Challenges for Nanomechanics of Biological Systems

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NNI Interagency Workshop : Instrumentation and Metrology for Nanotechnology Grand Challenge Workshop National Institute of Standards and Technology, Gaithersburg, MD Jan 27-29, 2004

Challenges for Nanomechanics of Biological Systems Christine Ortiz, Assistant Professor Massachusetts Institute of Technology Department of Materials Science and Engineering WWW : http://web.mit.edu/cortiz/www/ Biological systems are one of the most difficult classes of materials to study mechanically at the nanoscale.1-3 Their complex multilevel and multicomponent structures (e.g. tissues, cells, proteins) need to be highly purified and characterized with minimal sample preparation damage (if possible, with no polishing and chemical treatments) so as to still give information relevant to in vivo function. Nanomechanical testing can be challenging due to the need for near-physiological conditions (i.e. aqueous salt solutions, ionic strength=0.15M, pH=7.4), the existence of varied 3-dimensional geometries and multiple buried interfaces, and their dynamic and sometimes, extremely soft, fluid-like nature. Following is a summary of a few new areas which I believe represent the most significant new paths in this field. I. Integration of Nanomechanical Testing Methods with Nanoscale Chemical/Structural Characterization Techniques Down to the Single Molecule Level in Near-Phys iological Conditions. One promising example of this is the combination of atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS)(Figure 1).4-8 SERS is a higher resolution version of traditional Raman spectroscopy, in which the wavelength and intensity of inelastically scattered light from molecules is measured. These wavelengths are shifted from the incident light by the energies of molecular vibrations and hence, allow for chemical identification and characterization of substances. Since AFM- SERS should be able function in aqueous Figure 1 Schematic media, it will provide information about of AFM-SERS6 biomolecular identity, bonding, dynamics such as real time molecular conformational changes (e.g. mechanically induced single protein unfolding), orientation, the spatial distribution of proteins in living cell membranes, the quantification of normal and abnormal proteins in tissue, and chemical variations across nanoindentation sites in whole tissues. II. Integration of Nanomechanics Technologies with High Throughput Biological Arrays . High throughput biological arrays such as DNA7, catalytic RNA9, protein10, and live cell arrays11 have been developed in the context of rapid DNA sequence analysis, platforms for pharmaceutical drug development, fundamental tools to study cell fate and function, etc. Such arrays have not yet been fully exploited in the context of nanomechanics. For example, high-throughput testing of live, isolated, individual chondrocyte cells (Figure 212) can provide information on the the self-assembling and nanomechanical properties of the pre-cartilage tissue layer called the pericellular matrix which coats the chondrocyte. Selective deposition of different types of molecules and cells with higher spatial resolution and high Figure 2 micropatterned Si throughput nanomechanical experimental automation, data substrate with individual cartilage acquisition, analysis, and archiving are necessary. Once again, chondrocyte cells placed in 15 mm combining with nanoscale biochemical/structural assays will provide wells for nanomechanical testing.12 even further complementary information.

1 NNI Interagency Workshop : Instrumentation and Metrology for Nanotechnology Grand Challenge Workshop National Institute of Standards and Technology, Gaithersburg, MD Jan 27-29, 2004

III. High Resolution Chemical Characterization of Bioactive, Chemically Functionalized Nanosized Probe Tips. Probe tips functionalized with proteins, ligands and receptors, cells, and nanotubes have enabled studies of biologically relevant intermolecular interactions.13-16 Probe tip functionalization has been achieved by covalent immobilization, nonspecific physisorption, and conventional adhesives for larger structures (= 1 mm).17-21 However, attachment of smaller structures, such as macromolecules, to the apex of a probe tip with a prespecified orientation, conformation, and density is difficult due to the small surface area involved and, for polyelectrolytes, the presence of fixed charge groups. Functionalization and subsequent characterization of parameters such as the polymer chain grafting density, in the vicinity of the probe tip apex are also difficult and critical to the interpretation of nanomechanical data relating interaction. One effort in this direction have been the use of fluorescence microscopy directly on functionalized cantilever probe tips to detect the presence of bound biomolecule s (Figure 3a22). Another more quantitative method recently developed (Figure 3b23) is the use of chemical force mic roscopy for this specific application. For example, the nanomechanical interaction between a biochemically functionalized probe tip and a surface of known nanomechanical properties is fit to a standard theoretical model where one or two of the fitting parameters are obtained which represent the density and/or orientation of the molecules on the probe tip.23 10 0.5 Electrically modified probe tip Passively modified probe tip 8 Charged rod model; s = 6 nm 0.4 Charged rod model; s = 10 nm

