Langmuir 1994,10,3809-3814
3809
Imaging Adhesion Forces and Elasticity of Lysozyme Adsorbed on Mica with the Atomic Force Microscope Manfred Radmacher,*t?Monika Fritz,??*Jason P. Cleveland,? Deron A. Walters,? and Paul K. Hansmat Department of Physics, University of California, Santa Barbara, California 93106,a n d Marine Science Institute, University of California, Santa Barbara, California 93106 Received April 18, 1994. I n Final Form: July 11, 1994@ We have investigated the adsorption of lysozyme molecules on mica substrates in buffer by atomic force microscopy (AFM). The stability of the adsorbate is influenced by buffer conditions. At pH 4 single molecules can be imaged stably, while at pH 6.4only aggregates of molecules are observed. Maps of the tip-sample interaction were obtained by recording laterally resolved force curves. These force curves showed that the adhesion of the tip on the lysozyme is smaller than on the mica. The intrinsic viscoelastic properties of lysozyme could be calculated by analyzing these force curves.
Introduction The atomic force microscope (AF'M), invented in 1986,l has become a common scanning probe tool for biological applications. In AFM, a sharp tip, which is mounted on a soft cantilever, is raster scanned over a sample. The deflection ofthe cantilever is measured by an optical lever and used for generating images of the sample's surface topography. The microscope's ability to operate in a liquid environment, especially physiological buffers, makes it a promising tool for studying the native structure of biological One way to measure the interaction forces between the tip and sample in AF'M is recording force The deflection of the cantilever is recorded while the sample is approached, brought in contact with the tip, and then retracted again. Force curves (see for instance figures 2 and 5 ) contain information about surface properties such as topography, adhesion,llJ2 e1asticity,l3-l5 and interactions between single m o l e ~ u l e s . ~ ~We J ' have recorded force curves as a function of lateral position to generate maps of adhesion18-20and other properties of the sample. + Department of Physics, University of California.
* Marine Science Institute, University of California.
@Abstractpublished in Advance ACS Abstracts, September 1, 1994. (1)Binnig, G.; Quate, C. F.; Gerber, C. Phys. Reu.Lett. 1986,56,930. (2)Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Albrecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. K. Science 1989,243, 1586-1589. (3) Rugar, D.; Hansma, P. K. Phys. Today 1990,43, 23-30. (4) Radmacher, M.; Tillmann, R. W.; Fritz, M.; Gaub, H. E. Science 1992,257, 1900-1905. (5) Hansma, H. G.; Hoh, J. H. Annu. Rev. Biophys. Biophys. Chem. 1994,23, 115-139. (6) Henderson, E. Prog. Surf. Sci. 1994, 46, 39-60. (7) Weisenhorn, A. L.; Hansma, P. K.; Albrecht, T. R.; Quate, C. F. Appl. Phys. Lett. 1989,54,2651-2653. (8) Bumham, N. A.;Colton, R. J. J . Vm. Sci. Technol. 1989, A7 (4), 2906-2913. (9) Weisenhorn, A. L.; Maivald, P.; Butt, H.-J.; Hansma, P. K. Phys. Reu. B: Condens. Matter 1992,45, 11226-11232. (10) Burnham, N. A.; Colton, R. J.;Pollock, H.M. J . Vm.Sci. Technol. 1991, A9,2548-2556. (11) Hoh, J. H.; Cleveland, J. P.; Prater, C. B.; Revel, J. P.; Hansma, P. K. J . Am. Chem. SOC.1992,114, 4917-4918. (12) Hartmann, U. Phys. Rev. B 1991,43, 2404-2407. (13) Tao, N. J.;Lindsay, N. M.; Lees, S. Biophys. J . 1992,63,11651169. (14) Weisenhorn, A. L.; Khorsandi, M.; Kasas, S.; Gotozos, V.; Celio, M. R.; Butt, H. J. Nanotechnology 1993, 4 , 106-113. (15)Fritz, M.; Radmacher, M.; Gaub, H.E. Biophys. J . 1994, 66, 1328-1334. (16) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994,264, 415417. (17) Lee, G. U.;Kidwell, D. A.; ColtonR. J. Langmuir 1994,10,354. 0743-7463/94I2410-3809$04.50l0
Lysozyme, a mucopeptide glycohydrolase, is a common and abundant enzyme in eucaryotes. Its biological function is to hydrolyze polysaccharides of the peptidoglycan layer: the major component of bacterial cell walls (for an overview see Ossermann et a1.21 and Imoto et a1.221. It is very well studied and was the first enzyme whose atomic structure was determined by X-ray diffraction of crystals.23 We used hen egg white lysozyme, which has an isoelectric point of pH 11.1. Protein adsorption was controlled by changing the electrostatic interaction forces between the negatively charged mica surface and the lysozyme. At pH 6.4 the adsorption was weak: only aggregates of lysozyme could be imaged stably by AFM. At pH 4,the highly positive charged protein adsorbs very tightly to mica. In the latter case single molecules can be imaged stably by AFM. In this study we examine the viscoelastic properties of protein aggregates and single proteins.
