Langmuir 1994,10, 4295-4306
4295
Probing Biopolymers with Scanning Force Methods: Adsorption, Structure, Properties, and Transformation of Gelatin on Mica Greg Haugstad and Wayne L. Gladfelter* Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455-0431
Elizabeth B. Weberg, Rolf T. Weberg, and Timothy D. Weatherill Medical Products Division, E. I. du Pont de Nemours and Company Received August 26, 1994@ Scanning force microscopy of thin gelatin films on mica reveals two distinct film components with characteristic frictional, morphological, and adsorptive signatures. A high-friction continuous film 1-4 nm thick stronglyadheres to mica, while a low-friction component is more weakly adsorbed as large islands on top of, or small domains within, the high-friction layer. The low-friction component exhibits a porous morphology and fluid-like character and is selectively destroyed when the film is heated sufficiently. A high-force scanning procedure remarkably transforms the molecularly-rough high-friction film into the molecularly-smoothlow-friction component if a sufficient amount of water is present in or on the film. The nanostructure of both the high- and low-friction components is imaged using a nanometer-scale asperity of gelatin attached to the SFM tip. "he anticipated network structure of gelatin is observed on the highfriction layer. The low-friction material is interpreted as moieties of intramolecularly-folded gelatin, with thickness (1.5& 0.2nm) equal to the diameter of the collagen-fold triple helix, containing substantial structural water. Analysis suggests that differences in viscoelasticity account for the component-specific frictional dissipation.
1. Introduction Scanning force microscopy (SFM) is emerging as a premier tool for characterizing organic The unprecedented capability toprobe with a single instrument trib~logical'-~ and mechanica18-12properties, long- and ~~
Abstract published inAdvance ACSAbstracts, October 1,1994. (1) Meyer, E.; Ovemey, R.; Brodbeck, D.; Howald, L.; Luthi, R.; Frommer, J.; Guntherodt, H. Phys. Rev.Lett. 1992, 69, 1777. (2) Overney, R. M.; Meyer, E.; Frommer, J.; Brodbeck, D.; Liithi, R.; Howald, L.; G ~ t h e r o d tH.; , Fujihira, M.; Takano, H.; Gotoh, Y.Nature 1992, 359, 133. (3) Meyer, E.; Ovemey, R.; Liithi, R.; Brodbeck, D.; Howald, L.; Frommer, J.; Giintherodt, H.; Wolter, 0.; Fujihira, M.; Takano, H.; Gotoh, Y. Thin Solid Films 1992,220, 132. (4) Haugstad, G.; Gladfelter, W. L.; Weberg, E. B. Langmuir 1993, 9, 3717. ( 5 ) Yuba, T.; Kakimoto, M.; Imai, Y.; Shigeno, M. Chem. Lett. 1993, @
1 fiS5
(6)Mate, C. M. Phys. Rev. Lett. 1992, 68, 3323. (7) O'Shea, S. J.; Welland, M. E.; Rayment, T. Langmuir 1993, 9, 1826. (8)Radmacher, M.; Tillmann, R. W.; Gaub, H. E. Biophys. J. 1993, 64.735. (9) Joyce, S. A.; Thomas, R. C.; Houston, J. E.; Michalske, T. A.; Crooks, R. M. Phys. Rev.Lett. 1992, 68, 2790. (10) Thomas, R. C.; Houston, J. E.; Michalske, T. A.; Crooks, R. M. Science 1993,259, 1883. (11) Salmeron, M.;Neubauer, G.: Folch, A.; Tomitori, M.; Odetree, D. F.; Sautet, P. Langmuir 1993, 9, 3600. (12) Haupstad. G.: Gladfelter. W. L. Ultramicroscoav 1994.54. 31. (13) B d a m , ' N.'A.; Dominbez, D. D.; Mowery, R: L.; Colton, R. J. Phys. Rev. Lett. 1990, 64, 1931. (14) Blackman, G. S.; Mate, C. M.; Philpott, M. R. Phys. Rev.Lett. 1990,65,2270. (15) Tsao, Y.; Evans, D. F.; Wennerstrom, H. Science 1993,262,547. (16) Schwartz, D. K.; Viswanathan, R.; Zasadzinski, J.A. N . L a -m u i r 1993, 9, 1384. (17) Bourdieu, L.; Ronsin, 0.;Chatenay, D. Science 1993,259,798. (18)Alves, C. A.; Smith, E. L.; Porter, M. D. J.Am. Chem. SOC.1992, 114, 1222. (19) Tsao, Y.; Yang, S. X.; Evans, D. F.; Wennerstrom, H. Lungmuir 1991, 7, 3154. (20) Hillier, A C.; Ward, M. D. Science 1994,263, 1261. (21) Dietz, P.; Hansma, P. K; Ihn,K. J.;Motamedi, F.; Smith, P. J. Mater. Sci. 1993,28, 1372. (22) Devaud, G.;Furcinitti, P. S.; Fleming, J. C.; Lyon, M. K.; Douglas, K. Biophys. J. 1992, 63, 630.
