Biomacromolecules 2004, 5, 1356-1361
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Zein Adsorption to Hydrophilic and Hydrophobic Surfaces Investigated by Surface Plasmon Resonance Qin Wang,† Jin-Feng Wang,‡ Phillip H. Geil,‡ and Graciela W. Padua*,† Department of Food Science and Human Nutrition and Department of Materials Science and Engineering, University of Illinois, 382/D AESB, 1304 West Pennsylvania Avenue, Urbana, Illinois 61801 Received January 11, 2004; Revised Manuscript Received April 16, 2004
Zein, the prolamine of corn, has been investigated for its potential as an industrial biopolymer. In previous research, zein was plasticized with oleic acid and formed into sheets/films. Physical properties of films were affected by film structure and controlled in turn by zein-oleic acid interactions. The nature of such interactions is not well understood. Thus, protein-fatty acid interactions were investigated in this work by the use of surface plasmon resonance (SPR). Zein adsorption from 75% aqueous 2-propanol solutions, 0.05% to 0.5% w/v, onto hydrophilic and hydrophobic self-assembled monolayers (SAMs) formed by 11mercaptoundecanoic acid and 1-octanethiol, respectively, was monitored by high time resolution SPR. Initial adsorption rate and ultimate surface coverage increased with bulk protein concentration for both surfaces. The initial slope of plotted adsorption isotherms was higher on 11-mercaptoundecanoic acid than on 1-octanethiol, indicating higher zein affinity for hydrophilic SAMs. Also, maximum adsorption values were higher for zein on hydrophilic than on hydrophobic SAMs. Flushing off loosely bound zein in the SPR cell allowed estimation of apparent monolayer values. Differences in monolayer values for hydrophobic and hydrophilic surfaces were explained in terms of zein adsorption footprint. Introduction Zein comprises a group of alcohol soluble proteins (prolamines) found in corn endosperm. Zein has been actively investigated for its film forming ability and its potential to produce novel biobased polymeric films.1,2 Plasticizers were used to overcome film brittleness and impart flexibility.1 Di Gioia and Gilbert pointed out that effectiveness of plasticizers was affected by their ability to interact with zein.3 However, the nature of those interactions is still not well understood. The isoform R-zein, which accounts for ∼85% of zein in the kernel, has a unique amino acid sequence containing more than 50% nonpolar amino acids.4 The average hydrophobicity of zein is reported at 1263 kcal/mol, which is nearly 50 times higher than that of albumin, γ-globulin, and fibrinogen of bovine blood.5,6 Unlike other proteins (i.e., membrane proteins), zein is not soluble in pure water nor in pure alcohol. It requires high percentage alcohol aqueous systems (i.e., 40-80%) for dispersion.7 The secondary and tertiary structure of zein were investigated by Argos and co-workers.8 They reported a possible structure containing 9 or 10 R-helix segments folded upon each other in an antiparallel fashion. According to that model, helical segments are arranged in a ring of “pencils” held together, side-by-side, by hydrogen bonds and linked at each end by glutamine-rich turns or loops. Matsushima et al. proposed that the helical segments were aligned to form a compact rectangular prism rather than an open ring.9 They reported the dimensions of the zein * To whom correspondence should be addressed. Telephone: (217) 3339336. Fax: (217) 333-9329. E-mail:
[email protected]. † Department of Food Science and Human Nutrition. ‡ Department of Materials Science and Engineering.
