- Nano Express
- Open Access
A Novel Amperometric Glutamate Biosensor Based on Glutamate Oxidase Adsorbed on Silicalite
© The Author(s). 2017
- Received: 29 December 2016
- Accepted: 29 March 2017
- Published: 7 April 2017
In this work, we developed a new amperometric biosensor for glutamate detection using a typical method of glutamate oxidase (GlOx) immobilization via adsorption on silicalite particles. The disc platinum electrode (d = 0.4 mm) was used as the amperometric sensor. The procedure of biosensor preparation was optimized. The main parameters of modifying amperometric transducers with a silicalite layer were determined along with the procedure of GlOx adsorption on this layer. The biosensors based on GlOx adsorbed on silicalite demonstrated high sensitivity to glutamate. The linear range of detection was from 2.5 to 450 μM, and the limit of glutamate detection was 1 μM. It was shown that the proposed biosensors were characterized by good response reproducibility during hours of continuous work and operational stability for several days. The developed biosensors could be applied for determination of glutamate in real samples.
- Glutamate oxidase
Glutamate (glutamic acid) plays an important role in vital activity of humans and other mammals, especially in the functioning of the central nervous system. In particular, glutamate is the major excitatory neurotransmitter in the central nervous system of mammals. It also has a significant effect on nitrogen metabolism. The concentration of glutamate in certain parts of the body may influence the development of heart attacks, strokes, and various neuropathological states [1, 2].
Glutamate is part of many pharmaceuticals due to its ability to sensitize the taste receptors and stimulate the brain activity. A lot of foodstuff contains small amounts of glutamate [3, 4], which gives food “beef” taste. Therefore, glutamate is often used as a flavor enhancer. This is why it is rather problematic to completely eliminate glutamate from the diet. In glutamate-sensitive people, the so-called “Chinese restaurant syndrome” may develop [5, 6]. Glutamate badly affects the retina and can contribute to vision loss.
Determination of glutamate is of significance in clinical biochemistry when diagnosing the diseases associated with abrupt changes of glutamate level in the body, including diseases of liver and cardiovascular system [5, 7]. In clinical laboratories, glutamate is used to determine the activity of some aminotransferases.
The scope of practical application of glutamate is continuously growing. The methods of accurate and rapid detection of glutamate are required in neurophysiology and neuropathology, fundamental and clinical medicine, pharmaceutical and food industries, and in analytical biochemistry and biotechnology [1, 5, 7].
Additionally, chemiluminiscence can be also used, which includes the application of luminol, potassium ferricyanide, and luminophotometer . Oxygen consumption at the glutamate oxidation can be fixed with an oxygen fiber optic sensor, which registers the changes in luminescence of a deposited layer sensitive to the oxygen concentration . The method used for glutamate determination in meat and meat products is based on two enzymatic reactions resulting in the glutamate oxidation and formation of a colored compound formazan, the concentration of which is measured with a spectrophotometer.
The disadvantage of the above methods is the requirement of rather difficult pretreatment of analyzed samples and their unsuitability for rapid analysis of large amount of samples and for real-time monitoring. New bioanalytical devices, biosensors, can be considered as a promising alternative to the methods mentioned .
Among electrochemical biosensors, the amperometric ones are considered to be the most promising, and they are most often used to determine glutamate [1, 4, 5, 7, 12]. Besides, the potentiometric electrodes (NH4+ detection) can be an alternative; however, they are less sensitive. For selective determination of glutamate in the brain, it was developed the system of platinum microelectrodes covered with electropolymerised hyperoxidated polypyrrole, which were immobilized on ceramics . An automatic flow-injection system in biosensor devices can be useful in the monitoring of glutamate determination in real-time [5, 14]. The multi-channel biosensor system was created for dynamic identification of several components (including glutamate) in food production. Modified graphite electrodes with stabilizing additives were used to provide stable function of the biosensor at long-term storage . In most studies on the development of glutamate biosensors, the enzyme L-glutamate oxidase of different origin is used [16–18]. The enzymes glutamate decarboxylase, glutamate dehydrogenase, and glutamate synthetase are also used , but glutamate oxidase far exceeds them in characteristics.
