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Andriana Surleva,Journal Annaof Chemical Marinova, Technology Vladislava and Ivanova, Metallurgy, Robert 52, Gradinaru,3, 2017, 513-525 Stela Georgieva ENZYME BASED METHODS FOR LACTATE DETERMINATION Andriana Surleva1, Anna Marinova1, Vladislava Ivanova2, Robert Gradinaru3, Stela Georgieva1 1 Analytical Chemistry Department Received 07 November 2016 University of Chemical Technology and Metallurgy Accepted 22 January 2017 8 Kl. Ohridski, 1756 Sofia, Bulgaria E-mail: [email protected] 2Physics Department University of Chemical Technology and Metallurgy 8 Kl. Ohridski, 1756 Sofia, Bulgaria 3 Biochemistry group, Faculty of Chemistry “Alexandru Ioan Cuza” University of Iasi Iasi, Romania ABSTRACT Development of biosensors for lactate determination has gained an increased research interest because of their wide application in clinical analysis and control of fermentation processes in food industry. Lactate biosensors with enzyme modified modules are a perspective alternative to the conventional methods in clinical analysis for their fast response, applicability to continuous mode of analysis, low cost and easy automation. Food and beverage indus- try can also benefit of the advantages of lactate biosensors for monitoring of fermentation processes or control of foods labeled as “low lactate content”. The quantification of low lactate levels demands for sensitive and reliable analytical methods for lactate quantification in complex matrices. This review discusses the enzymatic methods for lactate determination. Recent advances in lactate biosensors development are summarized. Analytical bioreactors and biosensors for application in flow injection analysis are specially emphasized. Keywords: lactate, biosensors, bioreactors, flow injection analysis. INTRODUCTION 2 mmol L-1. Lactate concentrations higher than 4 mmol L-1 are considered referring to acute sepsi, while those Development of biosensors for lactate determina- of 7 - 8 mmol L-1 are reported as lethal Аnastomotic tion has gained increased research interest because of bowel leak is one of the difficulties in urgent surgery. It their wide application in clinical analysis and control of demands for fast diagnostics and urgent action to prevent fermentation processes in food industry [1, 2]. Lactate is sepsis [3, 4]. Biomarkers tests and X-ray analysis are a mediator in metabolism of carbohydrates. The blood mainly used [5]. The lactate level is one of the biomark- levels of lactate depend on the rate of its formation in ers for diagnostics of urgent surgery states or cancer and muscles and erythrocytes and the rate of its metabo- on-site analytical methods of lactate determination will lism in liver. Low lactate levels are related to different benefit the prevention a lot [6 - 8]. Lactate biosensors pathological mechanisms: low oxidation rate in tissue, with enzyme modified modules are perspective alterna- liver malfunction, drug intoxication or genetic metabolic tive to the conventional methods for their fast response, malformations. The high level of lactate is a symptom of applicability to continuous mode of analysis, low cost an organ failure, cancer malformations, drug intoxication and easy automation [1, 9]. Food and beverage industry or sepsis. The normal level of lactate in blood is less than are commonly using lactate biosensors for monitoring 513 Journal of Chemical Technology and Metallurgy, 52, 3, 2017 fermentation processes or controlling foods labeled as on addition of free enzyme LDH is recently reported “low lactate content” [10]. Therefore, the quantification [16]. This method is appropriate for lactate determina- of low lactate levels demands for sensitive and reliable tion in heterogeneous, turbid and colored samples and analytical methods in complex matrices. is applied for both L– and D-lactate determination in This review is focused on enzymatic methods for milk, wine and beer. A flow injection amperometric lactate determination. The recent advances in lactate method based on injection of free LOx in the electrode biosensors development are summarized. Analytical cell shows working range up to 0.1 mmol L-1 lactate at bioreactors and biosensors for application in flow injec- LOx concentration of 22 U/mL [17]. tion analysis are specially emphasized. The first reported method for lactate determination Enzymatic methods for lactate determination based on iodometric titration was published in 1927 by based on immobilized enzymes T. E. Friedemann. It has been followed by a number of Enzyme immobilization consists in enzyme fixation methods of considerably improved analytical charac- on an inert support (carrier matrix). Usually inexpen- teristics. They are summarized in a recently published sive, inert and physical stable polymers or inorganic review