pubs.acs.org/Langmuir © 2009 American Chemical Society
Reduction of the Damping on an AFM Cantilever in Fluid by the Use of Micropillars )
Masaru Kawakami,*,†,‡ Yukinori Taniguchi,†,§ Yuichi Hiratsuka,†,‡ Masahiko Shimoike,† and D. Alastair Smith
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† School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa, Japan, ‡PRESTO of Japan Science and Technology Corporation, 4-1-8 Honcho Kawaguchi, Saitama Japan, §Japan Society for the Promotion of Science (JSPS), 8 Ichibancho, Chiyoda-ku, Tokyo, and Avacta Group plc, Biocentre, York Science Park, Heslington, York YO10 5NY, United Kingdom
Received July 8, 2009. Revised Manuscript Received August 31, 2009 In single molecule force measurements with soft atomic force microscope (AFM) cantilevers, the force sensitivity is limited by the Brownian motion of the cantilever. When a cantilever is close to the surface, the hydrodynamic interaction between the cantilever beam and the surface, called the “squeezing effect”, becomes significant, and the resonance peak of the thermal oscillation of the cantilever is heavily broadened and shifted to lower frequency which makes it difficult to eliminate the thermal noise by low-pass filtering. In this study, we propose an easy and low-cost method to improve the force sensitivity. We demonstrate that by bringing a tip of a cantilever onto the edge of a micropillar structure a significant reduction of the damping and an enhancement of force sensitivity are achieved.
Introduction The atomic force microscope (AFM)1 has been used as a powerful tool to probe the forces working on a single molecule. The molecule of interest is tethered between the cantilever and substrate using site-specific chemical modification or nonspecific interaction (physisorption). To date, single molecule force spectroscopy with an AFM has provided mechanical and dynamical properties of a wide range of molecules such as synthesized polymers,2,3 polysaccharides,4-6 DNA,7,8 and protein molecules.9-12 In single molecule force spectroscopy with an AFM, usually a very soft cantilever is used to gain high force sensitivity. However, according to the equipartition theorem, a soft cantilever has a big thermal fluctuation (Æxæ = (kBT/k)1/2) which reduces the S/N ratio in the force signal. Even so, if the frequency of the thermal noise peak is much higher than a frequency at which most of single molecule events (for example, mechanical protein unfolding) can be monitored, use of a low-pass filter is effective in eliminating most of the thermal noise which lies above the cutoff frequency. However, for soft cantilevers immersed in aqueous solution, the resonance frequency of thermally driven oscillation is typically at around 10 kHz or below and the resonance peak is heavily broadened by a hydrodynamic drag by the surrounding solvent; it then becomes difficult to eliminate the thermal noise from the force signal. One of the approaches to *Corresponding author. Telephone & fax: þ81-761-51-1593. E-mail:
[email protected] (1) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930. (2) Begum, R.; Matsuura, H. J. Chem. Soc. 1997, 93, 3839. (3) Oesterhelt, F.; Rief, M.; Gaub, H. E. New J. Phys. 1999, 1, 6.1–6.11. (4) Fisher, T. E.; Marszalek, P. E.; Oberhauser, A. F.; Carrion-Vazquez, M.; Fernandez, J. M. J. Physiol. 1999, 520, 5. (5) Marszalek, P. E.; Oberhauser, A. F.; Pang, Y. P.; Fernandez, J. M. Nature 1998, 396, 661. (6) Marszalek, P. E.; Li, H.; Fernandez, J. M. Nat. Biotechnol. 2001, 19, 258. (7) Rief, M.; Clausen-Schaumann, H.; et al. Nat. Struct. Biol. 1999, 6, 346. (8) Clausen-Schaumann, H.; Rief, M.; et al. Biophys. J. 2000, 78, 1997. (9) Best, R. B.; Clarke, J. Chem. Commun. 2002, 3, 183. (10) Forman, J. R.; Clarke, J. Curr. Opin. Struct. Biol. 2007, 17, 58. (11) Mitsui, K.; Hara, M.; Ikai, A. FEBS Lett. 1996, 385, 29. (12) Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; M€uller, D. J. Science 2000, 288, 143.
