ENZYME BASED METHODS for LACTATE DETERMINATION Andriana Surleva1, Anna Marinova1, Vladislava Ivanova2, Robert Gradinaru3, Stela Georgieva1

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 industry 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 bifunctional agents. The main drawback namide adenine dinucleotide (NAD+). The quantity of is the enzyme activity decrease caused by modification NADH obtained in the enzymatic reaction is determined of the active conformation or the biomolecules active by the oxidation current recorded [6, 30, 31, 36]: sites. In covalent immobilization the enzymes can be bounded to the support via some functional groups which are not involved in the catalysis. The support is initially activated by multifunctional reagents. The high stability of the immobilized enzymes obtained by covalent NADH based biosensors are characterized by high bounding is their main advantage. The high consumption sensitivity and a low detection limit, but also by low se- of reagents, low reproducibility of the sensor technology lectivity and stability of NADH electrochemical determi- and the enzyme activity decrease can be mentioned as nation due to its high oxidation potential [11,30,31,37]. drawbacks of covalent immobilization [9, 20]. One of the modern approaches refers to using carbon nanotubes able to decrease considerably this oxidation Lactate biosensors potential [30, 31, 35, 37]. Several mediators are also Recently Sassolas et al. [9] have defineda biosensor proposed: Meldola‘s Blue [33, 37], Variamine blue [35], as a device based on two associated elements: a biore- Neutral red [38]. An enzyme amperometric electrode ceptor – an element that recognises the analyte, and a for lactate determination based on co-immobilization transducer – an element that converts the biochemical of LDH and a mediator on multi-wall carbon nanotubes signal to an electrical one. The enzymes are the most paste electrode is proposed by Pereira et al. [37]. The widely used bioreceptors, while the electrochemical obtained LDH biosensors show high sensitivity, good cells are the most widely used transducers in lactate working stability (96 % of the sensor activity is preserved biosensors due to their low price, simple design and after 300 determinations) and a wide linear concentration small dimensions [22, 23]. range (0.1 - 10) mmol L-1 lactate. Some limitations can be encountered irrespective of the high selectivity of Electrochemical biosensors for lactate determination LDH (as oxygen is not included in the reaction). Due to Enzymes biosensors with electrochemical trans- the high oxidation potential of NADH, interference in ducers are known as enzyme electrodes. The latter are real samples can be expected, which demands additional the most intensively developed electrodes due to their optimization of the analysis [11, 31, 37]. Co-enzyme, a selective and fast response to a specific substrate [24]. second enzyme and a mediator are often necessary for The low detection limit (10 nmol L-1) and the wide linear efficient lactate determination by LDH based biosensors. concentration range (3 - 6 decades) make the enzyme This increases the complexity and the price of analysis amperometric electrodes the most attractive biosensors [39]. Therefore, LOx is preferred in lactate determina- [20]. Their response is based on the catalytic activity of tion as the reaction and the sensors design are simpler LOx and LDH. compared to those of LDH based biosensors [11, 39].