by Kumar and Kumar [11]. The enzyme based materials are used as carrier matrices. Nevertheless the methods emerged as a valuable technique for lactate application of immobilized enzymes for analysis or in determination. The most widely used enzymes refer biotechnological processes, the preservation of their to lactate oxidase (LOx) and lactate dehydrogenase stable structure and biocatalytic activity is a challenge. (LDH). LOx catalyzes the aerobic oxidation of lactate From analytical point of view, the immobilized enzymes to pyruvate and hydrogen peroxide. LDH catalyzes the offer some important advantages: the enzyme stability anaerobic oxidation of lactate to pyruvate in the presence is improved, the enzyme-carrier complex can be easily of the oxidized form of nicotinamide adenine dinucleo- separated from the sample and the sample is not “con- tide (NAD+). Two approaches are mainly applied in the taminated” by the enzyme [18, 19]. These advantages enzyme based methods: (1) addition of a free enzyme convert the immobilized enzyme into an integral part and (2) enzymes immobilization. The first approach is of the analytical instrument [20]. However, the applica- applied relatively rarely because of the high enzyme con- tion of immobilized enzymes in analytical techniques sumption. The second approach allows using the enzyme demands an additional decrease from matrix components several times. The constant enzyme activity is the main interference. prerequisite in both approaches. Thus, the analytical Enzymes immobilization provokes the researchers’ signal depends only on the analyte concentration under interest because of the possibility of enzymes reuse. the experimental conditions applied [12]. This decreases considerably the analysis cost. Moreover, through the development of appropriate immobilization Enzymatic methods for lactate determination techniques the enzyme activity, the product inhibition based on a free enzyme and the enzyme specificity can be positively influenced. The enzyme methods for lactate determination are The techniques used so far and the trends in the devel- well known for the high sensitivity of the bioelement and opment of analytical methods and chemical sensors are the simple analytical procedure [13]. The most widely discussed in details in a number of recent reviews [7, used method is the Trinder’s method based on addition 9, 21]. The immobilization by physical adsorption is of free enzymes LOx and peroxidase to the analyte con- enzyme fixation by Van der Waal’s forces, electrostatic taining solution [14]. A new spectrophotometric method and/or hydrophobic interactions on an inert support. The for lactate determination based on L-lactate oxidation carrier is expected to have high affinity and adsorption by the enzyme flavocytochrome 2 in presence of nitro- capacity towards the biomolecule. The advantage of tetrazolium blue is proposed by Smutok et al. [15]. The this method in respect to LOx and LDH immobiliza- 푏 working range of the described method comprises 2.5 tion refers to the enzymes activity preservation, but low decades (0.005 mmol L-1 - 0.14 mmol L-1 lactate) with working and long-term stability are reported [9]. Immo- a low limit of detection of 2.0 mol L-1 of L-lactate. A bilization by entrapment is an inclusion of the enzyme fluorimetric method without sample pretreatment based used and the corresponding additives in a single layer 휇 514 Andriana Surleva, Anna Marinova, Vladislava Ivanova, Robert Gradinaru, Stela Georgieva of a gel or a polymer. The enzyme activity is preserved (a) Biosensors based on immobilized lactate dehy- as the quaternary structure of the biomolecule is not drogenase affected. However, some small matrix molecules can Lactate dehydrogenases are homogeneous or hetero- diffuse into the reaction layer and provoke interfering geneous tetrameric enzymes. These biomolecules occur reactions. Тhe entrapped enzymes are often character- in the cells of animals, plants or microorganisms. Rabbit ized by better operational stability compared to that of muscle derived enzyme has been the most used LDH in enzymes immobilized by physical adsorption. However, lactate detection [25 - 32]. Pig, bovine and human heart the biocomponent leaching still poses some limitations LDH are also good options [33]. In addition, enzymes to their analytical performance [9]. Immobilization by derived from microorganisms (Sphyraena argentea, B. cross-linking results in strong chemical bonds formation stearothermophilus) are used for this purpose [34, 35]. between the molecules. The enzymes can cross-link with LDH catalyzes the anaerobic oxidation of lactate each other or in the presence of inert proteins by glutaral- to pyruvate in presence of the oxidized form of nicoti- dehyde or other

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