Biological Applications of Fluorescence Polarization Methods

Data

Official data in SubjectManager for the following academic year: 2023-2024

Topic

The property of some materials that they are showing light emission under irradiation made considerable development in studies performed on complex biological systems. This is because considering that only a part of materials produces fluorescence and this fluorescence is characteristic for the given material and also by the nanosecond timeframe of the fluorescence, the related methods show high selectivity and applicable also to study the most molecular motions too. The polarization of light which is associated to the main characteristics of the materials offers additional selectivity for the measuring techniques, therefore applicable for analytical purposes too. In the frame of this course further the fundamentals of fluorescence polarization and the related equipments the following topics will be discussed: immune-fluorescence, fluorescence-activated cell-selection, resonance energy transfer, applications of polarization of the intrinsic and extrinsic protein fluorescence, investigation of the interactions and transfers of some selected toxins and drugs (antibiotics, cytotoxic drugs) by fluorescence polarization measurements, applications in HPLC detectors and fluorescent flow cytometry. Students will be known through the most recent scientific results about the pharmaceutical applications of the methods listed above, will have ability for successful application of the related measuring and data-evaluating methods. The curse gives considerable support to earn the drug-designer attitudes of pharmacists.

Lectures

• 1. General overview on the applicability of fluorescence polarization methods in biology, experiments raises the necessity of the applications. Anomalies obtained in measurements based on the intensity changes of fluorescence signal. - Dr. Kunsági-Máté Sándor
• 2. Rotation of molecules, rotational diffusion. Characteristic changes obtained during the adsorption of small molecules onto the large surfaces. - Dr. Kunsági-Máté Sándor
• 3. Fundamentals of the fluorescence processes, Jablonski diagram. Effects disturbs the fluorescence measurements: Rayleigh-scattering, Raman-scattering, inner filter effect. - Dr. Kunsági-Máté Sándor
• 4. Emission surfaces: fluorescence characteristics of Tripaflavin, Rheonin A, Aurophospin and of the Rodamine B. - Dr. Kunsági-Máté Sándor
• 5. Dependence of the fluorescence polarization on the molecular environment: the role of the viscosity and the temperature. - Dr. Kunsági-Máté Sándor
• 6. Correlation between the polaroization of light and the absorption and emission transitions associated to the molecular skeleton and the related absorption and emission oscillators. - Dr. Kunsági-Máté Sándor
• 7. The Perrin-Ljovsin and Szvesnyikov-Feofilov equations. Expectation of the real polarization. Relationnship between the anisotropy and the degree of polarization. - Dr. Kunsági-Máté Sándor
• 8. Methods to determine the real polarization spectra: extrapolation from measurements of two samples having different viscosities and/or temperatures. - Dr. Kunsági-Máté Sándor
• 9. Detectors implemented in HPLC equipments and in the cytometers to measure the fluorescence polarization. - Dr. Kunsági-Máté Sándor
• 10. Circumstances disturbing the measurements: consideration of the inner filter effect and possibilities of their elimination. Protein dynamics in cases of highly light-scattering samples. - Dr. Kunsági-Máté Sándor
• 11. Calculation the corrected polarization spectra. Consequences of Kasha-rule on fluorescence polarization, the inner quantum efficiency. - Dr. Kunsági-Máté Sándor
• 12. Fine-structure of polarization spectra, connection to the vibration spectra of molecules. - Dr. Kunsági-Máté Sándor
• 13. Possibilities according to the determination of theoretical polarization spectra, connections to the quantum chemistry. - Dr. Kunsági-Máté Sándor
• 14. Measuring the polarization spectra. Right angle and front face optical rearrangements, consequences on measurements of biological samples. - Dr. Kunsági-Máté Sándor
• 15. Fluorescence of conjugated aromatic molecular units, relationship between the spectral shift and the degree of conjugation. - Dr. Kunsági-Máté Sándor
• 16. Experimental and theoretical determination of the polarization spectra of anthracene: comparative study. - Dr. Kunsági-Máté Sándor
• 17. Protein dynamics: fluorescence of amino acids and advantages of the fluorescence polarization measurements. Exeptional fluorescence and fluorescence polarization behaviors of azulene. - Dr. Kunsági-Máté Sándor
• 18. Inclusion complexes of aromatic amino acids formed by cavity-shaped molecules, the measurements of anisotropy lifetime. - Dr. Kunsági-Máté Sándor
• 19. Effect of microsolvation on the interactions of serum albumins with cavity-shaped molecules applied for molecular packing of different drug molecules. - Dr. Kunsági-Máté Sándor
• 20. Determination of cell-membrane fluidity by measuring the fluorescence polarization and anisotropy decay. - Dr. Kunsági-Máté Sándor
• 21. Fluorescent molecular packers: determination of the microsolvation by solvent relaxation measurements. - Dr. Kunsági-Máté Sándor
• 22. Determination of the molecular adsorption of Citrinine onto the human serum albumin molecules by fluorescence polarization methods. - Dr. Kunsági-Máté Sándor
• 23. Competition of the Citrinine molecules with drugs for the binding sites of albumins. - Dr. Kunsági-Máté Sándor
• 24. Determination of the molecular adsorption of Ochratoxin A onto the human serum albumin molecules by fluorescence polarization methods. - Dr. Kunsági-Máté Sándor
• 25. Competition of the Ochratoxin A molecules with drugs for the binding sites of albumins. - Dr. Kunsági-Máté Sándor
• 26. Exchange reactions of polyphenols with toxins bonded to proteins, signal changes obtained in fluorescence polarization. - Dr. Kunsági-Máté Sándor
• 27. Extraction of antibiotics from vaste waters by carbon nanotubes, process followed by the fluorescence polarization. - Dr. Kunsági-Máté Sándor
• 28. Extraction of antibiotics from vaste waters by carbon nanotubes, process followed by the fluorescence polarization. - Dr. Kunsági-Máté Sándor

