Biotechnological applications of bioluminescence and chemiluminescence Aldo Roda, Patrizia Pasini, Mara Mirasoli, Elisa Michelini and Massimo Guardigli Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy Recent progress in molecular biology has made avail- able several biotechnological tools that take advantage of the high detectability and rapidity of biolumines- cence and chemiluminescence spectroscopy. These developments provide inroads to in vitro and in vivo continuous monitoring of biological processes (e.g. gene expression, protein – protein interaction and disease progression), with clinical, diagnostic and drug discovery applications. Furthermore, combining lumi- nescent enzymes or photoproteins with biospecific rec- ognition elements at the genetic level has led to the development of ultrasensitive and selective bioanalyti- cal tools, such as recombinant whole-cell biosensors, immunoassays and nucleic acid hybridization assays. The high detectability of the luminescence analytical signal makes it appropriate for miniaturized bioanalyti- cal devices (e.g. microarrays, microfluidic devices and high-density-well microtiter plates) for the high- throughput screening of genes and proteins in small sample volumes. Since the first transgenic tobacco plant expressing the firefly luciferase gene (luc gene) was developed in Marlene DeLuca’s laboratory , 20 years ago [1], a new era in applied bioluminescence has come about. The main advan- tage of bioluminescence and chemiluminescence (BL/CL) is the high detectability of the luminescence signal, which can be measured down to a few emitted photons using either a photomultiplier tube or a charge-coupled device (CCD) imager. BL and, to a lesser extent, CL reactions are characterized by high quantum efficiency, in the order of 0.5–0.8 and 0.1–0.001, respectively. Therefore, BL/CL enables detection of a few molecules and achieves detection limits down to 10 218 –10 221 mol, which are significantly lower than those of other spectroscopic techniques. BL/CL assays have been developed for many analytes of diagnostic interest that are present at micro- or nanomolar levels in biological samples. This has been achieved by the coupling of BL/CL to many enzyme-catalyzed reactions, such as those involving oxidases, dehydrogenases and kinases [2]. However, luminescence-based analysis is still underutilized as a diagnostic tool, particularly for multi- analyte purposes. If the full potential of BL/CL were to be exploited, many substances could be measured in small sample volumes by relatively inexpensive, reliable devices, even in point-of-care environments [3]. Progress in molecular biology and biotechnology con- tinuously produces new BL/CL reagents, including recom- binant and mutated enzymes and genes that can be used as reporters or probes. The availability of these tools, along with new highly efficient CL enzyme substrates, has prompted the development of innovative bioanalytical methods for the ultrasensitive detection of a variety of target analytes. New BL/CL biotechnological tools New BL/CL reporter genes Reporter gene technology represents one of the major recent achievements of molecular biology. Reporter genes are DNA sequences that encode an easily detectable protein or enzyme. They can be artificially introduced into a cell to monitor gene expression, to obtain whole-cell biosensors or for cell-localization purposes. The principle of reporter gene technology applied to the development of BL/CL whole-cell biosensors is shown in Figure 1a. Currently, bacterial luciferase genes (e.g. the lux genes of terrestrial Photorhabdus luminescens and marine Vibrio harveyi bacteria), as well as the eukaryotic luc and ruc genes from firefly (Photinus pyralis) and sea pansy (Renilla reniformis), represent the most widely used BL reporter genes. In addition, cDNAs for new genes have recently been cloned and expressed [4]. Interesting new genes, such as those of the red- and green-light-emitting luciferases from the railroad worm Phrixothrix hirtus [5], as well as mutated firefly luciferases with different emission wavelengths [6], could have analytical appli- cations. Properties of luciferases from different organisms are reported in Table 1. In addition, the recent cloning and purification of recombinant horseradish peroxidase (HRP) has made available a new biotechnological tool for which numerous applications are expected in the near future [7]. Indeed, HRP is one of the most widely used enzymes with CL detection, because very efficient substrates are commercially available. Dual-reporter systems In multiple luminescent assays, the signals are simul- taneously generated and independently measured, either by differences in the kinetic profile of the luminescent Corresponding author: Aldo Roda (aldo.roda@unibo.it). Review TRENDS in Biotechnology Vol.22 No.6 June 2004 www.sciencedirect.com 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.03.011