short-range surface f ~ r c e s , ~and J - ~even ~ the chemical as well as atomic- or molecular-scale surface structure,16-22is manifest in recent SFM studies. A landmark achievement was the first resolution of chemically dissimilar surface regions by Meyer, Overney, and co-workers via differencesin the frictional interaction with the SFM The major role of gelatin in the photographic, pharmaceutical, and food industries, as well as in holography, binding, and coating, attests to its general utility. Despite well-established product applications, gel research remains extremely a c t i ~ e , primarily ~ ~ . ~ ~ because of the complexity and richness ofthe subject. Gelatin is derived from collagen, the primary protein component of animal connective tissue, and comprises the ubiquitous binding matrix in photographic emulsions. Our recent characterization of the AgBrlgelatin interface with SFM4sZ5 followed studies of silver halide surface structure by several research groups in the photographic science community.26-28 Gelatin gels in photographic products are stabilized and strengthened by added hardening agents which couple select functional groups.29 In our current research we are applying SFM to probe the mesoscopic- to molecular-scale structure and properties of dry and water-swollen gelatin films, and their dependence on both intrinsic and extrinsic molecular coupling mechanisms. (23) See: Djabourov,M.Contemp.Phys. 1988,29,273and references therein. (24) See: Clark, A. H.; Ross-Murphy, S. B. Adu. Polym. Sci. 1987, 83, 57 and references therein. (25) Haugstad, G.; Gladfelter, W. L.; Keyes, M. P.; Weberg, E. B. Langmuir 1993, 9, 1594. (26) Haefke, H.; Meyer, E.; Howald, L.; Schwarz,U.; Gerth,G.; Krohn, M. Ultramicroscopy 1992, 42-44, 290. (27) Hegenbart, G.; Mussig, T. Surf. Sci. Lett. 1992,275, L655. (28)Keyes, M. P.; Phillips, E. C.; Gladfelter, W. L. J. Zmug. Sci. Technol. 1992,36, 268. (29) Curme, H. G. In The Theory ofthe Photographic Process; 4th ed.; Mees, C. E. K , James, T. H., Eds.; MacMillan Publishing Co.: New York, 1966; 45.
0743-7463l94I2410-4295$04.5QlO 0 1994 American Chemical Society
4296 Langmuir, Vol. 10, No. 11, 1994
Haugstad et al.
Our attempts to maximize the resolution of structure in gelatin films led to the present study of thin films on a rigid hydrophilic substrate (mica). This architecture also allows us to determine layer thickness precisely and probe the strength of film adhesion by selectivelyremoving portions of the film with high-force scanning. Our results reveal two film components with distinctly different frictional, morphological, and adsorptive character. A high-force scanning procedure remarkablytransforms the high-friction, primary component into a low-friction material. Comparison of the properties of both types of low-friction moieties (as-deposited and scan-transformed from high-friction component)indicatesthat the materials are the same. We present molecular interpretations of these findings on the basis of protein folding, apparently induced by high-force scanning. 2. Experimental Details Aqueous 10-3 w t % gelatin solution was prepared by slowly heating ( ~ h)2 a 1 w t % mixture of gelatin (Kind and Knox photographic grade, type 2688)in distilledldeionizedwater (DW) to ~ 4 “C, 0 followed by dilution with DW at 20 “C. Freshlycleavedmuscovite mice (UnionMica Corp.) substrates were rinsed wt % aqueous gelatin solution (at in DW, immersed in the least 2 h old initially) for 3 h, rinsed in a DW bath, carefully extracted to retain a residual puddle of water, covered, and allowed to dry slowly overnight in moderate-humidity (35% < RH -= 60 %) conditions. The films were initially imaged in air the following day. Repeated imaging over a period of several months reproduced the same qualitative film morphology. The Nanoscope I11 (Digital Instruments) SFM, a beam-deflection instrument,3O was used for all film characterization. Topographic and frictional force images were simultaneously collected at constant vertical cantilever deflection using triangular microfabricated lOOpm cantilevers (springconstant = 0.58 N/m) with pyramidal Si3N4 tips. Some of these tips were coated with Cr at the manufacturer; in air these native chromium oxide tips were briefly used to comparemeasurements with those employing Si3N4 tips. All of the results reported here were obtained with the Si3N4 tips unless specified otherwise. Contact forces during imaging were in the 0.2-50 nNrange. We attempted to minimize this force when possible; however, applying negative loads to offset tip-sample attractive forces often resulted in the loss of contact at surface asperities. The 12315 scanner with lateral/ vertical scanning ranges of 16014.7 pm was used. Images were collected with the tip scanning left-to-right in the sample’sinertial frame; note that in the laboratory frame the tip remains fmed while the sample is correspondingly scanned right-to-left. Friction-actuated cantilever torsion was enabled by choosing a fastscan direction perpendicular to the primary cantilever axis. Multiple imaging locations were systematically investigated on each sample to ensure reproducible and representative results. Friction loop data were collected in the “y-disabled” mode, where scanning is performed along the fast (x), but not the slow (y), scanning axis. Region-specificrelative frictional forces were measured by collecting a single topography/friction trace over a left-to-rightlright-to-left scanning cycle containing all surface regions to be compared. Friction was quantified as the difference of lateral forces sensed upon scanning in opposite