2. Molecular interactions observed in a real time Bogdan Walkowiak Department of Biophysics Technical University of Lodz and Department of Molecular and Medical Biophysics, Medical University of Lodz – possibilities and limitations of SPR technology

3. Endpoint assay

4. Introducing a binding profile

5. TIR – Total Internal Reflection

6. SPR – Surface Plasmon Resonanse k x =2  n 1 sin(  / k sp =(n g 2 n 2 2 /n g 2 +n 2 2 ) 0.5

7. SPR Detection System Sensor chip with gold film Prism Flow channel Light source Polarized light Optical detection unit Reflected light I II I I Sensorgram

8. Microfluidic system IFC Liquid Handling

9. Gold-Dextran Surfaces Biocompatible Low non-specific binding More than 100 runs on the same surface Specific layer Dextran layer Linker layer Gold film Glass

10. 12 14 16 The Sensorgram Resonance Signal (KRU) Dissociation Regeneration Association Kinetics Concentration 100200300400500600 Time (s) 18

11. BiaCore sensor

12. BiaCore sensor family

13. Correlation between SPR response and surface concentration Signal proportional to mass Same specific response for different proteins

14. pH 6.8 pH 4.0 pH 2.8 Protein concentration at the sensor surface

15. Coupling Chemistries

16. Protein immobilization at the sensor surface activation concentration and immobilization saturation

17. Szemraj J., Pietrucha T., Nowak P., Walkowiak B. In preparation 2002 Recombinant protein mediating fibrynolyitc process

18. pH-dependence of MBP and DNAK interaction Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

19. Final presentation of pH-dependence for MBP and DNAK Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

20. Specific antibody binding to immobilized MBP protein Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

21. Specific binding inhibition by Mab against MBP protein Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

22. Acid glycoprotein binding to immobilized PAI-1 Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001

23. Vitronectin binding to immobilized PAI-1 Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001

24. Glass plate one side covered with gold film Carbon deposition by RF CVPD technology Preparation of sensor surface for NCD-plasma proteins interaction Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

25. Raman spectrometry analysis of prepared sensor surface Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

26. Adsorbtion of plasma proteins to the NCD surface Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

27. Sensor for detection of platelet adhesion to immobilized fibrinogen Immobilization of rabbit anti-fibrinogen at the sensor surface Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

28. fibrinogensuspension of blood platelets bound fibrinogen adhered platelets regeneration Adhesion of blood platelets to the sensor with immobilized fibrinogen Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

29. Cells interaction with immobilized ligands

30. Effect of high salt concentration on antibody-ligand interaction

31. Effect of high salt concentration on antibody-ligand interaction

32. Sample Recovery and Fractionation 563 Fractionate dissociating analyte for - re-injection - sequencing - mass spectrometry - electrophoresis - PCR Recover eluted material for - enzymatic transfer assays - on-surface cleavage processes - alterations due to binding or regeneration Recover excess sample during injection - save expensive sample and reagents

33. BiaCore - MS

34. Limitations Molecular weight of analyte not less than 200 Limitet chemical resistivity to organic solvents Residual interaction of analyte with dextran matrix Active for measuremet distance from a sensor surface is limited to 300 nm

35. Thank you very much for your attention