Currently, a number of biosensors and biosensor systems have been developed for glutamate determination in various real samples—foods and pharmaceuticals [1, 7], cell cultures [12, 19], blood serum and urine [1, 5], microdialyzates at neurophysiological studies [2, 20], and for monitoring fermentation in the food industry [21, 22]. However, many of these biosensors are based on a complex and time-consuming method of immobilization, often with the use of toxic reagents. Neither of them has not been commercialized so far. Therefore, the elaboration of new methods of creating glutamate-sensitive biosensors with improved analytical characteristics is an actual challenge.
This study is aimed at creation of the amperometric biosensor for glutamate determination, which allows faster and more accurate analysis and can be suitable for mass production in future. The problem is supposed to be solved using a new method of enzyme immobilization, the glutamate oxidase adsorption on transducers covered with a silicalite layer.
For the first time, the efficiency of various types of zeolites as carriers for the enzyme immobilization has been shown when developing conductometric biosensors [23–27]. The procedure of enzyme adsorption on silicalite was tested for a number of enzymes—acetylcholinesterase , urease [23, 25], recombinant urease , and butyrylcholinesterase . Additionally, the effectiveness of this technique has been shown for enzyme biosensors based on pH-sensitive field-effect transistors [28–32]. Moreover, it was revealed that some zeolites can be useful for glucose oxidase adsorption in amperometric biosensors for glucose determination . Therefore, an attempt was made to apply this method of immobilization for the development of amperometric glutamate-sensitive biosensor with improved analytical characteristics using GlOx adsorbed on silicalite.
Glutamate oxidase (GlOx, EC 220.127.116.11) from Streptomyces sp., activity 7 U/mg (Yamasa Corporation, Tokyo, Japan) was used in biorecognition elements of biosensors. Bovine serum albumin (BSA, fraction V), glycerol, ascorbic acid, HEPES, and 50% aqueous solution of glutaraldehyde (GA) have been received from Sigma-Aldrich Chemie (Germany). L-glutamate was from Affymetrix (USA). All other chemicals were of analytical purity grade.
Synthesis of Silicalite Crystals
The molar composition of the clear solution used for synthesis of silicalite crystals is 1TPAOH: 4TEOS: 350H2O. Hydrolysing tetraethoxysilane (TEOS) with tetrapropylammonium hydroxide (TPAOH) at a constant stirring for 6 h at room temperature, we obtained a homogeneous solution. The solution was introduced into Teflon-lined autoclaves. The crystallization took place at 125 °C during 1 day. The material, which did not react, was removed from the solution by centrifugation. The size of silicalite particles was approximately 400 nm.
Characterization of Silicalite
Characteristics of silicalite
Part. size (nm)b
Pore size (nm)c
S ext (m2/g)d
S total (m2/g)e
Pore volume (cc/g)c
Design of Amperometric Transducers
Drop-Coating Transducers with Silicalite
A silicalite layer was formed on the transducer surface by dip-coating. 2.5% silicalite suspension in 20 mM HEPES, pH 6.5, was used. 0.15 μl of the solution were deposited onto active zones of transducer, then it was heated during 1–1.5 min to 150 °C. This temperature had no effect on silicalite and did not influence the transducer working parameters.
Enzyme Adsorption on Silicalite
0.1 μl of 4% GlOx solution in 20 mM HEPES, pH 6.5, were deposited onto the active zone of transducer previously coated with silicalite, then the transducer was exposed to complete air-drying (for 5 min). Neither glutaraldehyde nor any other auxiliary compounds were used. Next, the transducers were submerged into the working buffer for 5–10 min to wash off the unbound enzyme. After the experiments, the transducer surface was cleaned from silicalite and adsorbed enzyme with ethanol-wetted cotton.
Experimental Setup for Amperometric Measurements
Measurements were carried out in 20 mM HEPES, pH 7.4, in a chronoamperometric mode (“amperometric detection”) at a constant potential of +0.6 V vs Ag/AgCl reference electrode in an open cell with vigorous stirring. The substrate concentration in the measuring cell was specified by the introduction of aliquots of the substrate standard stock solution to the working buffer. All experiments were performed in at least three series.
