Amperometric tetrathiafulvalene-mediated lactate electrode using lactate oxidase absorbed on carbon foil

The features of a new sensor for determining l-lactate are reported. The enzyme lactate oxidase and the mediator, tetrathiafulvalene (TTF), are absorbed on carbon foil disks previously bonded onto the ends of glass tubes. Linear calibration graphs were obtained in the range 10^?4?10^?3 M with physiological phosphate buffer (pH 7.35) and at 30°C with a response time of a few seconds. Calibration graphs in the range 10^?3?10^?2 M were also obtained and the difference in response times between these two ranges were investigated. The results are promising for assembling disposable lactate sensors for in vitro or for in or ex vivo measurements.     

Novel electron transfer mediators based on dichloroindophenol derivatives for lactate oxidase

Novel indophenol derivatives were synthesized and characterized electrochemically with respect to their abilities to act as electron-transfer mediators for lactate oxidase. These compounds showed suitable redox potentials and high second-order rate constants, k_med, in solution for electron-transfer from the reduced enzyme. A lactate sensor using these derivatives demonstrated high sensitivity and good substrate selectivity. This sensor could also achieve excellent durability which retained more than 70% residual activity and good linearity in the range from 0 to 16 mM lactate concentration even after 10 days.     

l-Glutamate enzyme electrode involving amplification by substrate recycling

An enzyme electrode with an amplified response for l-glutamate is constructed by co-immobilizing l-glutamate oxidase and glutamate-pyruvate transaminase on a platinum disk electrode. With l-alanine (1 mM) in the solution, the sensitivity of the electrode to l-glutamate is greatly increased by the substrate recycling reaction between the two enzymes; the detection limit is 0.2 nM in the presence of l-alanine compared with 0.2 μM in its absence. The amplification factor for 1 μM l-glutamate is ca. 53.     

Design and characterization of a lactate biosensor based on immobilized lactate oxidase onto gold surfaces

The design and characterization of a lactate biosensor and its application to the determination of this analyte in wine and beer are described. The biosensor is developed through the immobilization of lactate oxidase (LOx) using two different strategies including direct adsorption and covalent binding. The characterization of the resulting lactate oxidase monolayers was performed in aqueous phosphate buffer solutions using atomic force microscopy (AFM) and quartz crystal microbalance (QCM) techniques. In presence of lactate and using hydroxymethylferrocene as a redox mediator, biosensors obtained by either direct adsorption or by covalent binding exhibit a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. Results obtained under these conditions give a linear current response versus lactate concentration up to 0.3 mM, with a detection limit of 10 μM of lactate and a sensitivity of 0.77 ± 0.08 μA mM⁻¹. Finally, biosensors were applied to the determination of lactate in wine and beer. The results obtained are in good agreement with those obtained by a well-established enzymatic-spectrophotometric assay kit.     

Amperometric determination of serum lactate dehydrogenase activity using an ADP-modified graphite electrode

Graphite electrodes modified with a drop-coated layer of polyethyleneimine (PEI) and adenosine diphosphate (ADP) displayed an electrocatalytic response to NADH after the adenine moiety of ADP was electrochemically oxidised. NADH can be detected amperometrically in alkaline solution (pH 9.0) at low applied potentials (+50 mV (Ag/AgCl)). Using a stationary electrode arrangement, linear response for NADH concentrations between 1.0×10⁻⁸ and 1.0×10⁻⁴ M was found, with a response time of 12 s and a detection limit of 8×10⁻⁹ M. The electrode was applied to the amperometric monitoring of the reaction between lactate and NAD⁺ catalysed by lactate dehydrogenase (LDH). A flow injection-amperometric method for the determination of LDH activity in human serum was developed. The method allows a fast and accurate discrimination between pathological and normal LDH activity levels, with a sampling rate of 40 h⁻¹. Quantitative results for a random set of human serum samples were found to be in good agreement with the standard spectrophotometric method.     

