Tel: 44-1494-676161 Fax: 44-1494-679331/3. Copyright © 2000 PerkinElmer, Inc. PerkinElmer is a registered trademark of PerkinElmer, Inc. FLA-38.
FLUORESCENCE APPLICATIONS THE BIOLUMINESCENT DETERMINATION OF ATP USING THE MODEL LS-50 LUMINESCENCE SPECTROMETER ABSTRACT The PerkinElmer Model LS-50 luminescence spectrometer fitted with the total emission accessory has been used for the quantitative bioluminescent determination of ATP (Adenosine 5' -triphosphate). It was found that ATP could be detected at a level of 10 pg (2 x 10-14 moles) added to a microcuvette containing 0.5 mL of assay mix solution by measurement of light emitted from the ATP/firefly luciferase reaction. INTRODUCTION Chemiluminescence occurs when a molecule is raised to an excited state as a result of a chemical reaction. The decay of the excited molecule to the ground state results in the emission of light. When chemiluminescence is found in biological systems, where an enzyme increases the efficiency of the luminescent reaction, the process is termed bioluminescence. Much work has been done recently in the field of luminescence and the technique has found a variety of applications in the detection and analysis of biological compounds, rapid detection of bacteria, analysis of phagocytic cell function, analysis of cell metabolism, assay of metal ions and luminescent immunoassay (1-7). Many of these applications are based on one of the following three reactions: 1. NAD(P)H/FMNH bacterial luciferase reaction. OXIDOREDUCTASE NAD(P)H + FMN + H+
FMNH2 + NAD(P)+ EBL
FMNH2 + O2 + RCHO
FMN + RCOOH + H2O + LIGHT
EBL = bacterial luciferase Since many enzymic systems require electron transport in the form of either NAD(P)H or FMNH2, this reaction has a wide range of application to substrate and enzyme assays (8-10). 2. Chemiluminescent assays. Some compounds produce luminescence on oxidation (10-15). Systems involving these compounds may be used to investigate a number of processes e.g. the luminol (5-amino-2,3-dihydro-1,4-phthalazine dione) reaction may be used to follow a number of enzymic processes via estimation of levels of hydrogen peroxide: GLUCOSE OXIDASE GLUCOSE + O2 + H2O
GLUCONIC ACID + H2O FERRICYANIDE
H2O2 + LUMINOL + OH-
1-AMINOPHTHALATE + N2 + 2H2O + LIGHT
3. ATP/firefly luciferase reaction. FIREFLY LUCIFERASE ATP + LUCIFERIN ADENYL-LUCIFERIN + O2
ADENYL-LUCIFERIN + PPi OXYLUCIFERIN + AMP + CO2 + LIGHT
This is the most extensively studied bioluminescent system. One of the main applications of the reaction is concerned with cell counting. Since all living cells contain ATP at a level that is relatively constant for a given cell type, the quantity of ATP present is proportional to the number of cells. This is particularly important in microbial assays such as antibiotic sensitivity testing. Coupling other reactions to the luciferase reaction allows the measurement of a large number of compounds, including glucose, antibiotics, nucleotides, coenzyme A, creatine phosphate, pyrophosphate and glycerol (16-21). This investigation uses the ATP/firefly luciferase system as a model for general bioluminescence and chemiluminescence applications.