directions, averaged over all data points pertainingto a single surface region. Asperity-related contributions to cantilever torsion averaged to approximately zero over a complete cycle, leaving only the nonconservative dissipative term.4 The applied load was varied by changing the vertical cantilever deflectionmaintained during scanning. Unless specified otherwise, lateral scanning was performed a t a frequency of 10 Hz;tip speed is then given by 2(scan length)(10 Hz). Region-specificcharacterization of force versus distance was achieved in “force-displacement”measurements, i.e. vertical cantilever deflection as a function of vertical sample displacement toward and away from the chosen surface region. Tip-sample “adhesive” forces (including those due to capillary condensation) were quantified from the maximum cantilever deflection toward the sample during withdrawal, (30) Meyer, G.; h e r , N. M. Appl. Phys. Lett. 1988,53,1045.
Figure 1. Representative topography (left) and frictional force (right) images of an as-prepared gelatin film; higher elevation or frictional force is rendered brighter: (a)20 000 x 20 000 nm region displaying scattered low-friction islands; (b) a typical magnified view (3000 x 3000 nm) of two islands with lower and upper surfaces approximately 1.5 and 6-10 nm above the continuous “first-layer” film; (c) a 2000 x 2000 nm region displaying low-friction domains Ffi,t layer > Fmica 'Fislanabansfomed, observed in all cases examined independent of tip, humidity, and film age. After regions like that in Figure 6 were produced, we measured frictional force versus load on multiple surface types simultaneously. Representative results a t applied loads producing no wear are presented in Figure 7, for the surfaces of first-layer gelatin (solid squares), a low-friction island (solid diamonds), a transformed film (open diamonds), and bare mica (solid triangles). A calibration of absolute frictional force was determined with a method described in the Appendix. A linear fit of each data set is included (solid lines). The slope of each fit yields an approximate coefficient of friction:
hand
*
/%amformed
* O*'
The island and transformed film values were identical within experimental uncertainty. The above ratio of /kland/transfonned to pi&layer (0.2) represents the lower bound of values measured on numerous samples with several SFM tips in variable humidity. The same ratio from the data in Figure 2a (0.3)is representative of the upper bound.
V
-30
-20
-10
0
10
20
Applied Load (nN) Figure7. Frictional force at applied loads not producing wear; data collected simultaneously on four surface regions: firstlayer gelatin (solid squares), a low-friction island (solid diamonds),a transformed film (open diamonds), and bare mica (solid triangles). A linear fit of each data set is shown (solid lines), the slope of which quantifies the frictional coefficient. The absolute frictional force calibration was obtained with a method described in the Appendix.
An understanding of the contribution of tip condition, film water content, scanning speed, etc. to this variance is the subject of ongoing work. We have not extracted a definitive coeacient of friction on scan-disrupted regions because of substantial variability among the cases studied and difficulties with sporadicfurther plastic deformation and clinging of gelatin to the tip. In general, the frictional force on these regions during stable scanning was more than double that on unmodified first-layer gelatin. 3.3. Film Nanostructure. We have found that during force-displacement measurements, by the repeated ramping of 2 to yield contact forces of at least several hundred nanonewtons, the tip-sample adhesive force decreases gradually over a period of 2-3 min from several tens of nanonewtons to as small as w2 nN. A tear in the gelatin film extending to the mica substrate results a t the site of this procedure. Subsequent force-displacement measurements at unperturbed locations (and on different samples) reveal an identically small adhesive force, indicating a change of tip state.32 Figure 8 compares representative force-displacement data collected during withdrawal from unperturbed gelatin initially (line) and af'ter the above 2-ramping procedure (solid circles); the maximum attractive forces are a40 and a2.5 nN, respectively. Approach data were subtracted from the raw withdrawaldata to remove shallow, long-rangeoscillations due to optical instrumental effects.33 The sample displacement scale is zeroed a t the jump-to-contact location; the initial withdrawal data (line) are off scale a t 2 = 2565 nm. With the initial tip, the loss of contact during withdrawal occurred suddenly, when the cantilever spring force exceeded the attractive tip-sample interaction. For measurements in ambient conditions (asin our case) the required force presumably contains a large contribution (32)If the initial adhesive force is more than a150 nN, a reduction does not take place at the highest force attainable (a1.5pN). In this case the implicitly large contact area nets a force unit area (stress) apparentlytoo small to exceed the "activationbarrier"for contaminant removal or gelatin deposition. Note that an approximately15"cantilever incline results in slight lateral tip displacement in the contact regime during force-displacement measurements; this is clearly evident in linear gouges lying in the XY plane produced by rampingto very high forces. The critical quantity for tip modification therefore may be a large shear stress. (33)Weisenhorn, A. L.; Maivold, P.; Butt,H.; Hansma, P. K. Phys. Rev. B 1992,45, 11226.
