Analytical Applications of Very Near-IR Fluorimetry

Abstract

Fluorescence spectrometry has been accepted for many years as a major technique for trace analysis, and it is applied routinely and successfully to such diverse fields as the detection of solutes in flowing systems (e.g. in HPLC, CZE, and FIA); the monitoring of biospecific reactions, as in immunoassays and DNA-probe and enzyme assays; the detection of inorganic ions such as H⁺ and Ca²⁺ in cellular and other samples; the detection of components separated by laminar methods such as TLC and zone electrophoresis; and the study of molecular interactions, such as ligand-protein binding. It is noteworthy that in most of these assays it is not the intrinsic fluorescence of the determinant which is measured (polynuclear aromatic hydrocarbons are the major exception to this generalization): in most applications fluorescent labels or probes are used to provide the desired optical and molecular properties. [In this paper the term label is used to describe a covalently bound fluorophore group, while probe means a fluorophore, usually non-covalently bound to a protein or other surface, whose environment dependent fluorescence properties give information on the polarity of its binding sites]. The major advantages of fluorimetry in such fields are increased selectivity compared with UV-Visible absorption spectrometry, the great variety of sample handling methods available, and most of all the exceptional limits of detection accessible in favourable circumstances.