At the first stage of this work, the method of enzyme adsorption on silicalite was optimized for creating amperometric GlOx-based biosensor for glutamate determination. The enzyme amount adsorbed on a transducer depends in the first place on the amount of sorbent (silicalite). The size of silicalite layer is a function of both its concentration in solution and the time of layer formation.
The next task was to find the optimal conditions of GlOx adsorption on silicalite, i.e., the time of procedure and enzyme concentration. The adsorption efficiency was assessed by measuring the biosensor responses. Despite our assumption about significant dependence of the adsorption efficiency on the time, the value of biosensor responses was about the same at the adsorption time ranging from 2 to 30 min. Five minutes was taken as optimal value because it was enough for complete drying of the enzyme drop deposited on the transducer.
To study the operational stability of the biosensor, nine responses to 1 mM glutamate were measured step-by-step daily during 4 days. All the time between measurements, the biosensor remained in the buffer at continuous stirring; after a series of nine measurements, the biosensor was dried and placed in a refrigerator (+4 °C). As seen in Fig. 9b, the biosensor was characterized by good operational stability over 4 days.
A new amperometric glutamate-sensitive biosensor has been developed on the basis of GlOx adsorption on the amperometric disk platinum electrode coated with a layer of silicalite. The optimal procedures of deposition of a silicalite layer on platinum electrode and GlOx adsorption on silicalite have been elaborated. It has been shown that the biosensor created in compliance with optimized conditions of immobilization has high sensitivity to glutamate (the minimum detection limit—0.5–1 μM, wide linear range of operation (2.5–400 μM) and is characterized by good reproducibility (error did not exceed 7%) and operational stability during 4 days. Summarizing all the results obtained, the conclusion can be made that the developed amperometric biosensor based on GlOx adsorbed on silicalite is a promising device for further successful application for glutamate analysis in real biological fluids.
The authors gratefully acknowledge the financial support of this study by the STCU Project 6055. Furthermore, this study was partly supported by the National Academy of Sciences of Ukraine in the frame of Scientific and Technical Government Program “Sensor systems for medico-ecological and industrial-technological requirement: metrological support and experimental operation.”
OVS and OOS optimized methods of drop-coating silicalite onto amperometric transducers and GlOx adsorption on silicalite. DYK and ISK studied analytical characteristics of obtained glutamate biosensor. BOK synthesized silicalite and took part in the deposition of silicalite onto the transducers surface. BAK planned the experiments, controlled the silicalite synthesis by electron microscopy, and made XRD spectrum. OVS, OOS, and BOK processed the obtained results, wrote, and arranged the article. SVD proposed the idea of the development new amperometric biosensor based on GlOx adsorbed on silicalite-modified electrodes. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Villarta RL, Cunnigham DD, Guilbault GG (1991) Amperometric enzyme electrodes for the determination of L-glutamate. Talanta 38(1):49–55View ArticleGoogle Scholar
- Berners MOM, Boutelle MG, Fillenz M (1994) Online measurement of brain glutamate with an enzyme/polymer-coated tubular electrode. Anal Chem 66(13):2017–2021View ArticleGoogle Scholar
