Scanning Probe Microscopy of Polymers - American Chemical Society

Chapter 21

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 29, 2016 | http://pubs.acs.org Publication Date: July 7, 1998 | doi: 10.1021/bk-1998-0694.ch021

Recognition and Nanolithography with the Atomic Force Microscope T. Boland, E. E. Johnston, A . Huber, and Buddy D. Ratner

Departments of Chemical Engineering and Bioengineering, University of Washington, Seattle, W A 98195 The abilities of the atomic force microscope (AFM) to apply forces in precisely defined surface regions and to measure forces in the piconewton range have been applied to spatially distribute and specifically recognize biomolecules on surfaces. First, an RF -plasmadeposited 7 0 Åthick triglyme film on a gold substrate was AFM-patterned and the pattern filled with a self-assembled adenine layer. Thymine modified AFM tips were used to probe the pattern. Second, a plasma deposited film was patterned with a short chain, carboxylic acid terminated thiol. The usefulness of this pattern as a template for protein adsorption was investigated.

Living biological systems routinely make use of recognition events that are based upon biomolecules in ordered (oriented) assemblies with spacings in the range from 0.05|im to 10 (im. Technologies that can create materials and surfaces with precise incorporation of spatial information at this scale represent a powerful approach to the engineering control of biological systems. Control of chemistry at surfaces in this mezzo-scale regime is technically and intellectually challenging and has led, in recent years, to a veritable flood of literature exploring molecular self-assembly and surface patterning at the nanometer and micron scales. Self-assembled monolayers of alkane thiols, where the n-alkane chain is 11 or more carbons in length, have been widely explored as a readily implementable means to precision ordered surfaces [1]. Films prepared by spontaneous assembly of mixed alkyl thiols segregate into islands [2]. However, there was no direct control of the size and shape of the islands other than by varying the relative solution concentration. Some methods have recently been developed to pattern selfassembled films including soft X-ray lithography [3-6], near-UV irradiation through a Cr-glass mask [7], or using elastomeric stamps [8]. Usually, the masking techniques will produce features with dimensions in the range of 1-10 microns that are well defined chemically and spatially. Higher resolution in patterning can be achieved with scanning probe instruments. Many studies using the scanning tunneling microscope for direct spatial 342

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Ratner and Tsukruk; Scanning Probe Microscopy of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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modification of surfaces have been published [e.g., 9-11]. Most studies address semiconductor or conductor surface modification. Atomic force microscopy (AFM) has also been used for spatially controlled surface modification [12], but its application to the nanopatterning of organic films is novel. By slightly increasing the scanning force, A F M has been used to manipulate adsorbed organic layers [13]. Increasing the A F M loading force even further has been shown to damage overlayer films and expose bare substrate [14]. It is possible by A F M to rupture amorphous, weakly crosslinked films such as those resulting from glow discharge plasma deposition of organic precursors. Films of this type have been extensively studied and characterized by ESCA, static SIMS, contact angle, and A F M measurements [13-16]. By using patterns created by film removal as templates for self-assembled monolayers, this couples the chemical precision available through self-assembly with the durable film properties of plasma deposits. The precursor molecule to create the plasma deposited films in the studies reported here was triethylene glycol dimethyl ether (triglyme). Layers formed from this precursor have been shown to exhibit unusually low protein adsorption and cell adhesion [13,16]. Two examples that illustrate how surface patterning of plasma deposited films and self-assembled monolayers may be used are presented here [17]. First, a plasma deposited triglyme film on a gold substrate was AFM-patterned and the pattern filled with self-assembling adenine. Chemically sensitive, thymine modified A F M tips were used to probe the pattern. Second, a plasma deposited film was patterned with a short chain, carboxylic acid terminated thiol. The usefulness of this pattern as a template for protein adsorption was investigated. Experimental A Nanoscope II A F M instrument (Digital Instruments, Santa Barbara, CA) equipped with a 10 |jm x 10 jxm scanner and fluid cell was used for all experiments. The set-up included cantilevers possessing spring constants ranging from 0.6 to 0.06 N/m with integrated tips (r - 50 nm) (commercially available Digital Instruments, Santa Barbara, CA). Spring constants were calibrated using the room temperature oscillation method [17, 18]. The N I H Image software package (version 1.49) was employed in image analysis and particle quantification. In the first experiment, gold surfaces were prepared by gold coating freshly cleaved mica using an argon plasma sputtering technique (dc, 200 mtorr, 40 W, 90 sec.) followed by annealing at 300°C for 7 minutes on a hot plate. Triglyme was deposited as a 8-10 nm thick film on these gold samples using the radio-frequency plasma-deposition technique previously described [19,20]. The A F M tip, mounted in the instrument, was then used to rupture the film, and expose micron sized areas of the underlying substrate, with loading forces exceeding 100 nN [14]. The removal of plasma deposited material was visualized by imaging larger areas, at loading below 10 nN. The AFM-spatially etched gold surfaces were subsequently used as substrates for spontaneous self-assembly of purines, pyrimidines and dithiodipropionic acid (Aldrich Chemical) dissolved in ethanol at a concentration of 1 mM [17, 21]. Functionalized A F M tips were then used to scan the surface. The functionalized tips were prepared by gold coating with the technique described above, followed by immersion in dilute solutions of adenine and thymine (Sigma Chemical) in absolute ethanol for several days to deposit a self-assembled nucleotide monolayer [22].



