Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 309
Figure 8.53. Three-dimensional structure of yellow fluorescent protein
YFP-H48Q. The solid line is the peptide backbone, and the green
structure is the native chromophore. The red and blue surfaces show
the location of bound iodide ions. Reprinted with permission from
[132].
As discussed in Chapter 19, a wide variety of fluorophores
are available that can be used for intracellular
sensing. It is difficult to use these probes in an intracellular
environment because they are likely to bind to macromolecules
that can shift the analyte calibration curves. GFP and
its variants are promising intracellular sensors because the
chromophore is buried in the β-barrel and unable to interact
directly with biomolecules. With this consideration in mind,
YFP-H148Q was used to measure the intracellular pH of
Swiss 3T3 fibroblasts. 133 An advantage of GFP is that the
probe is synthesized by the cell itself, and not added to the
cells. The fibroblast were transfected with the gene for
YFP-H148Q, which appeared as a bright green fluorescence
in the epifluorescence microscope (Figure 8.55). The
response of the protein to pH and chloride was tested using
ionophores to modify the internal ion concentrations. 133
When excited at 480 nm the emission intensity decreased
with increasing pH and decreased with increasing chloride
Figure 8.54. Absorption spectra of YFP-H148Q in the presence of
NaCl, pH 6.4. Binding of chloride is thought to stabilize the right side
structure in the upper panel. Revised from [133].
concentrations. These remarkable results show that it is
now possible to insert genes that cause the cells to synthesize
their own fluorophores, and the genetically engineered
protein-fluorophore can be designed to have sensitivity to a
desired analyte.
8.14.4. Amplified Quenching
Collisional quenching was useful for sensing and imaging
of chloride because of its high physiological concentration.
However, quenching does not usually provide high sensitivity
detection. 134 However, it is now possible to amplify
quenching to obtain higher sensitivity detection. 135 Amplification
is accomplished in two ways. The fluorophore is
bound to a fluorescent conjugated polymer (Figure 8.56). In
this case the polymer contained conjugated stilbene
residues. This polymer was found to be highly sensitive to
quenching by the PET acceptor methylviologen (MV 2+ ).
The Stern-Volmer quenching constant is near 10 7 M –1 ,
which is clearly too large for collisional quenching.
There are two reasons for the large quenching constant.
The positively charged MV 2+ binds electrostatically to the
polymer. Secondly, the excited state migrates rapidly
between the stilbene residues. If any one residue is excited
this energy migrates until it encounters the bound MV 2+ .
One MV 2+ quenches the emission from about 1000 stil-