nescence and therefore any interference results in effects observable as a loss of luminescence. Recently published applications of the luminescence biosensor exploiting immobilized cells are summarized in Table 1.

Because the entire metabolism involved in bioluminescence is intracellular, effects on bioluminescence are likely to occur as a response to toxic compounds available in a form, which can be transported into the cell. As a result of the transport processes, correlation between concentrations of active compound is not always satisfactory and is usually very specific to each microorganism-toxic compound system.

Immobilization techniques used to immobilize bioluminescent cells are in general any gentle immobilization techniques used in other fields that can preserve the viability of the immobilized cells. Another crucial requirement is optical property of an immobilization material resulting in minimal attenuation of bioluminescence. The use of several immobilization materials and techniques was reported: alginate (19), agar (25), porous sol-gel glass (28), immobilization on a gold surface (29), and several reports of immobilization on polyamide membranes (30-32).

Because of their relative simplicity and capability to detect contaminants dangerous to humans, bioluminescence biosensors have great potential in the environment monitoring. Main advantages that make bioluminescence methods so attractive are: extreme sensitivity and speed compared to other methods, ease of implementation, and potential to be used in portable field devices.

2. Materials

2.1. Instrumentation

1. Bio-Orbit 1253 Luminometer (Bio-Orbit, Turku, Finland) equipped with a recorder or a computer for data collection.

2. High-precision analytical balance.

1. Monitoring kit (1243-114) containing following reagents, delivered by Bio-Orbit.

2. 1243-251 Saline: 0.9% w/v NaCl in double distilled water, sterile.

3. 1243-244 Diluent: 100 mM Tris-acetate buffer, 2 mM ethylene diamine tetraacetic acid (EDTA), pH 7.75 at 25°C, sterile.

4. 1243-250 ATP Releasing Buffer (ionic surfactant for releasing biological ATP).

5. 1243-209 ATP Monitoring Reagent (firefly luciferase, D-luciferin, bovine serum albumin, magnesium acetate, inorganic pyrophosphate).

6. 1243-118 ATP Standard, if ATP content is to be measured.

2.3. Bioluminometric Assay

1. Transparent cylinder-shaped 4 mL polystyrene cuvets, disposable (see Note 3).

2. Micropipets with disposable tips (volumes 10-500 ||L, see Note 3).

3. Methods

3.1. Bioluminometric Biomass Determination

1. Weigh an empty measuring cuvet.

2. Take about 5 to 10 gel beads with a 2- to 3-mm diameter (or an equivalent corresponding to 1 to 80 mg of dry cell weight), taken either immediately after immobilization or removed from a cultivation flask, and dry them quickly with a piece of blotting paper (see Note 4).

3. Place them into the cuvet and weigh again.

4. Add 500 |L ATP releasing buffer (ATP-RB) to the cuvet to extract intracellular ATP from cells.

5. Mix by modest spinning for about 20 s.

6. Insert the cuvet into the measuring chamber of the luminometer and record background light emission (baseline).

7. Add 500 |L of ATP monitoring reagent (ATP-MR), mix quickly, and place the cuvet back into the measurement chamber and measure light output. The light output (LO) is given in relative light units (RLU), and the response corresponds directly to the ATP level present in the sample (see Fig. 1).

3.2. Evaluation of Biomass Concentration

1. Calibration of the method must be performed for every cell type separately. 250 |L of standards containing between 1 and 80 mg 100% active cell biomass suspended in 1 mL of re-distilled water, are measured by luminometer, followed the procedure given in Subheading 3.1. Calibration line showing the relation between the response and the biomass concentration in a standard is then constructed.

Bioorbit Luminometer
Fig. 1. Scheme for measurement and evaluation of extracelullar ATP for active biomass detection.

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