Design and optimization of a lactate amperometric biosensor based on lactate oxidase cross-linked with polymeric matrixes

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Sensors and Actuators B 131 (2008) 590–595

Design and optimization of a lactate amperometric biosensor based on lactate oxidase cross-linked with polymeric matrixes Marcelo Ricardo Romero, Fernando Garay, Ana M. Baruzzi ∗ INFIQC, Dpto. de F´ısico Qu´ımica, Fac. de Ciencias Qu´ımicas, UNC, Pab. Argentina, Ala 1, 2◦ piso, Ciudad Univ., 5000 C´ordoba, Argentina Received 24 October 2007; received in revised form 13 December 2007; accepted 17 December 2007 Available online 27 December 2007

Abstract The design and characterization of a lactate biosensor is described. The biosensor is developed through the immobilization of lactate oxidase (LOD) in an albumin and mucin composed hydrogel. The enzyme is then cross-linked with glutaraldehyde to the polymeric matrix and entrapped between two polycarbonate membranes. The hydrogen peroxide produced by the reaction of lactate and LOD is detected on a Pt electrode operated at 0.65 V versus Ag|AgCl. The performance of the biosensor was evaluated in matrixes with different amounts of albumin, mucin and glutaraldehyde. The response time of the sensor to 10 ␮M lactate required 90 s to give a 100% steady-state response of 0.079 ␮A. Linear behavior was obtained for 0.7 ␮M < cLac < 1.5 mM. The detection limit calculated from the signal to noise ratio was 0.7 ␮M. Only 0.1 U of enzyme was necessary to get a biosensor with a relatively high current flow and an excellent stability over a storage period of 30 days. High reproducibility in the response was obtained when several biosensors were prepared with the same composition. © 2008 Elsevier B.V. All rights reserved. Keywords: Biosensor; Electrochemical sensor; Enzyme electrode; l-Lactate; Lactate oxidase; Mucin; Albumin

1. Introduction During the past four decades there has been increasing research on the development of lactate biosensors mainly because of the association of lactate with several severe clinical conditions. Elevated blood lactate concentration can predict multiple organ failure and death of patient with septic shock. Lactic acidosis is known to accompany decreased tissue oxygenation, left ventricular failure, and drug toxicity [1]. Analysis of lactate in saliva can be used as a preliminary diagnostic for cystic fibrosis. Gauging blood lactate is also relevant for the results of exercise and athletic performance. In addition to the relevance in clinical diagnosis, the determination of lactate is very important in other areas such as fermentation and food analysis [2]. Most lactate amperometric biosensors reported in literature are based on immobilized lactate dehydrogenase (LDH) or lactate oxidase (LOD) [3–10]. In the latter configuration, LOD ∗

Corresponding author. Tel.: +54 3514334169; fax: +54 3514334188. E-mail addresses: [email protected], [email protected] (A.M. Baruzzi). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.12.044

catalyzes the conversion of lactate to pyruvate and hydrogen peroxide, which can be oxidized at the electrode surface, according to the following reactions: l-lactate + LODox → pyruvate + LODred

(1)

LODred + O2 → LODox + H2 O2

(2)

H2 O2 → O2 + 2e−

(3)

Two of the major problems that concern to most biosensors, not only those related to lactate, are operational and storage stability as well as sensor-to-sensor reproducibility. The stability is of great importance for the success of these devices as analytical instruments, and is mainly dependent on the lifetime, or the rate of denaturation or inactivation, of the enzyme employed [2,10,11]. The use of novel immobilization methods [8,9,12–16] or media [17–21] is usually employed in order to enhance the enzyme stability by optimizing the surrounding microenvironment. The sensor-to-sensor reproducibility is also a major problem concerning the reliability of these devices. This issue is rarely addressed [22,23], and when relevant data are presented the numbers are rather discouraging [10,24–26].