prism, measured by small-angle X-ray scattering (SAXS), as 16 × 4.6 × 1.2 nm3. According to this model, the exterior of the helical segments forming the lateral faces of the prism have a hydrophobic character, whereas the top and bottom surfaces containing the glutamine-rich loops are hydrophilic. Zein films were drawn from moldable zein resins containing oleic acid as a plasticizer.10 Resins were prepared by stirring the proper amounts of zein and oleic acid in warm 75% aqueous alcohol then pouring the mixture into ice water to form a dough-like mass. Structure characterization of zein films utilizing SAXS suggested a film structure consisting of staggered zein planes alternating with oleic acid layers.11 However, no conclusion could be made on the interaction between zein and oleic acid. Previous research (unpublished) suggested that the carboxylic group in oleic acid was critical to zein plasticization. Moreover, Wang and Padua suggested that plasticization in zein films was the result of surface controlled interactions between zein and oleic acid leading to film structure development.12 Therefore, a study of zein adsorption onto chemically different surfaces was undertaken to gain understanding of fundamental interactions between protein and fatty acid existing in zein films.13 The mechanism of globular protein adsorption to solid/ water interfaces has been reviewed by Haynes and Norde.14 Surface hydrophobicity is particularly important since it plays a role in many biomaterial applications.14-16 The use of surface plasmon resonance (SPR) as a biosensor has emerged over recent years as a valuable tool for probing adsorption processes at a surface in real time. Owing to its high sensitivity and label-free technique, SPR has been widely used to monitor the kinetics of biomolecular interactions such
10.1021/bm049965r CCC: $27.50 © 2004 American Chemical Society Published on Web 06/12/2004
Zein Adsorption at Solid-Liquid Interfaces
as protein/polymer adsorption, DNA hybridization, and lipid membrane-protein interactions. SPR principles and various biotechnology applications have been reviewed by several researchers.17-19 Silin and co-workers reported SPR studies of nonspecific binding of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) from aqueous solutions onto surfaces with different hydrophobicity.20 They observed that, in general, both proteins showed higher surface concentration on hydrophobic than that on hydrophilic surfaces. Wang and co-workers conducted SPR experiments to investigate zein adsorption to fixed hydrophilic and hydrophobic self-assembled monolayers (SAMs).13 They observed zein interaction with both surfaces and obtained estimates of monolayer values. However, their experimental setup did not permit observations on the kinetics of adsorption processes. Thus, the present study was undertaken to observe the dynamic adsorption of zein onto different surfaces. Protein adsorption was monitored by SPR. The surface topography of zein deposits was examined by atomic force microscope (AFM). Materials and Methods Chemicals. Zein (regular grade F4000, lot # F4000318C) was from Freeman Industries, Inc. (Tuckahoe, NY). The reported protein content for the F4000 Zein was 90-96% (dry base). Oleic acid (C18:1, 90%, technical grade), 11mercaptoundecanoic acid, and 1-octanethiol were from Aldrich Co., Inc. (Milwaukee, WI). Ethyl alcohol and isopropyl alcohol were from Midwest Grain Products (Pekin, IL). Dithiothreitol (DTT) and guanidine hydrochloride were from Fisher Scientific (Pittsburgh, PA). Sodium dodecyl sulfate (SDS) was from Bio-Rad Laboratories (Hercules, CA). SPR Sample Preparation. Zein solutions of 0.05, 0.10, 0.30, and 0.50% zein w/v were prepared by stirring the proper amount of zein in aqueous 75% 2-propanol, a known solvent for zein.21 SPR substrates were gold-coated glass microscope slides. Before metal evaporation, glass slides (Fisher Scientific, Pittsburgh, PA) were thoroughly cleaned with a mixture of concentrated sulfuric acid and 30% hydrogen peroxide (2:1 volume ratio) at 60 °C for 1 h. The dried slides were then coated with a 2 nm chromium (Kurt J. Lesker, Clairton, PA) adhesion layer at 0.02 nm/s and a 49-50 nm gold (99.99% purity) overlayer at 0.05 nm/s by resistive evaporation under a pressure of 4 × 10-6 Torr. Chromium was applied to ensure good adhesion of the gold layer since gold coatings easily peel off from glass. For the preparation of carboxylic acid terminated or methyl terminated SAMs, gold-coated slides were immersed overnight in 2 mM ethanolic solutions of 11-mercaptoundecanoic acid (COOH(CH2)10SH) or 1-octanethiol (CH3(CH2)7SH). Slides thus prepared were rinsed with ethanol and dried with filtered nitrogen. 11-mercaptoundecanoic acid and 1-octanethiol were selected because they are commercially available compounds that represent respectively the carboxylic and methyl end groups of oleic acid. To prevent dissociation of carboxylic groups of the 11-mercaptoundecanoic acid SAM, the pH of aqueous alcohol used to prepare zein solutions was previously adjusted to 3.55-3.85 with chloroacetic acid.