Materials and Methods Lysozyme Grade I (from chicken egg white, purchased at Sigma, St. Louis, MO) was dissolved in 66 mM KHzPO4 (pH 6.4) at a concentration of 0 . 1 mg/mL and adsorbed on freshly cleaved mica. After 20 min of incubation the solution was exchanged against 10 mM KHzPOl (pH 6.4) without any lysozyme. This
procedure resulted in stable aggregates oflysozyme,which could be imaged by AFM. The single molecules adsorbed on mica were prepared using a 10 mM KHzPOl (pH 4) buffer solution with a lysozyme concentration of 1 &mL. After 20 min incubation the solution was exchanged against 10 mM KH2P04 (pH 4) without any lysozyme. Incubation of the protein solution was performed inside the mounted fluid cell of the AFM. Before incubation the mica surface was first verified to be free of any contaminant layer. The sample was flat and free of contaminant on the micrometer scale as found by imaging with the AFM. We also checked for low adhesion and a clean jump off from the surface during retract in the force curve and for very good atomic resolution. These four phenomenological criteria were used to check the absence of contaminants. A commercialAFM (Nanoscope11,Digital Instruments, Santa Barbara, CA) was used in this study. Commercially available, (18)Mizes,H,A.;Loh,K.-G.;Miller,R. J.D.;Ahuja,S.K,;Grabowski, E.F.Appl. Phys. Lett. 1991,59, 2901-2903. (19) Baselt, D. R.; Baldeschwieler,J. D. J.Appl. Phys. 1994, 76 (l),
-- --. .?.?-RR
(20) Radmacher, M.; Cleveland, J. P.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Biophys. J . 1994, 66, 2159-2165. (21) Lysozyme; Osserman, E. F., Canfield, R. E., Beychok, S., Eds.; Academic Press, Inc.: New York, 1974. (22) Imoto, T.; Johnson, L. N.; North, A. C. T.;Phillips, D. C.;Rupley, J. A.In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1972; Vol. VII; pp 665-868. (23) Blake,C.C.F.;Koenig,D.F.;Mair,G.A.;North,A.C.T.;Phillips, D. C.; Sarma, V. R. Nature 1965,206, 757-761.
0 1994 American Chemical Society
3810 Langmuir, Vol. 10, No. 10, 1994
Radmacher et al.
Figure 1. AFM image of aggregates of lysozyme adsorbed on mica (a). The lysozyme solution (0.1 mg/mL in 66 mM KH2P04 at pH 6.4)was incubated for 20 min on the mica slide. Then the solution was exchanged against 10 mM KH2P04 (pH 6.4) without free lysozyme. This resulted in moderately stable aggregates of lysozyme which could be imaged by AFM. Figure l b shows an AFM image of individual lysozyme molecules on mica. The lysozyme solution (1pg/mL in 10 mM KH2PO4 at pH 4)was incubated for 20 min. Then the solution was exchanged against 10 mM KH2P04 (pH 4) without free lysozyme. 200 pm long Si3N4 cantilevers with integrated, oxide sharpened tips (Nanoprobes, Digital Instruments, Santa Barbara, CA) were used. Since we did not directly measure the force constant ofthe cantilever we used, we can only estimate it by a typical value for this particular wafer: 50 pN/nm (&20%).This force constant was calibrated from the measured resonance frequency of other cantilevers on the same wafer.24 Force curve images were recorded by using the Nanoscope only for creating the xy-scan voltages. The z-voltage was generated by an external function generator (for the sweep) and a high precision power supply (for the bias). Data were recorded using a separate computer (Macintosh Quadra 840AV) equipped with a data acquisition board (Mac Adios, GWI Instruments, MA) and run by custom software. The images were generated and exported to a public domain image processing program (NIH Image, Wayne Rasband, NIH, Bethesda, MD). For details of image generation see Radmacher et a1.20 Data analysis was done with a commercial program (IGOR, Wavemetrics, Lake Oswego, OR).
