Residence Time, Loading Force, pH, and Ionic Strength Affect

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Residence Time, Loading Force, pH, and Ionic Strength Affect Adhesion Forces between Colloids and Biopolymer-Coated Surfaces Li-Chong Xu, Virginia Vadillo-Rodriguez, and Bruce E. Logan* Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802 Received April 6, 2005 Exopolymers are thought to influence bacterial adhesion to surfaces, but the time-dependent nature of molecular-scale interactions of biopolymers with a surface are poorly understood. In this study, the adhesion forces between two proteins and a polysaccharide [Bovine serum albumin (BSA), lysozyme, or dextran] and colloids (uncoated or BSA-coated carboxylated latex microspheres) were analyzed using colloid probe atomic force microscopy (AFM). Increasing the residence time of an uncoated or BSA-coated microsphere on a surface consistently increased the adhesion force measured during retraction of the colloid from the surface, demonstrating the important contribution of polymer rearrangement to increased adhesion force. Increasing the force applied on the colloid (loading force) also increased the adhesion force. For example, at a lower loading force of ∼0.6 nN there was little adhesion (less than -0.47 nN) measured between a microsphere and the BSA surface for an exposure time up to 10 s. Increasing the loading force to 5.4 nN increased the adhesion force to -4.1 nN for an uncoated microsphere to a BSA surface and to as much as -7.5 nN for a BSA-coated microsphere to a BSA-coated glass surface for a residence time of 10 s. Adhesion forces between colloids and biopolymer surfaces decreased inversely with pH over a pH range of 4.5-10.6, suggesting that hydrogen bonding and a reduction of electrostatic repulsion were dominant mechanisms of adhesion in lower pH solutions. Larger adhesion forces were observed at low (1 mM) versus high ionic strength (100 mM), consistent with previous AFM findings. These results show the importance of polymers for colloid adhesion to surfaces by demonstrating that adhesion forces increase with applied force and detention time, and that changes in the adhesion forces reflect changes in solution chemistry.

Introduction An understanding of the chemical and physical factors influencing bacterial adhesion is important to bioremediation of contaminated soil,1 biofilm formation on orthopaedic implants,2,3 and biofouling of ship hulls in marine systems.4 The initial bacterial attachment (reversible adhesion) to a surface depends on the physicochemical characteristics of the bacterium, the fluid interface, and the substratum characteristics.5,6 These factors affect one or more of the many forces that govern the approach and adhesion of a particle to the surface, such as electrostatics,7,8 van der Waals,9,10 hydrophobic,11-13 hydration,14,15 and specific chemicals forces.3 Several investigators have observed that bacterial adhesion and desorption are residence-time-dependent.7,16,17 The time dependence of this process can be regarded as the result of interaction and attraction of bacterial exopolymers (e.g., proteins, lipopolysaccharides, and other biopolymers) with the surface and the formation of weak and strong chemical bonds of the polymers with the surface. Such interactions can depend on specific protein acceptor-donor interactions as well as on specific * Corresponding author. Phone: 814-863-7908. Fax: 814-8637304. E-mail: [email protected]. (1) Li, Q.; Logan, B. E. Water Res. 1999, 33, 1090-1100. (2) Sheehan, E.; McKenna, J.; Mulhall, K. J.; Marks, P.; McCormack, D. J. Orthop. Res. 2004, 22, 39-43. (3) Speranza, G.; Gottardi, G.; Pederzolli, C.; Lunelli, L.; Canteri, R.; Pasquardini, L.; Carli, E.; Lui, A.; Maniglio, D.; Brugnara, M.; Anderle, M. Biomaterials 2004, 25, 2029-2037. (4) Schmidt, D. L.; Brady, R. F.; Lam, K.; Schmidt, D. C.; Chaudhury, M. K. Langmuir 2004, 2830-2836. (5) Gristina, A. G. Science 1987, 4822, 1588-1595. (6) Iwabuchi, N.; Sunairi, M.; Anzai, H.; Morisaki, H.; Nakajima, M. Colloids Surf., B 2003, 30, 51-60.