- Dremel BAA, Schmid RD (1991) Comparison of two fibre-optic L-glutamate biosensors based on the detection of oxygen or carbon dioxide and their application in combination with flow-injection analysis to the determination of glutamate. Anal Chim Acta 248:351–359View ArticleGoogle Scholar
- Vahien W, Bradley J, Bilitewski U, Schmid RD (1991) Mediated enzyme electrode for the determination of L-glutamate. Anal Lett 24(8):1445–1452View ArticleGoogle Scholar
- Ghobadi S, Csöregi E, Marko-Varga G, Gorton L (1996) Bienzyme carbon paste electrodes for L-glutamate determination. Curr Sep 14(3/4):94–102Google Scholar
- Suleiman AA, Villarte RL, Guilbault GG (1992) Analytical applications of glutamate oxidase based amperometric electrodes. Bull Electro-chem 8(4):189–192Google Scholar
- Almeida NF, Mulchandani AK (1993) A mediated amperometric enzyme electrode using tetrathiafulvalene and L-glutamate oxidase for the determination of L-glutamic acid. Anal Chim Acta 282(2):353–361View ArticleGoogle Scholar
- Herbreteau B, Lafosse M, Morin-Allory L, Dreux M (1990) Automatic sugar analysis in the beet industry. Part I: parameter optimization of a reversed phase HPLC carbohydrate determination. J Sep Sci 13(4):239–243Google Scholar
- Vercellotti SV, Clarke MA (1994) Comparison of modern and traditional methods of sugar analysis. Int Sugar J 96:437–445Google Scholar
- Murachi T, Tabata M (1987) Use of a bioreactor consisting of a sequentially aligned L-glutamate dehydrogenase and L-glutamate oxidase for the determination of ammonia by chemiluminescence. Biotechnol Appl Biochem 9(4):303–309View ArticleGoogle Scholar
- Soldatkin AP, Dzyadevych SV, Korpan YI, Sergeyeva TA, Arkhypova VN, Biloivan OA, Soldatkin OO, Shkotova LV, Zinchenko OA, Peshkova VM, Saiapina OY, Marchenko SV, El’skaya AV (2013) Biosensors. A quarter of a century of R&D experience. Biopolym Cell 29(3):188–206View ArticleGoogle Scholar
- Wollenberger U, Scheller FW, Hintsche R, Bohm K (1989) Verfahren zur amperometrischen bestimmung von L-glutamat. Pat DD 272:478, A1Google Scholar
- Walker E, Wang J, Hamdi N, Monbouquette H, Maidment N (2007) Selective detection of extracellular glutamate in brain tissue using microelectrode arrays coated with over-oxidized polypyrrole. Analyst 132:1107–1111View ArticleGoogle Scholar
- Stalikas CD, Karayanis MI, Tzouwara-Karayanni SM (1993) Immobilization of glutamate oxidase on non-porous glass beads. Automated flow injection system for the assay of glutamic acid in food samples and pharmaceuticals. Analyst 118:723–726View ArticleGoogle Scholar
- Gibson TD, Hulbert JN, Parker SM, Woodward JR, Higgins IJ (1992) Extended shelf life of enzyme-based biosensors using a novel stabilization system. Biosens Bioelectron 7:701–708View ArticleGoogle Scholar
- Wollenberger U, Scheller FW, Renneberg R, Boehmer A, Wollenberger U, Mueller H-G (1988) Biosensor zur bestimmung von glutamat und substanzen die in glutamat umgewan-delt werden. Pat DD 257272:A1Google Scholar
- Ye BC, Li Q-S, Li Y-R, Li X-B, Yu J-T (1995) L-Glutamate biosensor using a novel L-glutamate oxidase and its application to flow injection analysis system. J Biotechnol 42:45–52View ArticleGoogle Scholar
- Chen CY, Su YC (1991) Amperometric L-glutamate sensor using a novel L-glutamate oxidase from Streptomyces platensis NTU 304. Anal Chim Acta 243:9–15View ArticleGoogle Scholar
- White SF, Turner APF, Bilitewski U, Schmid RD, Bradley J (1994) Lactate, glutamate and glutamine biosensors based on rhodinized carbon electrodes. Anal Chim Acta 295:243–251View ArticleGoogle Scholar