Immobilization of lactate dehydrogenase on tetraethylorthosilicate-derived sol-gel films for application to lactate biosensor

Tetraethylorthosilicate (TEOS)-derived sol-gel films were utilized for the immobilization of lactate dehydrogenase (LDH) by physical adsorption and sol-gel/LDH/sol-gel sandwich configuration. An attempt was made to ascertain the optimum pH and temperature for the immobilized LDH. It was shown that TEOS-derived sol-gel films containing physically adsorbed LDH exhibited linearity from 0.5 to 4 mM, whereas those containing LDH in sandwich configuration showed linearity from 0.5 to 3 mM l-lactate. These sol-gel films, immobilized with LDH, were found to be stable for about 4 weeks at 4–10°C.     

Effect of methyltin trichloride on the activity of lactate dehydrogenase

The effect of MeSnCl₃, which is a highly toxic compound, on the activity of L-lactate:NAD oxidoreductase (lactate dehydrogenase) in the extract from the liver of Russian sturgeon (Asipenser gueldenstaedtiB.). Noncompetitive inhibition of the enzymatic reaction was discovered. This can be due to a change in the enzyme conformation caused by the action on the thiol groups, important for enzyme activity.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.     

Liquid—liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes

An extract from porcine muscle containing soluble enzymes has been partitioned between the two liquid phases of an aqueous, biphasic system consisting of dextran, polyethylene glycol, and water. The influence of polymer-bound triazine dyes (Cibacron blue F3G-A and Procion yellow HE-3G) on the partition of lactate dehydrogenase and total protein was studied. The effects of pH and concentrations of polymers and buffer on this so-called affinity partitioning were also determined. The two-phase systems were used in extraction procedures for purification of lactate dehydrogenase to a specific activity of 456–494 U (7.6–8.4 μkat) per mg protein. The use of these systems for extraction of enzymes in technical scale is discussed.     

A Biofuel Cell Based on Biocatalytic Reactions of Lactate on Both Anode and Cathode Electrodes – Extracting Electrical Power from Human Sweat

Electrodes composed of carbon fibers were modified with graphene nano‐sheets in order to increase their surface area and facilitate electrochemical reactions. Electrocatalytic species, such as Meldola's blue (MB) and hemin were immobilized on the graphene surface due to their π‐π stacking and then used for electrocatalytic oxidation of NADH and reduction of H₂O₂, respectively. Further modification of these electrodes with enzymes producing NADH and H₂O₂ in situ (lactate dehydrogenase, LDH, and lactate oxidase, LOx, respectively), allowed assembling of a biofuel cell operating in the presence of lactate, oxygen and NAD⁺. The cathode of the biofuel cell required lactate and O₂ for its operation, while the anode operated in the presence of lactate and NAD⁺. Notably, both bioelectrocatalytic electrodes operated in the presence of lactate, one producing H₂O₂ in the reaction catalyzed by LOx in the presence of O₂, second producing NADH in the reaction catalyzed by LDH in the presence of NAD⁺. Both reactions were performed in the biofuel cell without separation of the cathodic and anodic solutions and with no need of a membrane. The biofuel cell was tested in solutions mimicking human sweat and then in real human sweat samples, demonstrating substantial power release being able to activate electronic devices.     

Partitioning of lactate dehydrogenase from bovine heart crude extract by polyethylene glycol-citrate aqueous two-phase systems

Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG)-citrate have been used for enzyme partitioning studies. The behavior of lactate dehydrogenase (LDH) from bovine heart crude extract was analyzed using a two-level factorial design in which the PEG molar mass and concentration, the citrate concentration were selected as independent variables, while the purification factor, the partition coefficient (K) and the activity yield were selected as responses. The statistical analysis revealed the effect of PEG molar mass on K. LDH exhibited a better partitioning toward PEG-rich phase and the highest K value (1079.81) was obtained with 42% (w/w) PEG 400 and 7.5% (w/w) citrate concentration. PEG molar mass also influenced the purification factor of the enzyme in the top phase. Possibly these ATPS remove inhibitors present in the extract affording higher enzyme yield.     

