Electrodes lactate dehydrogenase

Multienzyme electrodes can increase sensitivity from micromolar to nanomolar detection levels (53,57). In this case the substrate is converted to a detectable product by one enzyme, then that product is recycled into the initial substrate by another enzyme resulting in an amplification of the response signal. For example, using lactate oxidase and lactate dehydrogenase immobilized in poly(vinyl chloride), an amplification of 250 was obtained for the detection oflactate (61). 

Many dehydrogenase enzymes catalyze oxidation/reduction reactions with the aid of nicotinamide cofactors. The electrochemical oxidation of nicotinamide adeniiw dinucleotide, NADH, has been studied in depthThe direct oxidation of NADH has been used to determine concentration of ethanol i s-isv, i62) lactate 157,160,162,163) pyTuvate 1 ), glucose-6-phosphate lactate dehydrogenase 159,161) alanine The direct oxidation often entails such complications as electrode surface pretreatment, interferences due to electrode operation at very positive potentials, and electrode fouling due to adsorption. Subsequent reaction of the NADH with peroxidase allows quantitation via the well established Clark electrode. 

Simon et al. [92] investigated a biocatalytic anode based on lactate oxidation by lactate dehydrogenase (LDH). The anodic current is generated by the oxidation of NADH (produced by NAD+ and substrate) while LDH catalyzes the electro-oxidation of lactate into pyruvate. As previously mentioned, the oxidation of NADH at bare electrodes requires a large overpotential, so these authors used poly(aniline) films doped with polyanions to catalyze NADH oxidation. Subsequent research by this group focused on targeting mutants of LDH that are amenable to immobilization on the polyaniline surface [93],... 

Suaud-Chagny and Gonon [3] presented a new procedure for protein immobilization adapted to carbon microelectrode characteristics. The principle of this method of immobilization is based on the association of the protein with an inert porous film immobilized around the active tip of the electrode. For this purpose the carbon was coated with an inert, electrochemically obtained protein sheath (bovine serum albumin, BSA) a few micrometers thick. Then the sheath around the fiber was impregnated with lactate dehydrogenase (LDH), which could be immobilized onto the electrode and resulted in an electrode sensitive to pyruvate. 

The successful synthetic application of this electroenzymatic system has first been shown for the in-situ electroenzymatic reduction of pyruvate to D-lactate using the NADH-dependent D-lactate dehydrogenase. Electrolysis at — 0.6 V vs a Ag/AgCl-reference electrode of 50 mL of a 0.1 M tris-HCL buffer of pH 7.5 containing pentamethylcyclopentadienyl-2,2 -bipyridinechloro-rhodium(III) (1 x 10 3 M), NAD+ (2 x 10 3 M), pyruvate (2 x 10 2 M), 1300 units D-lactate dehydrogenase (divided cell, carbon foil electrode) after 3 h resulted in the formation of D-lactate (1.4 x 10 2 M) with an enantiomeric excess of 93.5%. This means that the reaction occurred at a rate of 5 turnovers per hour with respect to the mediator with a 70% turnover of the starting material. The current efficiency was 67% [67],... 

Avramescu et al. [24] d-Lactate Wines D-Lactate dehydrogenase/with NAD+ deposited onto the surface of the electrode and covered with polyethyleneimine-Nafion membrane Carbon electrode, modified with an insoluble salt of Meldola s Blue/-50mV vs. Ag/ AgCl Meldola s Blue... 

Kwan et al. [27] l-Lactate Yoghurt milk, soda, sport drinks, and healthy supplement Salicylate hydroxylase (SHL), L-lactate dehydrogenase (LDH), and pyruvate oxidase (PyOD)/entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane Clark-type oxygen electrode ... 

Katrlik et al. [63] L-Malate, l-lactate Wines L-Malate dehydrogenase or l-lactate dehydrogenase and diaphorase/covered by a dialysis membrane NAD+-modified graphite-2-hexadecanone composite electrode Hexacyanoferrate(III)... 

Sulfite modified enzyme electrode. (2) L-Lactate/L-malate/ sulfite multibiosensor L-lactate dehydrogenase/L-malate dehydrogenase/ sulfite oxidase surface-modified enzyme electrodes/enzymes were deposited on the composite electrodes and covered with a dialysis membrane ... 

All potentials vs. screen-printed pseudo Ag/AgCl reference, except values marked with double-dagger (J), which are vs. screen-printed Ag/Pd, and 02 those marked with asterisk ( ), which are vs. Ag/3M AgCl double-junction reference electrode. Abbreviations LDH lactate dehydrogenase, LOD lactate oxidase, PVA-SbQ styrlpyridinium-modified poly(vinyl alcohol), FIA flow injection analysis. aLinear ranges using a dialysis system. 

The commercial availability of various lactate-specific enzymes and the high demand for lactate analysis for clinical and food processing led to the construction of numerous lactate enzyme electrodes (Tkble 8). Depending on the selected enzyme—lactate oxidase (LOD), lactate mono-oxidase (LMOD), or lactate dehydrogenase (LDH)—several mechanistic approaches may be applied ... 

Co-immobilizing a second enzyme, NADH oxidase (280) or lactate dehydrogenase (288), permits the regeneration of NAD+. Measurements may be completed in Tris-HCl or carbonate buffers, but borate and glycine buffers inhibit L-alanine dehydrogenase (289). Highly selective L-histidine electrodes are available (290, 291) to determine histidine in urine (291). 