carbohydrate polymers.5,18,19 The bond between biopolymers and a surface may strengthen or weaken over time, leading to adsorption and desorption rates that vary between bacterial strains.16 Solution ionic strength (IS) and pH are well-known to influence the extent of bacterial adhesion to a surface. Increasing the IS compresses the thickness of the electrostatic double layer surrounding a bacterium and results in an increase in bacterial adhesion.20-22 This phenomenon (7) Bouttier, S.; Han, K. G.; Ntsama, C.; Bellon-Fontaine, M. N.; Fourmat, J. Colloids Surf., B 1994, 2, 57-65. (8) Mosley, L. M.; Hunter, K. A.; Ducker, W. A. Environ. Sci. Technol. 2003, 37, 3303-3308. (9) Hayashi, H.; Tsuneda, S.: Hirata, A.; Sasaki, H. Colloids Surf., B 2001, 22, 149-157. (10) Poortinga, A. T.; Bos, R.; Norde, W.; Busscher, H. J. Surf. Sci. Rep. 2002, 47, 1-32. (11) Rijnaarts, H. H. M.; Norde, W.; Bouwer, E.; Lyklema, J.; Zehnder, A. J. B. Environ. Sci. Technol. 1996, 30, 2877-2883. (12) Vadillo-Rodriguez, V.; Busscher, H. J.; Norde, W.; de Vries, J.; van der Mei, H. C. J. Bacteriol. 2004, 186, 6647-6650. (13) Salerno, M. B.; Logan, B. E.; Velegol, D. Langmuir 2004, 20, 10625-10629. (14) Busalmen, J. P.; de Sanchez, S. R. Int. Biodeterior. Biodegrad. 2003, 52, 13-19. (15) Valle-Delgado, J. J.; Molina-Bolı´var, J. A.; Galisteo-Gonza´lez, F.; Ga´lvez-Ruiz, M. J.; Feiler, A.; Rutland, M. W. J. Phys. Chem. 2004, 108, 5365-5371. (16) Meinders, J. M.; van der Mei, H. C.; Busscher, H. J. J. Colloid Interface Sci. 1995, 176, 329-341. (17) Switalski, L.; Butcher, W. G. Arch. Oral Biol. 1994, 39, 155161. (18) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368-1374. (19) Fu¨nfstu¨ck, R.; Franke, S.; Hellberg, M.; Ott, U.; Kno¨fel, B.; Straube, E.; Sommer, M.; Hacker, J. Int. J. Antimicrob. Agents 2001, 253-258. (20) Zita, A.; Hermansson, M. Appl. Environ. Microbiol. 1994, 60, 3041-3048. (21) Deshpande, P. A.; Shonnard, D. R. Water Resour. Res. 1999, 35 (5), 1619-1627.

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is qualitatively consistent with predictions based on DLVO theory as an increase in the IS will reduce the range of electrostatic repulsion between two negatively charged surfaces.9,23,24 Changes in solution pH are thought to influence bacterial adhesion by altering hydrogen bonding forces between polymers and the surface, as well as changing electrostatic interactions. For example, Ahimou et al.25 found that lowering the pH from 7 to 4 increased bacterial adhesion. They attributed the increased adhesion at the lower pH to increased formation of hydrogen bonds between the bacteria and surface, while at higher pH they attributed the reduction in adhesion to the increased electrostatic repulsion between the bacteria and the surface. A consistent change in bacterial adhesion with solution pH, however, is not always observed. For example, some investigators have found that pH has no effect on adhesion when the pH range is limited to a smaller, and near neutral, range of 5.5-7.26,27 The solution IS can also be a factor in the effect of pH. Busalmen and de Sa´nchez14 observed that bacterial adhesion increased with pH in a low-IS solution (0.1 M), but adhesion was consistently low over a pH range of 2-8 in a high-IS solution (0.6 M). Several macroscopic techniques have been used to study the kinetics of microbial adhesion to surfaces, ranging from well-defined systems such as parallel plate flow chambers28,29 and packed columns30,31 to simple adhesion tests based on exposing a surface to a bacterial suspension for a specific amount of time.32 While these techniques distinguish adhesion characteristics of bacteria or surfaces, they do not provide more detailed information on adhesion forces at nanoscale to molecular scale that influence bacterial adhesion and allow us to better understand the factors affecting adhesion. By using an atomic force microscope (AFM), it has been possible to directly measure the physicochemical properties of biopolymers on bacterial surfaces at molecular scales, including the interaction forces of these surfaces with the AFM tip or other surfaces due to electrostatic and steric interactions.33-38 It has been shown that the contact time of the AFM tip with a surface of either proteins or bacteria increases the adhesion force measured during tip retraction.18,39-41 It is (22) Rijnaarts, H. H. M.; Norde, W.; Lyklema, J.; Zehnder, A. J. B. Colloids Surf., B 1999, 14, 179-195. (23) Azeredo, J.; Visser, J.; Oliveira, R. Colloids Surf., B 1999, 14, 141-148. (24) Brant, J. A.; Childress, A. E. Environ. Eng. Sci. 2002, 19, 413427. (25) Ahimou, F.; Denis, F. A.; Touhami, A.; Dufrene, Y. Langmuir 2002, 18, 9937-9941. (26) Sadr Ghayeni, S. B.; Beatson, P. J.; Schneider, R. P.; Fane, A. G. J. Membr. Sci. 1998, 138, 29-42. (27) Jewett, D. G.; Hilbert, T. A.; Logan, B. E.; Arnold, R. G.; Bales, R. C. Water Res. 1995, 29, 1673-1680. (28) Meinders, J. M.; Noordmans, J.; Busscher, H. J. J. Colloid Interface Sci. 1992, 152, 265-280. (29) McClaine, J. W.; Ford, R. M. Biotechnol. Bioeng. 2002, 78, 179189. (30) Gross, M. J.; Albinger, O.; Jewett, D. G.; Logan, B. E.; Bales, R. C.; Arnold, R. G. Water Res. 1995, 29, 1151-1158. (31) Burks, G. A.; Velegol, S. B.; Paramonova, E.; Lindenmuth, B. E.; Feick, J. D.; Logan, B. E. Langmuir 2003, 19, 2366-2371. (32) Li, B. K.; Logan, B. E. Colloids Surf., B 2004, 36, 81-90. (33) Razatos, A.; Ong, Y. L.; Boulay, F.; Elbert, D.; Hubbell, J. A.; Sharma, M. M.; Georgiou, G. Langmuir 2000, 16, 9155-9158. (34) Fang, H. H. P.; Chan, K. Y.; Xu L.-C. J. Microbiol. Methods 2000, 40, 89-97. (35) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353 (6341), 239-241. (36) Camesano, T. A.; Abu-Lail, N. I. Biomacromolecules 2002, 3, 661-667. (37) Velegol, S.; Logan, B. E. Langumir 2002, 18, 5256-5262. (38) Emerson, R. J.; Camesano, T. A. Appl. Environ. Microbiol. 2004, 70, 6012-6022. (39) Hemmerle, J.; Altmann, S. M.; Maaloum, M.; Horbert, J. K. H.; Heinrich, L.; Voegel, J. C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 67056710.