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SPR Instrument. SPR readings were taken in a customconstructed instrument in the Kretschmann configuration based on a design reported by Kooyman et al.22 The light source was a laser diode (Thor Labs, Newton, NJ) made of 3 mW helium-neon with wavelength of 670 nm. Passing through a dichroic polarizer, the light was polarized along the p-polarization direction. A half-wave retarder plate (HWP) was placed in the beam path to rotate the polarization by 90° and switch between s-polarized and p-polarized light without variation in the intensity of incident light. An electronic optical scanner (Electrooptical Products Corp., Model SC-20, Fresh Meadows, NY) was utilized to allow an angular scan of the laser beam through various angles of incidence. The light was directed by a planocylindrical lens on the curved surface of a half-cylinder made of SF10 glass. A silicon photodetector (PDA5, Thor Labs, Newton, NJ) was used to monitor the angle-dependent light intensity of the reflected light. Index matching oil (No. 16242, Cargill, NJ) was used to obtain optical contact between the gold-coated substrate and the prism. A flow cell measuring 7 mm × 15 mm × 0.7 mm was machined in Teflon and mounted onto the gold substrate and prism using a Kalrez O-ring. Fluids were pumped through Teflon tubing with a peristaltic pump (P-07523-40 and P-77390-00, Cole-Parmer, IL) to the flow cell. Initially, 75% 2-propanol was pumped through to clean the surface. It was then flushed with a large amount of buffer solution (10 mM chloroacetic acid and 75% 2-propanol). Once a stable baseline was reached, the flow was switched to the zein solution. The solution flowed over the surface at a predefined flow rate of 0.2 mL/min. The temperature of the solution was around 20-22 °C. When a plateau in the adsorption of zein was reached, the surface was rinsed with buffer solution. SPR Data Analysis. Data were processed with the software IgorPro written in HPVee 4.0 (Agilent Technologies, CO). During a half-cycle (0 < t < T/2; T ) 16.64 ms) of the mirror scan, the SPR angle θp was traversed twice, and two minima were observed at t1 and t2 in the measured intensity of the reflected light. Typically, the driving voltage was set such that the mirror scanned an angular range of 0.83°. In addition to recording the reflectivity curve over the complete angular range, we used an analogue differentiator circuit and a digital timer (resolution ) 5 µs) to track the time difference (τ ) t2 - t1) between the two minima in the reflected intensity. This change in τ is proportional to the shift in the SPR angle and is thereby a direct measure of the adsorption at the gold-solution interface. Because an electronic optical scanner was used, the angular scan took approximately 16 ms, and processing and recording of each data point could be completed within approximately 400 ms. This is significantly faster than measurement times reported in past studies using reflectometry or ellipsometry and allows monitoring of the rapid rates of adsorption at high bulk protein concentrations. The reflectivity in the SPR experiment was fit to an optical model based on the Fresnel equations. A five-layer model was used for the glass, gold, SAMs, protein, and buffer system. The normalized reflectivity was obtained as the ratio of the p-polarized light to the s-polarized light. Because s-polarized light does not couple to the surface