Results Figure l a shows a standard height-mode AFM image of lysozyme aggregates on mica. The lysozyme solution (0.1 mg/mL, in a buffer containing 66 mmol of KH2P04a t pH 6.4) was incubated for 20 min on the mica slide. Then the solution was exchanged against pure buffer containing 10 mM KH2PO4 (pH 6.4). This resulted in moderately stable aggregates of lysozyme as shown in Figure 1. After continuous scanning, larger aggregates were formed by pushing the smaller ones together. The height of the aggregates was about 3-4 nm, consistent with the height of single molecules. Figure l b shows an AFM image of single lysozyme molecules adsorbed on mica. Here a lysozyme solution of 1pg/mL in buffer containing 10mmol of KH2P04 at pH 4 was incubated for 20 min on the mica slide. Then the solution was exchanged against pure buffer containing 10 mM KH2P04 (pH 4). The apparent size of the individualmolecules is about 20 nm, their height about 3 nm. Figure 2 shows two typical force curves, one on mica (Figure2a) and the other on an aggregatelysozyme (Figure 2b). The adhesion, measured as the hysteresis during retract, is much higher on mica than on lysozyme. There is also a slight deviation from linearity in the contact region of the curve on the lysozymedue to elastic deformation.13914 In the case of hard samples there should be no visible (24) Cleveland,J. P.;Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993,64,403-405.
indentation resulting in a line of slope 1 in the contact region of the force curve. In order to get a good estimate for this ideal contact line, it was calculated by averaging several force curves on mica. This averaged line was then subtracted from a single force curve to show the deviation in Figure 2c for the case of mica and in Figure 2d for the case of lysozyme, respectively. Note also that the liftoff occurs quickly on the mica but seems to be slowed down on the lysozyme aggregate. It takes more time than the liftoff of the mica even though the travel distance is much shorter. (Compare the two sections in parts a and b of Figure 2 marked with arrows.) This quantity, called liftoff speed, will be used to estimate the viscosity of the lysozyme. While scanning along a line of the sample of Figure 1 and sampling force curves (as in Figure 2) as a function of lateral position the following quantities were calculated: point of contact (Figure 3a), adhesion force (Figure 3b), and liftoff speed (Figure 3c). Three aggregates of lysozyme were scanned in this line, as indicated by the bar in Figure 3a. The algorithms which extract certain quantities from force curves can also be used to generate images when force curves are recorded along many lines. The lines together form a complete frame. Figure 4 displays images of the point of contact (a)and the adhesion (b) of a 350 x 350 nm sample region extracted from a set of 100 x 100 force curves. Lysozyme was incubated and imaged at pH 4. High sample spots appear bright in Figure 4a as do spots of high adhesion in Figure 4b. The apparent size of individual molecules is about 12-25 nm. This value is reasonable when the effect of tip broadening is accounted for. Figure 4 demonstrates that individual molecules can be detected not only by their height, as usually done in AFM, but also by their adhesion properties. Figure 5 shows two force curves from the dataset used for generating Figure 4. Similar to the case of aggregates the adhesion on individual molecules is smaller and the liftoff speed is smaller compared to mica. In Table 1we summarize the measured values of the indentation at a given loading force and the liftoff time. A total of 100 force curves were analyzed in both case (single lysozyme molecules and aggregates). The given error is the range of values found in the analyzed data.
Langmuir, Vol. 10, No. 10, 1994 3811
Imaging Adhesion Forces 40
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Loading Force [nN] Loading Force [nN] Figure 2. Force curves on the sample of Figure 1.Figure 2a shows a force curve on the substrate mica, Figure 2b on an aggregate of lysozyme. Note that the hysteresis during retract is larger on mica, indicating a higher adhesion force. However, the liftoff on the lysozyme is slow in comparison to the mica. This effect is probably due to viscous damping of the cantilever by the lysozyme (sweep frequency of the force curve, 77 Hz). Due t o elastic indentation the contact region of the force curve is not just a straight line but shows some deviation in the case of lysozyme (see fitted contact lines in parts a and b of Figure 2). The fitted line is in both images the same, where the slope was determined by analyzing 20 force curves on mica. Figure 2c shows this deviation from a straight line in the case of mica (Figure 2c) and lysozyme (Figure 2d) respectively. In the case of lysozyme the indentation as a function of loading force is clearly visible. Table 1
liftoff substrate mica
lysozyme (aggregate) lysozyme (single mol)
time h s ) 110 f 10 155 f 50 170 f 35
damping constant bNs/m) 5.5 f 0.5 7.8 f 2.5 8.5 f 2
indentation at 1 nN (nm) 0 f 0.25 1 zk 0.25
1 f 0.25
Discussion Here we report about force curves measured on lysozyme and showing differences compared to the curves measured on the substrate mica. One major prerequisite of this investigation is the knowledge on where the tip is positioned. It is possible, in principle, to image the sample and then zoom in on a certain area to take a force curve. But it turns out to be very hard in practice, especially on the scale of single molecules. This is mainly due to lateral
drift caused, e.g., by temperature differences and by creep of the piezo. A much easier approach is taking a twodimensional array of force curves, calculate the topography from the force curves, and use this topography map to determine which curves are taken on the substrate and which are taken on the protein. This advantage has been used in this study. Even then there is still quite a variability in the force curves, which can be seen in the noisiness of the topography and adhesion map constructed from a set of force curves (see Figure 4). But, since a two-dimensional array of force curves has been recorded, we can average the calculated quantities like adhesion force or elastic indentation to increase the signal to noise ratio. Averaging only helps to eliminate statistical errors, but not systematical errors. In order to get a clear idea of the uncertainty of our results, a discussion of the systematic
Radmacher et al.