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reasonable to assume that this increase in adhesion results from how the biopolymers on the bacterial surface interact with the surface over time. However, the time-dependent nature of biopolymer interactions with surfaces has not been sufficiently examined, especially as a function of solution chemistry and the force applied on a biopolymer. In this study, we used colloid probe AFM to measure interaction forces between a colloid mounted on an AFM tip with a surface, in the presence or absence of various polymers coating these surfaces. These interactions were examined both as a function of time and loading force (the force applied by the colloid probe), as well as solution chemistry (pH and IS). By using this approach of examining polymer interactions with surfaces, we hope to better understand the molecular-scale factors that affect biopolymer interactions with surfaces and, therefore, microbial adhesion to surfaces. Methods Colloid and Surfaces. Carboxylated polystyrene latex microspheres (4.5 µm, Polysciences, Warrington, PA) or glass beads (∼4.5 µm, Polysciences) were used as the colloid probe in all AFM experiments. Latex microspheres and glass beads were washed and rinsed three times with Milli-Q water by centrifugation prior to use. Glass cover slips (25 mm × 60 mm, Corning No.1 cover glass, Corning, NY) were rigorously cleaned,42 rinsed with copious amounts of ultrapure water (Milli-Q), and stored in ultrapure water at 4 °C before use. Covalent Bonding of Biopolymers to Glass and Latex Microsphere Surfaces. Bovine serum albumin (BSA, 69 kDa), lysozyme (14 kDa) from chicken egg white, and dextran (144 kDa; Sigma, St. Louis, MO), were used as model protein or polysaccharide biopolymers having different charges at pH 7.0. BSA is negatively charged, while lysozyme is positively charged. Dextran is a hydrophilic and neutrally charged polysaccharide at pH 7.0. Glass cover slips were modified by bonding an amino functional group to the glass cover slip and then covalently bonding a protein (BSA or lysozyme) to this amino group as previously described.43 BSA was covalently bonded to a carboxylated latex microsphere using a standard carbodiimide kit (Polysciences). To couple dextran to a glass surface, it was first modified with carboxyl functional groups using succinic anhydride and dimethyl sulfoxide (DMSO) as described elsewhere.44 Amino-functionalized glass surfaces were incubated in the succinoylated dextran solution (10 mg/mL) containing 1-[3-(dimethylamino)propy]-3ethylcarbodiimide hydrochloride (EDC, 400 mM) and sulfo-Nhydroxysulfosuccinimide (115 mM, Pierce Biotechnology Inc. Rockford, IL) in sodium phosphate buffer (10 mM, pH 7.5) and allowed to react overnight with gentle shaking at 50 rpm. After being coated with biopolymers, the slides were rinsed with Milli-Q water before being used in AFM experiments. The ζ potentials of uncoated colloids and colloids coated with BSA were measured 5 times using 25 cycles/analysis (ZetaPALS analyzer, Brookhaven Instruments Corp., Holtzville, NY). AFM Experiments. All AFM experiments were performed using a BioScope AFM (Digital Instrument, Santa Barbara, CA) with a Nanoscope IIIa control system (Version 4.32r3). Biopolymer coatings were imaged using tapping mode in liquid at a drive frequency of ∼8 kHz and a drive amplitude of 100 mV. Silicon nitride AFM tips (Veeco NanoProbe NP-20; narrow long cantilever) were used for imaging. Adhesion force measurements were performed in contact mode using a colloid probe. Colloid probes were prepared by gluing (40) Mondon, M.; Berger, S.; Ziegler, C. Anal. Bioanal. Chem. 2003, 375, 849-855. (41) Vadillo-Rodriguez, V.; Busscher, H. J.; Norde, W.; de Vries, J.; van der Mei, H. C. J. Colloid Interface Sci. 2004, 278, 251-254. (42) Camesano, T. A.; Natan, M. J.; Logan, B. E. Langmuir 2000, 16, 4563-4572. (43) Xu, L.-C.; Logan, B. E. Environ. Sci. Technol. 2005, 39, 35923600. (44) Stevens, M. M.; Allen, S.; Davies, M.; Roberts, C. J.; Schacht, E.; Tendler, S. J.; VanSteenkiste, S.; Williams, P. M. Langmuir 2002, 18, 6659-6665.

Colloid and Biopolymer-Coated Surface Adhesion

Figure 1. Colloid probe force curve drawn to show the time scale of the ramp time needed to achieve a loading force of 5.4 nN. (DEVCON epoxy S-31/31345) the latex microsphere to a tipless silicon nitride cantilever (Veeco NanoProbe NP-OW) as previously described.45 The spring constant of the cantilevers (all taken from the same wafer) was determined using the thermal tuning method (Nanoscope V6.12r2) as 0.0613 ( 0.0030 N/m. After preparation, the colloid probe was stored in the refrigerator (4 °C) prior to use. Each probe was inspected before an experiment using a microscope (Olympus IX70) to ensure probe integrity.