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plasmon wave, such normalization corrected the deviation from the optical components for the scanning of the laser. Nonlinear least-squares fitting was performed by a Levenberg-Marquardt algorithm using IgorPro software (WaveMetrics, OR). SPR results are given as averages of mass adsorbed per unit area. Viscosity of Zein Solutions. The intrinsic viscosity of various zein solutions was measured using an Ubbelohde viscometer (Cannon Instrument Company Inc., State College, PA) at 40 °C. Zein was dissolved (10 mg/ml) in aqueous ethanol (75%) at varying pH, 2.0, 4.0, 6.0, 8.0, 12.0, and 13.5. The pH was adjusted using proper amounts of 0.1 M HCl and 0.15 M NH4OH. In another set of experiments, zein solutions were added with DDT (0.04 M at pH ) 8 tris/ HCL buffer), guanidine hydrochloride (4 M), or SDS at three concentration levels (0.01, 0.1, and 0.2 M). AFM Analysis. AFM experiments were carried out with a Nanoscope IIIa microscope (Dimension 3100, Digital Instruments Inc., Santa Barbara, CA) where the tip motion is followed by deflection of a laser beam reflected off the rear side of the cantilever. The deflection is monitored with a position-sensitive detector. All AFM images were taken with an A-type scan head. Silicon nitride (Si3N4) probes attached to a microfabricated cantilever (200 µm, U shape base) with a scan frequency of 1.5 Hz were used. Measurements were collected in the contact force mode in which the deflection of the lever is kept constant by means of a feedback loop. In most experiments, images were taken repeatedly by changing the slide position. This procedure helps verify the observed structure and optimize the scan parameters and quality of recorded images. Contact mode scanning measures topography by sliding the probe tip across the sample surface. Samples of gold, SAMs, and zein deposits were examined by AFM.
Wang et al.
Figure 1. Zein adsorption kinetics from 75% 2-propanol solutions onto SAMs of 11-mercaptoundecanoid acid (1A) and 1-octanethiol (1B) for different zein bulk concentration levels: (a) Cb ) 0.05% w/v; (b) Cb ) 0.1% w/v; (c) Cb ) 0.3% w/v; (d) Cb ) 0.5% w/v.
Results and Discussion Zein adsorption from 2-propanol solutions (0.05-0.50% w/v) to 11-mercaptoundecanoic acid and 1-octanethiol SAMs was observed by SPR. Figure 1 presents the mass of zein deposited (Γ, mg/m2) as a function of time at different bulk protein concentrations (Cb, % w/v); Figure 1A,B corresponds to zein adsorption on carboxylic acid and methyl-ended SAMs, respectively. Initially, zein adsorption proceeded rapidly at the bare SAMs surface and Γ increased linearly with time. Later, when competition for space at the surface became dominant, the rate of adsorption decreased and eventually reached a plateau at maximum surface coverage (Γmax). The initial adsorption rate (before plateau region) of zein, [dΓ/dt]0, on both, hydrophilic and hydrophobic, surfaces increased with Cb, see Figure 2. The initial rate of adsorption on hydrophobic surfaces was proportional to Cb, [dΓ/dt]0 ) 0.0098Cb (R2 ) 0.929). This behavior is consistent with mass-transport-limited adsorption kinetics. Adsorption kinetics of aqueous solutions of albumin on hydrophobic surfaces was studied by Wertz and Santore.23 They observed that the initial rate was linear with Cb at low protein concentrations (0-0.01% w/v) and explained it in terms of transport-limited adsorption kinetics from the bulk to the bare surface. Zein
Figure 2. Zein initial rate of adsorption as a function of bulk concentration for hydrophobic and hydrophilic SAMs.
adsorption rate on hydrophilic surfaces did not follow the same trend, suggesting that adsorption was not masstransport-limited but surface controlled. In Figure 2, the initial adsorption rate was higher for hydrophilic than for hydrophobic surfaces, indicating that substrate hydrophobicity had a measurable effect on the rate of zein adsorption. Figure 1 also shows that maximum surface coverage on both hydrophilic and hydrophobic surfaces was affected by Cb. Adsorption isotherms are shown in Figure 3. Maximum zein adsorption, Γmax, increased rapidly with concentration up to 0.1% and then proceeded at a slower rate up to 0.5%
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Figure 3. Zein adsorption isotherm on hydrophilic and on hydrophobic SAMs.