344 The second example of nanopatterning involved the adsorption of the protein lysozyme from aqueous solution. A triglyme plasma coated gold surface was prepared as in the previous experiment. Using an A F M probe tip, two square regions, 1 |im on each side, were scraped into the surface. A lysozyme solution, with a final concentration of 100 |ig/ml, was prepared by dissolving a known amount of triply crystallized, dialyzed and lyophilized egg white lysozyme (Sigma, Lot 21F-8081) in 140 mM citrate PBS buffer at pH 7.4. This solution was adsorbed to the scraped surface for 30 minutes before rinsing with buffer, followed by repeated scans under buffer. The deposited lysozyme was then removed from the 1 iim square regions by scraping, re-exposing the underlying gold surface. A dithiodipropionic acid solution was introduced into the fluid cell, and allowed to react for 30 minutes, forming a thiol-gold bonded acidic film on the surface. The specimen was then rinsed with buffer. This dicarboxylic acid was chosen since lysozyme has a positive net charge at physiological pH, and thus would be expected to be strongly adsorptive to lysozyme. The surface was imaged once again by A F M . A final adsorption of lysozyme solution was performed followed by rinsing and A F M imaging. Results and Discussion An example demonstrating the efficacy of the A F M scraping technique can be seen in Figure 1. The image shows the result of removing a portion of the triglyme film with the A F M tip. Two 600 A parallel lines were "written" less than 1 |im apart. The width of the lines is directly related to the probe tip diameter. This shows the feasibility of using triglyme plasma deposited films on gold as substrates for patterning. Typically, triglyme films resist the adsorption of biomolecules [13]. Following are the results of two experiments demonstrating how these scratched tryglyme surfaces can be used to form templates for protein or nucleotide adsorption. In the first experiment, a 1 mM adenine solution in ethanol was adsorbed onto the scraped surface. The surface was then imaged with an unmodified tip and a thymine functionalized tip, as shown in Figure 2. When imaged with the unmodified silicon nitride tip (image A), a negative contrast (darker color) indicates the scraped region. Image B shows that there is an attraction between the thymine tip and the adenine surface because the scraped/adsorbed region appears light. This confirmed the presence of an adenine film in the scraped regions, and its absence on the plasma triglyme portion of the surface. Thus, A F M is useful for both topographic imaging and "recognition" imaging. The second experiment was used to show that the patterned samples could be used as templates for protein adsorption. The series of A F M images displayed in Figure 3 shows the various steps of the procedure. Image A displays the triglyme plasma surface with square regions of the gold substrate exposed by the scraping technique. The plasma film remaining in the unscraped areas is approximately 8 nm thick. This surface was adsorbed with lysozyme, and Figure 3B suggests that little protein is present on the plasma surface, but the exposed gold regions are covered with a thin protein film of approximately 6 nm (i.e., the scraped areas from Figure 3A are filled in to approximately the thickness of the triglyme film). The adsorbed proteins are then removed by the scraping technique as imaged in Figure 3C. A



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Figure 1. Patterning of a tryglyme plasma deposited thin film with A F M tip forces greater than 100 nN. After scraping, the images were acquired with a tip force of 10 nN. (A) The initials "UW." (B) Two 600A wide lines engraved less than l | i m apart. The etched features are approximately 70A deep.



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Figure 2. A F M images (2.8 jLim x 2.8 UMn) of a triglyme film deposited onto gold. The images were obtained using (a) an unmodified tip (silicon nitride) and (b) a thymine-coated tip. In both cases, the triglyme overlayer film was selectively removed with the A F M and the bare gold thus exposed was interacted with adenine.



347

Figure 3. A F M images (12 \im x 12 urn) of a triglyme film deposited onto gold. (A) Two square regions of film have been scraped off. (B) Lysozyme has been adsorbed into the scraped features. (C) Dithiodipropionic is adsorbed to the gold exposed by scaping the film. (D) High levels of adsorbed lysozyme are noted where the diacid has adsorbed.

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Figure 3. Continued.


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dithiodipropionic acid film is adsorbed to the surface followed by a repeat of the lysozyme adsorption. Figure 3D displays the preferred adsorption of the positively charged lysozyme to the carboxylic acid-derivatized areas over the plasma film. It has been observed previously that lysozyme adsorbs in amounts greater than monolayers onto acidic surfaces at physiological pH[23- 25].