Langmuir, Vol. 21, No. 16, 2005 7493 Multiple probes were made at the same time to obtain consistent responses of the probes between experiments, and each probe was tested for a consistent response to a clean glass surface prior to an experiment. Any probe that did not display behavior consistent with other probes from that batch was discarded. All force measurements were made in phosphate buffer solutions (PBS) with an IS of 1 or 100 mM at a scan rate of 1 Hz and z scan size of 1 µm. A series of PBS (IS, 1 and 100 mM) were prepared that had pH values of 4.5, 7.3, 8.8, and 10.6. To set the loading force applied by the colloid to a surface at values of 5.4, 3.6, 2.4, 1.2, and 0.6 nN, the trigger mode setting was used in the software with the threshold of the cantilever deflection set at the appropriate value. The ramp delay in the software was set at different times, producing different colloid residence times. The time needed for the tip to reach the desired loading force from the point of contacting sample is defined as the ramp time, while the residence time is defined as the time after the loading force has reached the set value until the tip begins to be retracted (Figure 1). Force measurements were performed at residence times of 0.001-50 s at four to five randomly selected locations over every sample studied. Ten force curves were obtained at each location and at a fixed residence time. To avoid changes in salt concentration due to evaporation, solution levels were examined and adjusted as needed with PBS during an experiment. Interaction force curves were shown to be reproducible for the same operating conditions by examining different locations on the surface. Nonreproducible curves were assumed to indicate tip contamination, and therefore the probe was discarded. Forces were reported as positive when the cantilever was deflected

Figure 2. AFM 3-D images of (a) bare glass, (b) BSA, (c) lysozyme, and (d) dextran on glass surfaces in 1 mM IS. (Scale: x, y, 100 nm/div; z, 10 nm/div).

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Figure 3. Section analysis of AFM topographic image of BSA in (a) 1 mM and (b) 100 mM IS solutions. upward from the sample and negative when the cantilever was drawn down to the sample. The adhesion force is the value measured at the point of maximum deflection during the colloid probe retraction from the surface. Model. To quantitatively relate the adhesion forces measured using the different biopolymer-coated surfaces and colloids with time, adhesion forces were modeled using a simple power-law relationship of

F ) - ATn

(1)

where F is the adhesion force, T is the residence time, and A and n are empirical coefficients determined from log-log plots.

Results Characteristics of Biopolymer Coatings and Colloids. In general, biopolymers were covalently bonded and uniformly adsorbed on the glass surfaces (Figure 2). Uniform coatings were also confirmed from force measurements with colloid probes by the observation of consistent interaction forces in force curves from different locations of each sample. The presence of the biopolymer coating on a surface is indicated by surface roughness. For bare glass, the roughness (Rq) was only 0.060 nm, while surfaces coated with BSA, lysozyme, and dextran had average roughness values of 0.50, 0.30, and 1.50 nm (IS ) 1 mM). On the basis of the surface roughness, the thickness of the biopolymer coatings was calculated as follows: BSA, 5.2 nm; lysozyme, 3.0 nm; and dextran, 11.1 nm. Solution IS changed the conformation of biopolymers. In high-IS solution (100 mM) polymers appeared to become more folded and compressed, resulting in the

reduction of the effective diameter of the polymers as evidenced by a reduction in the thickness and roughness of the surface. This phenomenon was consistently observed for each biopolymer. For example, in 100 mM solution the thickness of the BSA coating was 2.0 nm and the roughness was 0.23 nm. As a result, the average diameter of the BSA was calculated to be 12.9 ( 3.6 nm in the 100 mM IS solution versus 18.6 ( 5.3 nm in the 1 mM IS solution (Figure 3). A carboxylated latex microsphere is a “soft” microparticle with the elastic modulus of 3 × 109 N/m2 and Poisson ratio of 0.35, while the glass surface is rigid and had a elastic modulus of 64.7 × 109 N/m2 and a Poisson ratio of 0.19.43 When a microsphere was coated with BSA, there was an average of (1.61 ( 0.22) × 10-4 ng of BSA on a surface. The ζ potential of uncoated carboxylated latex microspheres was -107 ( 1.7 mV in 1 mM solution and -44.6 ( 1.8 mV in 100 mM IS solution (pH ) 7.3). Coating the colloid with BSA decreased the ζ potential of the latex microspheres to -85.6 ( 0.8 and -24.6 ( 1.4 mV in 1 and 100 mM IS solutions, respectively. This decrease in ζ potential likely results from coverage of the colloid surface with BSA that has a lower negative charge than the surface at neutral pH Loading Force. Interaction distances and adhesion forces between an uncoated microsphere colloid probe and a glass surface consistently increased with loading force in the presence of a biopolymer regardless of biopolymer type. Examples of retraction curves measured at three different loading forces for a BSA-coated microsphere interacting with a BSA-coated glass, shown in Figure 4,


Figure 4. Representative approach and retraction force curves of BSA-coated microspheres to BSA-coated glass surfaces at different loading forces (IS ) 100 mM; pH ) 7.3).