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Therefore, immobilization of enzyme on electrode surface is a key step for the construction and performance of biosensors. It worth to mention that one of the best analyte detection limit was achieved for a sol–gel enzyme electrodes that immobilizes the enzyme into an albumin matrix [27,28]. Albumin is well characterized globular protein has several amino groups that could be linked to other species by using bi-functional molecules such as glutaraldehyde [29,30]. Recently, biomaterials such as mucin and chitosan have attracted increasing attention for immobilizing enzymes through the formation of polyelectrolyte complexes between the enzymes and polysaccharide chains [29–32]. These polymers form hydrogels that stabilize the three-dimensional structure of the enzyme by allowing for a sufficient amount of water to be present and by mimicking cytoplasm or cytosol properties [27]. With regards to mucin, it corresponds in fact to a group of proteins that are major components of the mucous that coats the surface of cells lining the respiratory and digestive tracts [33]. They have polypeptide chains with domains rich in threonine and/or serine whose hydroxyl groups are in O-glycosidic linkage with oligosaccharides [33]. These extracellular glycoproteins consist in a proteic core surrounded by diverse polysaccharide groups which molecular weight ranges from 0.5 to 20 MDa. Irrespectively of their size, approximately the 80% of the molecular mass corresponds to oligosaccharide chains, providing suitable environment to stabilize the three-dimensional structure of enzymes [34,35]. In this study, albumin–mucin hydrogel is employed as LOD host matrix. The enzyme is cross-linked to the matrix with glutaraldehyde and entrapped between two polycarbonate membranes. The effect of different parameters related to the composition of the proposed biosensor are presented and discussed. Finally, characteristics related to the storage stability and the sensor-to-sensor reproducibility are analyzed for the resulting lactate biosensor prototype.

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2.2. Apparatus All electrochemical experiments were performed with an Autolab PGSTAT30 Electrochemical Analyzer (Eco Chemie, Netherlands). The measurements were carried out using a conventional three-electrode system with a Pt wire as the counter electrode, an Ag|AgCl|KCl (3 M) as the reference electrode, and a homemade 3 mm diameter Pt disc as the working electrode. The working electrode was made from a Pt rod fixed into a 10 mm diameter Teflon cylinder. Amperometric detection was obtained under batch conditions with stirring of 120 rpm at a desired working potential after a preconditioning time of 2000 s. 2.3. Preparation of the enzymatic electrode A total mass of 6.0 mg composed by different amounts of mucin and albumin was dissolved in 40 ␮L of base electrolyte. Proteins were mixed during 3 min until a colloidal suspension was obtained, and then transferred into an eppendorf containing 1 U of LOD. The 60 ␮L of the LOD-matrix system were then mixed during 3 min and saved at 4 ◦ C. To prepare the enzymatic electrode, aliquots of 6 ␮L of the LOD-matrix system and 3 ␮L of glutaraldehyde were entrapped between two membranes of polycarbonate that were previously wet with buffer solution pH 7. After this, the enzymatic matrix was placed on the working electrode surface and fixed to it with a suitable cap. After waiting 5 min, buffer solution was added to stop the cross-linkage of the system. 3. Results and discussion 3.1. pH-effect Fig. 1 shows normalized enzyme electrode responses in 1.0 mM lactate solutions. The LOD-matrix system was made with a hydrogel composed by 100% of mucin cross-linked

2. Experimental 2.1. Reagents All solutions were prepared with ultra pure water (18 M cm−1 ) from a Millipore Milli-Q system. The base electrolyte solution (0.11 M) consisted in 0.01 M HK2 PO4 /H2 KPO4 and 0.1 M KNO3 (all from Merck). This solution was renewed weekly and small amounts of H2 SO4 (Baker) or KOH (Merck) were used to fix it at pH 7.0. A stock solution of 0.01 M lactate (Sigma) was prepared in the base electrolyte. Different mother solutions of glutaraldehyde (Sigma) were also made in the base electrolyte. 50 U LOD from Pediococus species (Sigma) were diluted in 1000 ␮L of the base electrolyte. After dissolution, aliquots of 20 ␮L of enzyme were separated into 50 eppendorf and saved at −20 ◦ C. Thus, every aliquot bears 1 U of LOD. Mucin (Sigma) was powdered and saved as a dry powder at 4 ◦ C. Bovine serum albumin (Sigma) was used as received. All solutions were stored at 4 ◦ C. All chemical reagents were of analytical grade and used as received.

Fig. 1. Normalized electrochemical response of the enzymatic electrode to 1.0 mM lactate solutions at different pH values. LOD-matrix system: mucin and LOD cross-linked with glutaraldehyde (1%, v/v).

