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Characteristic Protein Adhesion Forces on Glass and Polystyrene Substrates by Atomic Force Microscopy G. Sagvolden,† I. Giaever,†,‡ and J. Feder*,† Department of Physics, University of Oslo, Box 1048 Blindern, N-0316 Oslo, Norway, and School of Science, Renesselaer Polytechnic Institute, Troy, New York 12180-3590 Received March 6, 1998. In Final Form: August 17, 1998 The force acting between a protein molecule and a nonbiological surface is of great importance in biotechnology. We adsorb protein-covered microspheres on glass and polystyrene substrates in Trisbuffered saline. Using a novel atomic force microscopy technique, we show that proteins adsorb better on hydrophobic than hydrophilic surfaces and that the microspheres adhere with a force characteristic of the particular protein and substrate. The adhesion force on the hydrophobic polystyrene substrate is shown to depend on the structural rigidity of the protein, while on the hydrophilic glass surface, protein and surface charge is more important.
The adsorption of proteins to artificial surfaces offers many interesting scientific challenges and is also an important practical problem in biotechnology.1,2 Many immunology tests, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), depend on immobilized protein at solid surfaces, as do attachment of cells in tissue culture. Thus, quantitative methods to measure protein adsorption would supply valuable data for guiding scientists seeking better biocompatible materials and for constructing better theoretical models. In this paper we describe a novel method capable of measuring the adhesion forces of proteins to solid surfaces. Proteins are first attached to microspheres that are subsequently adsorbed to various substrates. After a relaxation time, the spheres are dislodged using the cantilever of an atomic force microscope (AFM) we developed for this task (Figure 1) that measures the force as a function of displacement (Figure 2). An AFM3 measures forces by the deflection of a cantilever with known stiffness. Sensitivities of the order of pico newtons are routinely obtained. In a liquid environment, the AFM has successfully provided detailed information on specific forces that bond pairs of biomolecules,4-7 and recently on mechanical properties of single proteins8 and protein adhesion to hydrophobic polystyrene.9 In these experiments, the test molecule bridges the cantilever and substrate surfaces. However, protein adsorption may develop over time on the order of hours.10-13 Thus, limitations in the mechanical and thermal stability of the AFM may prohibit adsorption from * To whom correspondence should be addressed. E-mail:
[email protected]. † University of Oslo. ‡ Renesselaer Polytechnic Institute. (1) Claesson, P. M.; Blomberg, E.; Fro¨berg, J. C.; Nylander, T.; Arnebrandt, T. Adv. Colloid Interface Sci. 1995, 57, 161-227. (2) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-340. (3) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930-933. (4) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354357. (5) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771773. (6) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415-417. (7) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Gu¨ntherodt, H.-J. Biophys. J. 1996, 70, 2437-2441. (8) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109-1112. (9) Chen, X.; Davies, M.; Roberts, C.; Tendler, S.; Williams, P.; Davies, J.; Dawkes, A.; Edwards, J. Langmuir 1997, 13, 4106-4111.
Figure 1. The instrument (see Materials and Methods).
fully saturating. With the proteins allowed to bind microspheres to a substrate, prolonged contact with the cantilever was avoided, allowing long adsorption times without mechanical interference. Materials and Methods Glass substrates (Mentzel) were cleaned by heating to 300 °C. Hydrophilic (PS-phil) and hydrophobic (PS-phob) substrates were cut from sterile polystyrene culture dishes (Nalge Nunc). The liquid cell was made by gluing a Viton O-ring (Busak & Shamban) to the substrate, and closed by the glass slide holding the cantilever (Figure 1). A small gap between the O-ring and the glass slide was held in place by surface tension and allowed for the relative movement of cantilever and sample. The cantilever (Digital Instruments) was glued to a standard glass microscope slide using a laser for precise angular alignment. A water-filled chamber was placed on top the liquid cell to improve the focus at the cantilever. A fiberoptic laser (Scha¨fter & Kirchhoff), a beam splitter cube (Melles Griot), and a split diode (Advanced Photonics) in an autocollimator arrangement was used to detect the cantilever deflection. The diode signal was amplified, (10) Tripp, B. C.; Magda, J. J.; Andrade, J. D. J. Colloid Interface Sci. 1995, 173, 16-27. (11) van der Vegt, W.; Norde, W.; van der Mei, H. C.; Busscher, H. C. J. Colloid Interface Sci. 1996, 179, 57-65. (12) Ma˚rtensson, J.; Arwin, H.; Nygren, H.; Lundstro¨m, I. J. Colloid Interface Sci. 1995, 174, 79-85. (13) Suttiprasit, P.; Krisdhasima, V.; McGuire, J. J. Colloid Interface Sci. 1992, 154, 316-326.
