Abstract. Fluorescence spectroscopy in is numerous variations has become a most powerful bioanalytical and diagnostic tool in the past 20 years and - in terms of versatility - seems to be second only to nmr spectroscopy, but with an entirely different field of application. This development reflects the transition of fluorescence spectroscopy from a merely academic area of research into a highly practical tool and was paralleled by a gradual move of fluorescence spectroscopy from physics to chemistry and biology. We also note an increasingly better understanding of the basic work (that had been performed by physicists) by chemists and biologists. As a result, fluorescence has become highly interdisciplinary, and its main applications are clearly in the biosciences. One of the exciting fields is in biosensor technology, and specific examples are given on how the numerous methods known in fluorescence spectroscopy have been implemented to design biosensors.
The commercially most successful application of fluorometry is in luminescence immunoassay, now followed by the diverse applications of fluorescence activated cell sorting (FACS) and other studies on the function of cells. Other exciting novel areas include fluorescence correlation spectroscopy which enables the detection of single molecules, multi-photon excitation with its inherent advantages over conventional excitation, fluorescence imaging and sensing. This contribution will focus on bioanalytical and sensing aspects of fluorescence and - in a wider sense - luminescence.
Biosensors can be based on a variety of detection schemes. Among those, optical sensors form a major group and display features that can make them advantageous over other systems such as electrochemical, mass-sensitive, thermal, acoustic, or other devices. Typical optical schemes are based on absorption spectrocopy (from the UV to the deep infrared), Raman and conventional fluorescence spectroscopy and imaging, but also on more sophisticated methods such as surface plasmon resonance, evanescent wave and near-field spectroscopy, fiber optic spectroscopy, correllation spectroscopy, and - last not least - luminescence lifetime, polarization and energy transfer.
Luminescence spectroscopy knows numerous parameters to be determined. In analytical applications these include measurement of light intensity, its decay time, polarization, quantum yield and quenching efficiency, radiative and non-radiative energy transfer, and numerous combinations thereof. Luminescence can be excited by light (including laser light), electrochemically, by (bio)chemical energy, pressure or sound. Luminescence can be measured of gaseous, liquid and solid samples, of tissue and cells, by directly illuminating the object or by using waveguide optics including optical fibres. Its versatility is obvious. Luminescence can be presented in Cartesian format, but also in multidimensional form to result in so-called excitation-emission matrices or contour plots. Fluorescence lends itself to imaging which has become the basis for exciting studies in cell biology, dermatology, and aviation research, but also for remote sensing, e.g. from satellites.
Labels are preferably attached to the species of interest by covalent binding via a reactive group that forms a chemical bonds with other groups such as amino, hydroxy, sulfhydryl or carboxy. Labels are expected to be inert to other chemical species present in the environment, for example to pH. In order to reduce background luminescence of biological matter, labels preferably have long-wave excitation and emission, and/or long decay times so that background luminescence decays much faster than the luminescence of the label. As a result, there is a substantial interest in the design of longwave and long-decay luminescent labels.
The co-enzyme FAD is another strongly luminescent species but has found less wide applications because both the oxidized (FAD) and the reduced form (FADH2) display fluorescence so that they are less useful for monitoring the course of a biochemical reaction. Their excitation is at around 450 nm, and fluorescence peaks at 512 nm. Both NADH and FAD have been shown to be useful for purposes of chemical sensing using immobilized reagents and, in some cases, using fiber optics waveguides. However, their practical applications have been been confined mainly to conventional cuvette tests so far.
Optical sensing of oxygen is almost exclusively accomplished on the basis of the quenching effect which it exerts on numerous luminophores. Both luminescence intensity and decay time may be measured, the latter being preferred for reasons of system stability, precision and accuracy. The pH of blood and related biological matter is determined via classical pH indicators such as the fluoresceins which are immobilized on solid supports (or inside polymeric matrices) and undergo a pH-induced spectral change. Numerous schemes exist that are based on measurement of either luminescence intensity, decay time, or energy transfer efficiency. Fiber optic sensors for measurement of oxygen, pH, carbon dioxide and hematocrit are on the market, as are microsensors (with ~10 Ám thin tips) for measurement of oxygen in algae and in the deep sea.
glucose + oxygen ===> gluconic acid + hydrogen peroxide (1)
It can be seen that such a reaction can be monitored kinetically in various ways, namely by measureing the consumption of oxygen (via the above fluorescent oxygen sensor), the formation of the acid (via the above pH sensor), or the formation of hydrogen peroxide (via a respective sensor). Since 2 - 4% of caucasians suffer from Diabetes mellitus, there is a substantial need for such sensors. Numerous other enzyme based biosensors have been described in the past 10 years, and typical examples will be given.
Enzymatic reactions can be inhibited by toxic species including organophosphates and heavy metals. This is the basis for so-called inhibition biosensors which respond to environmentally harmful species like herbicides, warfare agents, and heavy metals including lead and cadmium. Respective examples will be presented as well.