Conclusions These experiments offer insights useful in the development of nanopatterned of surfaces. Because the triglyme plasma film is known to resist the adsorption of organic molecules, selective removal of the triglyme enabled controlled patterning on the surface. The addition of a functionalized tip in the first experiment allowed for biorecognition information at the molecular level. In the second experiment, precision spatial control of protein adsorption was shown. Biology normally uses biorecognition and spatially localized receptor events to control processes in living systems. Thus, this nanotechnology may ultimately be useful for precision control of biological processes at interfaces with applications for biomaterials, cell culture, affinity separations and biosensors. Acknowledgement Generous support from the NIH (NESAC/BIO grant RR01296), the National Science Foundation (UWEB ERC grant EEC-9529161) and the Center for Process Analytical Chemistry, University of Washington is acknowledged. References 1.

Bain, C.D.; Whitesides, G . M . Science, 1988, 240, pp. 62-63.

2. Lopez, G.P.; Biebuyck, H.A.; Whitesides, G . M . Langmuir, 1993, 9(6), pp. 1513-1516. 3. Dressick, W.J.; Calvert, J.M. Jpn. J. of Appl. Physics, 1993, 32(12B), pp. 5829-5839. 4.

Calvert, J.M.; et al. SPIE, 1993, pp. 30-41.

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Calvert, J.M.; et al. Optical Engineering, 1993. 32(10), pp. 2437-2445.

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Dulcey, C.S., et al. SPIE, 1993. 1925(657-666).

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Frisbie, C.D., et al. J. Vac. Sci. Technol. A, 1993. 11(4), pp. 2368-2372.

8. Kumar, A.; Whitesides, G . M . Appl. Phys. Lett., 1993. 63(14), pp. 20022004. 9. Zeppenfeld, P.; Lutz, C.P.; Eigler, D . M . Ultramicroscopy, 1992. 4244(pt.A), pp. 128-133. 10.

Kobayashi, A.; et al. Science, 1993, 259(5102), pp. 128-133.


350 11. Yang, R.; Evans, D.F.; Hedrickson, W.A. Langmuir, 1995, 11(1), pp. 211-213. 12.

Kim, Y . ; Lieber, C . M . Science, 1992. 256(5068), pp. 375-377.

13. Lopez, G.P.; Ratner, B.D.; Tidwell, C.D.; Haycox, C.L.; Rapoza, R.J. Horbett,T.A. J. Biomed. Mater. Res., 1992, 26, pp. 415-439.


14. Favia, P.; Perez-Luna, V . H . ; Boland, T.; Castner, D.G.; Ratner, B . D . Plasmas and Polymers, 1996, 1(4), pp. 299-326. 15.

Clark, D.T.; Abu, S.M.M. J. Polym. Sci. A, 1984, 22(1), pp. 1-16.

16. Lopez, G.P.; Rapoza, R.J.; Ratner, B.D.; Horbett, T.A., Preparation of non-fouling biomaterial surfaces by plasma deposition of poly(ethylene glycol) oligomers and precursors. In Extended Abstracts of the 2nd Topical Conference on Emerging Technologies in Materials, AIChE: San Francisco, C A , 1989. 17. Boland, T., Analysis and Engineering of Two-Dimensional Assemblies of Purines and Pyrimidines on Gold, Ph.D. Thesis in Chemical Engineering, University of Washington: Seattle, Washington, 1995. 18.

Hutter, J.L.; Bechhofer, J. Rev. Sci. Instrum., 1993. 64, pp. 1868.

19. Lopez, G.P., Effect of precursor adsorption on film chemistry and plasma deposition of organics., Ph.D. Thesis in Chemical Engineering, University of Washington: Seattle, Washington, 1991. 20. Ratner, B.D.; Chilkoti, Α.; Lopez, G.P, Plasma deposition and treatment for biomaterial applications., In Plasma-Materials Interactions; Auciello, O.; Flamm, D.L.., Eds.; Academic Press: San Diego, C A , 1990, pp. 463-516. 21.

Boland, T.; Ratner, B.D. Langmuir, 1994, 10, pp. 3845-3852.

22. Boland, T.; Ratner, B.D. Proc. Matl. Acad. Sci. (USA), 1995, 92(12), pp. 5297-5301. 23. Horsley, D., et al., Human and hen lysozyme adsorption: a comparative study using total internal reflection fluorescence spectroscopy and molecular graphics, In Proteins at Interfaces: physiochemical and biochemical studies., Brash, J.L.; Horbett, T.A., Eds.; American Chemical Society: Washington, D.C. 1987, pp. 290-305. 24. Schmidt, C.F.; Zimmerman, R . M . ; Gaub, H.E. Biophys. J., 1990, 57(3), pp. 577-588. 25. Bohnert, J.L.; Horbett, T.A.; Ratner, B . D . ; Royce, F . H . Invest. Ophthalom. Vis. Sci., 1988, 29(3), pp. 362-373.


Scanning Probe Microscopy of Polymers - American Chemical Society

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