Chemiluminescence as diagnostic tool. A review

Abstract

The principles of chemiluminescence and its applications as diagnostic tool are reviewed. After an introduction to the theoretical aspects of luminescence and energy transfer, the different classes of chemiluminogenic labels including luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives are described emphasizing the molecules which best fulfil the requirements of today’s clinical chemistry. Applications of chemiluminescence and enhanced chemiluminescence to immunoassays, receptor assays, DNA probes, biosensors and oxygen metabolism are discussed as well as the role of enzymes in the selectivity and the sensitivity of these reactions.

Introduction

In clinical chemistry, most of the compounds of interest are present in the body fluids at concentrations so low that common analytical methods are not efficient for their determination. In 1959, Yalow and Berson proposed to determine these products using antibody antigen reactions after radiolabeling of one of the partners, the antigen (insulin) in order to discriminate bound and free components [1]. This method is now widely used but known drawbacks of radioisotopes e.g. health hazard, waste disposal problems, short half-life, conjugate radiolysis and legislative bias have induced intensive search for alternative labels [2], [3], [4]. Moreover, the widespread opinion that radioactive labels are unsuitable for non-separation protocols increases the need for compounds allowing non-isotopic detection although homogeneous immunoassays have been described, using low-energy as well as high-energy radioisotopes, since the past decade [5], [6]. Luminescence and especially chemiluminescence is one of these alternatives.

Luminescence is a term used to describe the emission light, which occurs when a molecule in an excited state relaxes to its ground state. The various types of luminescence differ from the source of energy to obtain the excited state. This energy can be supplied by electromagnetic radiation (photoluminescence also termed as fluorescence or phosphorescence), by heat (pyroluminescence), by frictional forces (triboluminescence), by electron impact (cathodoluminescence) or by crystallization (crystalloluminescence). In chemiluminescence, the energy is produced by a chemical reaction [7]. Since excitation is not required for sample radiation, problems frequently encountered in photoluminescence as light scattering or source instability are absent in chemiluminescence. High backgrounds due to unselective photoexcitation are absent too: there is no need for time resolved detection. Consequently, luminometers based on a rough light detection by photomultiplier tubes are among the cheapest devices in the field [8], [9], [10].

In an excellent paper, Rongen et al. have reviewed the main advantages of chemiluminescence labeling and detection in immunoassays [11]. They have pointed out the large linear response reaching up to six orders of magnitude, the fast emission of light especially when it is generated in a single flash, the high stability of several reagents and most of the conjugates (increased stability is often observed after conjugation), the low consumption of expensive reagents. They have also noted the short incubation times owing to the high sensitivity generally achieved, the full compatibility with homogeneous or heterogeneous, competitive or not competitive, direct or indirect immunoassays or immunometric assays developed in one step as well as two steps formats and finally the absence of toxicity. These statements valid until 1994, especially the last one, remain true at the present time.

Kricka and his co-workers have also published several review articles dealing with chemiluminescence. Some of these cover the early developments of chemiluminescence [12] or applications to all fields which can benefit from chemiluminescence [13] while others published regularly up to 1997 are devoted to recent advances of this method in clinical chemistry [14], [15], [16], [17], [18], [19], [20], [21].

Although chemiluminescence has been widely used as detection method in many fields as flow injection analysis [22], high performance liquid chromatography [23], capillary electrophoresis [24] and thin layer chromatography [25], this paper will focus on applications in the field of diagnostic.