ＡＦＭ Ｐｒｏｂｉｎｇ Ｏｐｉｏｄ Ｓｉｇｎａｌｏｓｏｍｅ Ｃｏｍｐｌｅｘ

1. ＡＦＭ　Ｐｒｏｂｉｎｇ　Ｏｐｉｏｄ　Ｓｉｇｎａｌｏｓｏｍｅ　Ｃｏｍｐｌｅｘ

2. ＡＦＭ　ｓｃｈｅｍａｔｉｃ deflection (nm) =[sensitivity (nm/V)] × [deflection(V)] force = (cantilever spring constant) × (deflection)

3. O O N N O O OH O O OH O O O O C C C C OH O C NH NH O O C C CH3 CH3 CH3 CH3 CH3 CH3 S S S S S S CH3 + S S S S SH SH S S + Thiols Au Au Au Au Functionalization of AFM tips antibody MT(PEG4) CT(PEG12) Antibody solution NHS/EDC

4. T7 T7 Anti-T7 SHSY5Y neuroblastoma cell line Detection of antibody-receptor recognition - SHSY5Y cell line stably expresses the MOPR tagged with T7. - The T7-tagged receptor can be identified by AFM probe modified with T7-antibody

5. approach contact retract Force measurements by AFM Forces between the tip and receptor were monitored by continuously indenting and retracting the tip at constant lateral positions. Force curve

6. Three typical force curves demonstrating the interaction events between the tip and the receptor nonspecific unbinding interaction specific unbinding interaction no interaction deflection (nm) =[sensitivity (nm/V)] × [deflection(V)] force = (cantilever spring constant) × (deflection)

7. Ｆｕｚｚｙ　ｌｏｇｉｃ Fuzzy logic is an approach to computing based on "degrees of truth" rather than the usual "true or false" (1 or 0) Boolean logic. Fuzzy logic includes 0 and 1 as extreme cases of truth but also includes the various states of truth in between so that, for example, someone whose age is 60 might be assigned a membership of 0.25 in the fuzzy set of old people.

8. Reading the Force volume file and calibrating the force curves Loading a new force curve YES Detection of all the vertical segments on force curve Has the whole FC been analyzed? Analysis of each force curve NO Higher than the threshold? NO YES Fuzz logic analysis of the corresponding FC segment Attribution of a grading to the event Building the histogram Flow chart of detecting events algorithm

9. Convolution • The integral of two functions f and g. • The resulting amount overlap that occurs when g is shifted over f is the convolution of f and g. • f * g ≡ ∫ f (τ ) g (t − τ )dτ

10. Convolution between force curves and the filter (difference)

11. Convolution with different filters The convolution of the FC with three different filters gives three new curves that present peaks at locations where steep vertical segments, right angles, or v shapes exist on the original FC curve.

12. Ｃｈａｒａｃｔｅｒｉｓｔｉｃ　Ｒｅｔｒａｃｔ　ｃｕｒｖｅＣｈａｒａｃｔｅｒｉｓｔｉｃ　Ｒｅｔｒａｃｔ　ｃｕｒｖｅ

13. (-0.03,-0.03, 0.5, 0)

14. Unbinding force distribution histogramsＰｅａｋ＝123.1ｐＮ，　Ａｖｅｒａｇｅ＝195.3ｐＮ

15. Determining elastic modulus Elastic modulus is the measure of a substance's tendency to be deformed elastically when a force is applied to it. E=stress/strain

16. The Hertz Model Indentation = z-x The data obtained by indentation experiments are usually plots of force against piezo displacement rather than tip sample separation. In applying the Hertz model, curves should eb converted to Force indentaion curve

17. Elastic Modulus • wave             E • 0 241.46 • 7 298.3 • 15 259.37 • 23    313.26 • 31 332.96 • 39 276.49 • 47 158.73 • 55 275.02 • 63 294.34        • Average 267.0838

19. F1 n1 F2 n2 F = F1+ F2 Results Poisson statistical method: The number of receptor-antibody pairs (n) is finite and follows a Poisson distribution. µn= σn2 F= measured force for n pairs, F = nFs , Fs= single pair force µF= µnFs, σF2 = σn2 Fs2, Fs = σF2 / µF If there are nonspecific interactions (Fn), µF= µnFs + Fn, σF2 = σn2 Fs2 = (µF- Fn) Fs= µF Fs - Fn Fs

20. Variance versus mean of the unbinding forces Computed forces based on Poisson Statistical method Fs= 123 pN Fn= 93 pN

21. Conclusion • Based on the Poisson statistical method, the force required to unbind a single ligand and its pair is 123 pN and the nonspecific unbinding force is 93 pN. The unbinding force calculated is consistent with those obtained for antigen-antibody pairs by others. • The potential of the sensor for localizing individual receptor sites can be used in future simultaneous studies on the effect of agonist stimulation on the receptor distribution and its association to changes in biophysical properties.

22. Appendix

23. µ Opiod receptor (MOPR) major target of pain treatment A Few Concepts Ｏｐｉｏｄ　ｓｉｇｎａｌｏｓｏｍｅ permits the encounter of thereceptors, ligands, membrane proteins and effectorsinitiating events leading to any of ligand’s effect. LIGANDS are Chemicals that bind to receptors. EFFECTORS translate the drug-receptor interaction into a change in cellular activity. AGONISTS is a drug capable of fully activating the effector system when it binds to the receptor. RECEPTORS interact with specific ligands to produce changes in the function of the system.