S0743-7463(98)00271-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/19/1998
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Figure 3. Cumulative force distributions for protein-covered microspheres on glass and hydrophobic (PS-phob) and hydrophilic polystyrene (PS-phil). By use of a ∼0.3 N/m cantilever for all measurements, the lower limit of peak resolution was 25 pN. The upper limit was given by the dynamical range of the instrument, corresponding to about 200 nN. Samples outside this range are not shown. The position of each distribution was characterized by its median, given in Table 1. Table 1. Protein Data protein
pI
N
MW
BSAa
4.6 5.4 7.0 11.0
607 4176 153 129
66 200 476 000 16 900 13 900
FERb MYOa LYSa
Figure 2. Displacement of microspheres. (A) In each sample, displacement measurements were carried out on several microspheres. For each measurement, the cantilever was aligned with the microsphere before sample translation and data logging started. No force was detectable as the cantilever traveled through the buffer (a). When the cantilever interacted with the sphere, the force increased (b) and the sphere started to roll (c). When all bonds were broken, the sphere was translated at zero force (d). For each peak, the maximal force ∆F and the peak width ∆x were found. (B) An illustration of sphere displacement. (C) At low forces, the force peaks are no longer continuous but consist of a group of sharper peaks. This peak shape is consistent with a sphere, which rolls upon interaction with the cantilever, but is held back by a few proteins attached to the substrate. measured with a multimeter (Keithley) at a rate of 1 kHz, and logged by a PC. The sample was translated by a geared motor (Halstrup) that drove a hydraulic micromanipulator (Narishige) continuously at 0.83 µm/s. The optical detection system was calibrated by displacing the cantilever using a 100 µm gold wire glued to each sample holder, while the translation was calibrated using a high-resolution CCD camera (Photometrix) attached to the microscope. The cantilever stiffness was measured to 0.30 N/m using a 13 µm gold wire as standard. Microspheres. Glass microspheres (4 µm) (Bangs Labs) were washed and exposed to a 5% (γ -amminopropyl)triethoxysilane (Fluka) solution for 2 h and then to a 2.5% glutaraldehyde (Fluka) solution for 2 h. Bovine serum albumin (BSA) (Sigma), horse spleen ferritin (FER) (Sigma), hen egg white lysozyme (LYS) (Sigma), or horse skeletal muscle myoglobin (MYO) (Sigma) at a concentration of 1 mg/mL in Tris (33 mM) (Sigma) buffered saline (100 mM) (TBS), pH 8.0, were adsorbed on the microspheres. The microspheres were washed after each step and continuously stirred. Preparation. A small amount of protein-covered spheres was injected into the TBS, pH 7.4, filled sample holder to give
a Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed.; Worth Publishers: New York, Chapter 6. b The isoelectric point and the molecular weight were estimated from protein sequence data. The estimation program is available at http://www.expasy.ch. The protein sequence was obtained from http://srs.ebi.ac.uk.
about 1 sphere per 100 µm2 of substrate area. After 30 min, 0.5 mg/mL protein was added to the buffer to prevent displaced spheres from reattaching. The experiment started 1 h after sphere injection and lasted for about 1 h. Measurement. Each force peak was identified from the digitized signal consisting of typically 4000 measurements by a computer program. The raw data were filtered using WienerFourier filter attenuating components above 45 Hz. The force peak was identified by noting the position at which the signal rose above 3 times the noise level observed in a sliding averaging window having a width corresponding to 8 nm translation. Peaks separated by less than 0.1 µm were treated as a single peak, and data from the peak with the largest area were collected.