3812 Langmuir, Vol. 10,No. 10,1994 Mica Lysozyme 8
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points. Therefore it only causes problems while discussing differences between approach and retract curve but not while discussing the center portion of one trace. So, we can conclude the discussion of errors by summing the known systematic errors (20%for the piezo calibration and 20% for the force constant calibration) with the apparent variability in the datasets (-25%) to get a combined error in the order of 50%. This is a conservative estimate and the actual accuracy can be much better in our case. Figures 2 and 5 show force curves on aggregates and individual molecules of lysozyme. One interesting feature in these data is the slope in the contact region. There is a deviation from a straight line due to elastic deformation, which can be analyzed assuming a Hertzian contact between the tip and the sample.26Since the elastic moduli of the tip material and the substrate are very high, the only detectable indentation will be the indentation of a soft material-like protein adsorbed on the mica. Therefore we neglect the elastic indentation of the tip and the mica in our case. There are several limitations in this model, like the assumption of isotropic elastic properties and no adhesion forces. To address the second point, we could use a more elaborate but to limit the number of necessary assumptions, we use the simpler model. In the case of AFM, where the tip can be assumed as a sphere of radius R with a very much higher Young modulus than the flat sample, the Hertzian model leads to the following expression28
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Figure 3. Quantities extracted from force curves while the samplewas scannedlaterally. (Figure2 showedtwo forcecurves out of the complete dataset of 100 force curves along one scan line used for this figure.) Part a shows the calculated point of contact, which is the topography. The lower parts correspond to the substrate and the higher parts t o the proteins. Part b shows that the adhesion force is lower on the protein. Part c shows that speed of the IiRoff on the protein is significantly slower than on mica (length of line, 700 nm).
errors is necessary: the calibration of the force constant has been addressed above and has an accuracy of about 20%. The same error is true for the calibration of the piezo. We actually were able to calibrate the piezo to better than 10% at the nanometer scale (using steps on mica) but prefer to be more conservative here. Nonlinearity of the piezo is only a problem near to the turn around points of the curves, but not in the center region. Note that the size of the sweep is about 3%of the overall scan range of the used piezo tubescanner. Thermal drift of the sample or the microscope is, to our experience, a couple of angstroms per second while imaging in liquids. This leads to a negligible drift on the time scale of the force curves (the sweep frequency was 77 Hz). On the other hand this high frequency leads to a notable seperation of the approach and retract curve while the cantilever is off the surface (horizontal part on the right of each force curve) due to hydrodynamic drag.25 This drag is constant during the entire force curve, but changes sign at the turnaround
where v is the Poisson ratio, Fo the loading force, d the indentation, and E the Young's modulus, respectively. Assuming a typical Poisson ratio of 1/3, we can calculate the Young's modulus from the indentation versus force curves of parts c and d of Figure 2. The only assumption entering this calculation is the radius of the tip, which is known to be of order 20 nm for the tips used in this study. Fitting the functional dependence of eq 1to several force curves gives the following value for the Young's modulus of lysozyme: 0.5 f 0.2 GPa. The other interesting feature in the force curves is the slowing down of the cantilever during liftoff due to a viscous effect, either of the medium between the tip and sample or the sample itself. Since all the experiments were carried out in water, there is a measurable damping of the liftoff in the force curves even on bare mica. However, Table 1 clearly shows that additional damping occurs on the lysozyme. The damping on the aggregates and the individual molecules is the same within our experimental accuracy. For quantifying the damping, we model the tiphample system as a dampened harmonic oscillator, where k is the force constant of the tip, D is the effective damping constant of the sample, and m is the mass of both the tip and the part of the protein which is moved
m*x + D * x + k * x = O
(2)
Using the Ansatz x = xo * e-t/zto solve this equation leads (25)Hoh, J.; Engel, A. Langmuir 1993,9,3310-3312. (26)Hertz, H. J. Reine Angew. Mathematik 1881,92,156. (27)Johnson, K. L.;Kendall, K.; Roberts, A. D. Proc. R. SOC.London, Ser. A 1971,324,301-313. (28)Radmacher, M.;Tillman, R. W.; Gaub, H. E. Biophys. J. 1993, 64,735-742.
