1. Copyright © 2010 ISS, Inc. All Rights Reserved. Ewald Terpetschnig Ph.D. Newly designed Fluorescent Probes & Labels enabling Lifetime-Based Sensors Analytica 2010

2. Copyright © 2010 ISS, Inc. All Rights Reserved. Two Ways To Measure Fluorescence Lifetime

3. Copyright © 2010 ISS, Inc. All Rights Reserved. Time-Domain I t =  e – t/  TCSPC Excitation with pulse - Measurement of photons time-correlated Time-to-amplitude Converter

4. Copyright © 2010 ISS, Inc. All Rights Reserved. Frequency Domain Phase Shift Demodulation tan  = 

5. Copyright © 2010 ISS, Inc. All Rights Reserved.

7.  Reactive group for covalent attachment to biomolecules  Lifetime-sensitive probe  Good overall brightness Fluorescence Lifetime Labels  High chemo- and photostability

8. Copyright © 2010 ISS, Inc. All Rights Reserved. Lifetime-Sensitive Cyanine Dye: Seta-633 Study interaction between low-molecular-weight  Study interaction between low-molecular-weight analytes and proteins analytes and proteins  Fluorescence lifetime as the readout parameter Model-System:  Biotinylated label with anti-biotin antibody 2009 Bioconjugate Chem. 2009, 20, 1907–1912

9. Copyright © 2010 ISS, Inc. All Rights Reserved. Measurement Principle  Lifetime of labeled species changes upon binding to protein  Lifetime – More robust parameter compared to intensity –Independent of probe concentration –Unaffected by probe volume and to some extend photobleaching  Lifetime – More robust parameter compared to intensity – Independent of probe concentration – Unaffected by probe volume and to some extend photobleaching Antibody- Antigen-Complex   AbAb

10. Copyright © 2010 ISS, Inc. All Rights Reserved. Comparison With Commercially Available Dyes  Cyanine dyes like Cy5 and Alexa 647 exhibit smaller lifetimechanges (factor < 2) upon binding to proteins  Cyanine dyes like Cy5 and Alexa 647 exhibit smaller lifetime changes (factor < 2) upon binding to proteins  Seta-633 exhibits a 10-fold lifetime change upon binding to BSA and a 6-fold change with IgG

11. Copyright © 2010 ISS, Inc. All Rights Reserved. Synthesis of D-Biotin Conjugate

12. Copyright © 2010 ISS, Inc. All Rights Reserved. Competitive Immunoassay  Titrate fixed [Seta-633-biotin] concentration with increasing amounts of [Anti-biotin] and record phase- increasing amounts of [Anti-biotin] and record phase- modulation data modulation data

13. Copyright © 2010 ISS, Inc. All Rights Reserved.  Free analyte replaces labeled analyte – lifetime change  FD measurements – phase angle changes at single frequency Competitive Binding Study  FD measurements – accuracy of measurement of  = 0.1 deg

14. Copyright © 2010 ISS, Inc. All Rights Reserved.  Ratiometric Measurement  Lifetime of Tracer determines MW of Analyte in FPA Fluorescence Polarization  Short Lifetime – Low MW Analyte Long Lifetime – High MW Analyte Long Lifetime – High MW Analyte

15. Copyright © 2010 ISS, Inc. All Rights Reserved. Fluorescence Polarization A Race between Emission and Molecular Motion Fluorescence Polarization A Race between Emission and Molecular Motion Polarized light excites molecules aligned with plane of polarization  fl <<  rot  fl >>  rot Excited molecules remain aligned. Fluorescence is polarized. Orientation of excited molecules randomizes. Fluorescence is depolarized.

16. Copyright © 2010 ISS, Inc. All Rights Reserved. How is Polarization measured?

17. Copyright © 2010 ISS, Inc. All Rights Reserved. Polarization (P) = I v - I h / I v + I h Anisotropy (r) = I v - I h / I v + 2 I h P = 3 r / 2 + r r = 2 P / 3 -P

18. Copyright © 2010 ISS, Inc. All Rights Reserved.  fl ~ 300 ns  fl = 4ns  HSA = 40 ns Measurement of High-MW-Analytes in FPIA

19. Copyright © 2010 ISS, Inc. All Rights Reserved.  For best results lifetime  fl should be in the range of rotational correlation time  of antigen that is labeled  fl = 26 ns SeTau-425 TG-404  fl = 9 ns  Lifetime  fl of the label is a measure for MW of the species that can be measured in a FPIA Commercially available Lifetime Dyes:

20. Copyright © 2010 ISS, Inc. All Rights Reserved.  Loading of Cells with Florescent Probes cannot be controlled  Ratiometric Measurements Fluorescence Sensing in Cells  FLIM (Fluorescence Lifetime Imaging)  Fluorescent Probes will accumulate differently at various locations in the cell Quantitative Measurements:

21. Copyright © 2010 ISS, Inc. All Rights Reserved. Measurement Mode  Qualitative or quantitative measurements. Ion-probes showing spectral shifts - radiometric measurements Are light source available? How to Choose the Correct Fluorescent Probe Dissociation Constant (K d )  Calibration: Kd of probe is dependent on pH, temperature, viscosity, ionic strength etc……. Indicator Form  Influences cell loading and distribution of the probe Salts and dextran-conjugates - microinjection, electroporation, AM-esters - passively loaded and cleaved by intracellular esterases

22. Copyright © 2010 ISS, Inc. All Rights Reserved.  pKa in the physiolocical pH-range pH-range  Absorption (653 nm) and emission (671 nm) in the emission (671 nm) in the red region red region Seta-650-pH Dual-Ratiometric pH-Measurements in Cells Seta-650-pH Dual-Ratiometric pH-Measurements in Cells  Excitation and emission ratiometric probe ratiometric probe Anal. Biochem. 390, 136–140 (2009)

23. Copyright © 2010 ISS, Inc. All Rights Reserved.  Excitation and Emission Ratiometric Measurements  Excitation-ratiometric measurement λ em = 710 nm λ em = 710 nm pH-Sensing in Cells  Emission-ratiometric measurement λ ex = 590 nm λ ex = 590 nm

24. Copyright © 2010 ISS, Inc. All Rights Reserved.  Seta-650-pH probe also shows pH- sensitive lifetime changes pH- sensitive lifetime changes  Phase angle changes ~ 16 deg between protonated and between protonated and deprotonated forms at 100 MHz deprotonated forms at 100 MHz FLIM pH-Measurements in Cells  pH-Label applicable for FLIM

25. Copyright © 2010 ISS, Inc. All Rights Reserved.  Seta-650-pH labeled E.Coli is internalized in Cell is internalized in Cell  Inside Cell pH of certain compartments is lower pH ~ 5 compartments is lower pH ~ 5 pH-Sensing in Cells - Phagocytosis  Fluorescence Intensity or Lifetime will reflect these pH changes and will reflect these pH changes and are utilized as parameters for are utilized as parameters for visualization visualization Anal. Biochem. 390, 136–140 (2009)

26. Copyright © 2010 ISS, Inc. All Rights Reserved. Acknowledgment: Y. Povrozin ISC, Kharkov, Ukraine A. Tatarets ISC, Kharkov, Ukraine A. Tatarets ISC, Kharkov, Ukraine L. PatsenkerISC, Kharkov, Ukraine and L. PatsenkerISC, Kharkov, Ukraine and Seta BioMedicals, Urbana IL, USA

27. Copyright © 2010 ISS, Inc. All Rights Reserved.