Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 627
Figure 19.7. Jablonski diagram for the free (F) and bound (B) forms
of a sensing probe. From [13].
Another mechanism for sensing is available when the
fluorophore can exist in two states, if the fractions in each
state depend on the analyte concentration (Figure 19.7).
Typically there is equilibrium between the fluorophore free
in solution and the fluorophore bound to analyte. One form
can be nonfluorescent, in which case emission is only seen
in the absence or presence of analyte, depending on which
form is fluorescent. Probes that act in this manner are not
wavelength-ratiometric or lifetime probes. Alternatively,
both forms may be fluorescent but display different quantum
yields or emission spectra. This type of behavior is
often seen for pH probes, where ionization of the probe
results in distinct absorption and/or emission spectra. Spectral
shifts are also seen for probes that bind specific cations
such as calcium. Such probes allow wavelength-ratiometric
measurements. In this case the change in intensity or shift
in the emission spectrum is used to determine the analyte
concentration. Probes that bind specific analytes are often
referred to as probes of analyte recognition. 19
There are many mechanisms that can be used to design
probes that exhibit changes in fluorescence in response to
analytes. Fluorescence probes can form twisted intramolecular
charge-transfer (TICT) states. 20 Another mechanism is
photoinduced electron transfer (PET), which has been used
to develop sensors for metal ions. 21–23 These sensors often
rely on the well known quenching by amines due to PET.
Figure 19.8 shows a PET-based zinc sensor. In the absence
of zinc the anthracene is quenched by exciplex formation
with the amino groups. Upon binding of zinc, the nitrogen
lone pair of electrons is no longer available for PET. As a
result charge transfer no longer occurs, and the anthracene
becomes fluorescent. 21 While the mechanism of this particular
sensor is understood, this is not true of all sensors. In
many cases spectral changes are seen but the mechanism is
not certain. In the following sections we describe examples
Figure 19.8. A zinc probe based on photoinduced electron transfer.
Revised from [21].
of each type of sensor (collisional, RET or analyte recognition).
19.4. SENSING BY COLLISIONAL QUENCHING
19.4.1. Oxygen Sensing
Use of collisional quenching as the sensing mechanism
requires the fluorescent probe to be sensitive to quenching
by the desired analyte. Collisional quenching results in a
decrease in intensity and lifetime, which is described by the
Stern-Volmer equation:
F 0
F τ 0
τ 1 k qτ 0Q 1 K Q
(19.1)
In this equation F 0 (τ 0 ) and F(τ) are the intensities (lifetimes)
in the absence and presence of the quencher, respectively,
K is the Stern-Volmer quenching constant, and k q is
the bimolecular quenching constant. The most obvious
application of collisional quenching is oxygen sensing. In
order to obtain sensitivity to low concentrations of oxygen,
fluorophores are typically chosen that have long lifetimes in
the absence of oxygen (τ 0 ). Long lifetimes are a property of
transition metal complexes 24 (Chapter 20), and such complexes
have been frequently used in oxygen sensors. 25–31
For use as an oxygen sensor the metal–ligand complexes
(MLCs) are usually dissolved in silicone, in which oxygen
is rather soluble and freely diffusing. The silicone also
serves as a barrier to prevent other interfering molecules
from interactions with the fluorophores and affect the intensity
or lifetime.
The high sensitivity of the long-lifetime MLCs to oxygen
is shown by the Stern-Volmer plots (Figure 19.9). The
compound [Ru(Ph 2 phen) 3 ] 2+ is more strongly quenched