1002 DOI: 10.1021/la902472h
reduce the hydrodynamic effect and keep the resonance frequency high is to make the cantilever small. However, when the device size is decreased, the ratio of surface area versus device volume and mass of the device decrease. Consequently, the quality factor of the small cantilevers in fluid is significantly decreased, and this is the main problem in using microcantilevers. Nevertheless, this approach has had some successful results,13,14 but in order to make such cantilevers we require microfabrication13 or FIB milling equipment.15 In addition, a special AFM probe head with a small spot might be required.16 Some soft and small cantilevers (biolever from Olympus, for example) are commercially available, but the hydrodynamic drag is very high when the tip is approached near the surface (typically within a micrometer), as will be demonstrated in this paper. Usually, the length of molecules measured in the single molecule force spectroscopy ranges from 10 nm (proteins) to 1 μm (synthesized polymers, nucleic acids, or polysaccharides), so that elimination of the thermal noise does not work effectively with such small cantilevers. In dynamic force single molecule spectroscopy, the thermal noise or magnetically driven oscillation of a cantilever which is pulling a molecule of interest is measured. We have been developing this technique17-20 and found that the uncertainty in viscoelasticity of single molecules depends on that of a free cantilever, as the single molecule viscoelasticity is extracted from (13) Viani, M. B.; Schaeffer, T. E.; Chand, A.; Rief, M.; Gaub, H. E.; Hansma, P. K. J. Appl. Phys. 1999, 86, 2258. (14) Viani, M. B.; Pietrasanta, L. I.; Thompson, J. B.; Chand, A.; Gebeshuber, I. C.; Kindt, J. H.; Richter, M.; Hansma, H. G.; Hansma, P. K. Nat. Struct. Biol. 2000, 7, 644. (15) Maali, A.; Cohen-Bouhacina, T.; Jai, C.; Hurth, C.; Boisgard, R.; Aime, J. P.; Mariolle, D.; Bertin, F. J. Appl. Phys. 2006, 99, 024908. (16) Viani, M. B.; Schaffer, T. E.; Paloczi, G. T.; Pietrasanta, L. I.; Smith, B. L.; Thompson, J. B.; Ritcher, M.; Rief, M.; Gaub, H. E.; Palxco, K. W.; Cleland, A. N.; Hansma, H. G.; Hansma, P. K. Rev. Sci. Instrum. 1999, 70, 4300. (17) Kawakami, M.; Byrne, K.; Khatri, B.; McLeish, T. C.; Radford, S. E.; Smith, D. A. Langmuir 2004, 20, 9299. (18) Kawakami, M.; Byrne, K.; Khatri, B.; McLeish, T. C.; Radford, S. E.; Smith, D. A. Langmuir 2005, 21, 4765. (19) Kawakami, M.; Byrne, K.; Khatri, B.; McLeish, T. C.; Smith, D. A. ChemPhysChem 2006, 7, 1710. (20) Kawakami, M.; Byrne, K.; Brockwell, D. J.; Radford, S. E.; Smith, D. A. Biophys. J. 2006, 91, L16.
Published on Web 09/28/2009
Langmuir 2010, 26(2), 1002–1007
Kawakami et al.
Article
Figure 1. (a) Schematic of the fabrication process of a PDMS micropillar mold (top) and gold-sputtered micropillar structure on a glass coverslip (bottom). The micropillar is easily replicated by repeating the bottom line of the process with a commercial UV trans illuminator for curing the adhesive and use of a sputtering instrument. (b) Mask pattern for fabricating the array of micropillars. (c) SEM micrograph of a fabricated micropillar. (d) Optical micrograph of a short biolever brought above the edge of a micropillar stage. The cantilever is the dark rectangular structure on the right side of the image, and the square is a micropillar’s top stage.
that of a cantilever-molecule system by subtracting the contribution of a free cantilever. Therefore, if the damping of the cantilever itself is reduced, an improved S/N ratio in the measurement of single molecule viscosity is expected. In this Article, we propose a more simple approach. We introduce a use of a micropillar which enables a cantilever tip to contact the surface (on top of the micropillar) while separating a cantilever beam far from the flat surface. Significant reduction of the hydrodynamic damping on a cantilever was obtained especially within a few micrometers from the surface, and improvement of force sensitivity with a DSP low-pass filter was achieved.
Experimental Section Fabrication of the Micropillar Structures on a Coverslip. In this study, we have introduced a microreplica molding method21 for the fabrication of the micropillar structure so that structures with identical shape and distribution can be repeatedly and easily obtained at low cost. Details of the fabrication process are described below. A schematic diagram of the fabrication process is shown in Figure 1a. We made a template of micropillar structures by a standard method. A stirred and degassed (Thinky, Japan) poly(dimethylsiloxane) (PDMS, prepolymer with curing agent (Sylgard 184, Dow Corning)) was poured on to the template mold and incubated for 12 h at 65 C. After curing, the PDMS sheet was carefully peeled off from the template, and we obtained a mold of micropillar structures (shown as the end of first line in Figure 1a). Details are given in the Supporting Information. The key of this approach is that once we get this PDMS mold, no further lithography or etching processes are required to obtain (21) Xia, Y. N.; Whitesides, G. M. Angew. Chem. Int. Ed. 1998, 37, 551.