for both surfaces. However, the initial slope was higher for hydrophilic than that for hydrophobic SAMs. According to Haynes and Nordes, the magnitude of the initial slope of the adsorption isotherm is an unambiguous indicator of protein affinity for a specific surface.14 Sorption isotherms in Figure 3 suggest that zein has a higher affinity for the 11mercaptoundecanoic acid surface than for the one with 1octanethiol. In general, proteins in aqueous systems show higher affinity for hydrophobic than for hydrophilic surfaces.20,24-26 However, Wertz and Santore observed higher Γmax for albumin and fibrinogen on hydrophilic than on hydrophobic SAMs.23,27 They interpreted their results as the dependence of Γmax on the spreading rate of adsorbed proteins, which increased with substrate hydrophobicity. It appears that aqueous-soluble proteins use their hydrophilic sites to associate with polar solvent molecules leaving the hydrophobic sites unoccupied. Therefore, they tend to adsorb more readily on hydrophobic surfaces than hydrophilic ones. The opposite argument could be applied to zein. Figure 1A,B also shows the desorption effect caused by flushing the SPR cell with buffer solution (at 1200 s). Flushing was thought to have removed loosely bound or overlaid zein adsorbed above SAMs surface saturation. The amount of adsorbed zein decreased from Γmax to a lower value, Γflushed, that was similar for all concentration curves in each figure. Γflushed was interpreted as representing the amount of zein adsorbed directly onto SAMs forming a monolayer. For the lowest concentration curves (0.05%), flushing had a minimum desorption effect possibly due to the absence of excess adsorption at that level. The average Γflushed value from the curves corresponding to 0.1%, 0.3%, and 0.5% zein was higher for hydrophilic (0.54 mg/m2) than for hydrophobic (0.11 mg/m2) surfaces. Wertz and Santore discussed lower maximum coverage of albumin and fibrinogen on hydrophobic than on hydrophilic SAMs in terms of protein relaxation and unfolding.23,27 Protein relaxation increases with time and determines ultimate footprint and surface coverage. When a protein initially adheres to a surface, it may occupy a region whose size is similar to that in solution and later the molecule may increase its number of segment-surface contacts and unfold or reorient on the surface to occupy a larger area. However, Norde noted that globular proteins are often highly ordered and the thickness of adsorbed layers is comparable to the dimensions of native proteins in solution.28
Figure 4. (a) Intrinsic viscosity of zein at different pH levels. The lines are included to guide the eye only. (b) Intrinsic viscosity of zein treated with SDS. The lines are included to guide the eye only.
The intrinsic viscosity of zein solutions with added denaturing agents was followed in order to gain understanding on the dimensional stability of zein. Data obtained from ethanolic solutions was considered useful for this purpose since it had been previously observed that SPR experiments yielded similar data when adsorption had taken place from ethanol or 2-propanol solutions. Viscosity of zein solutions at different pH levels is shown in Figure 4a. Viscosity changes were not apparent except at extremely low (2.0) or high (13.5) pH levels. Low viscosity at extreme pH may be attributed to protein degradation.29 DTT and guanidine hydrochloride are reducing agents often used to break disulfide bonds in proteins. Viscosity of zein solutions treated with DTT decreased only slightly from 13.0 to 12.3 mL/g. Similarly, the viscosity of zein solutions treated with 4 M guanidine hydrochloride decreased from 13.0 to 11.7 mL/g. Zein was also treated with SDS, an effective denaturant for many proteins. Measured viscosity at three different concentrations (0.01, 0.1, and 0.2 M) is shown in Figure 4b. Changes were only observed at high SDS concentrations. The viscosity of zein solutions was not substantially altered by pH (except at the extreme pH ) 13) or by the addition of DTT, guanidine hydrochloride, or SDS, thus suggesting that zein is quite stable against those denaturing agents. Higher Γflushed values observed in Figure 1 for zein adsorbed on hydrophilic surfaces maybe due to a smaller footprint for zein on hydrophilic than on hydrophobic surfaces. According to the structural model by Matsushima et al.,9 zein consists of repeats of helical segments aligned in antiparallel fashion forming a rectangular prism measuring
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Wang et al.