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Fig. 2. Calibration curves for lactate when LOD is in different hydrogel systems. Curves (a) matrixes composed by mucin and/or albumin (100 × massmucin /masstotal ): (A) 100; (B) 70 and (C) 0. Curves (b) matrixes composed by mucin and/or carbopol (100 × massmucin /masstotal ): (A) 90; (B) 70 and (C) 0. All matrixes were cross-linked during 5 min. with 0.1 U of LOD employing 3 ␮L of glutaraldehyde 1.5%. The data correspond to the oxidation current of H2 O2 at 0.65 V and pH 7.0.

to LOD with glutaraldehyde (1%, v/v). These activity measurements exhibit a rapid increase in LOD catalytic behavior from pH 5.0–7.0, after which slow decrease in enzyme activity is observed. The highest sensitivity corresponds to the data obtained at pH 7.0, which is in agreement with other previously reported LOD-based electrochemical sensors [8–10,12,36,37]. 3.2. Optimization of the LOD-matrix system Fig. 2 shows calibration curves obtained with identical LOD concentration in different hydrogel systems. Curve 2A (a), corresponds to the performance of LOD entrapped into a mucin matrix. Although relatively good sensitivity is found, data show important variability when high lactate concentrations are added. Commonly, this kind of sensor loses the linear dependence between current and lactate concentration (cLac ) when cLac > 0.6 mM. Conversely, results obtained with matrixes composed only with albumin exhibited low dispersion of data, curve 2C (a). The combination of both polymers not only provided very low dispersion of results, with respect to the average calibration curve, but also increased the range in which linear behavior was found, curve 2B (a). The best performance of the LOD-sensor was found when mucin/albumin mass ratio was 70/30. Under these conditions, linear relationship between current and cLac extends from 0.7 ␮M < cLac < 1.5 mM and the mean sensitivity is 0.79 mA M−1 . Other mixtures of polymers were evaluated, but significant diminution of current was observed. Curves 2 (b) show calibration curves obtained when LOD is cross-linked with diverse amounts of mucin and/or carbopol. The presence of carbopol diminishes the sensitivity of the LOD-sensor practically to zero. This was an unexpected behavior, since very promising results were obtained when this polymer was used for the development of oxalate biosensors [29,30]. This effect can be explained considering that the position N(5) of flavins is avid to form adducts with the anionic forms of mono- and dicarboxylic acids [38]. Carbopol is a species of polyacrylic acid in which a huge amount of carboxylic groups are present to form adducts that would inhibit the catalytic properties of enzymes like oxalate

oxidase and LOD. Oxalate oxidase based biosensors, however, have their highest sensitivity in solutions with pH close to 2.8, wherein carboxylic acids are mostly protonated [29]. On the contrary, the adducts formation would be important between flavins and carboxylate substituents of carbopol when neutral solutions are required to provide a suitable environment for enzymes like LOD. Fig. 3 exhibits the effect of using different concentrations of glutaraldehyde to cross-link LOD with the other polymers of the matrix. The cross-linking time was kept equal to 5 min because this time allowed us to build the sensor properly. Provided low concentrations of glutaraldehyde, also low LOD-sensor sensitivity is observed, curve (a). The sensitivity and the linear behavior of the biosensor are both maxima when 1.5% of glutaraldehyde is used to cross-link the enzymatic matrix. For higher concentrations of glutaraldehyde diminution of the sensitivity is found, curves (d and e). Possibly, this is due to the high cross-linking

Fig. 3. Calibration curves for lactate when LOD is cross-linked in matrixes with 70/30 mucin/albumin mass ratio with 3 ␮L of glutaraldehyde of concentration (%, v/v): (a) 0.15; (b) 1; (c) 1.5; (d) 2.5; (d) 5. All matrixes were cross-linked during 5 min. With 0.1 U of LOD and the data correspond to the oxidation current of H2 O2 at 0.65 V and pH 7.0.

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between LOD and the other polymers of the matrix. It is important to keep in mind that LOD is a rather labile enzyme with several amino groups that are avid to be cross-linked by glutaraldehyde and whose catalytic activity could be lost by slight changes in its structure [6,12]. On the contrary, if the concentration of the cross-linker species is low, the enzyme and the other components of the hydrogel matrix may be not fixed properly and they would eventually escape from the membranes that conform the biosensor. These are the main reasons to have rigorous control of concentration and time of the cross-linker species. The slope values corresponding to calibration curves prepared with different concentrations of glutaraldehyde and albumin are presented in Fig. 4A. As can be observed, the addition of solutions with more than 2% of glutaraldehyde would not be recommended if the cross-linking time were equal (or higher) to 5 min. The slopes found for matrixes composed by mixtures of mucin and albumin are similar to those of biosensors prepared only with albumin. In this regard, the sensitivity of LOD-sensors build simply with mucin and employing 1% of glutaraldehyde would present the highest sensitivity from the analyzed set of sensors. On the contrary, the range in which the

