An amperometric biosensor based on lactate oxidase immobilized in laponite–chitosan hydrogel on a glassy carbon electrode. Application to the analysis of l-lactate in food samples

Abstract A biosensor based on the immobilization of lactate oxidase (LOx) on a glassy carbon electrode modified with laponite/chitosan hydrogels for the quantification of l -lactate in alcoholic beverages and dairy products is presented. Ferrocene–methanol (FcMe) is used as artificial mediator. The purpose of this work is to determine the best hydrogel composition from the analytical point of view. The characterization of the hydrogels was carried out by CV, amperometry and EIS. According to permeabilities and charge transfer resistances for ferrocyanide (used as molecular probe) as well as the enzymatic behavior of the enzyme for l -lactate, the best laponite/chitosan mass ratio found was 25/50. The distinct features of the bioelectrode are its long stability, its ability to reject or minimize most interferents including ascorbic acid, and its excellent analytical response, which allowed the reduction of the enzyme content below 0.5 U, for a sensitivity of (0.326 ± 0.003) A cm −2  M −1 , with a time response lower than 5 s and a detection limit of (3.8 ± 0.2) × 10 −6  M. Our l -lactate biosensor was validated by comparison with a standard spectroscopic method.

[1]  S. Cosnier,et al.  Amperometric phenol biosensor based on laponite clay-chitosan nanocomposite matrix. , 2007, Biosensors & bioelectronics.

[2]  A. Arnaud,et al.  Purification and properties of a malolactic enzyme from Leuconostoc oenos ATCC 23278 , 1990, Journal of basic microbiology.

[3]  B. Boukamp.  A Nonlinear Least Squares Fit procedure for analysis of immittance data of electrochemical systems , 1986 .

[4]  M. Morales,et al.  Ion-exclusion chromatographic determination of organic acids in vinegars , 1998 .

[5]  Chang Ming Li,et al.  Highly sensitive lactate biosensor by engineering chitosan/PVI-Os/CNT/LOD network nanocomposite. , 2007, Biosensors & bioelectronics.

[6]  Hitoshi Yamato,et al.  Design of novel electron transfer mediators based on indophenol derivatives for lactate sensor. , 2002, Biosensors & bioelectronics.

[7]  Félix Pariente,et al.  Design and characterization of a lactate biosensor based on immobilized lactate oxidase onto gold surfaces , 2006 .

[8]  Pierre Labbe,et al.  An L‐Lactate Amperometric Enzyme Electrode Based on L‐Lactate Oxidase Immobilized in a Laponite Gel on a Glassy Carbon Electrode. Application to Dairy Products and Red Wine , 2010 .

[9]  César Fernández-Sánchez,et al.  Electrochemical impedance spectroscopy studies of polymer degradation: application to biosensor development , 2005 .

[10]  Maogen Zhang,et al.  Coupling the lactate oxidase to electrodes by ionotropic gelation of biopolymer. , 2003, Analytical chemistry.

[11]  Osvaldo N. Oliveira,et al.  Controlled fabrication of gold nanoparticles biomediated by glucose oxidase immobilized on chitosan layer-by-layer films , 2009 .

[12]  S. Poyard,et al.  Optimization of an inorganic/bio-organic matrix for the development of new glucose biosensor membranes , 1998 .

[13]  J. Pingarrón,et al.  Graphite-Teflon composite bienzyme electrodes for the determination of L-lactate: application to food samples. , 1999, Biosensors & bioelectronics.

[14]  Rahul Singhal,et al.  Development of a lactate biosensor based on conducting copolymer bound lactate oxidase , 2005 .

[15]  M. Kawasaki,et al.  Electrode coatings based on chitosan scaffolds. , 2000, Analytical chemistry.

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

[17]  H. Yamato,et al.  Novel electron transfer mediators, indoaniline derivatives for amperometric lactate sensor , 2002 .

[18]  A. Bard,et al.  Clay-modified electrodes , 1983 .

[19]  Hongyuan Chen,et al.  Biocomposite of cobalt phthalocyanine and lactate oxidase for lactate biosensing with MnO2 nanoparticles as an eliminator of ascorbic acid interference , 2006 .

[20]  L. Chen,et al.  Bioinorganic composites for enzyme electrodes. , 2001, Analytical chemistry.

[21]  P. Labbé,et al.  Amplification of amperometric biosensor responses by electrochemical substrate recycling: Part II. Experimental study of the catechol–polyphenol oxidase system immobilized in a laponite clay matrix , 1999 .

[22]  Xin Wei,et al.  Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films. , 2002, Analytical chemistry.

[23]  A. Domb,et al.  Chitosan chemistry and pharmaceutical perspectives. , 2004, Chemical reviews.

[24]  C. Iriondo,et al.  Determination of acids and volatile compounds in red Txakoli wine by high-performance liquid chromatography and gas chromatography. , 1998, Journal of chromatography. A.

[25]  S. Cosnier,et al.  A Composite Clay Glucose Biosensor Based on an Electrically Connected HRP , 2000 .

[26]  Nabil El Murr,et al.  Enzymatic electrode for the determination of L-lactate , 1996 .

[27]  Q. Fu,et al.  Largely improved tensile properties of chitosan film via unique synergistic reinforcing effect of carbon nanotube and clay. , 2008, The journal of physical chemistry. B.

[28]  P. Yáñez‐Sedeño,et al.  Gold nanoparticle-based electrochemical biosensors , 2005, Analytical and bioanalytical chemistry.

[29]  Y. Tsai,et al.  Immobilization of lactate dehydrogenase within multiwalled carbon nanotube-chitosan nanocomposite for application to lactate biosensors , 2007 .

[30]  H. Yamato,et al.  Novel electron transfer mediators based on p-phenylenediamine for lactate oxidase , 2001 .

[31]  S. Cosnier,et al.  HRP/[Zn-Cr-ABTS] redox clay-based biosensor: design and optimization for cyanide detection. , 2004, Biosensors & bioelectronics.

[32]  P. Labbé,et al.  Characterization of organosilasesquioxane-intercalated-laponite-clay modified electrodes and (bio)electrochemical applications , 1998 .

[33]  M. Nuti,et al.  Properties of Malolactic Activity Purified from Leuconostoc oenos ML34 by Affinity Chromatography , 1984, Applied and environmental microbiology.

[34]  Thomas S. Collins,et al.  Artifactual Signal Splitting in the Capillary Electrophoresis Analysis of Organic Acids in Wine , 1997 .

[35]  A. Simonian,et al.  Glucose, lactate, and pyruvate biosensor arrays based on redox polymer/oxidoreductase nanocomposite thin-films deposited on photolithographically patterned gold microelectrodes , 2002 .