Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 629
Lifetime-based sensing is valuable for probes that are
subject to collisional quenching and do not display wavelength-ratiometric
behavior. The capability for ratiometric
measurements can be designed into oxygen sensors by
including a nanosecond-lifetime fluorophore in the supporting
media. This fluorophore can provide a reference that is
not sensitive to the oxygen concentration. For in-vivo applications
MLCs are known to have longer absorption and
emission wavelengths. 34 These MLCs have been used to
measure lifetimes and oxygen concentrations through
skin. 35
19.4.3. Mechanism of Oxygen Selectivity
An important consideration for any sensor is selectivity.
For oxygen, the selectivity is provided by a unique combination
of the fluorophore and the supporting media. Almost
all fluorophores are collisionally quenched by oxygen, so
that no fluorophore is completely specific for oxygen. However,
the extent of quenching is proportional to the unquenched
lifetime τ 0 (eq. 19.1). For fluorophores in aqueous
solution with decay times under 5 ns, the extent of
quenching by dissolved oxygen from the atmosphere is
negligible. Hence, one reason for the apparent oxygen
selectivity of [Ru(Ph 2 phen) 3 ] 2+ is its long lifetime near 5
µs, which results in extensive quenching by atmospheric
oxygen.
Selectivity of the MLC oxygen sensor is also due to the
silicone support. Silicone is impermeable to most polar
species, so most possible interferants cannot penetrate the
silicon to interact with the probe. Fortunately, oxygen dissolves
readily in silicon, so that the support is uniquely permeable
to the desired analyte. Finally, there are no other
substances in air which act as collisional quenchers. NO is
also a quencher but is not usually found in the air. Hence,
the sensor is selective for O 2 because of a combination of
the long lifetime of the MLC probe and the exclusion of
potential interferants from the nonpolar silicone support.
19.4.4. Other Oxygen Sensors
While [Ru(Ph2phen) 3 ] 2+ is the most commonly used fluorophore
in oxygen sensors, other probes are available.
Almost any long-lived fluorophore can be used as an oxygen
sensor, particularly when dissolved in an organic solvent.
Because of the long decay times, phosphorescence
can be used to detect oxygen. For many applications, such
as oxygen sensing in blood or through skin, it is useful to
have probes that can be excited with red or NIR wavelengths.
Several porphyrin derivatives are known that dis-
Figure 19.12. Absorption and emission spectra of a phosphorescent
porphyrin ketone. Revised and reprinted with permission from [36].
Copyright © 1995, American Chemical Society.
play oxygen-sensitive phosphorescence. 36–37 One example
is platinum (II) octaethylporphyrin ketone (Figure 19.12),
which can be excited at 600 nm. This molecule shows a surprisingly
large Stokes shift, with the emission maximum at
758 nm. The lifetime of 61.4 µs results in oxygen-sensitive
emission even when the probe is embedded in polystyrene.
Simple instrumentation can be constructed for lifetimebased
sensing with long-lifetime emitters. Figure 19.13
(top) shows a schematic for a simple device for lifetimebased
sensing of oxygen. 38 The fluorophore is platinum (II)
Figure 19.13. Lifetime-based sensing of oxygen using platinum (II)
octaethylporphyrin ketone in a polymer membrane. The light modulation
frequency is 3907 Hz. The gaseous oxygen concentrations were
0, 0.1, 5.1, 9.96, and 20.55%. Revised from [39].