Fluorescence Sensing

Abstract

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 monographs6–8 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 105 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-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.