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sampled current depends linearly on the concentration of lactate is very low in the case of biosensors prepared only with mucin, Fig. 4B. This behavior can be also observed from Fig. 2 where diverse calibration curves were presented. Thus, although LOD-sensors developed merely with mucin would have quite good sensitivity, calibration curves obtained with this kind of hydrogel usually have less reproducible and desirable behavior than those matrixes in which albumin has been utilized. The best performance was found for LOD-sensors assembled with a 70/30 mucin/albumin mass ratio, which is cross-linked with 3 ␮L of 1.5% glutaraldehyde. This electrode provided very good reproducibility and linearity regarding consecutive additions of lactate standard concentrations. Fig. 5 shows a current–time profile obtained after the addition of 5 ␮L from mother lactate solutions of 10 and 100 mM to a sample of 5 mL. It was necessary a waiting time of 2500 s to get a proper base line from which the limit of detection was calculated. In this regard, standard deviation was calculated from data collected during the last 300 s of the blank signal providing a limit of detection equal to 0.7 ␮M. From these experiments linear response was obtained up to concentrations of 1.5 mM. An average response time of 90 s was necessary to get the 100% of the signal current. 3.3. Long-term stability The performance of the LOD-sensor prepared with 0.1 U of LOD and 70/30 mucin/albumin mass ratio is shown in Fig. 6. Calibration curves were collected two times per week during almost 1 month to analyze the storage stability of the biosensor. In the meantime, the sensor was stored in buffer solution at 4 ◦ C. During these 27 days, calibration curves kept their linear behavior up to 1.1 mM while the limit of detection presented no change. The slopes calculated from these calibration curves pointed that the sensitivity decreased less than 13% of its initial value. Bearing in mind the very low amount of LOD required

Fig. 4. Dependence of sensitivity “I(cLac )−1 ” (A) and of the linear zone of calibration curves “cLac–max ” (B) on the percentages of glutaraldehyde and albumin used to build the lactate biosensor. All matrixes were cross-linked during 5 min. With 0.1 U of LOD and the data correspond to the oxidation current of H2 O2 at 0.65 V and pH 7.0.

Fig. 5. Current–time curve for a LOD-sensor upon successive additions of lactate standard concentrations. The sensor was prepared with 0.1 U of LOD, 70/30 mucin/albumin mass ratio cross-linked during 5 min. with 3 ␮L of glutaraldehyde 1.5%. The data correspond to the oxidation current of H2 O2 at 0.65 V and pH 7.0.

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of polycarbonate is placed over the enzymatic matrix only after 1 min of cross-linking, then the weight of the second membrane displaces the excess of matrix and provides reproducible LOD-sensors. 4. Conclusion

Fig. 6. Long-term stability of a LOD-sensor prepared with 0.1 U of LOD, 70/30 mucin/albumin mass ratio. Cross-linking: 3 ␮L of glutaraldehyde 1.5% during 5 min. The data correspond to the oxidation current of H2 O2 at 0.65 V and pH 7.0.

for building this sensor, it could be considered the replacement of membranes together with the LOD-matrix system after this diminution of the sensor activity. 3.4. Sensor-to-sensor reproducibility Fig. 7 shows calibration curves obtained for three different sensors constructed with equivalent matrix composition, but from aliquots of enzyme corresponding to diverse eppendorfs saved at −20 ◦ C (see Section 2.1). The standard deviation of these curves was <1% pointing out very good sensor-tosensor reproducibility. It is very difficult to fix the thickness of the enzymatic matrix to a given value, and because of this, it was expected that it would be not easy to get good reproducibility of the sensor. Nevertheless, if the second membrane

Fig. 7. Calibration curves corresponding to different sensors prepared with 0.1 U of LOD, 70/30 mucin/albumin mass ratio cross-linked during 5 min. with 3 ␮L of glutaraldehyde 1.5%. The data correspond to the oxidation current of H2 O2 at 0.65 V and pH 7.0.