Figure 5. AFM image of gold substrate on glass slide.
Figure 7. (a) AFM image of zein deposited on hydrophilic SAMs after washing with buffer solution. (b) AFM image of zein deposited on hydrophobic SAMs after washing with buffer solution.
Figure 6. (a) AFM image of zein deposited on hydrophilic surface. (b) AFM image of zein deposited on hydrophobic surface.
17 nm × 4.6 nm × 1.2 nm. They considered that the 17 nm × 4.6 nm surfaces corresponded to the exterior of R-helix segments and were largely hydrophobic. The 17 nm × 1.2 nm surfaces contain glutamine loops or turns, which present sites for polar interaction. If zein is thought of as being dimensionally stable, it will maintain its prismatic structure after adsorption to SAM surfaces. It is possible that zein binds to hydrophilic and hydrophobic SAMs using different sides of its molecule. The 17 nm × 4.6 nm faces of the prism would be used to adsorb to hydrophobic SAMs, whereas the 17 nm × 1.2 nm faces containing glutamine loops would be used to adsorb to hydrophilic SAMs. This would result in
different zein footprint size and different monolayer values for each surface. The topography of SPR gold substrates, SAMs, and zein deposits was examined by AFM. To avoid regions of abnormal adsorption at the sides of channels, all images were taken within the center of the SPR flow channel. A typical image of a gold substrate is presented in Figure 5. Its roughness, calculated based on the root-mean-square (RMS) deviation from the average height of peaks above the background, is 1.84 nm. Images of zein adsorbed on carboxylic acid terminated thiol and methyl-terminated alkanethiol SAMs on gold-coated slides are shown in Figure 6a,b, respectively. Figure 6a presents a surface populated by distinct circular and relatively tall structures, 40 nm high, presumably resulting from overlaying zein molecules. Surface roughness was calculated at 6.97 nm. By comparison, zein adsorbed to the hydrophobic surface appears featureless with higher uniformity and a lower roughness value (1.88 nm). Images of zein layers remaining after flushing the SPR cell with buffer solution are presented in Figure 7a,b, for adsorption on hydrophilic and hydrophobic surfaces, respectively. Surfaces appeared covered with grains ∼200 nm in diameter. Both surfaces showed complete and uniform coverage. Zein impurities could sometimes be detected by AFM (images not shown) as particles of different size (larger) and morphology (rough edge) than zein.
Zein Adsorption at Solid-Liquid Interfaces
Conclusions SPR experiments indicated that zein was adsorbed to 1-octanethiol SAMs (hydrophobic) as well as to 11-mercaptoundecanoic acid SAMs (hydrophilic). However, the initial adsorption rate was higher for zein on 11-mercaptoundecanoic acid than on 1-octanethiol suggesting that different adsorption mechanisms operated in each case. Moreover, zein affinity for hydrophilic SAMs was higher than for hydrophobic SAMs, as evidenced by the different initial slopes of the sorption isotherms. Flushing off loosely adsorbed zein allowed the observation of an apparent monolayer value which was larger for zein on 11-mercaptoundecanoic acid than for zein on 1-octanethiol. This observation may be explained in terms of footprint size which according to a current model for the molecular structure of zein would be larger for zein binding to hydrophobic surfaces than for hydrophilic ones. Zein may have adsorbed to hydrophobic or hydrophilic SAMs utilizing different surfaces of its molecule. Acknowledgment. This work was supported in part by the Illinois Corn Marketing Board. We appreciate the help from the Center for Microanalysis of Materials, University of Illinois, which is partially supported by the U.S. Department of Energy under Grant DEFG02-91-ER45439. Sincere thanks are also extended to Dr. Antony R. Crofts, Dr. YuWen Huang, and Derrick R. Kolling for their helpful suggestions on SPR experiments. References and Notes (1) Reiners, R. A.; Wall, J. S.; Inglett, G. E. In Industrial Uses of Cereals; Pomeranz, Y., Ed.; Proceedings of the 58th Annual Meeting of the AACC Symposium; St. Paul, MN, 1973; pp 285-302.