Figure 5. Effect of loading force on the adhesion of biopolymers and surfaces: (a) interactions between BSA and a microsphere or BSA-coated microsphere (residence time ) 1 ms; pH ) 7.3); (b) interactions between lysozyme or dextran and uncoated or BSA-coated microsphere (IS ) 100 mM; residence time ) 1 ms).

demonstrate that both the maximum retraction forces and maximum pull-off distances increased with the force applied by the colloid. Changing the applied loading force did not affect approach curves. The maximum pull-off distance was 105 ( 45 nm at loading force 0.6 nN, while it reached 145 ( 30 and 184 ( 49 nm when the loading forces were increased to 2.4 and 5.4 nN, respectively. The increase in adhesion with a range of applied forces is shown in Figure 5a for both uncoated and BSA-coated microspheres interacting with a BSA-coated surface in solutions with a low (1 mM) and high (100 mM) IS. The maximum adhesion force between a BSA-coated microsphere and a BSA-coated glass surface was -2.1 ( 0.5 nN in a 1 mM IS solution, with a slightly lower maximum adhesion force of -1.4 ( 0.3 nN in a 100 mM solution (5.4 nN applied force). Similar trends between the measured adhesion force and the loading force were observed when lysozyme

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or dextran were investigated at IS ) 100 mM (Figure 5b). The increase in the adhesion force is thought to result from an increase in the number of chemical bonds formed between the biopolymers when the surfaces are forced into greater contact using higher loading forces. The solution IS affected the measured adhesion forces, but only at the higher loading forces. No significant effect of IS was found for the adhesion forces between two surfaces at loading forces < 3.6 nN, when both surfaces were coated with BSA (for example, see Figure 5a). However, at a higher loading force of 5.4 nN, the adhesion force was larger at the low-IS (1 mM) solution for BSAcoated surfaces and the colloid (uncoated or BSA-coated) than at high IS (100 mM). This finding of adhesion being inversely related to IS has been previously observed,43 but only a single high loading force of ∼6.0 nN was previously examined. Residence Time. The adhesion force measured between a colloid and biopolymer-coated surfaces increased with residence time over a range of 0.001-50 s (fixed pH ) 7.3 and IS ) 100 mM) (Figure 6). The largest changes in adhesion force were generally observed when the residence time was in the range of 1-20 s. This increase in adhesion with residence time in the presence of polymers has been noted by others.39-41 Increasing the loading force produced greater adhesion forces over most of the range of residence times examined (Figure 6). At the lower loading force of 0.6 nN, there was only a slight increase in the adhesion forces for residence times up to 5 s. For example, the adhesion force of an uncoated microsphere to a BSA-coated surface was only -0.15 nN within a residence time of 5 s, but the force increased to -0.47 and -0.84 nN when residence time was increased to 10 and 20 s, respectively. At a higher loading force of 5.4 nN, the adhesion forces were -3.3, -4.1, and -4.8 nN at residence times of 5, 10, and 20 s (Figure 6a). For the BSA-coated microsphere and BSA system, the adhesion forces increased to as much as -7.5 nN at a residence time of 10 s, while it is only -1.36 nN at a residence time 0.1 s. These results reinforce the above finding that the loading force significantly influences the adhesion between biopolymers and surfaces and shows that this phenomenon can be generalized to different residence times of the colloid at biopolymer-coated surfaces. Adhesion forces increased when both a colloid and the flat surface were coated with biopolymers compared to forces measured between an uncoated microsphere and a biopolymer-coated surface for the range of residence times examined here (Figure 6). At the shortest residence time (0.001 s), the adhesion forces measured decreased in the order BSA-lysozyme > BSA-dextran > BSA-BSA (loading force of 5.4 nN). However, the maximum adhesion forces measured at the longest residence times were larger for BSA-coated microspheres interacting with BSA-coated surfaces, than those for BSA-coated microspheres and lysozyme- or dextran-coated surfaces (loading force of 5.4 nN). Recall that BSA, lysozyme, and dextran are negatively, positively, and neutrally charged polymers at neutral pH. This suggests that electrostatic interactions were dominant during short contact times, while the chemical bonding between polymer molecules, polymer rearrangement, and water exclusion became more important to the measured adhesion force with increasing residence times. The adhesion force between uncoated microspheres and a bare glass surface was also larger than that observed for an uncoated glass bead and a glass (45) Li, X.; Logan, B. E. Langmuir 2004, 20, 8817-8822.

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Figure 6. Effect of residence time on the adhesion forces of different biopolymers with uncoated (a, c, and e) and BSA-coated microspheres (b, d, and f) for three different loading forces (9, 5.4 nN; b, 2.4 nN; 2, 0.6 nN) in 100 mM IS at pH 7.3.

Figure 7. Time-dependent adhesion forces of glass surface with uncoated microspheres or glass beads in 1 or 100 mM IS (pH ) 7.3; loading force ) 5.4 nN).

