1. ＡＦＭ Ｐｒｏｂｉｎｇ Ｏｐｉｏｄ Ｓｉｇｎａ ｌｏｓｏｍｅ Ｃｏｍｐ ｌｅｘ

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

3. Functionalization of AFM tips NHS/EDC Antibody solution SH CH 3 Thiols+ + OH SH O C CT(PEG 12 ) MT(PEG 4 ) Au S O C OH S O C S CH 3 S Au S O C O O N O S CH 3 S S O C O O N O Au S CH 3 S NH S O C S O C Au antibody

4. Detection of antibody-receptor recognition - SHSY5Y cell line stably expresses the MOPR tagged with T7. SHSY5Y neuroblastoma cell line T7 Anti-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 specific unbinding interaction nonspecific 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 Detection of all the vertical segments on force curve Analysis of each force curve Higher than the threshold? Fuzz logic analysis of the corresponding FC segment Attribution of a grading to the event Building the histogram YES Has the whole FC been analyzed? NO YES 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 ｃｕｒｖｅ

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

14. Unbinding force distribution histograms Ｐｅａｋ＝ 123.1 ｐＮ， Ａｖｅｒ ａｇｅ＝ 195.3 ｐＮ

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. Results Poisson statistical method: The number of receptor-antibody pairs (n) is finite and follows a Poisson distribution. µ n = σ n 2 F= measured force for n pairs, F = nF s, F s = single pair force µ F = µ n F s, σ F 2 = σ n 2 F s 2, F s = σ F 2 / µ F If there are nonspecific interactions (F n ), µ F = µ n F s + F n, σ F 2 = σ n 2 F s 2 = (µ F - F n ) F s = µ F F s - F n F s n1n1 n2n2 F = F 1 + F 2 F1F1 F2F2

20. Variance versus mean of the unbinding forces Mean Forc e (pN) Force varian ce (pN 2 ) N 149.44 1 7355.42188 178.37 54 10596.7293 190.96 46 11350.1291 158.10 89 9043.40893 100.18 09 868.3233107 126.67 18 3337.373106 F s = 123 pN F n = 93 pN Computed forces based on Poisson Statistical method

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 RECEPTORS interact with specific ligands to produce changes in the function of the system. Ｏｐｉｏｄ ｓｉｇｎａｌｏ ｓｏｍｅ permits the encounter of the receptors, ligands, membrane proteins and effectors initiating 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. A Few Concepts