Morphology, Adhesion, Friction and Wear Properties of Biomolecules for BioMEMS/NEMS

1. Morphology, Adhesion, Friction and Wear Properties of Biomolecules for BioMEMS/NEMS bhushan.2@osu.edu Dr. Dharma R. Tokachichu Prof. Bharat Bhushan NLIM Prof. Stephen C. Lee Matthew Keener Heart and Lung Research Institute Nanotribology Laboratory for Information Storage and MEMS/NEMS

2. Outline • Background • Various types of biosensors • Points of friction and wear • Surface modified by SAMs to reduce bioadhesion of polymers • Objectives • Experimental • Sample preparation nanopatterning and chemical linker method • AFM fluid cell • Morphology, Adhesion, Friction and Wear of Biomolecules on Si-based Surfaces • Morphological changes as a function of duration of buffer treatment physisorbed STA as a function of solution concentration with nanopatterning and chemical linker method • Adhesion measurements various surfaces in PBS solution effect of pH of PBS solution • Coefficient of friction of various surfaces • Wear property of various surfaces • SAMs to reduce bioadhesion of polymers • Contact angle measurements • Adhesion of uncoated and SAM coated PMMA and PDMS

3. Biomolecules, such as proteins, on silicon based surfaces are of importance in various bioMEMS application including silicon microimplants and biosensors Background Various types of biosensors BioFET sensor Cantilever type biosensor • The gate metal in MOSFET is replaced by a layer of receptor biomolecules. • Works based upon the stimulus produced by biomolecular interactions. • Coated with a specific biomolecule or bacteria • Works based upon the shift in the natural frequency of the cantilever and kinetics of the system.

4. Points of friction and wear • The implanted bioMEMS such as sensors and drug delivery devices experience relative motion which causes friction and wear between the biomolecules and tissues resulting in degradation of the biomolecular function.

5. In biosensors, the adhesion between the immobilized biomolecule and bioMEMS surface is a key factor which govern its performance and reliability. • For implanted devices, friction and wear issues as well as biofouling need to be addressed. • The adhesion, friction and wear are scale dependent. Study of these properties need to be carried out at nanoscale.

6. Surface modification by SAM to reduce bioadhesion of polymers Polymers in BioMEMS • To facilitate biofluid flow through nanochannels, surfaces with low bioadhesion are required. • SAM coated surfaces are expected to provide low adhesion compared to virgin surfaces as they are highly hydrophobic.

7. Objectives • Study step by step morphological changes and adhesion, friction and wear of biomolecules using atomic force microscopy (AFM). • Study surface modification approaches like surface nanopatterning and chemical linker method to improve adhesion of biomolecules on silicon based surfaces. • Develop SAM coatings for PDMS and PMMA that gives low bioadhesion

8. Experimental Sample preparation DI water: De-ionized water PBS: Phosphate buffered silane STA: Streptavidin NHS: N-hydroxysuccinimido BSA: Bovine serum albumin

9. Schematic representation of deposition of streptavidin (STA) by chemical linker method Silanized silica Coated with sulfo-NHS-biotin Coated with BSA Covalently coated with STA • Based on molecular weights, STA is 50 times larger than biotin, • and BSA is 5 times larger than STA.

10. AFM fluid cell • Fluid cell is used for imaging in liquid. • Special optical glass which allows the laser pass through without any refraction. • Working principle is similar to an AFM holder used for tapping mode AFM in air expect the manner it is excited. • The exciting frequencies lie in between 8-12 kHz. Schematic of an AFM fluid cell

11. Morphology, adhesion, friction and wear of biomolecules on Si-based surfaces Morphological changes as a function of duration of buffer treatment Silica soaked in PBS for 2 h and rinsed off salts Silica soaked in DI water for 2h Silica soaked in PBS for 2h Imaged in air Imaged in PBS • Increase in roughness of silica surface soaked in PBS and • imaged in air is due to salts residue on the surface.

12. Morphological changes of physisorbed STA as a function of solution concentration Concentration 10 mg/ml Concentration 1 mg/ml Concentration 100 mg/ml Imaged in PBS • STA forms multilayered structure if the concentration of STA • solution is higher than 10 mg/ml

13. Confirmation of protein distribution and density by enzyme-linked immunosorbent assay (ELISA) tests • ELISA tests prove that direct adsorption gives higher STA density • than chemical linker method. • Distribution is saturated at 10 µg/ml concentration, above this • STA probably forms multilayered structure.

