( 2.2 22.3 ( 2.2 17.9 ( 2.7

a Values of γ and R 2 rms are from AFM measurements for a 5 × 5 µm image window obtained from at least three independent experiments. γ (roughness factor) ≡ effective (actual) surface area/projected area obtained from AFM. c Rrms (root-mean-square roughness):

x∑ N

Rrms )

[

(Zn - Z)2/(N - 1)]

n)1

where Z h ) mean z height.

d

Rave (average roughness):

Rave )

N

|Zn - Z|

n)1

N

∑

R is obtained from a previous method.47,52 Mean horizontal, δH, and vertical, δV, length scales were obtained from more than 300 measurements of the depth (mean vertical distance from the top of the peak to the bottom of the groove) and the width (mean horizontal from peak to peak) for each membrane, respectively. f θ ) θM + R, where θ, θM, and R correspond to the corrected contact angle, the measured contact angle, and the surface slope ) tan-1(δV/δH), respectively. The values of the measured contact angles for each membrane were within (2° of the mean.

e

Figure 2. Changes in sessile captive air bubble contact angles, θM (measured, open circles) and θ (corrected, filled circles), and average roughness, Rave (filled triangles) as a function of the MWCO for the seven PES membranes (10, 30, 70, 100, 300, 500, and 1000 kDa MWCOs). The protocol for correcting θM used the same method as Belfort et al.47,52 The closed square on the left y axis is the contact angle for a smooth nonporous PES film.

For these model substrates, the surface roughness and surface chemical composition are decoupled because the membranes have the same surface chemistry but increasing nanometer-scale surface roughness. Protein Adsorption and Desorption. The adsorption kinetics of lysozyme from a 10 mg/mL PBS solution at pH 7.4 onto the seven different PES substrates showed typical convex curves reaching different saturation levels around 300 min (data not shown). The long-time saturation levels at 960 min were assumed to be the equilibrium adsorption levels [Qtot ads ) Γ/γ, where Γ is the adsorbed amount per unit projected surface area and γ is the roughness factor, which was used to convert the projected surface area to the actual surface area (Table 1)]. Thus, the total, Qtot ads, tot () Q Q and net, Qnet ), adsorbed amount, and des ads ads amount of lysozyme desorbed, Qdes, from washing after adsorption (in terms of milligrams per unit actual surface area) are plotted versus the MWCO, Rave, and γ in Figure 3a-c. Also plotted on these figures as horizontal lines is the range of calculated monolayer coverage of lysozyme depending on its adsorbed orientation (1.8-2.7 mg/m2).46,54

net tot Figure 3. (a) Total, Qtot ads, and net, Qads ()Qads - Qdes), adsorbed amount and amount of lysozyme desorbed, Qdes, from washing with deionized water five times after adsorption as a function of the MWCO. In part a, the open and closed circles denote Qtot ads and Qnet ads, respectively. The open triangles are for Qdes. The net net tot adsorbed amount, Qads ()Qads - Qdes), is plotted versus (b) Rave and (c) γ.

Except for the 100 kDa MWCO membrane, the Qnet ads values for all the other membranes fall within or are very close to the predicted range of monolayer coverage. Secondary Structural Changes on Adsorption. The secondary structure contents of lysozyme in the PBS solution such as R helix, β sheet, turns, and unordered structures as determined with ATR/FTIR49 are 0.40, 0.07, (54) Arai, T.; Norde W. Colloids Surf. 1990, 51, 17.

9872

Langmuir, Vol. 19, No. 23, 2003

Han et al.

recent report,42 where the secondary structural fractions of lysozyme change with increasing hydrophobicity of the substrate, we see no significant change here with the MWCO, Rave, or γ. Lysozyme, as expected, was perturbed from its free-solution secondary structure because the mean fractional values for all the substrates were changed to 0.36 ( 0.03, 0.16 ( 0.03, and 0.47 ( 0.04, from the free-solution values for the R helix, β sheet, and turns and unordered (combined) structures, respectively. Thus, for wild-type lysozyme adsorbed onto PES substrates for 5 h, the R-helix content dropped from 40 to about 36% (by ATR/FTIR) and the β-sheet content increased from 7 to about 16%. These results are consistent with how lysozyme adapts conformationally in the presence of lateral interactions.56,57 Conclusions

Figure 4. Fraction of secondary structure components of adsorbed lysozyme versus (a) the MWCO, (b) Rave, and (c) γ of the PES membranes for duplicate sets of measurements. Ordered helix (R helix; open and filled diamonds), sheet (β sheet; open and filled circles), and random/turns (unordered, unordered helix and turns; open and filled squares) for lysozyme adsorbed onto seven PES membranes in PBS solution at pH 7.4 and 25 °C for 300 min. Mean fractional values are represented by the dotted lines. The arrows show the values of the fraction of secondary structure components as measured by ATR/FTIR for lysozyme in free solution: ordered helix (0.40; R helix), sheet (0.07; β sheet), and random/turns (0.55; unordered, unordered helix and turns). The confidence limits for the structure estimates is (0.02.

0.40, and 0.14, respectively, and with X-ray55 are 0.46, 0.07, 0.43, and 0.05, respectively. Thus, for wild-type lysozyme in free solution, the R-helix content is approximately 40% (by ATR/FTIR) and the β-sheet content is about 7%. The structure fractions for lysozyme adsorbed onto the different PES substrates together with those for free solution are shown in Figure 4. In contrast to our (55) Frishman, D.; Argos, P. Proteins 1995, 23, 566.

This work provides strong evidence that, at the nanolength scales considered here (5 < Rave < 60 nm), the roughness has little effect on the amount of adsorbed protein when considering the actual surface area of the substrate for adsorption. Also, hen egg lysozyme, although structurally disturbed through adsorption to PES substrates, displays no significant changes in its secondary structure with increasing roughness. We speculate that only when the average length scale of the substrate surface is of the same size or smaller than the dimension of the adsorbate (protein) would the surface roughness likely influence the adsorption kinetics and amount adsorbed. Clearly, for cases when the scale of the roughness exceeds that of the protein dimension, the chemical composition of the substrate surface has a dominant influence on the adsorption process of the proteins and on secondary structural changes of a particular protein resulting from adsorption. Acknowledgment. Pall Filtron Corporation is thanked for donating the PES membranes, and Masahide Taniguchi is acknowledged for helpful advice and discussions. This research has been supported by the U.S. Department of Energy, Basic Chemical Sciences Division (Grant DE-FG02-90ER14114) and the National Science Foundation, Division of Chemical and Thermal Systems (Grant CTS-9400610). LA030132G (56) Roth, C. M.; Lenhoff, A. M. Langmuir 1994, 9, 962. (57) Johnson, C. A.; Wu, P.; Lenhoff, A. M. Langmuir 1994, 10, 3705.