515 Journal of Chemical Technology and Metallurgy, 52, 3, 2017

(b) biosensors based on immobilized lactate oxidase [1, 67 - 70], ferrocene and its derivatives [54, 63, 64, 66], Lactate oxidases (LOx) belong to the flavoenzyme ferricyanide [65], cobalt phthalocyanine [71], variamine family. They can be extracted and purified from various blue [35] аre studied as mediators in lactate biosensors. microorganisms. LOx from Pediococcus sp, an FMN The benefits of applying iron(III) hexacyanoferrate(II) (flavin mononucleotide)-dependent enzyme, is suc- as a mediator in an electrochemical process in order to cessfully used to design several lactate sensor devices. allow selective reduction of hydrogen peroxide at low More than 15 research papers [40-54] deal with this potentials is thoroughly reviewed by Ricci et al. [70]. enzyme. However, enzymes derived from other bacteria Hirst et al. [1] report a lactate biosensor obtained by (lactate monooxidase from Mycobacterium smegmatis immobilization of LOx on poly(ethyleneimine) impreg- and lactate oxidase from Streptococcus sp) are used so nated with Prussian blue. The obtained biosensor, based far [55-57]. In the past decade an oxidase isolated from on screen printed carbon electrode, is reported to show Aerococcus viridans is introduced to create an electro- improved selectivity in the presence of ascorbic acid. It chemical biosensor [58 - 60]. LOx catalyzes the lactate is applied for lactate determination in drainage liquids oxidation to pyruvate and hydrogen peroxide. The latter with sensitivity adequate to differentiate dangerous sur- generates the analytical signal of the amperometric sen- gery states (7 mmol L-1 lactate) from normal postsurgery sor [2, 51, 61, 62]: concentrations of lactate (3 mmol L-1). Gold, carbon or platinum electrodes modified by nanoparticles are used as transducers for amperometric lactate biosensors. Nanomaterials and especially nanotubes enhance the direct electron transfer between the electrode surface and enzymes active sites [11]. Nanoparticles modification of amperometric electrodes considerably improve lactate sensors conductivity, stability and electrocatalytic The detection is based on monitoring: (1) the de- activity [35, 42, 72 - 74]. Carbon nanotubes affect posi- crease of consumed oxygen concentration or (2) the tively biosensor behavior as the quantity of immobilized increase of the generated hydrogen peroxide concentra- enzyme increases, the mass to volume ratio and the tion in the amperometric cell. The limitation of the first electrocatalytic activity increase significantly. Lactate method refers to its low selectivity in complex samples: bioelectrodes based on LOx and modified with platinum other substrates may compete with the quantified analyte nanoparticles or nanowires [65, 73, 75], and diamond for the enzyme active sites thus affecting the oxygen nanoparticles [76] have been recently reported. New consumption. The main problem of the second method is materials for amperometric transducers as nanostruc- connected with the interference from electroactive com- tured and 3D macroporous gold electrodes are proved ponents such as ascorbic acid, uric acid, and glutathione to show improved electrocatalytic activity compared to which oxidize at relatively low potentials decreasing thus that of policrystal gold electrodes [63, 64]. the inherent selectivity of the enzyme sensor [1, 38]. 2) Limit the electroactive components access to the Two approaches to decrease the interference effects on electrode surface of an amperometric sensor by semiper- LOx based lactate biosensors with electrodes sensitive to meable membranes. The experiments so far show that a hydrogen peroxide are described in the recent literature: Nafion membrane successfully eliminates such type of

1) Decrease of H2O2 reaction redox potential by elec- interference [35, 38, 77 - 79]. trode surface modification. The low reduction potential The main challenge in electrochemical biosensors allows on one hand to preserve the high specificity and development, including LOx based ones, is to obtain sensitivity of the enzyme reaction, while on the other hand reusable biosensor of improved operational and long provides to avoid possible interference redox reactions term stability [80]. The analytical characteristics refer- with the participation of the matrix components. Media- ring to the working concentration range, the sensitivity, tors decrease the potential of hydrogen peroxide reaction the selectivity, the response time, the long-term stability and thus increase the selectivity of lactate biosensors [1, strongly depend on the enzyme immobilization and the 63 - 67]. Iron(III) hexacyanoferrate(II) (Prussian blue) electrode preparation procedures [2, 9, 24]. The analyti- 516 Andriana Surleva, Anna Marinova, Vladislava Ivanova, Robert Gradinaru, Stela Georgieva

Table 1. Analytical characteristics of LOx based enzyme electrodes. Table 1. Analytical characteristics of LOx based enzyme electrodes. linear range, LOD, carrier and support stability references mmol/L µmol/L LOx immobilized by physical adsorption mesoporous conjugate silica 0.02 - 2.0 9 months [69] layer on modified with Resydrol polymer 0.004–0.5 - decrease by [39] platinum electrode 2.5% daily silica sol–gel film on multi- 0.2–2.0 10 4 weeks [42] walled carbon nanotubes/platinum nanoparticle modified glassy carbon electrode three-dimensional 0.016 - 0.06 16 1 month [64] ordered macroporous gold film modified electrodes LOx immobilized by cross-linking albumin and mucin hydrogel 0.007 - 1.5 30 days [2] modified with Prussian blue and 0.005-0.2 3.5 [69] copper platinum microelectrode LOx immobilized by entrapment polypyrrole polymeric matrix up to 12 - - [24] with polyanion/PEG conjugate electrochemically polymerized 0.05–1.6 - decrease by 3 [39] poly(3,4- % daily ethylenedioxythiophene) on platimun eletrode laponite–chitosan hydrogel on a 0.002 - 3.6 3.8 22-30 days [54,81] glassy carbon electrode double layer hydrogel matrix 0.05–30 one month [82] LOx immobilized by covalent immobilization electropolymerized film 0.1-5.5 100 26 days [51] self-arranging layer of dithiobis- up to 1.3 3.93 > 1 month [63,64] N-succinimidyl propionate on nanostructured or 3D macrospore gold electrodes carbon electrode modified by 0.01 - 2.0 6.9 3 months [75] platinum nanoparticles gold electrodes modified with up to 0.3 10 - [66] hydroxymethylferrocene cal characteristics of amperometric lactate biosensors ibility of a series of biosensors is achieved by cross-link- based on lactate oxidase immobilized by different ap- ing LOx in a hydrogel based on albumin and mucin [2]. proaches are summarized in Table 1. The efficiency of High selectivity in the presence of ascorbic acid, glucose, two approaches for LOx immobilization on platinum gallic acid, citric acid, and quercetin is demonstrated by electrode, i.e. physical adsorption and entrapment in an an enzyme electrode based on LOx cross-linked with electropolymerized matrix, is compared [39]. The results glutaraldehyde on Prussian blue modified with copper obtained show that the immobilization procedure does platinum microelectrode [69]. Synthetic or bio-polymers not affect the biosensor stability. Good signal reproduc- with appropriate pore size are proposed for overcoming