Amperometric enzyme electrodes of glucose and lactate based on poly(diallyldimethylammonium)-alginate-metal ion-enzyme biocomposites

Sodium alginate (AlgNa) and poly(diallyldimethylammonium chloride) (PDDA) were mixed to obtain an interpenetrating polymer composite via electrostatic interaction and then cast on an Au electrode surface, followed by incorporation of metal ions (e.g. Fe³⁺ or Ca²⁺, to form AlgFe or AlgCa hydrogel) and glucose oxidase (GOx) (or lactate oxidase (LOx)), to prepare amperometric enzyme electrodes. The interactions of PDDA, Alg, and Fe³⁺ are studied by visual inspection as well as microscopic and electrochemical methods. Under optimized conditions, the PDDA-AlgFe-enzyme/Au and PDDA-AlgCa-enzyme/Au electrodes can give good analytical performance (e.g. nM-scale limit of detection of glucose or lactate, and sensitivities > 50 μA cm⁻² mM⁻¹) in the first-generation biosensing mode, which are better than the reported analogs using typical polysaccharide biopolymers as enzyme-immobilization matrices. The enzyme electrodes also worked well in the second-generation biosensing mode in the coexistence of p-benzoquione or ferrocene monocarboxylic acid artificial mediator. Biofuel cells (BFCs) with the enzyme electrodes as the bioanodes and glucose (or lactate) as the biofuel were also fabricated with satisfactory results. The proposed protocols for preparation of high performance Alg-based biocomposites may find wide applications in bioanalysis.     

Studies on the effects of Al(III) on the lactate dehydrogenase activity by differential pulse voltammetry

In this paper, differential-pulse voltammetry (DPV) was applied to study the effects of aluminum (Al(III)) on the lactate dehydrogenase (LDH) activity, and ν_max in the enzyme promoting catalytic reaction of “” by monitoring DPV reduction current of NAD⁺. The changes of Al's influence on the LDH activities caused by the concentration of LDH, pH, temperature as well as Al speciation including Al hydroxide (Al-OH), Al fluoride (Al-F) and organically complexing Al (Al-Org) compounds have been investigated. The results showed that the effects of Al on the LDH activity exhibited two kinds of behaviors under different conditions, i.e. inhibitory effects or slightly increased LDH activity at low concentrations and inhibited at high concentrations. To analyze the values of and ν_max of LDH reaction system in the absence and presence of 0.04 mmol/L Al(III), it was found that the types of the inhibition of Al(III) varied with experimental conditions. The comparisons of effects of Al(III) with Pb(II), Cd(II) and Cr(III) on the LDH activities were also inspected.     

Gamma-irradiation immobilization of lactate oxidase in poly(vinyl alcohol) on platinized graphite electrodes

A novel method for enzyme immobilization in a polymer matrix was examined with lactate oxidase (LOD) to make a sensor for lactate. Poly(vinyl alcohol) (PVAL) and LOD were applied in layers on platinized graphite electrodes and cross-linked by exposure to a ⁶⁰Co gamma radiation source. When the sensor is dipped in lactate solution, the product of the enzymatic reaction, hydrogen peroxide, is detected at +300 mV vs. Ag/AgCl. The LOD-PVAL lactate sensor exhibits a fast response (10–50 s), a linear range between 26 μM and 1.7 mM, a detection limit of 13 μM and a sensitivity of 2.94 μA mmol^?1. The sensitivity and the linearity of the electrode were improved considerably by bubbling oxygen continuously through the lactate solution. Optimum response to lactate was obtained with a radiation dose of 3–10 Mrad. LOD was found to be active in the presence of the polymer under radiation doses as high as 40 Mrad. Repeated use of the sensors under various conditions showed a stable and reproducible response to lactate for over 80 days.     