MATERIALS An Adenosine 5' -triphosphate (ATP) bioluminescence assay kit (Product Number FL-AA) was obtained from Sigma Chemical Company, Poole UK. The kit contained ATP assay mix, ATP assay mix dilution buffer and an ATP standard. The kit was stored below 0 °C. Deionized water was used throughout the investigation. METHOD The ATP assay mix was dissolved in 5.0 mL water. The solution was dispensed into 1.0 mL aliquots which were frozen. The ATP standard was dissolved in 10.0 mL water to produce a solution of concentration 0.1 mg/mL. This solution was diluted to obtain a range of standards from 10 µg/mL to 1 ng/mL. Data were collected under the following instrumental conditions:
Phosphorescence mode: Source OFF Emission filter NONE
Delay Time = 0 ms Cycle Time = 200 ms
Gate Time = 180 ms Flash Count = 1
Response = 4.0 s
Interval = 1 s
Time-drive parameters: Slits Ex/Em 15/20 nm
The total emission accessory (Part No. L225-0101) was placed in the emission beam and the photomultiplier tube voltage was set to 900 V for maximum sensitivity. The sample compartment was fitted with a microcell adaptor (L225-0139). 0.5 mL of the ATP assay mix were placed in a 5 mm pathlength microcuvette. The solution was left to equilibrate in the instrument before commencing the assay. RESULTS Data were collected in the form of time-drives. A 10 µL aliquot of the ATP standard was injected into the assay mix through the septum injector. A duplicate sample was injected once the luminescence signal had returned to the baseline level. This procedure was repeated for each ATP standard and for a blank. Figure 1 shows the time-drive for duplicate injections of the 100 ng/mL (i.e. 1 ng ATP, injected at 50 and 340 seconds), 10 ng/mL (i.e. 0.1 ng ATP, at 660 and 860 seconds) and blank at (1010 and 1110 seconds) standards. The total luminescence signal obtained for injections of each standard was calculated as the area of the peak corresponding to the standard, this was determined for each standard and the areas obtained were used to construct a calibration graph (Figure 2).
CONCLUSIONS The LS-50 fitted with a total emission accessory and set at maximum photomultiplier sensitivity can detect ATP at a level of 10 pg (2 x 10-14 moles) injected into 0.5 mL undiluted ATP assay mix. REFERENCES 1. Whitehead, T.P., Kricka, L.J., Carter, T.J.N., Thorpe, G.H.G., Clin Chem., 25, 1531, (1979). 2. David, J.L., Herion, F., Adv. Exp. Mod. Biol., 34, 341, (1972). 3. Messeri, G., Clin. Chem. 30, 653, (1984). 4. Hara, T., Chem. Soc. Jap., 56, 2965, (1983). 5. Wannlund, J., Anal. Biochem., 122, 385, (1982). 6. Kricka, L., Analyst, 108, 1273, (1983). 7. Wienhausen, G., Anal. Biochem, 129, 162, (1983). 8. Karp, M.T., Anal. Biochem., 121, 151, (1982). 9. Vogin, B., Anal. Chim. Acta., 142, 293, (1982). 10. Gorus, F., Schram, E., Clin. Chem., 25, 512, (1979). 11. De Boever, J., Kohen, F., Vandekerckhove, D., Clin. Chem., 29, 2068, (1983). 12. Woodhead, T.P., J. Clin. Immun., 7, 82, (1984). 13. Auses, J. P., Cook, S. L., Maloy, J.T., Anal. Chem., 47, 244, (1975). 14. Montano, L.A., Anal. Chem., 51, 919, (1979). 15. Schroeder, H., Anal. Chem., 50, 1114, (1978). 16. Hercules, D., Anal. Chem., 50, 22 , (1978). 17. Leach, F.R., J. Appl. Biochem., 3, 473, (1981). 18. Leach, F.R., Webster, J.J., Methods in Enzymology, 133, 51-70, (1986). 19. Lin, H., Cohen, H.P., Anal. Biochem., 24, 531, (1968). 20. Denburg, J. L., McElroy, W. D., Arch. Biochem. Biophys., 141, 668, (1970). 21. Holmson, H., Holmson, I., Bernhardson, A., Anal. Biochem., 17, 456, (1966).
PerkinElmer Instruments: 761 Main Avenue, Norwalk, CT 06859-0243, U.S.A. Tel: 1-800-762-4000 or 1-203-762-4000 Fax: 1-203-761-2882 Internet: http://www.perkinelmer.com PerkinElmer Ltd: Post Office Lane, Beaconsfield, Bucks, HP9 1QA, England Tel: 44-1494-676161 Fax: 44-1494-679331/3 FLA-38
Copyright © 2000 PerkinElmer, Inc. PerkinElmer is a registered trademark of PerkinElmer, Inc.