517 Journal of Chemical Technology and Metallurgy, 52, 3, 2017 Table 2

Enzyme amperometric electrodes for lactate determination in flow injection mode Table 2 . Enzyme amperometric electrodes for lactate determination in flow injection mode. signal working range, LOD, enzyme transducer precision, stability reference mol/L mmol/L Sr , % carbon electrode 1x10-6 – 8x10-5 LOx modified with silicate and 0.5 3 2 weeks [38] nanoparticales dotted 1x10-4 – 1x10-2 with neutral red composite graphite electrode modified by 1x10-6 – [36] LDH 0.87 2 - a polysulfone layer 1.2x10-5 and Meldola’s Blue screen printed carbon electrode modified by 5.5x10-4 – LDH 550 4 17 days [33] Meldola’s Blue- 1x10-2 Reinecke Salt screen-printed carbon electrode modified by LDH 2x10-4 –1x10-3 - - - [35] carbon nanotubes and Varamine blue LOx oxygen electrode 2x10-4 – 0.44 200 2 one year [89] glassy carbon 4x10-6 – LOx electrode modified by 0.84 2 14-days [45] 2.8x10-4 Prussian Blue HRP screen-printed carbon and 1x10-5 – 2x10-4 10 3 4 weeks [85] electrode LOx the leaching of LOx immobilized by entrapment [54, 82]. (c) Lactate biosensors based on co-immobilization The enzyme can be additionally stabilized by cross-linking of enzymes or covalent bounding [54, 81]. A lactate biosensor based The advantage of co-immobilized enzymes refers on a double layer hydrogel matrix as a carrier for LOx to the analytical signal amplification by recycling the immobilization by entrapment shows remarkable stabil- analytes. LOx and LDH are co-immobilized by physi- ity: the authors report no signal change after one month cal adsorption on a polyaniline film [83]. LOx catalyzes of continuous measurements [82]. Higher sensors stability oxidation of lactate to pyruvate. The obtained pyruvate is achieved by covalent bounding of enzyme. Inorganic is a substrate for LDH enzymatic reaction. Thus a materials as pore glass, SiO2 nanoparticles, carbon, plati- lactate is generated from pyruvate in the presence of num or gold nanoparticles, natural materials or synthetic NADH. The lactate regeneration through substrate re- polymers are used as LOx based biosensors supports [51, cycling allows multiplication of the detector signal and 63, 64, 75]. The enzyme can be covalently immobilized determination of low lactate concentrations. Hydrogen directly onto the transducer’s surface as a thin membrane peroxide formed in LOx catalyzed reaction is detected or in the vicinity of the electrode surface. Covalent im- amperometrically. The detection limit is 0.5 m mol L-1 mobilization of LOx allows reuse of a biosensor modi- L-lactate. The biosensor prepared is stable for 3 months fied with platinum nanoparticles for several consecutive in case stored at 4°C. A second enzyme – peroxidase, measurements. It is proved to be an effective alternative combined with a mediator (ferrocene), can be included of solid electrodes [75]. Improved working range and sen- to decrease the H2O2 oxidation potential in a LOx based sivity are reported for a biosensor based on a monolayer detection system [84]. All components are incorporated of covalent immobilized LOx on a self-arranging layer of in a graphite composite electrode. A biosensor based dithiobis-N-succinimidyl propionate on nanostructured or on a screen printed carbon electrode with immobilized 3D macrospore gold electrodes [63, 64]. LOx and horseradish peroxidase (HRP) is proposed by 518 Andriana Surleva, Anna Marinova, Vladislava Ivanova, Robert Gradinaru, Stela Georgieva

