Journal of Zhejiang University SCIENCE
(ISSN 1009-3095, Monthly)
2002   Vol. 3   No. 5   p.563-566

Fiber optic biosensor of immobilized firefly luciferase

CAI Jin(蔡谨)(College of Material & Chemical Engineering, Zhejiang University, Hangzhou 310027,China)  
MENG Wen-fang(孟文芳)(Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China)  
JI Xin-song(吉鑫松)(Institute of Biochemistry and Cell Biology, The Chinese Academy of Sciences, Shanghai 200031, China)  

Abstract:Luciferase from firefly lantern extract was immobilized on CNBr-activated Sepharose 4B. The kinetic properties of immobilized luciferase were extensively studied. The Km′ for D-luciferin is 11.9 μmol/L, the optimum pH and temperature for Sepharose-bound enzyme were 7.8 and 25℃ respectively. A luminescence fiber optic biosensor, making use of immobilized crude luciferase, was developed for assay of ATP. The peak light intensity was linear with respect to ATP concentration in range of 10-9-10-5 mol/L. A biological application was also demonstrated with the determination of serum ATP from rats bred in low versus normal oxygen environments.
Keywords:Firefly luciferase, ATP, Immobilized enzyme, Fiber optic biosensor

CLC Number:Q814.3  Document ID:A

Author Resume:CAI Jin(蔡谨),E-mail: caij@che.zju.edu.cn

References:

[1]Blum, L.J., Gautier, S.M., Coulet, P.R., 1988. Luminescence fiber optic biosensor. Anal. Lett., 21: 717-726.
[2]Lagido, C., Pettitt, J., Porter, A.J.R., Paton, G.I., Glover, L.A., 2001. Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors. FEBS Letters, 493:36-39.
[3]Leach, F.R., 1981. ATP determination with firefly luciferase. J. Appl. Biochem., 3: 473-478.
[4]Lee,Y., Jablonski, I., Deluca, M., 1981. Immobilization of firefly luciferase on glass rods: Properties of the immobilized enzyme. Anal. Biochem., 80:496-500.
[5]Ji, X.S., Li, H.X., Yuan, Z.Y., Liu S.H., 1984. Immobilization of NAD kinase. Enzyme Engineering 7, Anal. N. Y. Acad. Sci., 434:264-266.
[6], Maria, P. X., Begona, V., Maria, D. M., 2000. Fiber optic monitoring of carbamate pesticides using porous glass with covalently bound chlorophenol red. Biosensors & Bioelectronics, 14:895-905.
[7]Michel, P.E., Gautier-Sauvigne, S.M., Blum, L.J., 1998. A transient enzymatic inhibition as an efficient tool for the discriminating bioluminescent analysis of three adenylic nucleotides with a fiberoptic sensor based on a compartmentalized tri-enzymatic sensing layer. Analytica Chimica Acta, 360: 89-99.
[8]Neuberg, C., 1944. Convenient method for deproteinization. Arch. Biochem. 4:101-105.
[9]Tservistas, M., Koneke, R., Comte, A. Scheper T., 2001. Oxygen monitoring in supercritical carbon dioxide using a fibre optic sensor. Enzyme and Microbial Technology, 28:637-634.
[10]Vangelis, G. A., Yannis, D. C., 2002. A portable fiber-optic pesticide biosensor based on immobilized cholinesterase and sol-gel entrapped bromcresol purple for in-field use. Biosensor & Bioelectronics, 17:61-69.


Manuscript Received:2002 March 6

Manuscript Revised:2002 June 30

Published:2002 Dec. 1