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Topics in Nanobiotechnology- 2004

Surface Plasmon Resonance SPR. Final presentation María Viviana Duarte- PhD student. Topics in Nanobiotechnology- 2004. Antigen!!. Do you remember Antibodies?. And Antigens?. Antigen!!. To learn how this union works is very important for Immunochemical studies, antibody development, etc.

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Topics in Nanobiotechnology- 2004

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  1. Surface Plasmon Resonance SPR Final presentation María Viviana Duarte- PhD student Topics in Nanobiotechnology- 2004

  2. Antigen!! Do you remember Antibodies? And Antigens?

  3. Antigen!! To learn how this union works is very important for Immunochemical studies, antibody development, etc

  4. How to study biomolecular interactions as they occur ? SPR!!!

  5. Main objectives • What is SPR ? • How does it work ? • Let´s do the experiment! • Which applications are possible?? • Final comments…

  6. What is SPR ? Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index. SPR causes a reduction in the intensity of reflected light at a specific angle of reflection. This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side).

  7. Intensity at a specific angle • How does it work ? Glass slide. Non absorbing Media N1 “SPR – Light reflected from” Conducting film- Gold layer- 50 nm thick Sample side. n2 SPR =f (refractive index in the opposite from the reflected side; n2) =f (binging molecules)!!!!!! 1RU = 0,0001º; change in concentration of 1 pg/mm2 = f (system)

  8. When molecules in the sample bind to the sensor surface, the concentration and therefore the refractive index at the surface changes and a SPR response is detected.

  9. What is happening inside? Which is the signal? video

  10. 1- Prepare the sample – CHIP technology • Let´s do the experiment! What is SPR ?

  11. 1- Prepare the sample – CHIP technology 2- Prepare the sample- Microfluid system • Let´s do the experiment! What is SPR ?

  12. 1- Prepare the sample – CHIP technology 2- Prepare the sample- Microfluid system 3- Check the solutions • Let´s do the experiment! What is SPR ?

  13. 1- Prepare the sample – CHIP technology 2- Prepare the sample- Microfluid system 3- Check the solutions 4- Do the measurements • Let´s do the experiment!

  14. Diphenylurea Kon Antigen + Antibody Antigen_Antibody Koff • Which applycations are possible ? Diphenylurea hapten sensing with a monoclonal antibody and its Fab fragment: Kinetic and thermodynamic investigations Walter F.M. Sto»ckleina,*, Axel Warsinkea, Burkhard Micheelb, Gerhard Kempterc, Wolfgang Ho»hned, Frieder W. Schellera. Analytica Chimica Acta 362 (1998) 101-111

  15. What information did they take? How did they convert it into kinetic info? Kon

  16. Final comments… The Advantages • Real time, label free measurement • Choice of reusable sensor chips with different chemistries • Simultaneous use of multiple flow cells • An easy and fast way of obtaining real time binding kinetics

  17. Final comments… Points to Consider... • Highly skill-dependant operation: need for extensive training and hands-on experience • It is easy to draw wrong conclusions from incorrectly set experiment • Cost

  18. Questions and discussion time…

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