Ghamouss et al. [85]. The experimental results obtained working concentration range and the sensitivity. Biologi- show high reproducibility of the sensor preparation cal systems and processes are very sensitive to changes protocol when HRP is preliminary oxidized by iodate in optimal conditions. In some cases their thermo and ions and both enzymes are simultaneously immobilized. chemical stability can be considerably limited. These particularities limit the biosensors application [20]. One Optical biosensors for lactate determination modern solution refers to biosensors incorporation in Optical lactate biosensors are based on optic fibers flow injection analytical systems. with immobilized LOx or LDH. Lactate determination is based on chemiluminecence or electrochemilumine- Lactate biosensors and bioreactors in flow injec- cence. The optical biosensors design, their characteris- tion analysis tics and their application to clinical, bioorganic and phar- Flow injection analysis (FIA) is an analytical tech- maceutical analysis are recently discussed in the review nique well known for its susceptibility to automation, by Ispas et al. [7]. The developed sensors are studied in miniaturization, and easy modification of the analytical a conventional and a flow injection mode of analysis system. Additional benefits of using FIA refer to the de- [86]. The most intensively studied optical biosensors for creased operator effect on the chemical system by work- lactate are based on electrochemiluminescence (EChL) ing in a closed system and the decreased interference reaction of luminol and hydrogen peroxide. To improve effect by incorporating semipermeable membranes in the the electrocatalytic activity some modified electrodes are manifold. Possibility for multicomponent analysis and advanced. A sensitive chemiluminescence reaction is a low consumption of reagents should also by outlined applied to lactate and glucose determination by measur- [12]. The immobilized enzymes can be incorporated in ing the luminescence related to the generated hydrogen the flow injection system as biosensors or as bioreactors. peroxide amount. The enzymes are initially immobilized using an appropriate carrier and deposited on a glassy А) biosensors carbon electrode. The luminescence is detected by an Biosensors for FIA should possess high sensors optical fiber sensor. Based on this principle a number stability and high resistivity against “leaching” of ac- of electro-optical lactate sensors are developed [77, 86, tive component combined with an appropriate geometry 87]. A biosensor with immobilized LOx based on ZnO for incorporation in the flow through detector cell. The nanoparticles on multiwall carbon tubes (fixed on glassy recently reported lactate biosensors based on LDH and carbon electrode) is proposed for electrochemilumi- LOx are summarized in Table 2. The biosensors devel- nescence determination of lactate [77]. A Nafion mem- oped are used for direct lactate determination in: serum brane is additionally immobilized on the electrode. The [33], yogurt [35] or milk products [85]. To lower the electrode shows a high electrocatalytic activity towards matrix effects and to minimize the sample pretreatment, electrochemiluminescence reaction of luminol. The limit permselective membranes or separation units are effec- of detection is very low, 4 nmol L-1 lactate, but two linear tively combined with newly developed enzyme elec- concentration intervals are observe - (0.01-10) μmol L-1 trodes in a flow injection mode. A biosensor [85] based and (10-200) μmol L-1. A flow injection procedure is on an immobilized LDH and covered with a cellulose reported for the determination of L-lactate based on its acetate layer as a permselective membrane is applied enhancement on tris(2,2′-bipyridyl)ruthenium(III) chemi- for direct measurement of lactate in a serum without luminescence using immobilized lactate dehydrogenase pre-treatment [85]. A newly developed screen-printed [88]. The linear working range refers to 0.4 mmol L-1 - 5 carbon electrode with immobilized LOx and preliminary mmol L-1 with a limit of detection of 1x10−7 mol L-1 and a oxidized HRP is applied for lactate determination in significant sample throughput of 60 per hour. milks products. No significant matrix effect is observed Irrespective of the advantages of the electrochemical due to the high dilution of the samples and the good per- or the optical lactate biosensors, it should be taken into formance of the biosensor at low lactate concentrations account that both the specific activity of the biomolecule [85].The flow injection analysis offers also the advantage and the nature support material determine the analytical to shorten the biosensor contact with interfering compo- characteristics of the biosensor: the time response, the nents through flow velocity optimization. 519 Journal of Chemical Technology and Metallurgy, 52, 3, 2017