Langmuir, Vol. 10,No. 10, 1994 3813
Imaging Adhesion Forces
Figure 4. Images reconstructed from a set of 100 x 100 force curves showing single molecules of lysozyme adsorbed on mica. Certain properties, like topography and adhesion force in this case, can be extracted and used for generating images, where these quantities are encoded in gray shades. Part a shows the topography and part b the adhesion. The adhesion on the individual lysozyme molecules is smaller than on the mica, as was the case with the aggregates (image size, 350 nm; force curve sweep frequency, 77 Hz).
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Figure 5. Part a is a force curve on the substrate mica and part b on a single lysozyme molecule. These curves show the same qualitative behavior as the curves on the aggregates: the adhesion force is higher on the mica and the liftoff is more slowed down on the protein due to some viscous damping.
to the characteristic equation
(3) which gives the following expression for the damping constant D:
The damping is the sum of two different terms, one containing the mass of the system and the other the force constant. From the experimental data (the relaxation time z is about 160,us and the force constant is 50 mN/m) we can calculate both terms. The mass of the protein moved is negligible compared to the mass of the tip (approximately 2 ng). This gives 2 x N s/m for m/z and 8 x N s/m for kz. Thus the mass does not play a major role in this system and we can estimate the viscosity. In Table 1 we summarize the apparent damping constant calculated from the observed liftoff time. The damping of the lysozyme itself will be the apparent
damping minus the viscous damping on the mica, giving numbers of about 2.3 ,uN s/m for the aggregates and 3 ,uN s/m for the single molecules. The viscosity itself can be estimated by taking into account the geometry of the system. For an isotropic system the viscosity is defined as D * l/A, where 1 is the length of the strained system and A the area. Since the geometry of the contact between tip and sample on the molecular level is not known, only very crude estimates can be made for these two parameters. It makes sense to use the diameter of the molecule itself, about 3 nm, for the length 1. The area of contact can be estimated by calculating the Hertzian deformation of the sample under a given load.26 The radius a0 of a contact site is given in the Hertzian model as
a o = 3J
& --Y ~ ~ o
where Y is the Poisson ratio, E is the Young's modulus, R the tip radius, and Fo the loading force. The loading force can be estimated by the apparent adhesion force of the lysozyme,visible in Figure 2b, giving
3814 Langmuir, Vol. 10, No. 10, 1994
a value of 250 pN. The tip radius is estimated to be 20 nm. With the above value for the Young's modulus, eq 5 yields a value of 9 nm2 for the contact area. Using the value for the contact radius and the size of the molecule, we estimate the intrinsic viscosity to be 800 f 400 Pa s. This value is similar to values for typical polymers: polystyrene with a molecular weight between 8900 and 581 000 has a viscosity of 200 Pa s in a frequency range of 1 Hz to 10 ~ H Z . ~ ~
Radmacher et al. were able to detect single proteins in the adhesion maps as well as in the reconstructed topography from the force curves with a resolution of about 15 nm. The Young's modulus of lysozyme molecules is estimated to be about 0.5 f0.2 GPa and the viscosity to be 800 f 400 Pa s. The value for the elasticity is in good agreement with values recently obtained by measuring the macroscopic compressibility of wet crystals (0.2-1 GPaX3O
We have demonstrated the application of a new imaging mode in AFM in which force curves are recorded as a function of lateral position. Maps of the adhesion and other properties, here the intrinsic viscosity and elasticity of proteins, can be constructed from these data sets. We
Acknowledgment. This work was supported by the Office of Naval Research (M.R., M.F., J.P.C.), the Materials Research Division of the National Science Foundation under Grant NSF-DMR-91230948 (D.A.W.) and the National Science Foundation under awards No. DMR 9221781 (P.K.H.). We thank P. Hillner for helpful discussions. Equipment was supplied by Digital Instruments.
(29) Onogi,S.; Masuda, T.; Kitagawa, K. Macromolecules 1970, 3, 109-116.
(30) Morozov, V. N.; Morozova, T. Y. J. Biomol. Struct. Dyn. 1993, 11,459-481.
Conclusions