Biomacromolecules, Vol. 5, No. 4, 2004 1361 (2) Gennadios, A.; Weller, C. L. Food Technol. 1990, 44, 63. (3) Di Gioia, L.; Guilbert, S. J. Agric. Food Chem. 1999, 47, 1254. (4) Pomes, A. F. Encyclopedia of Polymer Science and Technology; Mark, H., Ed.; Wiley: New York, 1971; Vol. 15, pp 125-132. (5) Belitz, H.-D.; Kieffer, R.; Seilmeier, W.; Wieser, H. Cereal Chem. 1986, 63, 336. (6) Baszkin, A.; Lyman, D. J. J. Biomed. Mater. Res. 1980, 14, 393. (7) Mosse´, J. Ann. Physiol. 1961, 3, 105 (in French). (8) Argos, P.; Pederson, K.; Marks, M. D.; Larkins, B. A. J. Biol. Chem. 1982, 257, 9984. (9) Matsushima, N.; Danno, G. I.; Takezawa, H.; Izumi, Y. Biochim. Biophys. Acta 1997, 1339, 14 (10) Lai, H.-M.; Padua, G. W. Cereal Chem. 1997, 74, 771. (11) Lai, H.-M.; Geil, P. H.; Padua, G. W. J. Appl. Polym. Sci. 1999, 71, 1267. (12) Wang, Q.; Padua, G. W. J. Polym. EnViron. 2003, Submitted. (13) Wang, Q.; Crofts, A. R.; Padua, G. W. J. Agric. Food Chem. 2003, 51, 7439-7444. (14) Haynes, C. A.; Norde, W. Colloids Surf., B: Biointerfaces 1994, 2, 517. (15) Prime, K. L.; Whitesides, G. M Science 1991, 2, 1164. (16) Schakenraad, J. M.; Busscher, H. J. Colloids Surf. 1989, 42, 331. (17) Salamon, Z.; Macleod, A. H.; Tollin, G. Biochim. Biophys. Acta 1997, 1331, 117. (18) Salamon, Z.; Macleod, A. H.; Tollin, G. Biochim. Biophys. Acta 1997, 1331, 131. (19) Silin, V.; Plant, A. Trends Biotechnol. 1997, 15, 353. (20) Silin, V.; Weetall, H.; Vanderah, D. J. Colloid Interface Sci. 1997, 185, 94. (21) Manley, R. H.; Evans, C. D. Ind. Eng. Chem. 1943, 35, 661. (22) Kooyman, R. P. H.; Lenferink, A. T. M.; Eenink, R. G.; Greve, J. J. Anal. Chem. 1991, 63, 83. (23) Wertz, C. F.; Santore, M. M. Langmuir 1999, 15, 8884. (24) Su, T. J.; Green, R. J.; Wang, Y.; Murphy, E. F.; Lu, J. R. Langmuir 2000, 16, 4999. (25) Yang, Z.; Galloway, J. A.; Yu, H. Langmuir 1999, 15, 8405. (26) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464. (27) Wertz, C. F.; Santore, M. M. Langmuir 2001, 17, 3006. (28) Norde, W. AdV. Colloid Interface Sci. 1986, 25, 267. (29) Shukla, R.; Cheryan, M. Ind. Crops Prod. 2001, 13, 171.
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