surface (Figure 7) but smaller than that observed for biopolymer coated surfaces. For example, the adhesion force of an uncoated microsphere and a glass surface was only -2.1 ( 0.7 nN at a residence time of 10 s (IS ) 100 mM), compared to adhesion forces to BSA, lysozyme, and dextran of -4.1 ( 0.7, -4.7 ( 1.2, and -4.0 ( 0.4 nN, respectively. The adhesion force between a microsphere and an uncoated glass surface also reached a maximum

value sooner (within ∼10 s; Figure 7) than observed for biopolymer-coated surfaces (>20-50 s). The adhesion force between the uncoated latex microsphere and glass was larger for an IS of 1 mM than an IS of 100 mM, at the longest residence time, consistent with previous findings on the effect of IS on adhesion.43 There was no change in the adhesion force measured between an uncoated glass bead and an uncoated glass surface up to a residence time of 50 s (Figure 7). We attribute the lack of a change in the adhesion force of the glass-glass system to the absence of polymers on the glass surface. The observation that adhesion force increased with the uncoated latex microsphere with an increase in residence time suggests that there are polymers on the latex microsphere itself. This indicates the latex microsphere is not a truly “hard surface” in comparison to the glass bead. pH. The solution pH influenced both the approach and retraction force curves. A long-range repulsive force observed in approach curves at the two higher pHs (8.8 and 10.6) was absent at the lower pH of 4.5, for both the uncoated and BSA-coated microspheres with a BSA-coated glass surface (Figure 8). This lowest pH is below the isoelectric point of BSA (∼6.1). A long-range repulsive force was also observed for lysozyme (isoelectric point ∼ 9.0) with an uncoated microsphere at pHs of 8.8 and 10.6 and with a BSA-coated microsphere at pHs of 7.3-10.6. For dextran, a repulsive force was observed over the pH range of 7.3-10.6 in interactions with either uncoated- or


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Figure 8. Representative approach and retraction force curves of colloids to BSA-coated glass surfaces at different pH (IS ) 100 mM).

BSA-coated microspheres. The observation of long-range repulsive force at higher pH is consistent with the finding in the interaction of two different biopolymers (BSA and apoferritin) and silica sphere by Valle-Delgado et al.,15,46 who showed that the presence of hydration forces at high pH and a high salt concentration resulted in non-DLVO repulsive interactions. Retraction curves in low-pH solutions exhibited longrange, sawlike patterns that are indicative of multiple bonds being broken between the colloid and polymers as the probe is retracted from a surface (Figure 8). The adhesion forces are larger when both surfaces were coated by biopolymers than those when only one surface was coated. For example, the adhesion forces were as large as -1.9 ( 0.5 nN at a pH 4.5 when both surfaces were BSAcoated and decreased to -0.6 ( 0.2 nN when an uncoated microsphere was used (see Figure 9a). Similar results were observed with lysozyme and dextran (Figure 9b, c). Results showed that the adhesion force of a microsphere (uncoated or BSA-coated) to biopolymer-coated surfaces always decreased with an increase in pH at IS ) 100 mM. Note that, for all the cases, the adhesion forces between biopolymers and uncoated microspheres were significantly smaller than those between biopolymers and BSA-coated microspheres in the more acidic solution (pH of 4.5). In contrast, there was no significant difference in the adhesion forces in the interactions of microspheres with biopolymer-coated surfaces in the higher pH solutions (pH of 8.8 or 10.6). This relatively larger adhesion force at the lower pH could be due to either increased hydrogen bonding which results from the lower pH conditions or increased electrostatic repulsive forces at the higher pHs.25 Ionic Strength. A comparison of the adhesion data on the basis of IS reveals that adhesion force was inversely related to IS for both uncoated- and BSA-coated microspheres and all three different biopolymer coated surfaces (p < 10-5 for all cases, Analysis of Variance test; Figure 10). This finding is in contrast to what we would expect on the basis of a DLVO model for two like-charged surfaces. It is expected that as the IS decreases, two surfaces would have less adhesion due to an increase in the repulsive layer thickness between the two surfaces. Similarly, it is well-known that bacterial adhesion generally increases with ionic strength.20-22 On the basis of the opposite finding here, that adhesion decreased with IS, we surmise that conformational changes (Figure 3) of biopolymers due to the different IS produced unanticipated changes in molecular-scale interactions between the biopolymers and surfaces, resulting in lower adhesion forces at high IS than at lower IS.42 (46) Valle-Delgado, J. J.; Molina-Bolı´var, J. A.; Galisteo-Gonza´lez, F.; Ga´lvez-Ruiz, M. J. M.; Feiler, A.; Rutland, M. W. J. Phys.: Condens. Matter 2004, 16, S2383-S2392.

Figure 9. Effect of pH and type of biopolymer on adhesion forces for uncoated and BSA-coated microspheres (IS ) 100 mM): (a) BSA, (b) lysozyme, and (c) dextran.

Model. Examples of several colloid-surface adhesion force interactions are shown in Figures 11 and 12, with model results from all experiments shown in Table 1. The parameter n using this model was nearly constant for the various conditions, with an average value of 0.29 ( 0.06 ((SD), and all values within (2 SD except for those with uncoated microspheres and BSA- or lysozyme-coated surfaces at the lowest loading force of 0.6 nN. The magnitude of A varied for the different experimental conditions, but in general larger values of A were associated with higher loading forces, low pH, or low IS (Table 1). Discussion The presence of a biopolymer at a surface consistently produced an increase in the adhesion force between the colloid and the surface over time. This change in adhesion with the residence time was observed for all three biopolymer types, suggesting that the increase in adhesion was less of a function of the type of polymer and more a consequence of the physical presence of the biopolymer in the space between the surfaces. Using an empirical model to correlate residence time and adhesion data, it was shown that the model constant, n, was the same for almost all

