Principles of Fluorescence Spectroscopy

19
Fluorescence sensing of chemical and biochemical analytes
is an active area of research. 1–8 These efforts are driven by
the desire to eliminate the use of radioactive tracers, which
are costly to use and dispose of. There is also a need for
rapid and low-cost testing methods for a wide range of clinical,
bioprocess, and environmental applications. During
the past decade numerous methods based on high-sensitivity
fluorescence detection have been introduced, including
DNA sequencing, DNA fragment analysis, fluorescence
staining of gels following electrophoretic separation, and a
variety of fluorescence immunoassays. Many of these analytical
applications can be traced to the early reports by
Undenfriend and coworkers, 9 which anticipated many of
today's applications of fluorescence. The more recent
monographs 6–8 have summarized the numerous analytical
applications of fluorescence.
Why is fluorescence rather than absorption used for
high-sensitivity detection? Fluorescence is more sensitive
because of the different ways of measuring absorbance and
fluorescence. Light absorbance is measured as the difference
in intensity between light passing through the reference
and the sample. In fluorescence the intensity is measured
directly, without comparison with a reference beam.
Consider a 10 –10 M solution of a substance with a molar
extinction coefficient of 10 5 M –1 cm –1 . The absorbance will
be 10 –5 per cm, which is equivalent to a percentage transmission
of 99.9977%. Even with exceptional optics and
electronics, it will be very difficult to detect the small percentage
of absorbed light, 0.0023%. Even if the electronics
allow measurement of such a low optical density, the
cuvettes will show some variability in transmission and surface
reflection, which will probably exceed the intensity
difference due to an absorbance of 10 –5 . In contrast, fluorescence
detection at 10 –10 M is readily accomplished with
most fluorometers. This advantage is due to measurement
of the fluorescence relative to a dark background, as compared
to the bright reference beam in an absorbance meas-
Fluorescence
Sensing
urement. It is relatively easy to detect low levels of light,
and the electronic impulses due to single photons are measurable
with most photomultiplier tubes.
In this chapter we describe the various approaches to
fluorescence sensing, which include essentially all the phenomenon
discussed in previous chapters. Fluorescence
sensing is described mostly within the framework of the
medical applications, but it is clear that fluorescence detection
is also widely used in biochemical, chemical, environmental
and forensic analysis.
19.1. OPTICAL CLINICAL CHEMISTRY AND
SPECTRAL OBSERVABLE
One long-range goal of fluorescence sensing is noninvasive
monitoring of clinically relevant species and physiological
parameters (Figure 19.1). A suitable portable device would
measure the clinical values of interest, then store and/or
transmit them to the physician. At present we are rather distant
from the ultimate goal of noninvasive testing with
devices similar to cell phones. The limitation is not in optics
or electronics, but rather due to the lack of stable and biocompatible
methods of in-vivo sensing. It is already possible
to measure fluorescence through skin, and the measurements
can accurately return pH and ion concentrations. In
the near term we are likely to see devices similar to PDAs
(Figure 19.2) that contain the chemistry and optics needed
to perform clinical assays using body fluid samples. Such
portable devices would find widespread usefulness, especially
in emergency situations. The sensor array could be
exposed to blood, and the results would be immediately
available. This concept of rapid point-of-care clinical chemistry
is driving the rapid development of numerous fluorescence
sensing devices. In the following sections we
describe the principles of fluorescence sensing, and illustrate
how such devices can provide analytical data.
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