The biosensors main limitation refers to the different component can be determined in the presence of another. optimal operating conditions required by the biomole- A microflow bioreactor with immobilized LOx followed cule, the support and the transducer components [12, 20]. by chemiluminescence detection is proposed for lactate The biological component is very sensitive to deviations determination in a serum [92]. The microflow system from the optimal medium conditions and its stability is offers low sample consumption (0.2 μL), short response often considerably limited. Thus the optimization of the time (30 s), a good dynamic concentration range (0.5- experimental conditions is a matter of compromises. 5.0) mmol L-1 lactate with a signal precision of 3.2 %. A bioreactor based on immobilized thermostable LOx is B) bioreactors proposed for L-lactate determination in dairy products As thoroughly reviewed in ref.[90] the bioreactors [93]. The enzyme is immobilized in a thin layer cell incorporated in flow injection systems allow overcom- and incorporated in a flow injection system with an ing the main biosensors limitations described above. amperometric detector. A linear working range (0.1 - 1) First, the working conditions of the detector and the mmol L-1 L-lactate with a limit of determination of 50 biocomponent can be independently optimized. The m mol L-1 and a sample frequency of 40 samples/h are enzymes can be included in different channels of the reported. The flow injection signal is stable up to 800 flow injection manifold where optimal working condi- sample injections and diminishes slowly afterwards due tions can be created for each enzyme. Second, an array to decrease of the bioreactor enzyme activity. of bioreactors can be constructed and various analytes One of the limitations of enzymatic electrodes is can be simultaneously determined using the same de- the inhibition caused by high quantitates of hydrogen tector system. Third, the bioreactors can be changed peroxide produced in enzymatic reaction. In attempt to without changing the detector which is an independent overcome this drawback a flow injection system with part of the flow injection system. The bioreactors can a bioreactor and a bioelectrode (both with immobilized be combined with sensorless detection, which operates LOx) is proposed [94]. Around 60 % of the lactate is independently of preceding enzymatic reaction and even oxidized in the bioreactor, while the rest is oxidized in at conditions inappropriate for the enzymatic reaction. the electrode cell. Thus both the enzyme activity and the This represents an additional advantage. The increased method sensitivity are preserved. No enzyme inhibition dispersion, especially if column reactors packed with is found in case of 400 lactate injections. In another particles are used, can be treated as a bioreactors limita- work [95] the bioreactor is combined with sensorsless tion. However, some high effective bioreactors or tube detection in a spectrophotometric cell. A multichannel reactors are developed which decreasing considerably flow injection system is constructed to maintain differ- the sample dispersion. It should be taken into account ent working conditions appropriate for enzymatic and that incorporating a new element in a flow injection photometric reactions. Vojinovic [96] develops a flow system increases the complexity of the system and the injection method based on modified Trinder’s method cost of analysis. with crosslinked LOx and HRP on an activated pore А flow injection system with two parallel enzyme glass support. The bioreactor of a volume of 20 µL is LOx and GOD bioreactors is developed for simultaneous incorporated in a multichannel flow injection system. determination of lactate and glucose in blood plasma, urine and dairy products [91]. Hydrogen peroxide CONCLUSIONS AND TRENDS generated in enzymatic reactions is amperometrically detected using a platinum electrode modified by elec- The requirements to the modern methods for lac- tropolymerized m-phenylenediamine membrane aiming tate determination are not limited only to their high to achieve selectivity increase. Two consecutive peaks selectivity. A simple, an easy and a low cost analysis, whose height depends on the concentration of both ana- easy maintenance and reduced costs of the analytical lytes are obtained. The analysis is very simple, with low instruments, possibility of small devices incorporation sample consumption (Vsample = 20 µL), 3 months work- in portable instruments become increasingly important. ing stability, sample throughput of 50 samples/h, a two Additionally, the detector systems for lactate determina- decades linear working range, a good selectivity - one tion should have high sensitivity and selectivity in direct 520 Andriana Surleva, Anna Marinova, Vladislava Ivanova, Robert Gradinaru, Stela Georgieva

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