Langmuir, Vol. 10, No. 11, 1994 4301
Probing Biopolymers with SFM 5 withdrawal minus approach data
0 LL
-i ‘\
Effectof Z-ramping treatment on adhesive force I
-lo/
-15 0
\
I
40
I
1
I
1
I
80
120
160
200
Sample Displacement (nm) Figure 8. Typical processed force-displacement data (withdrawal minus approach) on unmodified first-layer gelatin characterizingthe SFM tip before (line)and after (solidcircles) repeated ramping of 2 to reach contact forces ’500 nN. The sample displacement scale is zeroed at the jump-to-contact location.
due to capillary interactions of water (contained in or on the gelatin film29)and possiblyinterfacial contaminant^.^^ Following the above treatment, the attractive forces fall off rapidly but continuously for the first 5 nm beyond the point of maximum attraction, and then very slowly for another =150 nm. A sudden cantilever jump away from the sample was only observed in cases where the gradient of attractive force exceeded the cantilever spring constant. The adhesive force for the modified tips often exhibits a strong dependence on Z cycling rate: a decrease of 3 orders of magnitude in rate can result in an increase of tip-sample adhesion by more than a factor of 5. No such rate dependence has been observed with tips in the initial state. Images collected with the modified tips display dramatically-enhanced resolution. This is illustrated in the representative images of Figure 9, each collected at contact forces of < 1 nN; low-pass Fourier filtering removed most of the small-wavelengthnoise comprising the streakiness normally visible along the fast-scan axis. Figure 9a contains a topographic image of a 300 x 300 nm region on first-layer gelatin prior to the 2 ramping procedure; a large negative load (%-20 nN)was applied to minimize the contact force. A granular morphology was imaged, the smallest “grains”being about 40 nm in diameter. The totalmeasured range of surface elevation is 2.1nm. Figure 9b shows a same-sized region of the same film imaged following the great reduction of the adhesive force; only a small negative applied load (*-2 nN) was needed to minimize the contact force. Here the imaged morphology appears fibrous (characteristic of gelatin23v24p29), with measured fiber width less than 10 nm. An amorphous network of fiber segments is revealed with typical segment lengths =20-30 nm. The total measured range of surface elevation is 3.0nm. Successive images collected on a 1000 x 1000nm region contained an identical, detailed network of fiber segments. A similar fibrous morphology was imaged on the recessed regions surrounding islands like that in Figure lb, and on the 1.5-nm thick island surfaces. The high-resolution imaging capability of the abovemodified tips is accompanied by a greater propensity for film disruption. Plastic deformation occurs in general at substantially lower contact forces; at a contact force of =150 nN, complete removal of first-layer gelatin from a (34) Thundat, T.; Zheng, X.; Chen, G. Y.; Sharp, S. L.; Warmack, R. J.; Schowalter, L. J. Appl. Phys. Lett. 1993,63,2150.
Figure 9. Comparison of representative small-scale topographic images (300 x 300 nm)of unmodified first-layergelatin collected at different locations (a)before and (b)after repeated ramping of 2 to reach contact forces > 500 nN. The measured ranges of surface elevations are (a) 2.1 and (b) 3.0 nm. Lowpass Fourier filteringwas employed to remove most ofthe smallwavelength noise comprising the streakiness along the fast scan axis.
Figure 10. Representative high-resolution topography/ frictional force images (1eWright)of a transformed film (300 x 300 nm),obtainedwith a tip modified via the repeated ramping of 2 to attain contact forces ’500 nN.
500 x 500 nm region is accomplished in just 2-3 raster scans compared to 5-10 raster scans prior to tip modification. We imaged the transformed films with the modified tips; Figure 10 contains 300 x 300 nm topography/frictionimages (lewright)collected at a contact force of