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Figure 10. Effect of ionic strength and type of biopolymer on adhesion forces to (a) uncoated microspheres and (b) BSA-coated microspheres (pH ) 7.3; loading force ) 6.0 nN). Table 1. Fitting Parameters for the Model Used To Relate Adhesion Force (F) to the Residence Time (T) sample loading force effect

pH effect

ionic strength effect

colloid

BSA

uncoated microsphere

BSA

BSA-coated microsphere

lysozyme


lysozyme


dextran


dextran


lysozyme


BSA


dextran


loading force (nN)

pH

IS (mM)

A

n

R2

5.4 2.4 0.6 5.4 2.4 0.6 5.4 2.4 0.6 5.4 2.4 0.6 5.4 2.4 0.6 5.4 2.4 0.6 2.4 2.4 5.4 5.4 5.4 5.4 2.4 2.4 0.6 0.6

7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 4.5 8.8 7.3 7.3 7.3 7.3 7.3 7.3 7.3

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 1 100 1 100 1

2.04 ( 0.14 1.39 ( 0.19 0.02 ( 0.003 3.45 ( 0.22 1.75 ( 0.12 0.34 ( 0.08 2.29 ( 0.27 1.20 ( 0.14 0.09 ( 0.01 3.88 ( 0.45 1.22 ( 0.08 0.38 ( 0.06 2.35 ( 0.16 1.81 ( 0.07 0.25 ( 0.03 2.96 ( 0.18 1.87 ( 0.14 1.21 ( 0.12 1.20 ( 0.14 1.30 ( 0.18 0.98 ( 0.12 3.45 ( 0.22 2.96 ( 0.18 5.69 ( 0.52 1.87 ( 0.14 4.86 ( 0.45 1.21 ( 0.12 2.86 ( 0.14

0.26 ( 0.02 0.33 ( 0.04 1.31 ( 0.05a 0.29 ( 0.02 0.35 ( 0.02 0.35 ( 0.08 0.34 ( 0.03 0.28 ( 0.04 0.48 ( 0.04a 0.24 ( 0.04 0.35 ( 0.02 0.38 ( 0.04 0.19 ( 0.02 0.22 ( 0.01 0.26 ( 0.04 0.25 ( 0.02 0.29 ( 0.02 0.37 ( 0.03 0.28 ( 0.04 0.31 ( 0.04 0.37 ( 0.04 0.29 ( 0.02 0.25 ( 0.02 0.21 ( 0.03 0.29 ( 0.02 0.25 ( 0.03 0.37 ( 0.03 0.26 ( 0.02 0.29 ( 0.06

0.98 0.98 0.91 0.99 0.99 0.90 0.94 0.95 0.98 0.83 0.99 0.97 0.95 0.99 0.92 0.98 0.96 0.98 0.95 0.94 0.98 0.99 0.98 0.93 0.96 0.96 0.98 0.99

av (mean ( SD) a

Not included in the calculation of the average value of n.

polymers under different conditions of IS and pH. We believe this consistent effect of residence time is primarily a result of two factors: water exclusion and biopolymer rearrangement. As the colloid is forced into contact with a surface, water is pushed out from between these surfaces. This change in water content should change the orientation of charged portions of the molecule with itself and the two surfaces. Over relatively long periods of time (∼20-50 s), the polymer molecules will rearrange, bridge, and then bind to the opposing surface until all bonds reach the lowest energy state for the imposed condition. Withdrawing the colloid from the surface after this biopolymer rearrangement therefore requires a greater force relative to that initially needed. The effect of residence time on the measured adhesion force produced by polymers is supported by previous studies where polymers are present, ranging from biopolymer-coated material surfaces to bacteria. For example, Mondon and co-workers40 observed the adhesion force between a protein-modified AFM tip and titanium surfaces

increased with interaction time, reaching a maximum adhesion force within ∼2 s. Vadillo-Rodriguez et al.41 found using bacteria that the adhesion force between the AFM tip and the cell surface increased with interaction time up to 100 s. They attributed this increase in adhesion force to the increased attachment of extracellular polymeric substances to the AFM tip over time. In experiments on interactions of a fibrinogen-coated AFM tip with a silica surface, Hemmerle et al.39 observed multiple consecutive ruptures during tip retraction and found that the mean number and strength of these ruptures increased steadily with interaction time. They suggested that the extent of bonding increased with retention time resulting in increased adhesion forces. It was found in the current study that, in the absence of an added biopolymer, the latex microsphere exhibited an increase in adhesion force over time to a glass surface, while the uncoated glass bead did not. Thus, we conclude that carboxylated functional groups on the microsphere surface function in the same manner as covalently bonded polymers on its surface. This finding


Langmuir, Vol. 21, No. 16, 2005 7499

Figure 12. Example of a best-fit model result for adhesion forces measured for BSA-coated surfaces to BSA-coated microspheres (IS ) 100 mM; pH ) 7.3). Lines shown are best fits based on eq 1 using constants listed in Table 1.

