Development of Optical Biosensors for Monitoring Proteins Activity

1. Development of Optical Biosensors for Monitoring Proteins Activity • Giorgi Shtenberg1, Naama Massad-Ivanir2, Michal Sharon3, Ljiljana Fruk4and Ester Segal2,5 Visit our web site: http://segallab.technion.ac.il/ • 1The Inter-Departmental Program of Biotechnology, Technion – Israel Institute of Technology, Haifa, Israel • 2Department of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa, Israel • 3Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel • 4Karlsruhe Institute of Technology, DFG – Center for Functional Nanostructures, Karlsruhe, Germany • 5The Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel

2. Proteases Enzymes that catalyze the hydrolytic breakdown of proteins into peptides or amino acids. Proteases clearly do a lot more than just digest your meals !!! 1. Trypsin 2. Chymotrypsin 3. Pepsin 1. Neurath H., Science, 1984. 2. http://fitnessegypt.blogspot.com/2010/11/digestive-system-for-human-in-nutrition.html

3. Proteases Not only hydrolyze the peptide bond ! • Specialized tasks: • Zymogene activation. • Sodium and blood pressure. • Controlling the degradation • of cytosolic and nuclear proteins. • Balancing the immune response • and apoptosis. • Transport of secretory • proteins across the membrane. 1. Neurath H., Science, 1984. 2. Turk B., Nature Reviews Drug Discovery, 2006.

4. Research Objective Design and construct a generic biosensor system for “real-time” monitoring of protease activity and to define substrate specificity of these enzymes. Silicon wafer Electrochemical etching Porous Si Thermal oxidation Porous SiO2

5. Research Objective Design and construct a generic biosensor system for “real-time” monitoring of protease activity and to define substrate specificity of these enzymes. Fabry-Pérot interference: Silicon wafer Electrochemical etching Porous Si Thermal oxidation Porous SiO2

6. Research Challenge Design and construct a generic biosensor system for “real-time” monitoring of protease activity and to define substrate specificity of these enzymes. PROTEASE Minute quantities • LARGE • Library of substrates

7. Previous Research 1. Killian K.A., ACS Nano, 2007. 2. Orosco M., Advanced Materials, 2006. 3. Orosco M., Nature Nanotechnology, 2009.

8. Limitations of the current state-of-the-art biosensing studies • Non reusable optical platforms. • Non reusable enzymes. • Intrinsic instability during enzyme immobilization. • Compatibility with proteomic analysis methods. 1. Killian K.A., ACS Nano, 2007. 2. Orosco M., Advanced Materials, 2006. 3. Orosco M., Nature Nanotechnology, 2009.

9. Research Concept DNA-protease protein-mix DNA Directed Immobilization (DDI) protease removal M/z spectrum peptide retrieval MS analysis

10. DNA Directed Immobilization Silane chemistry: Enzyme-DNA conjugate

11. DNA Directed Immobilization Optical measurements FRET/Fluorescence labeling Fabry-Pérot interference: Protein Hybrid ssDNA GluALD APTES Porous SiO2 Shtenberg G. et al., Nanoscale Research Letters, 2012.

12. Enzymatic Activity DNA-protease protein-mix protease removal M/z spectrum peptide retrieval MS analysis

13. Enzymatic Activity • Surface regeneration. • Enzyme reuse. • Minute samples • (220ng in 2µL). hybridization de-hybridization - HRP - Trypsin Rel. Activity in solution Hybridization Dehybridization Hybridization Dehybridization Cycle 1 Cycle 2 Shtenberg G. et al., Analytical Chemistry, in press.

14. Reflective Interferometric Fourier Transform Spectroscopy (RIFTS) protease removal

15. Reflective Interferometric Fourier Transform Spectroscopy (RIFTS)

16. Reflective Interferometric Fourier Transform Spectroscopy (RIFTS) (a) (b) (c) BSA BSA peptides • HEPES buffer • 1 mg mL-1 BSA in HEPES buffer • HEPES buffer Shtenberg G. et al., Analytical Chemistry, in press.

17. Mass Spectrometry Standard Procedure Sequence identification BSA peptides LC/MS/MS 1. http://news.thomasnet.com/fullstory/Triple-Quadrupole-MS-MS-Tool-enables-trace-level-screening-580688

18. Mass Spectrometry peptide retrieval MS analysis Shtenberg G. et al., Analytical Chemistry, in press.

19. Substrate ID by MS peptide retrieval MS analysis Shtenberg G. et al., Analytical Chemistry, in press.

20. Summary • Biosensing platform – proteolytic activity. • DNA directed immobilization – • Reusable Enzyme!!! • Compatibility with MS techniques: • TANDEM-MS, LC/MS/MS. 1. Shtenberg G. et al.,“Picking up the pieces: A generic porous Si biosensor for probing the proteolytic products of enzymes”,Analytical Chemistry, in press. 2. Shtenberg G. et al., “DNA-directed immobilization of enzymes onto porous Si optical transducers”, Nanoscale Research Letters, 2012.

21. Current Research • Size exclusion element, NANOREACTOR. • Proteasome: • Multicatalytic proteinase complex. Mechanism still not known !!! 20S proteasome • 1. http://www.tacc.utexas.edu/research/users/features/proteins.php

22. Acknowledgments Our team: Dr. Ljiljana Fruk Dr. Michal Sharon • Financial support: • NEVET program administered by the Russell Berrie Nanotechnology Institute (RNBI). • Marie Curie Reintegration Grant (NANOPACK) administered by the European Community. • Miriam and Aaron Gutwirth Memorial Fellowship for Technion Graduate Students award. • Sherman Interdisciplinary Fellowships for Technion Graduate Students excellence award.

23. Thanks for listening