The inerease in the activity of lactate dehydrogenase (LDH) on a GCE modified with a SWNTs film, compared to a bare GCE was also proposed [101], by using chronoamperometry in a convective system. The LDH-SWNT-modified GCE was more aetive than the plain GCE. The irreversible eatalytie oxidation of NADH was demonstrated by the 150 mV shifting of the anodie peak potential towards more negative values, compared to the plain GCE. The authors evaluated the SWNTs-modified electrode performanee in the reduction of pyruvate using NADH as coenzyme in 50 mM Tris-HCl buffer solution pH 7.5, by chronoamperometry in a forced conveetion system. 

Enzyme electrodes for lactate determination using immobilized lactate dehydrogenase 16), for urea determination using immobilized urease 17), for L-amino acids using immobilized L-amino acid oxidase 18), and for various amines using the appropriate immobilized deaminase system (19) have also been prepared. A urease electrode is commercially available from Beckman,... 

Figure 20. (A) The assembly of an integrated lactate dehydrogenase monolayer electrode by the cross-linking of an affinity complex formed between the enzyme and a PQQ-NAD monolayer-modified Au electrode. (B) Cyclic voltammograms of the integrated cross-linked PQQ-NAD / lactate dehydrogenase electrode (roughness factor ca. 15) (a) in the absence of lactate (b) with lactate, 20 mM. Recorded in 0.1 M Tris buffer, pH 8.0, in the presence of 10 mM CaCb, under Ar potential scan rate, 2 mV s . Inset amperometric responses of the integrated electrode at different concentrations of lactate upon application of potential 0.1 V vs. SCE.

An electrode for measuring urea has been described (Gll), consisting of a thin film of urease, immobilized in acrylamide gel, on the surface of a glass electrode responsive to NH. Conditions are carefully selected to ensure stability of the enzyme, and the potential developed is proportional to the logarithm of the urea concentration. Blood glucose and lactate have been determined with a membrane electrode in which the enzyme (glucose oxidase or lactate dehydrogenase) is trapped in a porous or jellied layer at the membrane surface (W20). 

Scheme 11. Idealized sketch showing the electroen matic oxidation of L-lactate at gold modified electrode surfaces, (a) Lactate dehydrogenase bound to CB-terminated alkylthiol SAMs prepared by covalent attachment of CB to 3-mercaptopropionic acid SAM derivatized with 1,4-diaminobutane. The electroenzymatic oxidation of lactate is observed only in the presence of soluble coenzyme (NAD" ") and a redox mediator (phenazine methosulfate) [215]. (b) Lactate deh3tdrogenase bound to NAD-terminated alkylthiol SAMs prepared by covalent attachment of Af -(2-aminoethyl)-NAD to a cystamine SAM derivatized with pjrrroloquinoline quinone. The reconstituted enzyme is electrically wired to the electrode surface via two NAD" -binding pockets involved in the affinity-binding surface reaction [242].

Fig. 1.5. Dependence of the electroenz3maatic response (Ay) of lactate dehydrogenase modified gold electrodes on the time of protein layer growth (a) mixed 3-mercaptopropamol and 3-mercaptopropionic acid SAM derivatized with 1,4-diamino-butane ligand fi-ee- ( ), CB- (O) and Ll-anchored (A) (b) cystamine SAM derivatized with fumaric acid ligand fi-ee- ( ), CB- (O) and Ll-anchored (A). The monolayers were incubated in a 0.36mgml enzyme solution in 50 mM Na-phosphate buffer, pH 7.0. Reproduced from [240] with permission.

Gorton et al. reported carbon paste electrodes based on Toluidine Blue O (TBO)-methacrylate co-polymers or ethylenediamine polymer derivative and NAD" " with yeast alcohol dehydrogenase for the analysis of ethanol [152,153] and with D-lactate dehydrogenase for the analysis of D-lactic acid [154]. Use of electrodes prepared with dye-modified polymeric electron transfer systems and NAD+/NADH to detect vitamin K and pyruvic acid has also been reported by Okamoto et al. [153]. Although these sensors showed acceptable performances, insensitivity to ambient oxygen concentration, sensor stability and lifetime still need to be improved to obtain optimal dehydrogenase based enzyme biosensors. 

Fry has used a similar system for the enzymatic reduction of pyruvate to L-lactate. In this case, the one-electron transfer redox catalyst, methyl viologen, and the lipoamide dehydrogenase are coimmobilized within a Nafion cation-exchange layer on the surface of a reticulated vitreous carbon electrode. As production enzyme, L-lactate dehydrogenase (LDH) was employed [48], which was later stabilized considerably in the form of cross-... 

Lactate dehydrogenase (LDH) has been immobilized on silica gel coated with niobium oxide carbon paste electrode for the development of lactate biosensor. This biosensor shows good sensitivity allows lactate estimation upto 6.5 x 10-6 mol L-1. Moreover, the biosensor shows linear range from 0.1 to 14 mmol L-l for lactate. [111]. 

This method was applied to assemble integrated electrically-contacted NAD(P)-dcpcndcnt enzyme electrodes. The direct electrochemical reduction of NAD(l ) cofactors or the electrochemical oxidation of NAD(P)H cofactors are kineticaUy unfavored. Different diffusional redox mediators such as quinones, phenazine, phenoxazine, ferrocene or Os-complexes were employed as electrocatalysts for the oxidation of NAD(P)H cofactors An effective electrocatalyst for the oxidation of the NAD(P)H is pyrroloquinoline quinone, PQQ, (7), and its immobilization on electrode surfaces led to efficient electrocatalytic interfaces (particularly in the presence of Ca ions) for the oxidation of the NAD(P)H cofactors. This observation led to the organization of integrated electrically contacted enzyme-electrodes as depicted in Fig. 3-20 for the organization of a lactate dehydrogenase electrode. 

Figure 3-20. Assembly of an integrated lactate dehydrogenase, LDI I. electrode for the...

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