A Biosensor Utilizing l -Glutamate Dehydrogenase and Diaphorase Immobilized on Nanocomposite Electrode for Determination
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A Biosensor Utilizing L-Glutamate Dehydrogenase and Diaphorase Immobilized on Nanocomposite Electrode for Determination of L-Glutamate in Food Samples Rastislav Monošík & Miroslav Streďanský & Ernest Šturdík Received: 25 April 2012 / Accepted: 21 June 2012 / Published online: 5 July 2012 # Springer Science+Business Media, LLC 2012
Abstract Amperometric biosensor utilizing bienzymatic composition consisting of L-glutamate dehydrogenase and diaphorase for the determination of L-glutamate has been developed. Enzymes were immobilized between chitosan layers onto the surface of planar nanocomposite electrodes consisting of multi-walled carbon nanotubes (diameter060– 100 nm; length05–15 μm, 95+% purity). Linear response was obtained from 10 to 3,495 μM in phosphate buffer solution of pH 9.0 and in the presence of enzyme cofactor NAD+ (2 mM) and mediator ferricyanide (5 mM). The limit of detection was 5.4 μM, and sensitivity was found to be 28 nA μM−1 cm−2. The biosensor showed a short response time (within 60 s), good storage (no loss of activity for at least 3 months), and operational (response ability above 90 % after 7 days since its first use) stability. Finally, the results obtained from measurements of the food samples were compared with those obtained with an enzymatic–spectrophotometric method and correlated well. Analytical performance of the biosensor indicated that the bienzyme system utilizing diaphorase as a secondary enzyme could be a general basis for other biosensors based on NAD+-dependent dehydrogenases.
R. Monošík (*) : E. Šturdík Department of Nutrition and Food Assessment, Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic e-mail: [email protected] M. Streďanský Biorealis, s.r.o., Dúbravska cesta 9, 841 04 Bratislava, Slovak Republic
Keywords analysis
L-glutamate
. Biosensor . Nanocomposite . Food
Introduction Glutamic acid (HOOC(CH2)2CHNH2COOH) is a nonessential proteinogenic amino acid. This amino acid is considered to be the major mediator of excitatory signals in the mammalian central nervous system and probably plays a role in most aspects of brain function including cognition, memory, and learning (Danbolt 2001). The commonly known flavor enhancer monosodium glutamate (MSG) was invented in 1908 by Dr. Kikunae Ikeda. His research was made for the purpose of commercializing the component of kombu (a type of seaweed) that produces its good taste as seasoning. This flavor is called “umai” in Japanese and is translated as “delicious” (available at: http:// www.jpo.go.jp/seido_e/rekishi_e/kikunae_ikeda.htm on 22 Feb 2012). According to some studies, the excessive intake of this salt in the diet may be a reason for initiation of several health disorders such as headache, stomach pain, and glutamatemediated excitation, often referred to as excitotoxicity. Also, it can act as a critical factor common to a variety of neurological disorders, including stroke, epile
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