Reagentless lactate electrodes based on electrocatalytic oxidation of flavocytochrome b2

Flavocytochrome b₂ (L-lactate :cytochrome c reductase, E.C. 1.1.2.3) from Hansenula anomala was entrapped on the surface of electrodes modified with various kinds of carbon black. The electrocatalytic oxidation of a reduced enzyme by the electroactive surface groups of carbon black enables this enzyme electrode to be used for the determination of lactate. The electrodes operate at ?0.2 to ?0.1 V vs. SCE (pH 7.0), which is low enough to avoid interference from ascorbic acid. Linear calibration graphs up to 0.5 mM lactate were obtained. Electrochemical measurements of lactate in human blood plasma and cell culture fluids showed good agreement with the results of spectrophotometric measurements.     

Amperometric flow-injection system with an immobilized enzyme reactor for the highly selective detection of phosphate and on-line amplification by substrate recycling

Bio-amperometric flow-injection systems are proposed for the highly selective and sensitive determination of phosphate. One system studied is based on the use of a co-immobilized purine nucleoside phosphorylase-xanthine oxidase reactor, which responds to phosphate with high selectivity, and a detection limit of 3 × 10^?7 M for a 20-μl injection. Another system with a co-immobilized purine nucleoside phosphorylase-xanthine oxidase-alkaline phosphatase reactor gives responses amplified by substrate recycling during passage through the enzyme reactor. Phosphate can be determined with 12 times the sensitivity in the latter system compared with the former, but the latter system responds to nucleotides and pyrophosphate in addition to orthophosphate.     

Amperometric l-lactate biosensor based on screen-printed carbon electrode containing cobalt phthalocyanine,coated with lactate oxidase-mesoporous silica conjugate layer

A novel amperometric biosensor for the measurement of l-lactate has been developed. The device comprises a screen-printed carbon electrode containing cobalt phthalocyanine (CoPC-SPCE), coated with lactate oxidase (LOD) that is immobilized in mesoporous silica (FSM8.0) using a polymer matrix of denatured polyvinyl alcohol; a Nafion layer on the electrode surface acts as a barrier to interferents. The sampling unit attached to the SPCE requires only a small sample volume of 100 μL for each measurement. The measurement of l-lactate is based on the signal produced by hydrogen peroxide, the product of the enzymatic reaction. The behavior of the biosensor, LOD-FSM8.0/Naf/CoPC-SPCE, was examined in terms of pH, applied potential, sensitivity and operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.4 and an applied potential of +450 mV. The determination range and the response time for l-lactate were 18.3 μM to 1.5 mM and approximately 90 s, respectively. In addition, the sensor exhibited high selectivity for l-lactate and was quite stable in storage, showing no noticeable change in its initial response after being stored for over 9 months. These results indicate that our method provides a simple, cost-effective, high-performance biosensor for l-lactate.     

An amperometric lactate sensor based on a NAD+-analog and lactate dehydrogenase coimmobilized on reticulated vitreous carbon

A NAD⁺-analog was coimmobilized with lactate dehydrogenase (LDH) on reticulated vitreous carbon (RVC) to give an amperometric lactate biosensor. Both LDH and the NAD⁺ -analog were bound covalently with carbodiimide to the surface of the porous RVC-material. The electrode was operated in a FIA-arrangement in the presence or absence of a soluble mediator. Meldola Blue. The stability was poor when the electrode was operated at +400 mV (vs. Ag/AgCl) in the absence of mediator but improved most significantly in the presence of 5 μM mediator, so that 65% of the original activity remained after 16 days. The amperometric currents were smaller with regeneration by mediator at −100 mV than with direct electrochemical oxidation at +400 mV, indicating that the additional steps slow down the reaction rate. Linear calibration plots were obtained from the detection limit, 1 μM, to 500 μM lactate with 5 μM mediator, reoxidized at −100 mV. The sample throughput was about 60 h⁻¹.