Results and Discussion Three common substrates were chosen for the experiments: glass, polystyrene, and a hydrophilic polystyrene. We selected proteins with different net charge, size, and structural rigidity. In every experiment, 100 microspheres were displaced, giving force and peak width distributions (Figure 3 and Table 2). Myoglobin (MYO) adheres with the strongest force on all surfaces and ferritin (FER) with the weakest. Bovine serum albumin (BSA) adheres with a strong force on the hydrophobic polystyrene surfaces, but with very weak forces on glass. Lysozyme (LYS) adheres with intermediate forces on all surfaces. The force difference when changing the surface is highest for BSA, decreasing 3
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Table 2. Force and Peak Widtha protein BSA
BSA & glu FER
FER & glu MYO
LYS
substrate
∆F (nN)
∆x (µm)
PS-phob PS-phil glass BSA & PS-phob PS-phob PS-phob PS-phil glass BSA & PS-phob FER & PS-phob PS-phob PS-phob PS-phil glass MYO & PS-phob PS-phob PS-phil glass LYS & PS-phob
99 1.8 0.07 0 25 3.1 0.22 0.04 0 0.06 32 163 18 4.6 3.2 41 4.3 1.0 7.9
1.49 0.69 0.05 0 AW 0.87 0.24 0.03 0 0.09 AW AW 1.14 0.76 0.85 AW AW AW AW
a
The distributions of maximal force and peak width were characterized by their median values ∆F and ∆x. For some samples, the peaks were arbitrarily wide (AW). PS-phil and PSphob are hydrophilic and hydrophobic polystyrene substrates. BSA & PS-phob is a BSA-covered hydrophobic polystyrene substrate, while BSA & glu are microspheres where the BSA was cross-linked using glutaraldehyde.
orders of magnitude, while for the other proteins, the force is reduced about 40-fold. As controls, hydrophobic polystyrene surfaces were covered with proteins prior to sphere adsorption. Since globular proteins normally do not adhere to globular proteins, we expect adsorption to be blocked if both surfaces are covered. The forces were reduced for all samples (Table 2). Adhesion forces were not measurable for BSA and ferritin in most cases, while myoglobin, at a pH close to its isoelectric point, and lysozyme interacts with the protein-covered substrate. This explains why the force peaks may be arbitrarily wide for myoglobin and lysozyme. These results agree with Chen et al.,9 who observed high adhesion of BSA on hydrophilic polystyrene, but low adhesion when the substrate was first covered with BSA. The protein bond length may be estimated from the peak width given the sphere translation mode. If the sphere moves without rolling, the maximal bond length is the same as the peak width, assuming that the protein attachment points to the surfaces are fixed. However, from simple geometric considerations, an ideal sphere of radius R attached by bonds of length l may roll a maximum distance
∆x ) 2x2Rl + l2,
l,R
which is much longer than l. The observed peak shapes are consistent with rolling spheres (Figure 2). This translation mode has been confirmed by displacing spheres with visible defects. The peak widths generally increase with the force (Table 2). For high forces, the observed peak widths give bond lengths larger than the folded protein size and surface roughness.14 This indicates that the proteins unfold under high strain, consistent with the results of Rief et al.8 Rolling in response to tangential forces has been proposed in a simulation model for cell detachment.15 The (14) The root mean square surface roughnesses were 8.1 nm for PSphob, 6.0 nm for PS-phil, and 10.1 nm for glass on a 1 µm2 area measured using a Topometix AFM. (15) Chang, K.-C.; Hammer, D. A. Langmuir 1996, 12, 2271-2282.