6 0.3

4 0.2 Force (nN) 2 0.1

probe Force / Radius (mN/m) tip 0 0 location 0 10 20 30 40 Distance (nm) a) b) Figure 3 a) Fluorescence microscope image of cantilever probe tip covalently functionalized with human serum albumin protein after tagging with a fluorescent compound22 and b) nanomechanical data for actively and passively functionalized glycosaminoglycan (polysaccharide) probe tips versus an OH-terminated self-assembling monolayer planar substrate compared to Poisson-Boltzmann based theoretical models for electrostatic double layer forces (aqueous solution IS=0.1M, pH=5.6).23 References (1) Shao, Z.; Yang, J. Quarterly Reviews of Biophysics 1995, 28, 195. (2) Shao, Z.; Mou, J.; Czajkowsky, D. M.; Yang, J.; Yuan, J.-Y. Advances in Physics 1996, 45, 1. (3) Sheiko, S. S. Adv. Polym. Sci. 2000, 151, 60-174. (4) Van Duyne, R. P.; Hulteen, J. C.; Treichel, D. A. J. Chem. Phys. 1993, 99, 2101-2115. (5) Hayazawa, N. J. Appl. Phys. 2002, 92, 6983. (6) http://www.ee.ic.ac.uk/wtp/RAFM.html. (7) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (8) Anderson, M. S. Appl. Phys. Lett. 2000, 76, 3130-3132. (9) Seetharaman, S.; Zivarts, M.; Sudarsan, N.; Breaker, R. R. Nat. Biotechnol. 2001, 19, 336-341. (10) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992-5996. (11) Ostuni, E.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 2828-2834. (12) Ng, L. J.; Grodzinsky, A. J.; Ortiz, C. 2004, unpublished data.

2 NNI Interagency Workshop : Instrumentation and Metrology for Nanotechnology Grand Challenge Workshop National Institute of Standards and Technology, Gaithersburg, MD Jan 27-29, 2004

(13) Florin, E. L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415- 417. (14) Hafner, J. H.; Cheung, C. L.; Woolley, A. T.; Lieber, C. M. Prog. Biophys. Mol. Biol. 2001, 77, 73-110. (15) Heinz, W. F.; Hoh, J. H. Trends Biotech. 1999, 17, 143-150. (16) Lower, S. K.; Hochella, M. F., Jr. ; Beveridge, T. J. Science 2001, 292, 1360-1363. (17) Strunz, T.; Oroszlan, K.; Schafer, R.; Guntherodt, H. J. Proc Natl Acad Sci U S A 1999, 96, 11277-11282. (18) McKendry, R.; Theoclitou, M. E.; Rayment, T.; Abell, C. Nature 1998, 391, 566-568. (19) Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275, 1295-1297. (20) Ducker, W. A.; Senden, T. J.; Parshley, R. M. Nature 1991, 353, 239-241. (21) Hugel, T.; Holland, N. B.; Cattani, A.; Moroder, L.; Seitz, M.; Gaub, H. E. Science 2002, 296, 1103- 1106. (22) Rixman, M. A.; Dean, D.; Macias, C. E.; Ortiz, C. Langmuir 2003, 19, 6202-6218 (*image takne in the Irvine Lab-MIT). (23) Seog, J.; Dean, D.; Frank, E.; Ortiz, C.; Grodzinsky, A. J. in press Macromolecules 2004.