4. 荧光偏振 - Shandong University

1 4.. If linear polarized light is used to excite an ensemble of fluorophores, only those fluorophores aligned with the plane of polarization will be excited. There are 2 scenarios for the emission. Perrin Weber Dandliker Jolley 1.. P A .. The fluorescence anisotropy of a fluorophore reflects the molecule's ability to rotate in its microenvironment, which depends on the viscosity of the solution, the fluorescence lifetime of the fluorophore, and the size and mass of the molecule to which the fluorophore is attached. I//-I I//-I . P= A=. I//+I I//+2I P = 3 A / (2 + A). Fluorescence polarization --p A = 2 P / (3 P). 2. Fluorescence anisotropy A or r (1) . A I//=I P=0 .. B I// I 0 P= 1 .. C I// I 0 0<P<1 .. 3. Fluorescence polarization (FP) can be considered a competition between the molecular motion and the lifetime of fluorophores in solution.

2 Rot is the rotational relaxation (correlation). time (the time required to rotate through an angle whose cosine is 1/e, or approximately ). fl is the fluorescence lifetime of the excited fluorescent probe 4. z Physical basis of fluorescence polarization assays. Dye molecules with their absorption transition vectors (arrows) aligned parallel to the electric vector of linearly polarized light (along the vertical page axis) are selectively excited. For dyes attached to small, rapidly rotating molecules, the initially photoselected orientational distribution becomes randomized prior to emission, resulting in low fluorescence polarization. Conversely, binding of the low molecular weight tracer to a large, slowly rotating molecule results in high fluorescence polarization. Fluorescence polarization therefore provides a direct readout of the extent of tracer binding to proteins, nucleic acids and other biopolymers.

3 5. (2) . A .. A P . B P . C .. V: the molecular volume of the molecule : the viscosity of the medium : the fluorescence lifetime of the fluorophore T: the temperature rotational relaxation time . 6.. 7. (3) . Steady-State Fluorescence Polarization Time-Resolved Fluorescence Polarization 8.. The Perrin equation (1926). (1/P)-(1/3)=((1/P0)-(1/3))(1+( / )). r = r0 / (1 + ( / )). The Stokes equation (for spherically symmetrical molecules). = V / RT. r = r0 / (1 + ( RT / V)). Corresponding expressions for spherically unsymmetrical and ellipsoidal molecules can be found in literatures. 9.. Following a pulse excitation, the fluorescence anisotropy of a spherical particle in a homogeneous isotropic medium decays exponentially r = r0 exp ( t / ). Non-spherical particles (most biopolymer): complex Chromophore (or a molecular domain) rotates around the bond linking it to the biopolymer: complex Anisotropic environment (like in phospholipid bilayers): complex 10.

4 Application Fluorescence polarization measurements have long been a valuable biophysical research tool for investigating processes such as membrane lipid mobility, myosin reorientation and protein protein interactions at the molecular level. Immunoassays that have been developed and used extensively for clinical diagnostics represent the largest group of bioanalytical applications. The more recent advent of microplate readers equipped with polarizing optics has led to the adoption of fluorescence polarization as a readout mode for high-throughput screening 11. Application z Immunoassay F. Ab Ag F-Ag F-Ag:Ab .. 12. Example Ab + Ag-F Ab:Ag-F.. (FITC) . paclitaxel . paclitaxel . (anti-pactaxel) .. 13.. 14.. Stokes shift) .. 15. Fluorescence Quenching Internal quenching due to intrinsic structural feature structural rearrangement.

5 External quenching interaction of the excited molecule with another molecule in the sample or absorption of exciting or emitted light by another chromophore in sample. 16. (quenching).. 3O 1O. 2 2.. 17.. I- Br- CNs- .. 18. Stern-Volmer equation Fluorescence quenching is a function of: The excited state lifetime The diffusion-limited quenching constant The concentration of the quencher Stern-Volmer equation F0/ F = 1 + s kQ [Q]. The Stern Volmer slope: KSV = s kQ. 19. Fluorescence Resonance Energy Transfer Known as fluorescence resonance energy transfer (FRET). or F rster energy transfer. It is the radiationless transfer of excitation energy from a donor to an acceptor. An important consequence of this transfer is that there is no emission of light by the donor. The acceptor may or may not be fluorescent.

6 FRET is a distance-dependent interaction where the energy transfer occurs typically over a distance of 1-10nm. The distance dependent nature of FRET is highlighted by the fact that it is proportional to the inverse sixth power of the intermolecular separation. 20. Fluorescence resonance energy transfer (FRET). If the fluorophores (extrinsic or intrinsic) have unique locations within the protein or complex, it is possible for emission light energy from A to be absorbed by B and to be emitted as part of B's emission spectrum Absorbance Fluor A emission A/Q. 1 2. Fluor B Absorbance emission A/Q. 2 3. (nm) 21. FRET is used as a spectroscopic ruler'. FRET is dependent on the distance, R, between the two fluors. Used to measure distances in proteins, membranes, & macromolecular assemblies 10 to 80 apart.

7 Efficiency of the energy transfer (E) from donor to acceptor (A to B) defined by E = R06. R06 + R6. R = distance between A & B, R0 is a constant calculated from absorption and emission spectra. E can be calculated from fluorescence intensity (F). E = Fda/Fd Fda = F in the presence of acceptor, Fd = F in the absence of acceptor. Once E is known, R can be calculated from the first equation if R0 is known. 22. Limitations Limited range (10 80 ) if R is > 8 nm E is very small Need 2 fluorophores, a donor & an acceptor. Assumptions are made in the calculation of R0 such as the orientation of the donor & the acceptor. 23. FRET optical shortcomings One major concern of FRET is related to the UV/vis- excitation nature of the energy donors, which normally are fluorescent dyes or proteins or semiconductor quantum dots.

8 In this excitation window, unfortunately, rather strong autofluorescence and scattering light always arise from biomolecules when the assay is conducted in biological sample matrixes. Another issue of FRET assays is the unexpected coexcitation of the energy donor and the acceptor because of the overlap of their excitation spectra, which at present is quite hard to eliminate due to the relatively small Stokes-shift of most down-converting fluorophores. 24.. (Tyndall) : .. (Tyndall) . (Rayleigh) : .. (Raman) : .. 25.. 1 . * * n . 2 .. 3 .. CH2. = =1. 26. (4) .. 10. C6H5 COOH 3. C6H5NO2 0. C6H5CH3 17. -OH - C6H5OH 18. NH2 -CN -OCH3 C6H5 OCH3 20. C6H5NH2 20. C6H5CN 20.. C6H5Cl 7. C6H5Br 5.. C6H5I 0. , .. 27.. C6H5OH 18. pH . C6H5O 10. pH . C6H5NH2 20.. +. C6H5NH3 0.. pH.

9 -- - -- .. 28.. 250nm .. 29. Absorption Fluorescence Luminescence 30.