1. Jeremy Colson, Boston University1 Resonant waveguide grating biosensor for living cell sensing Ye Fang, Ann M. Ferrie, Norman H. Fontaine, John Mauro, and Jitendra Balakrishnan Biochemical Technologies, Science and Technology Division, Corning Incorporated Biophysical Journal, June 2006

2. Jeremy Colson, Boston University2 Presentation Outline Motivation Background: assays, RWGs Methods –Vertical and horizontal mass distributions Results –Cell adhesion and spreading –Cell detachment Conclusion Where are they now?

3. Jeremy Colson, Boston University3

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5. 5 Assays Procedure to determine the concentration of a component part of a mixture Cell-based –More complex, less specific –Useful for functional information Pathway activation Toxicity Phenotypic responses –Need for label-free

6. Jeremy Colson, Boston University6 Resonant Waveguide Gratings & MRCAT

7. Jeremy Colson, Boston University7 Mass redistribution detection method Intensity as a function on incident angle –Resonant peaks Vertical mass redistribution –Shift in resonant peak Lateral mass redistribution –Changes in peak shape (PWHM)

8. Jeremy Colson, Boston University8 Effective refractive index 1 response unit = 5.82x10 -4

9. Jeremy Colson, Boston University9 Vertical mass redistribution Specific refractive index increment α =.0018 per 100ml/g (protein).0016 per 100ml/g (other, Na)

10. Jeremy Colson, Boston University10 Comments on ΔN 1.Primarily sensitive to vertical mass redistribution (DMR) 2.Directly a function of changes in protein concentration due to protein relocation (rather than ion mobilization) mediated by a stimulation 3.Relocation of a target or complex of certain mass near the sensor surface contributes more to the overall response than those further away 4.Optical signature is an integrated signal that is a sum of contributions from mass redistribution occurring at different distances away from sensor surface

11. Jeremy Colson, Boston University11 Horizontal mass movement Lateral inhomogeneity does not affect refractive index Lateral inhomogeneity does affect shape of resonant peaks

12. Jeremy Colson, Boston University12 Cell adhesion and spreading Human epidermoid carcinoma cells (A431) in 5% FBS 1.At room temp adhesion not optimal 2.Cells interact with surface through multiple steps 3.Spreading step increases the mass w/in sensing volume

13. Jeremy Colson, Boston University13 Cell spreading inhibitor added 100nM vincristine Reduced kinetics of cell spreading Initial steps primarily affected Are the effects of vincristine limited to first 14 hours?

14. Jeremy Colson, Boston University14 Cell detachment Trypsin – pancreatic serine protease with substrate specificity based on positively charged lysine and argenine side chains Used for cell detachment A431 cells, 95% confluency

15. Jeremy Colson, Boston University15 Low-doses: cell signaling Presence of P-DMR: cell signaling Slight N-DMR: insignificant cell detachment => activation of endogenous protease-activated receptors that lead to typical G q signaling

16. Jeremy Colson, Boston University16 Conclusions Optical signatures are integrated responses that can be used to examine cells in native environments label-free Systematic investigation of cell processes –Adhesion –Detachment –Cell signaling: EGFR and Bradykinin B2 receptors

17. Jeremy Colson, Boston University17 Back to Epic…

18. Jeremy Colson, Boston University18 Thank you!