Biology Reference
In-Depth Information
protein, protein/nucleic acid, protein/substrate, and biomembrane/biomolecule inter-
actions, which play crucial roles in the regulation of cellular pathways. This chapter
reviews the different fluorescence strategies that have been developed for sensing
molecular interactions in vitro at both steady- and pre-steady-state levels.
ABBREVIATIONS
ANS
1-anilinonaphthalene-8-sulfonate
AP5A a
,
g
-di[adenosine-5
0
] pentaphosphate
CPP
cell-penetrating peptide
FRET
fluorescence resonance energy transfer or F¨ rster resonance energy transfer
GFP
green fluorescent protein
GUV
giant unilamellar vesicles
LUV
large unilamellar vesicles
Mant
methylanthranylate
NBD
nitrobenzofurazan
RT
reverse transcriptase
SUV
small unilamellar vesicles
1. INTRODUCTION TO FLUORESCENCE
Over more than a century, fluorescence methodologies have under-
gone tremendous development, from physical and biological to clinical
applications. Indeed, since the first observations of fluorescence reported
by Sir John Frederick William Herschel in 1845 from a quinine solution
under sunlight,
1
the phenomenon of fluorescence has been continuously in-
vestigated. In 1852, Sir George G. Stokes proposed the term “fluorescence”
in honor of the blue-white fluorescent mineral fluorite/fluorspar.
2
Nowa-
days, fluorescence spectroscopy has become one of the most widely applied
technologies in the study of biological processes.
1.1. Fluorescence principle
The simplest definition of fluorescence could be formulated as the emission
of light by a substance that has absorbed light or electromagnetic radiation.
3
Today, fluorescence might be defined as a luminescent process in which sen-
sitive molecules emit light from electronically excited states induced by a
physical (absorption of light), mechanical (friction), or chemical mechanism.
On the basis of Herschel's and Stokes' observations, Jablonski was the first to
give a clear and scientific explanation of this phenomenon.
4
The Jablonski
diagram indicates the relationship between the ground state (S
0
), the excited
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