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

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 (2s) 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 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.

[1]  G. Palleschi,et al.  In vivo continuous monitoring of L-lactate coupling subcutaneous microdialysis and an electrochemical biocell , 1995 .

[2]  Chung-Yen Lin,et al.  An Enzymatic Microassay for Lactate Concentration in Blood and Hemolymph , 1999 .

[3]  P. Bartlett,et al.  Electrochemical immobilization of enzymes. Part VI. Microelectrodes for the detection of L-lactate based on flavocytochrome b2 immobilized in a poly(phenol) film , 1994 .

[4]  Jianjun Wei,et al.  Reagentless amperometric biosensor highly sensitive to hydrogen peroxide based on the incorporation of Meldola Blue, fumed-silica and horseradish peroxidase into carbon paste , 1997 .

[5]  F W Scheller,et al.  Enhancing biosensor performance using multienzyme systems. , 1993, Trends in biotechnology.

[6]  F. Mizutani,et al.  Amperometric determination of acetic acid with a trienzyme/poly(dimethylsiloxane)-bilayer-based sensor. , 2001, Analytical chemistry.

[7]  G. Palleschi,et al.  Amperometric biosensor for determination of lactate in sweat , 1993 .

[8]  A. Rocchini,et al.  Lactate blood levels in the perioperative period of orthotopic liver transplantation , 1997, International journal of clinical & laboratory research.

[9]  Phosphate Sensor Composed from Immobilized Pyruvate Oxidase and an Oxygen Electrode , 1991 .

[10]  G. Palleschi,et al.  Lactate and glucose electrochemical biosensors for the evaluation of the aerobic and anaerobic threshold in runners , 1990, Medical and Biological Engineering and Computing.

[11]  B. D. Malhotra,et al.  Immobilization of lactate dehydrogenase on tetraethylorthosilicate-derived sol-gel films for application to lactate biosensor , 2001, Applied biochemistry and biotechnology.

[12]  P. Zambonin,et al.  Flow injection analysis of L-lactate in milk and yoghurt by on-line microdialysis and amperometric detection at a disposable biosensor. , 2001, The Analyst.

[13]  R. Renneberg,et al.  An amperometric biosensor for determining amino acids using a bienzymatic system containing amino acid oxidase and protease , 2002, Biotechnology Letters.

[14]  F. Scheller,et al.  A bienzyme electrode for L-malate based on a novel and general design. , 1998, Journal of biotechnology.

[15]  J. Vincent,et al.  Serial blood lactate levels can predict the development of multiple organ failure following septic shock. , 1996, American journal of surgery.

[16]  M. Mascini,et al.  A lactate electrode with lactate oxidase immobilized on nylon net for blood serum samples in flow systems , 1984 .

[17]  R. Mccomb,et al.  Optimum reaction conditions for human lactate dehydrogenase isoenzymes as they affect total lactate dehydrogenase activity. , 1968, Clinical chemistry.

[18]  Ulla Wollenberger,et al.  An amperometric bi-enzyme sensor for determination of formate using cofactor regeneration. , 2003, Biosensors & bioelectronics.

[19]  Jean-Michel Kauffmann,et al.  Amperometric biosensor based on carbon paste mixed with enzyme, lipid and cytochrome c , 1994 .

[20]  B Danielsson,et al.  Urea and lactate determined in 1-microL whole-blood samples with a miniaturized thermal biosensor. , 1994, Clinical chemistry.

[21]  Frieder W. Scheller,et al.  Enzyme electrodes with substrate and co-enzyme amplification , 1985 .

[22]  David Gough,et al.  A Continuous, Implantable Lactate Sensor , 1995 .

[23]  J. Perdomo,et al.  Containment sensors for the determination of L-lactate and glucose. , 1999, Biosensors & bioelectronics.

[24]  F. Scheller,et al.  Coupled enzyme reactions for novel biosensors , 1986 .

[25]  M. Smyth,et al.  Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate. , 1997, Talanta.

[26]  A. J. Miranda-Ordieres,et al.  A bienzyme-poly-(o-phenylenediamine)-modified carbon paste electrode for the amperometric detection of l-lactate , 1997 .

[27]  M A Arnold,et al.  Fiber-optic biosensors based on the fluorometric detection of reduced nicotinamide adenine dinucleotide. , 1988, Analytical chemistry.

[28]  Fumio Mizutani,et al.  An enzyme electrode forl-lactate with a chemically-amplified response , 1985 .

[29]  P Atanasov,et al.  Needle-type lactate biosensor. , 1999, Biosensors & bioelectronics.

[30]  W. Cascio,et al.  A conducting salt-based amperometric biosensor for measurement of extracellular lactate accumulation in ischemic myocardium. , 1997, Analytical chemistry.

[31]  B. Möller,et al.  Amperometric lactate oxidase catheter for real-time lactate monitoring based on thin film technology. , 1997, Biosensors & bioelectronics.

[32]  D. Poole,et al.  Blood Lactate During Exercise: Time Course of Training Adaptation in Humans , 1988, International journal of sports medicine.