Langmuir 2010, 26(2), 1002–1007
micropillar structures. We only need to apply a very small quantity of liquid adhesive22 (NOA73, norland optical adhesive 73) onto the PDMS mold and cover it with a microscope cover glass slip. The adhesive NOA73 was cured by exposure to UV light, and the cover glass was carefully peeled from the PDMS mold. In this study, prior to the force experiments, the micropillar structures were sputtered with gold. The mask pattern used in this study is shown in Figure 1b, which is designed to arrange the micropillars in a large array, in which the orientation of the micropillars can easily be observed “by eye”. Knowing the orientation before setting the sample on the AFM state is quite important, since almost all commercially available AFM systems have only X-Y mechanical sample stages, and they cannot be “rotated”. A SEM image of the micropillar structure is shown in Figure 1c. The dimensions of the pillars were measured to be 50 x 50 μm (stage size) AFM Equipment. The instrumentation consists of a Picoforce AFM with a Nanoscope IIID controller (Veeco, CA)). A stereomicroscope with a K€ ohler illumination (SXZ16 and SZX2ILLC16, Olympus) was used for the adjustment of a cantilever position above a micropillar stage. A surface image of the pillar stage was then obtained, and the cantilever was exactly brought to the required position above the pillar. All measurements were performed under pure water, using a fluid cell cantilever holder from Veeco. For the thermal noise measurement, a PC equipped with an I/O board (PCI-6014, National Instruments) was used for collecting the thermal noise on the deflection signal with a sampling rate of 200 kHz. The distance between the cantilever and substrate was controlled by software for force measurements provided by Veeco (version 6.1). (22) Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Nano Lett. 2001, 1, 235.
DOI: 10.1021/la902472h
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Figure 2. Thermal noise power spectral density functions (gray traces) of a short biolever (k = 26 pN/nm) above (a) a flat surface and (b) a micropillar stage in fluid. The spectra are drawn with an offset. The solid lines are fits to the simple harmonic oscillator model given in eq 1. Spikes seen at around 4 kHz are due to mechanical noise from the piezo scanner. (c) Thermal noise of the short biolever separated from a flat surface and a micropillar in fluid. The traces are drawn with an offset. (d) Dependence of the effective damping ζ of the free short biolever in fluid on distance between the cantilever and a flat surface (þ) and a micropillar surface (O). All experiments were performed using Igor Pro software (Wavemetrics). All dynamic force measurements were carried out using soft cantilevers (Olympus biolever) whose nominal spring constants are 27 and 6 pN/nm for short and long levers, respectively. The actual spring constants for short and long biolevers used in this study were determined to be 26 and 9 pN/nm, respectively, by fitting of the Power Spectral Density (PSD) shown below. Fitting of PSD Spectra. Theoretical and experimental studies for the thermal vibration of an AFM cantilever23,24 near a surface25 have been carried out. We have described the details of the fitting and determining the viscoelastic quantities elsewhere.17,19 Fitting of the PSDs was performed using the simple harmonic oscillator (SHO) model which yields an expression for the PSD as follows:23,24,26 D E 2kB Tζ jxðνÞ2 j ¼ h þ DC ð1Þ i2 k - mð2πνÞ2 þ ζ2 ð2πνÞ2 where kB is Boltzmann’s constant, ν is the frequency in hertz, T is the absolute temperature, ζ is the effective damping, k is the elasticity, and m is the effective mass of the cantilever. An additional DC term in the fit was used to take into account the white noise or response of the cantilever’s spatial second harmonic.
Results and Discussion At first, we examined the damping effect between a cantilever and a flat surface in fluid. The cantilever used here is called (23) Sader, J. E. J. Appl. Phys. 1998, 84, 64. (24) Chon, J. W. M.; Mulvaney, P.; Sader, J. E. J. Appl. Phys. 2000, 87, 3978. (25) Roters, A.; Johannsmann, D. J. Phys.: Condens. Matter 1996, 8, 7561. (26) Schaffer, T. E.; Cleveland, J. P.; Ohnesorge, F.; Walters, D. A.; Hansma, P. K. J. Appl. Phys. 1996, 80, 3622.
1004 DOI: 10.1021/la902472h
“biolever”, a small and soft cantilever designed for force measurement and imaging of soft biomaterials. In fluid, the thermal noise signals of the cantilever were collected at various distances from a flat surface. Figure 2a shows PSD spectra of the thermal noise of a short biolever (60 μm length, 30 μm width) at different distances from a flat surface (drawn with dots). When the cantilever is separated by 10 μm from the surface, there is a distinct resonance peak around 7 kHz. However, as the cantilever is brought close to the surface (