- Zilkha E, Koshy A, Obrenovitch TP, Bennetto HP, Symon L (1994) Am-perometric biosensors for online monitoring of extracellular glucose and glutamate in the brain. Anal Lett 27(3):453–473View ArticleGoogle Scholar
- Li Q, Zhang S, Yu J (1996) Immobilization of L-glutamate oxidase and peroxidase for glutamate determination in flow injection analysis system. Appl Biochem Biotechnol 59(l):53–61View ArticleGoogle Scholar
- Chen RLC, Matsumoto K (1995) Sequential enzymatic monitoring of glucose, ethanol and glutamate in bioreactor fermentation broth containing a high salt concentration by a multichannel flow-injection analysis method. Anal Chim Acta 309:145–151View ArticleGoogle Scholar
- Kirdeciler SK, Soy E, Ozturk S, Kucherenko I, Soldatkin O, Dzyadevych S, Akata B (2011) A novel urea conductometric biosensor based on zeolite immobilized urease. Talanta 85:1435–1441View ArticleGoogle Scholar
- Saiapina OY, Pyeshkova VM, Soldatkin OO, Melnik VG, Kurc BA, Walcarius A, Dzyadevych SV, Jaffrezic-Renault N (2011) Conductometric enzyme biosensors based on natural zeolite clinoptilolite for urea determination. Mater Sci Eng C 31:1490–1497View ArticleGoogle Scholar
- Kucherenko IS, Soldatkin OO, Kasap B, Öztürk S, Akata B, Soldatkin AP, Dzyadevych SV (2012) Elaboration of urease adsorption on silicalite for biosensor creation. Electroanalysis 24:1380–1385View ArticleGoogle Scholar
- Velychko TP, Soldatkin OO, Melnyk VG, Marchenko SV, Kirdeciler SK, Akata B, Soldatkin AP, El’skaya AV, Dzyadevych SV (2016) A novel conductometric urea biosensor with improved analytical characteristic based on recombinant urease adsorbed on nanoparticle of silicalite. Nanoscale Res Lett 11:106View ArticleGoogle Scholar
- Kucherenko I, Soldatkin O, Ozansoy Kasap B, Kirdeciler SK, Akata Kurc B, Jaffrezic-Renault N, Soldatkin A, Lagarde F, Dzyadevych S (2015) Nanosized zeolites as a perspective material for conductometric biosensors creation. Nanoscale Res Lett 10:209View ArticleGoogle Scholar
- Soy E, Arkhypova V, Soldatkin O, Shelyakina M, Dzyadevych S, Warzywoda J, Sacco A Jr, Akata B (2012) Investigation of characteristics of urea and butyrylcholine chloride biosensors based on ion-selective field-effect transistors modified by the incorporation of heat-treated zeolite beta crystals. Mater Sci Eng C 32:1835–1842View ArticleGoogle Scholar
- Soldatkin OO, Kucherenko IS, Shelyakina M, Soy E, Kirdeciler K, Öztürk S, Akata B, Jaffrezic-Renault N, Dzyadevych SV, Soldatkin AP (2013) Application of different zeolite for improvement of the characteristics of a pH-FET biosensor based on immobilized urease. Electroanalysis 25(2):468–474View ArticleGoogle Scholar
- Soldatkin OO, Soy E, Errachid A, Jaffrezic-Renault N, Akata B, Soldatkin AP, Dzyadevych SV (2011) Influence of composition of zeolite/enzyme nanobiocomposites on analytical characteristics of urea biosensor based on ion-selective field-effect transistors. Sensor Lett 9:2320–2326View ArticleGoogle Scholar
- Shelyakina MK, Soldatkin OO, Arkhypova VM, Kasap BO, Akata B, Dzyadevych SV (2014) Study of zeolite influence on analytical characteristics of urea biosensor based on ion-selective field-effect transistors. Nanoscale Res Lett 9:124View ArticleGoogle Scholar
- Soldatkin OO, Shelyakina MK, Arkhypova VN, Soy E, Kirdeciler SK, Ozansoy Kasap B, Lagarde F, Jaffrezic-Renault N, Akata Kurç B, Soldatkin AP, Dzyadevych SV (2015) Nano- and microsized zeolites as a perspective material for potentiometric biosensors creation. Nanoscale Res Lett 10:59View ArticleGoogle Scholar
- Soldatkin OO, Ozansoy Kasap B, Akata Kurc B, Soldatkin AP, Dzyadevych SV, El’skaya AV (2014) Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation. Biopolym Cell 30(4):291–298View ArticleGoogle Scholar