critical force necessary to dislodge the cell depended on the direction of the applied force and the nature of the bonds to the surface. It is expected that these parameters also influence the forces measured in the present study. Adsorption is controlled by a competition between protein molecules and solvent molecules at the surface to minimize the free energy.1,2,16,17 At the hydrophobic surface, the results suggest that the cost of removing water from the surface is less than the free energy gained in adsorbing the protein molecule. Since proteins consist of both polar and nonpolar amino acids, it is advantageous to expose the nonpolar amino acids to the substrate and the polar ones to the solvent. The hydrophobicity of the surface and the structural rigidity of the protein will decide the extent to which this happens. BSA and ferritin are both globular proteins carrying a net negative charge at pH 7.4. Being globular proteins, they have an hydrophilic exterior in their native conformation, but ferritin is a more rigid molecule.18 The large difference in adhesion forces (Figure 3) supports the view that protein flexibility is important in deciding the adhesion strength. To check this, BSA and ferritin were cross-linked with glutaraldehyde before adsorption on the hydrophobic surface. This procedure leaves the proteins rigid and may change the nature of the interaction with the surface. The median adhesion force is reduced 4-fold for BSA, while the ferritin force increases to the same level (Table 2). The peaks are arbitrarily wide. Hence, the measured force is dominated by glutaraldehyde adhesion to the polystyrene substrate. Thus, by cross-linking BSA, it closely resembles ferritin. These results are in line with several other observations that proteins may change configuration on adsorption10,11,16,19-23 and adhere with high forces on hydrophobic substrates.1,2 BSA and ferritin adhere with very low forces to glass. The force peaks are no longer continuous, but broken up into groups of spikes (Figure 2C), suggesting that the spheres are held at a few sparsely distributed attachment points. On the other hand, lysozyme and myoglobin have continuous force curves, and therefore far more attachment points. Hence, there is a higher barrier to adsorption for the negatively charged BSA and ferritin, than for myoglobin and lysozyme. Since BSA and ferritin adhere with comparable forces, protein charge, rather than structural changes, seems to determine the adsorption behavior of proteins on glass, although structural changes on adsorption to hydrophilic surfaces have been observed by others.16,24,25 The hydrophilic polystyrene surface serves as an intermediate case. This substrate is less hydrophilic than the glass substrates. The forces for all proteins on this substrate are higher than that for glass and lower than that for the hydrophobic polystyrene. Thus, the adhesion force increases with hydrophobicity, although it should (16) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313-328. (17) Israelachvili, J.; Wennerstro¨m, H. Nature 1996, 379, 219-225. (18) Harrison, P. M. J. Mol. Biol. 1959, 1, 69-80. (19) Xu, S.; Damodaran, S. Langmuir 1992, 8, 2021-2027. (20) Maste, M. C.; Pap, E. H.; Hoek, A. v.; Norde, W.; Visser, A. J. J. Colloid Interface Sci. 1996, 180, 632-633. (21) Bekos, E. J.; Ranieri, J. P.; Aebischer, P.; Gardella, Joseph A., J.; Bright, F. V. Langmuir 1995, 11, 984-989. (22) Wu, H.; Fan, Y.; Sheng, J.; Sui, S.-F. Eur. Biophys. J. 1993, 22, 201-205. (23) Castelain, C.; Genot, C. Biochim. Biophys. Acta 1994, 1199, 5964. (24) Kondo, A.; Oku, S.; Higashianti, K. J. Colloid Interface Sci. 1991, 143, 214-221. (25) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87-93.
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be noted that the “hydrophobic force” is a combination of enthalpic and entropic terms,17 so substrate-specific interactions may be important in deciding the adhesion strength. We have shown that proteins adhere to substrates with a force characteristic of the protein and substrate. The strength of protein adhesion increases with the hydrophobicity of the surface. Loosely folded proteins adhere stronger to the hydrophobic polystyrene surface than rigid ones, while adhesion to glass is charge controlled. We have developed an instrument that provides a tool for systematically studying protein adhesion to transparent surfaces, with a potential of studying single proteins.
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The proteins are carried by spheres that may be prepared using readily available techniques. The method also allows for the study of long-term effects. In future experiments, we plan to study the adsorption kinetics of protein-covered microspheres. Acknowledgment. We gratefully acknowledge the financial support of the Norwegian Research Council, and Vista, a research collaboration between Den norske stats oljeselskap (Statoil) and the Norwegian Academy of Sciences and Letters. LA980271B
