Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor

Abstract

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD⁺. SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2 s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 μM lactate, with a detection limit of 4.3 μM. A good agreement (R²=0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.

Introduction

Over the last years, the measurement of lactate has been a main topic for different purposes. For intensive and sports medicine, the estimation of lactate in blood allows the determination of the anaerobic threshold under physical exercise and permits the success of training to be followed (Gaesser and Poole, 1988, Palleschi et al., 1990, Volpe et al., 1995). Besides, blood lactate is a clinically valuable diagnostic indicator. In adult elevated concentration of blood lactate can predict multiple organ failure and the death in patients with septic shock (Bakker et al., 1996) as well as the function of newly transplanted livers (DeGasperi et al., 1997). In food industry, microbial contamination of milk results in an increased lactate content as a consequence of homo- or hetero-lactate fermentation. Therefore, lactate determination is a very useful way to detect the microbial contamination (Palmisano et al., 2001).

Due to its importance, various methods have been used for the estimation of lactate concentration in different samples. Lactate is typically determined by enzymatic oxidation to pyruvate by lactate dehydrogenase (LDH) or lactate oxidase (LOD), followed by the detection of reduced form of nicotineamide adenine dinucleotide (NADH) (Wangsa and Arnold, 1998) or hydrogen peroxide (H₂O₂) (Perdomo et al., 1999), respectively. LOD/horseradish peroxide (HRP) quantifies lactate by catalyzing the oxidative condensation of 2,2′-azino-di-3-ethylbenzthiazoine sulfonate (ABTS) in the presence of hydrogen peroxide to a colored dye with a maximum absorbance at 405 nm (Lin et al., 1999). LOD has also been coupled with a system of liquid chromatography and an electrochemical detector for the determination of lactate (Yang et al., 1997). However, these conventional methods for lactate determination are tedious and time consuming. Spectrophotometric monitoring of NADH or colored dye generation is always interfered by other photometric active substances or suspended particles in the sample (Xie et al., 1994). Direct measurement of lactate at lower wavelength UV region also suffers from serious background interference (Yang et al., 1997).

The importance of enzyme-based, amperometric biosensors has increased considerably during the past decade due to high selectivity of the biorecognition element and the sensitivity of electrochemical signal transduction. This results in the development of rapid, accurate, and easy device for specific measurement of target analyte in complex matrices such as blood, food product, and environmental sample. Biosensors employed different enzymes have been developed for lactate monitoring. Cytochrome b₂ (Amine et al., 1994, Bartlett and Caruana, 1994), lactate monooxygenase (Mascini et al., 1984), and LOD (Baker and Gough, 1995, Faridnia et al., 1993, Pfeiffer et al., 1997) have been used for this purpose. Bienzyme systems, such as LOD/LDH (Mizutani et al., 1985), cytochrome b₂/LDH (Schubert et al., 1985) and glutamate pyruvate transaminase/LDH (Lobo-Castannon et al., 1997), have been investigated. Besides, mediated electrodes have been studied to reduce oxygen fluctuation effect in the oxidase-involved system (Liu et al., 1997, Park et al., 1997). Although various schemes for lactate measurements have been proposed, limitations have restricted the reliabilities of these biosensors in the measurements of samples. Hydrogen peroxide sensing lactate biosensors based on immobilized LOD always suffer from electroactive interferences, such as ascorbate in dairy samples (Palmisano et al., 2001). Those electro-oxidizable substances also contribute to current response at low potential in the mediated electrode systems. Besides, the leaking of redox mediator, most commonly ferrocene derviations and tetrathiafulvalene, reduces the stability and reliability of the mediated electrode systems (Marzouk et al., 1997). Although the best-investigated bienzyme system of LOD/LDH was very sensitive, yielding amplification factor of 48,000 (Wollenberger et al., 1993), it suffered from low reproducibility and stability (Renneberg et al., 1986). Also, the measuring range (nano-molar range) of this system was out of the range in practical analyses (milli-molar range).

The applications of nicotinamide adenine dinucleotide (NAD⁺) dependent dehydrogenases in amperometric biosensors are highly selective in many cases. Recently, the coimmobilization of NAD(P)⁺-dependent dehydrogenases with salicylate hydroxylase (SHL, E.C. 1.14.13.1) in front of a Clark electrode has been investigated for dehydrogenase-based biosensors (Gajovic et al., 1998, Mak et al., 2003, Yang et al., 1999). The sensitivity was improved by SHL which recycles the cofactor NAD⁺ from NADH effectively.

In this paper, amperometric determination of lactate with novel trienzyme system is described. The trienzyme sensor was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27) and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. LDH catalyzes the specific dehydrogenation of lactate using NAD⁺. SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate to form catechol in the presence of oxygen and NADH. PyOD decarboxylates the pyruvate to form hydrogen peroxide, carbon dioxide, and acetylphosphate in the presence of oxygen and phosphate. The SHL and PyOD keep the equilibrium of dehydrogenation of lactate to the product side by consuming the pyruvate and NADH generated by LDH. Dissolved oxygen acts as an essential material for the enzymatic activities of both PyOD and SHL, is consumed proportionally to the concentration of lactate during the measurements. A strong signal, caused by the consumptions of the dissolved oxygen by the two enzymes, was monitored at −600 mV versus Ag/AgCl by the Clark electrode. Signal amplification and regeneration of the cofactor were found in this trienzyme system (SHL/LDH/PyOD) when comparing with bienzyme systems (SHL/LDH and LDH/PyOD). All enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel, that was sandwiched between a dialysis membrane and a Teflon membrane. Electroactive interferences were eliminated by the oxygen membrane. The schematic diagram of the trienzyme system is shown in Fig. 1. The practicality of this biosensor has been studied by measuring the lactate content in beverage samples and comparing with the results obtained by using a commercial lactate testing kit.