Marian Navratil, Juraj Svitel, and Peter Gemeiner
Bioluminescence measurements have become extremely popular because of good sensitivity and the ability to quantitate a wide variety of analytes. Only recently have these measurements gained particular interest in relation to the study of gene expression and regulation. The firefly luciferase system is the most frequently used bioluminescent reaction, in which adenosine triphosphate (ATP) is used to generate light. ATP is often used as a measure of cell biomass, because the intracellular level of ATP is rather constant and is rapidly degraded upon cell death. The bioluminescence principle has been used successfully in a variety of biosensor applications, mostly for the environmental monitoring. In this chapter we describe detection of viable biomass in free and immobilized cells and outline a few applications, where this approach can be used for biosensor construction. We demonstrate the advantages of bioluminescence by two protocols for detection of ATP and viable biomass in free and immobilized cell samples, which have been proved very rapid and accurate. This indicates that bioluminescence measurements are a good indicator of cell viability and can be widely applied in a number of areas such as biotechnology and food industry.
Key Words: ATP; bioluminescence; biosensors; cell viability; entrapment; immobilization; luciferase.
The phenomenon of bioluminescence was first found more than a century ago in fireflies and the enzyme which catalyzes the oxidative light-emitting reaction was named luciferase. The biochemical reaction in its simplest form can be written as:
Luciferin + O2 luciferase) Oxyluciferin + CO2 + AMP + light (1)
From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ
Later many other bioluminescent organisms were found, including insects, fish, and bacteria. The luciferase reaction shown in Eq. (1) uses adenosine triphosphate (ATP) to generate light, however marine luminescent bacteria use reduced flavine instead of ATP. Bioluminescent bacteria can be found in nature in different habitats ranging from marine to terrestrial environments. In addition to naturally occurring microorganisms also genetically engineered microorganisms are used to develop bioluminescence-based biosensors. Gene coding enzymes needed for bioluminescence were mapped, isolated, and cloned. Genetic technology now enables the transfer of bioluminescence to a wide variety of microorganisms. Genetically engineered strains harboring bioluminescent reporter plasmids are now of growing importance for development of microbial luminescent biosensors (1).
The very early report of analytical application of bioluminescence appeared almost a hundred years ago, when Beijerinck described the detection of photo-synthetically formed oxygen in leaf extract, using luminuous bacteria. The phenomenon of bioluminescence was exploited to develop a wide variety of bioluminescent and chemiluminescent biosensor techniques. Bioluminescence found its practical applications mainly in development of instrumental methods in:
1. Measurement of microbial and other cells based on ATP content measurement.
2. Determination of active immobilized biomass including, and
3. Bioluminescence-based biosensors, mainly for environmental applications.
Bioluminescence principle has been extended from detection of luminescent cells to the detection of naturally nonluminescent organisms. Intracellular ATP, and possibly also other nucleotides, can be used to generate light after light-generating enzymes are being added to the system. The first works dealing with determination of biomass using bioluminescence principle are more than 30 yr old. Intracellular ATP content was studied in 19 species of Escherichia coli in detail and was found in the range 0.28-8.9 x 1010 ^g of ATP per organism (2). In principle, cell detection is conducted in vitro; it is based on efficient extraction of intracellular ATP with minimal ATP hydrolysis. Subsequently, light generating system is added (luciferin + luciferase) and emitted light is measured. Numerous agents to extract ATP have been tested, including organic solvents, inorganic acids, and surfactants. Therefore, the analysis of ATP can be utilized as a method to detect microbial cells at very low concentration. The method was adapted to the detection of various types of cells: bacterial content in fluids (2), protozoa (3), mycobacteria (4), yeasts (5), tumor cells (6), and other tissues. Luciferin and lu-ciferase required for the assay are commercially available, as well as commercial luminometers or other light-detecting instrumentation. A survey on commercially available luminometers and imaging devices for low-light level measurements was recently compiled (7).
Other methods use direct monitoring of cell number and their metabolic activity with incorporated chromosomal luxAB and gfp genes, encoding bacterial luciferase and green fluorescent protein, respectively. Because the metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells (8).
1.3. Bioluminescence-Based Determination oflmmobilized Biomass
The luminometric method for the determination of active immobilized biomass concentration is also based on measuring ATP content in cells. ATP serves as a carrier of chemical energy in cells, where available energy is stored in chemical bonds between terminal phosphate groups. After cell death, the ATP concentration rapidly decreases. Because ATP is a good indicator of cell viability, its concentration is dependent on active biomass amount (9). The concentration of ATP can conveniently be determined using bioluminescence method, as described above.
Extraction of the intracellular ATP is critical for a successful measurement. There are several extraction methods, and the choice of extractant depends on a particular microorganism. The extraction procedure should achieve quantitative release of nucleotides and simultaneously inactivate any nucleotide-converting enzymes which may be present in the immobilized cells. Wijffels et al. proved, that 90% of the living cells immobilized by entrapped in gel beads are located in a 140-^m thick outer layer of a 1-mm bead (10) and the cells retained more than 90% of their activity after the immobilization (11). Thus, the extraction of ATP from immobilized cells has been shown as quite effective.
Although attempts to measure free biomass by bioluminometry are widely described, only a few reports on bioluminescence-based estimation of immobilized biomass exist. The content of Beneckea natriegens cells immobilized on silica particles was tested by bioluminometry, and compared with the biomass viability in the free form (9,12). Determination of yeast biomass immobilized in a range of ionotropic hydrogels was also published, and as an example, this method is discussed in the experimental section in detail.
Biosensor techniques based on bioluminescence, and also on chemilumines-cence, have become very popular in the recent period mainly as a result of the advances in light detection. Light detection with the latest models of photomultipliers and charge-coupled device (CCD) cameras is very efficient and actual instruments are small and portable as well as relatively affordable compared to other techniques. Luminometry has some advantages over other optical techniques: extraordinary sensitivity and wide dynamic range. Luminometry is up to 105 times more sensitive than absorption spectrometry. Theoretically speaking, because single photon can be detected in photomultiplier a single molecule of analyte or a single cell can be detected. In real systems however light detection is not that efficient but still bioluminescent and chemiluminescent techniques can detect trace amounts of analytes or cells.
The major field of application of bioluminescence biosensors, exploiting immobilized intact cells, is environment monitoring. Applications of luminescence biosensors in the environment monitoring were extensively reviewed (26,27). Luminescence-based biosensor techniques have been extensively used for monitoring of various organic and inorganic chemical contaminants. Various toxic compounds and environmental contaminants can generally interfere with energetic metabolism and electron transfer system. These produce substrates for biolumi-
Bioluminescence-Based Microbial Biosensors
Analyte/Field of Application
Light generation microorganism
Hg2+, heavy metals
Vibrio fischeri (gen. modified)
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