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Fluorescence detection is one ofthe most popular detection methods used in bioanalytical analysis. Fluorescence detection methods offer the advantages ofhigh sensitivity combined with selectivity. Generally, the chromophore is attached to the species of interest and acts as a reporter molecule. Most fluorescence detection schemes involve the use ofvisible fluorophores, fluorescein being one ofthe most common. A problem often encountered with the used of fluorescence detection in the visible region of the spectrum is sample autofluorescence. Many compounds have intrinsic fluorescence in this region. Background fluorescence is present throughout the visible and UV region ofthe spectmm (fig 1). Consequently, background noise can be very high and, as a result, the overall sensitivity of the measurement is decreased. Very few molecules exhibit intrinsic fluorescence in the near infrared region of the spectrum (650-1 100 nm). As a result, the background noise associated with fluorescence detection in the visible region is nearly eliminated. Consequently, the near infrared region offers the potential for significant improvements in sensitivity, especially in situations where matrix autofluorescence is a concern. Near infrared fluorescence detection methods are therefor well suited to bioanalytical applications. Furthermore, light scatter, another source of interference, is decreased in the near infrared region due to its ii)dependence. Detection at 820 nm offers a six-fold reduction in light scatter over detection at 500 nm. Instrumentation associated with the use of near infrared dyes also offers some advantages over traditional instrumentation. Laser induced fluorescence is one ofthe most commonly used fluorescence detection methods, due to the high intensity associated with laser light. Lasers that operate in the visible region ofthe spectrum are often bulky, expensive, and have limited operational lifetimes. The advent of solid state diode lasers do not have any of the aforementioned disadvantages. They are mgged, inexpensive, compact, and have long operational lifetimes. The typical signal transducer for fluorescence measurements is the photomultiplier tube (PMT). However, PMT's make poor choices for signal transducers in the near infrared region due to their low quantum yields at these wavelengths. Consequently, Avalanche photodiodes (APD) are used. APD's offer high quantum efficiencies in the near infrared region. Additionally, they are rugged, cheap, and have long operational lifetimes.
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