14. Morphological changes with nanopatterning and chemical linker method STA coated by chemical linker method STA adsorption on patterned silica STA adsorption on unpatterned silica Imaged in PBS Streptavidin (STA) concentration 10 mg/ml • Adsorption on nanopatterned surface gives higher molecules density • compared to chemical linker method but has low bonding strength.

15. Adhesion measurements of various surfaces in PBS • Patterned silica surface exhibits higher adhesion compared to • unpatterned silica surface. Biotin coated surface exhibits further • high adhesion.

16. Effect of pH of PBS solution on adhesion as a function of concentration of streptavidin solution • Adhesion increases with increase in pH of PBS solution and increase in the concentration of streptavidin solution.

17. Coefficient of friction of various surfaces • Coefficient of friction decreases with increasing density of the streptavidin on silica surface in adsorption method due to lubrication effect. • Coefficient of friction increases with the increasing length of the chemical linker due to cushioning effect.

18. Wear property of various surfaces STA coated silica surface by adsorption as a function of load STA concentration 10 mg/ml • Wear depth increases with increasing normal load. • At lower normal loads wear depth appears due to damage • in the folding structure

19. STA coated silica surface by chemical linker method as a function of load STA concentration 10 mg/ml • Wear depth increases with increasing normal load. • Wear is higher compared to adsorption method because of larger thickness of the biomolecular film.

20. Summary • Liquid AFM has been successfully used to study morphology and adhesion of biomolecules like streptavidin on silicon based surface. • Buffer solutions like DI water and PBS make the silica surface rougher. • The density of immobilized molecules increases with the concentration of the solution until 10 µg/ml, above this increase in the molecules immobilized density is much low and forms multiple layers. • Chemical linker method provides higher adhesion compared to direct adsorption method. • Patterning of silica surface improved the adhesion between the silica surface and biomolecules. • Friction decrease with the increasing concentration of the streptavidin in the solution because protein acts as a lubricating film. • Friction increases with the increasing length of the chemical linker due to the cushioning effect and low lateral deformation. • Wear increases with the increasing normal load and is high in case of chemical linker method compared to adsorption method. • At lower normal loads the folding structure of the protein gets damaged and forms a wear mark.

21. SAMs to reduce bioadhesion of polymers Contact angle measurements Polymer surfaces need to have high contact angle to facilitate fluid flow. Surfaces can be coated with perfluorinated SAM (perfluorodecyltriethoxysilane or PFDTES). In order to improve adhesion of SAM to polymer, the surfaces were treated with oxygen plasma to create –OH bonds. • Contact angle of coated polymers is higher than that of corresponding • substrates

22. Adhesion of uncoated and SAM coated PMMA and PDMS • Adhesive force of coated polymers is • lower tan that of corresponding • substrates.

23. Summary • PDMS is more hydrophobic than PMMA but has high adhesion in ambient due to contribution from the electrostatic charge on the surface. • PFDTES coated PMMA and PDMS surfaces have same adhesion values, revealing that the electrostatic charge on virgin PDMS plays no role when the surface is coated. • Adhesive force is low for PFDTES coated PMMA and PDMS surfaces compared to virgin and oxygen plasma treated surfaces. • Adhesion values of PFDTES coated PMMA and PDMS surfaces are the same in all environments. This reveals that the surface chemistry of PFDTES does not change with the substrate surface. • PFDTES coated surfaces have very low bioadhesion, which is very useful in reducing biofouling and facilitating the biofluid through the micro/nanochannels.

24. Acknowledgements References • The financial support for this research was provided by the industrial membership of the Nanotribology Laboratory for Information Storage and MEMS/NEMS (NLIM). Nanopatterning of silica samples was conceived by Prof. L. J. Brillson and was done in his laboratory. • Bhushan, B. (2004), Springer Handbook of Nanotechnology, Springer-Verlag, Heidelberg, Germany. • Bhushan, B., Tokachichu D.R., Keener M. and Lee S.C. (2005), “Morphology and Adhesion of Biomolecules on Silicon Based Surfaces”, Acta Biomater., 1, 327-341. • Lee, S.C., Keener, M.T., Tokachichu, D.R., Bhushan, B., Barnes, P.D., Cipriany, B.R., Gao, M. and Brillson, L.J. (2005), “Protein binding on thermally grown silicon dioxide.” J. Vac. Sci. Technol. B. (In press). • Tokachichu D.R., Bhushan, B., Keener M. and Lee S.C. (2005), “Nanoscale adhesion, friction and wear studies of biomolecules on silicon based surfaces”, (Submitted) • Tokachichu, D.R. and Bhushan, B. (2005), “Bioadhesion of polymers for bioMEMS”, (Submitted). • http://rclsgi.eng.ohio-state.edu/nlim/