We have developed and characterized the performance of a lactate biosensor based on a hydrogel matrix composed by LODmucin-albumin cross-linked with glutaraldehyde and entrapped between two membranes of polycarbonate. Via a series of experiments, optimal experimental conditions for building the biosensor and detecting lactate have been studied. The proposed biosensor provides a medium response time, high sensitivity, high stability and high sensor-to-sensor reproducibility. It is well known that, a higher amount of LOD at the hydrogel matrix would enlarge the linear range of calibration curves and thus, the maximum concentration of lactate that could be quantified. However, this fact would also increase the cost of the biosensor. Instead of this, the proposed LOD-sensor presents linear behavior in a range higher than three orders of magnitude of lactate concentrations, providing suitable conditions to quantify lactate in real samples. It should be pointed out that, electroactive interferents such as ascorbic acid, uric acid, urea, glycine, and acetaminophen limit the practical application of this biosensor. However, future work will include evaluation of the effect of interference and the biosensor response in real samples such as serum and blood. Acknowledgement Financial support from CONICET, FONCYT and SecytUNC is gratefully acknowledged. A. Baruzzi and F. Garay are permanent research fellows of CONICET. References [1] J. Bakker, P. Gris, M. Coffernils, R.J. Kahn, J.L. Vincent, Serial blood lactate levels can predict the development of multiple organ failure following septic shock, Am. J. Surg. 171 (1996) 221–226. [2] F.K. Sartain, X. Yang, C.R. Lowe, Holographic lactate sensor, Anal. Chem. 78 (2006) 5664–5670. [3] J. Wang, Q. Chen, Enzyme microelectrode array strips for glucose and lactate, Anal. Chem. 66 (1994) 1007–1011. [4] J.M. Laval, C. Bourdillon, J. Moiroux, Enzymatic electrocatalysis: electrochemical regeneration of NAD+ with immobilized lactate dehydrogenase modified electrodes, J. Am. Chem. Soc. 106 (1984) 4701–4706. [5] Q. Yang, P. Atanasov, E. Wilkins, Needle-type lactate biosensor, Biosens. Bioelectron. 14 (1999) 203–210. [6] K. Wang, J.J. Xu, H.Y. Chen, Biocomposite of cobalt phthalocyanine and lactate oxidase for lactate biosensing with MnO2 nanoparticles as an eliminator of ascorbic acid interference, Sens. Actuators B: Chem. 114 (2006) 1052–1058. [7] S. Sumana, R. Singhal, A.L. Sharma, B.D. Malthotra, C.S. Pundir, Development of a lactate biosensor based on conducting copolymer bound lactate oxidase, Sens. Actuators B: Chem. 107 (2005) 768–772. [8] M. Gerard, K. Ramanathan, A. Chaubey, B.D. Malhotra, Immobilization of lactate dehydrogenase on electrochemically prepared polyaniline films, Electroanalysis 11 (1999) 450–452. [9] A. Chaubey, M. Gerard, R. Singhal, V.S. Singh, B.D. Malhotra, Immobilization of lactate dehydrogenase on electrochemically prepared

M.R. Romero et al. / Sensors and Actuators B 131 (2008) 590–595

[10]

[11] [12] [13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24]

[25]

[26]

