Massimo Guardigli

Chapter 5:“Classical” Applications of Chemiluminescence and Bioluminescence

Chemiluminescence and bioluminescence are powerful detection techniques used in such widely different areas as molecular biology, drug discovery, clinical diagnosis, food and hygiene control, environmental and forensic analysis. In this Chapter, analytical applications of chemiluminescence and bioluminescence in batch analytical formats (for examples, tubes or microtiter plates) are reviewed. The first part of the Chapter deals with applications of chemiluminescence, from the detection of hydrogen peroxide in environmental samples to the measurement of enzymes, enzyme substrates and enzyme inhibitors by means of coupled enzymatic reactions, the evaluation of reactive oxygen species production in cells and the assessment of the antioxidant activity of clinical and food samples. The second part of the Chapter is devoted to analytical bioluminescence, especially to ATP-related assays based on the firefly luciferase reaction. The central role of ATP in all living cells makes it possible to detect bacterial or other living cells, which is useful for example for rapid microbiology and hygiene monitoring, and to monitor a wide range of enzymatic reactions, thus allowing the development of diagnostic assays for enzymes and metabolites. Analytical applications of other luciferases, such as bacterial luciferase, are also briefly reported.

Lanthanide complexes of cage-type ligands as luminescent labels in fluoroimmunoassays

General aspects of immunological analyses are reported and the application of some lanthanide complexes in fluoroimmunoassays is discussed. The photophysical properties of the complexes of cage-type ligands, which, up to now, showed the most intense metal luminescence are reported. The luminescence intensity of these complexes is discussed considering the efficiency of the incident light/emitted light conversion, defined as the product of the absorption efficiency of the ligand and the metal luminescence quantum yield upon excitation in the ligand. It is illustrated how the luminescence intensity can be enhanced by adapting the ligands on basis of the previously obtained results.

Chapter 17:Recent Analytical Application Areas of Chemiluminescence and Bioluminescence

This Chapter deals with applications of CL and BL in topics of significant relevance, which are not described in the previous Chapters of this book. In particular, principle and limitations of the well-known luminol test, based on the catalysis of the luminol CL reaction by blood traces and utilised by forensic scientists in investigations involving violent crime for more than 40 years, are reviewed. Afterwards, the various CL-based methods employed for the rapid and sensitive detection of explosives, from portable explosive analyzers to gas chromatographic and HPLC techniques with CL detection and immunoassays, are described. Finally, examples of application of CL and BL in the study and conservation of cultural heritage are reported, including detection of biodeteriogen agents (bacteria, fungi, yeast, algae and lichens) on artworks by BL imaging of adenosine triphosphate (ATP) and immunolocalization of protein binders in painting cross-sections by CL imaging microscopy.

Chapter 11:Ultrasensitive Bioanalytical Imaging

Bio- and chemiluminescence imaging combines the high sensitivity of luminescence detection techniques with the possibility to localize and quantify signal emission down to the single-photon level, employing state-of-the art light imaging devices. These techniques have been employed for the development of sensitive analytical methods based on the evaluation of the spatial distribution of the photons emitted from the sample surface, both in macro- and microsamples. The use of imaging detection offers the possibility to exploit the analytical features of bio- and chemiluminescence for the rapid and precise analyte quantification in membrane-based assays and for the development of high-throughput methods in high-density microtiter plate formats.Furthermore, the high detectability of the bio- and chemiluminescent analytical signal also in small volumes makes it appropriate for miniaturized bioanalytical formats (e.g., microarrays, microfluidic devices) suitable for high-throughput screening assays and for point-of-care applications.Finally, bio- and chemiluminescence detection have also been employed for the sensitive localization and quantification of target analytes (metabolites, enzymes, proteins, and nucleic acids) in tissue sections and single cells, also in multiplexed formats and for the monitoring of biomolecular interactions.