Practices

Seminars

Reading material

Obligatory literature

David Freifelder: Physical Biochemistry

Literature developed by the Department

Slides and notes of all lectures are available electronically. All topics discussed will be associated with papers published recently. Those papers together with the related doctoral dissertations in English will also be offered to support the teaching.

Notes

Slides and notes of all lectures are available electronically. All topics discussed will be associated with papers published recently. Those papers together with the related doctoral dissertations in English will also be offered to support the teaching.

Recommended literature

Slides and notes of all lectures are available electronically. All topics discussed will be associated with papers published recently. Those papers together with the related doctoral dissertations in English will also be offered to support the teaching.

Conditions for acceptance of the semester

Maximum of 15 % absence allowed

Mid-term exams

One test in the 10th week, one summary prepared about the topic of lectures and one presentation prepared within the scope of the course.

Making up for missed classes

-

Exam topics/questions

Rotation of molecules, rotational diffusion. Characteristic changes obtained during the adsorption of small molecules onto the large surfaces.
Fundamentals of the fluorescence processes, Jablonski diagram. Effects disturbs the fluorescence measurements: Rayleigh-scattering, Raman-scattering, inner filter effect.
Emission surfaces: fluorescence characteristics of Tripaflavin, Rheonin A, Aurophospin and of the Rodamine B.
Dependence of the fluorescence polarization on the molecular environment: the role of the viscosity and the temperature.
Correlation between the polaroization of light and the absorption and emission transitions associated to the molecular skeleton and the related absorption and emission oscillators.
The Perrin-Ljovsin and Szvesnyikov-Feofilov equations. Expectation of the real polarization. Relationnship between the anisotropy and the degree of polarization.
Methods to determine the real polarization spectra: extrapolation from measurements of two samples having different viscosities and/or temperatures.
Detectors implemented in HPLC equipments and in the cytometers to measure the fluorescence polarization.
Circumstances disturbing the measurements: consideration of the inner filter effect and possibilities of their elimination. Protein dynamics in cases of highly light-scattering samples.
Calculation the corrected polarization spectra. Consequences of Kasha-rule on fluorescence polarization, the inner quantum efficiency.
Fine-structure of polarization spectra, connection to the vibration spectra of molecules.
Possibilities according to the determination of theoretical polarization spectra, connections to the quantum chemistry.
Measuring the polarization spectra. Right angle and front face optical rearrangements, consequences on measurements of biological samples.
Fluorescence of conjugated aromatic molecular units, relationship between the spectral shift and the degree of conjugation.
Experimental and theoretical determination of the polarization spectra of anthracene: comparative study.
Protein dynamics: fluorescence of amino acids and advantages of the fluorescence polarization measurements. Exeptional fluorescence and fluorescence polarization behaviors of azulene.
Inclusion complexes of aromatic amino acids formed by cavity-shaped molecules, the measurements of anisotropy lifetime.
Effect of microsolvation on the interactions of serum albumins with cavity-shaped molecules applied for molecular packing of different drug molecules.
Determination of cell-membrane fluidity by measuring the fluorescence polarization and anisotropy decay.
Fluorescent molecular packers: determination of the microsolvation by solvent relaxation measurements.
Determination of the molecular adsorption of Citrinine onto the human serum albumin molecules by fluorescence polarization methods.
Competition of the Citrinine molecules with drugs for the binding sites of albumins.
Determination of the molecular adsorption of Ochratoxin A onto the human serum albumin molecules by fluorescence polarization methods.
Competition of the Ochratoxin A molecules with drugs for the binding sites of albumins.
Exchange reactions of polyphenols with toxins bonded to proteins, signal changes obtained in fluorescence polarization.
Extraction of antibiotics from vaste waters by carbon nanotubes, process followed by the fluorescence polarization.

Examiners

Instructor / tutor of practices and seminars