Figure 11. Examples of log-log plots of adhesion force (F) versus time (T) for surfaces coated with different biopolymers: (a) lysozyme and uncoated microsphere (IS ) 100 mM; pH ) 7.3); (b) BSA and BSA-coated microsphere (IS ) 100 mM; loading force ) 5.4 nN); and (c) dextran and BSA-coated microsphere (pH 7.3). Lines shown are based on a linear regression.

of polymers at the microsphere surface is consistent with the findings of others of a “hairy” surface of the latex microspheres, and direct observations using AFM of surface heterogeneity of latex microsphere surfaces.47,48 The observation that the value of n in our model was constant for so many different conditions, combined with these other studies, is good evidence of the physical basis of this residence time effect. The magnitude of the adhesion force was a function of the type of the biopolymer, with the adhesion force varying with the AFM protocol (loading force) and the solution chemistry (pH and IS). The measured adhesion forces in the presence of a biopolymer increased by as much as 15× when the applied load to the colloid was increased from 0.6 to 5.4 nN (9×; Figure 5). The difference in the measured adhesion force with the various polymers and solution conditions is reflected in the large variations obtained for the coefficient A in our model. A high A value appears to be correlated with increased loading force. The large increase in the adhesion with loading force suggests that pushing the colloid into a polymer layer promotes increased binding of polymers with the introduced surface, resulting in stronger chemical bonds. The longer time

needed to apply a higher loading force (ramp time) cannot explain this trend with loading force as the additional time needed to achieve the highest loading force is only about 0.05 s, or half the time needed at the lower loading force (2.4 nN). The solution IS affected the adhesion force, as shown by a lower adhesion force with an increase in the IS (Figure 10). We believe that this reduction in adhesion is a result of the change in the initial conformation of the biopolymer in a solution at a fixed IS (Figure 3). In a low-IS solution, biopolymers will be in a more unfolded state and further expanded into solution, providing more potential binding sites when a surface is pressed onto the polymer.49,50 However, as IS is increased, the polymers will become more folded and compressed, forming a denser core and providing fewer interaction sites. The more condensed structure of a polymer at higher IS could result in the formation of weaker bonds with a surface, leading to a smaller adhesion force as observed here (Figure 11c). This speculation is supported by the observed changes in molecule conformation shown in Figure 3. Depletion of water within a polymer layer also has an important effect relative to IS because the concentration of cations at a charged interface, and therefore within the biopolymers and the colloid surface layer, increases with bulk salt concentration.50 Experiments by Vakarelski et al.51 showed that potassium ions adsorbed to silica and mica produced lower adhesion forces with increasing IS. Similarly, in a previous study we showed that adhesion forces between colloid and protein layer surface decreased as the salt concentration increased.43 The solution pH can also affect the adhesion of a biopolymer to a colloid surface. We believe that this result is primarily a function of the changes in hydrogen bonding and electrostatic interactions as a function of pH. The adhesion between two surfaces decreased when pH was increased over a range of 4.5-10.6, as shown by force values in Figure 9 and by values of A as shown in Table 1. At a low pH, hydrogen bonding between carboxyl groups in polymers should increase adhesion forces, while at high pH we expect long-range repulsive interactions to dominate due to the electrostatic double-layer forces between two negatively charged biopolymer and colloid surfaces. Repulsive forces were observed here in approach curves at high pH (see Figure 8). Our observations on the effect of pH on adhesion are consistent with the finding of Ahimou et al.25 In their study it was shown that there was a high adhesion force between an AFM tip and a bacterial cell surface at a low pH ) 4, with a repulsive force observed at a higher pH ) 10.

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Relevance of These Results to Bacterial Adhesion. Bacterial cell surfaces are composed of a complex mixture of proteins, phospholipids, polysaccharides, and other biopolymers. These exopolymers are known to be an important factor in bacterial adhesion to a surface. The current work with polymer-coated surfaces here highlights the importance of particle residence time on adhesion to a surface. In all cases examined here, increased residence time increased the adhesion force in the presence of a polymer. However, we found that increasing the IS decreased adhesion, a result that is opposite to that observed in most bacterial adhesion studies. This different finding may be a consequence of the way that AFM experiments are conducted versus how bacteria approach (47) Elimelech, M.; O’Melia, C. R. Colloids Surf. 1990, 44, 165-178. (48) Tan, S.; Sherman, R. L. Jr.; Qin, D.; Ford, W. T. Langmuir 2005, 21, 43-49. (49) Damaschun, G.; Damaschun, H.; Gast, K.; Zirwer, D. J. Mol. Biol. 1999, 291, 715-725. (50) Abu-Lail, N. I.; Camesano, T. A. Biomacromolecules 2003, 4, 1000-1012. (51) Vakarelski, I. U.; Ishimura, K.; Higashitani, K. J. Colloid Interface Sci. 2000, 227, 111-118. (52) Logan, B. E. Environmental Transport Processes; John Wiley & Sons: New York, 1999.

Xu et al.

and contact a surface. In an AFM experiment, the colloid probe is forced onto the biopolymer-coated surface. The main mechanism driving bacterial adhesion to a surface is normally diffusion, although convective flow and cell sedimentation are also important collision mechanisms.52 By forcing an AFM tip or colloid onto a surface, we may be missing an important component to the adhesion process, thereby underestimating the role of long-range interactions such as steric and electrostatic effects. Thus, while we can simulate the interactions between polymers and surfaces once a particle collides with a surface, we may currently be missing interactions, particularly at low IS, that occur on the approach of a particle to a surface that may prohibit collisions. The development of AFM protocols that better mimic the approach and probabilistic collision of a bacterium with a surface will help us to better study and understand the role of IS in bacterial adhesion. Acknowledgment. This material is based upon work supported by the National Science Foundation (NSF) Grants CHE-0089156 and CHE-0431328. LA0509091

Residence Time, Loading Force, pH, and Ionic Strength Affect

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