polypyrrole-polyvinylsulphonate composite films for application to lactate biosensors, Electrochim. Acta 46 (2000) 723–729. V.G. Gavalas, N.A. Chaniotakis, Polyelectrolyte stabilized oxidase based biosensors: effect of diethylaminoethyl-dextran on the stabilization of glucose and lactate oxidases into porous conductive carbon, Anal. Chim. Acta 404 (2000) 67–73. T.D. Gibson, J.R. Woodward, in: P.G. Eldman, J. Wang (Eds.), Biosensors and Chemical Sensors, ACS Books, 1992, p. 40. G.F. Khan, W. Wernet, Design of enzyme electrodes for extended use and storage life, Anal. Chem. 69 (1997) 2682–2687. C. Saby, F. Mizutani, S. Yabuki, Glucose sensor based on carbon paste electrode incorporating poly(ethylene glycol)-modified glucose oxidase and various mediators, Anal. Chim. Acta 304 (1995) 33–39. L. Doretti, D. Ferrara, P. Gattolin, S. Lora, Amperometric biosensor with physically immobilized glucose oxidase on a PVA cryogel membrane, Talanta 44 (1997) 859–866. H. Li, Z. Guo, H. Wang, D. Cui, X. Cai, An amperometric bienzyme biosensor for rapid measurement of alanine aminotransferase in whole blood, Sens. Actuators B: Chem. 119 (2006) 419–424. F. Palmisano, G.E. De Benedetto, C.G. Zambonin, Lactate amperometric biosensor based on an electrosynthesized bilayer film with covalently immobilized enzyme, Analyst 122 (1997) 365–369. B. Wang, B. Li, Q. Deng, S. Dong, Amperometric glucose biosensor based on sol–gel organic–inorganic hybrid material, Anal. Chem. 70 (1998) 3170–3174. S. Sampath, O. Lev, Renewable, reagentless glucose sensor based on a redox modified enzyme and carbon–silica composite, Electroanalysis 8 (1996) 1112–1116. B. Liu, R. Hu, J. Deng, Characterization of immobilization of an enzyme in a modified Y zeolite matrix and its application to an amperometric glucose biosensor, Anal. Chem. 69 (1997) 2343–2348. J. Wang, N. Naser, Improved performance of carbon paste amperometric biosensors through the incorporation of fumed silica, Electroanalysis 6 (1994) 571–575. J. Qian, Y. Liu, H. Liu, T. Yu, J. Deng, An amperometric new methylene blue N-mediating sensor for hydrogen peroxide based on regenerated silk fibroin as an immobilization matrix for peroxidase, Anal. Biochem. 236 (1996) 208–214. J.J. Gooding, V.G. Praig, E.A.H. Hall, Platinum-catalyzed enzyme electrodes immobilized on gold using self-assembled layers, Anal. Chem. 70 (1998) 2396–2402. J.J. Gooding, E.A.H. Hall, A fill-and-flow biosensor, Anal. Chem. 70 (1998) 3131–3136. S.A.M. Marzouk, V.V. Cosofret, R.P. Buck, H. Yang, W.E. Cascio, S.S.M. Hassan, A Conducting salt-based amperometric biosensor for measurement of extracellular lactate accumulation in ischemic myocardium, Anal. Chem. 69 (1997) 2646–2652. A. Lindgren, J. Emn´eus, T. Ruzgas, L. Gorton, G.M. Marko-Varga, Amperometric detection of phenols using peroxidase-modified graphite electrodes, Anal. Chim. Acta 347 (1997) 51–62. J. Svitel, M. Stredansky, A. Pizzarielo, S. Miertus, Composite biosensor for sulfite assay: use of water-insoluble hexacyanoferrate(III) salts as electrontransfer mediators, Electroanalysis 10 (1998) 591–596.

595

[27] A. Mueller, Enzyme electrodes for medical sensors, Mini-Rev. Med. Chem. 5 (2005) 231–239. [28] X. Chen, Y. Hu, G.S. Wilson, Glucose microbiosensor based on alumina sol–gel matrix/electropolymerized composite membrane, Biosens. Bioelectron. 17 (2002) 1005–1013. [29] R.H. Capra, M. Strumia, P.M. Vadgama, A.M. Baruzzi, Mucin/carbopol matrix to immobilize oxalate oxidase in a urine oxalate amperometric biosensor, Anal. Chim. Acta 530 (2005) 49–54. [30] R.H. Capra, A.M. Baruzzi, L.M. Quinzani, M.C. Strumia, Rheological, dielectric and diffusion analysis of mucin/carbopol matrices used in amperometric biosensors, Sens. Actuators B 124 (2007) 466–476. [31] B. Krajewska, Application of chitin- and chitosan-based materials for enzyme immobilizations: a review, Enzyme Microb. Technol. 35 (2004) 126–139. [32] Y. Liu, X. Qu, H. Guo, H. Chen, B. Liu, S. Dong, Facile preparation of amperometric lactate biosensor with multifunction based on the matrix of carbon nanotubes–chitosan composite, Biosens. Bioelectron. 21 (2006) 2195–2201. [33] J. Perez-Vilar, R.L. Hill, The structure and assembly of secreted mucins, J. Biol. Chem. 274 (1999) 31751–33175. [34] R. Bansil, B.S. Turner, Mucin structure, aggregation, physiological functions and biomedical applications, Curr. Opin. Colloid Interf. Sci. 11 (2006) 164–170. [35] N.A. Peppas, Y. Huang, Nanoscale technology of mucoadhesive interactions, Adv. Drug Deliv. Rev. 56 (2004) 1675–1687. [36] Y. Hu, Y. Zhang, G.S. Wilson, A needle-type enzyme-based lactate sensor for in vivo monitoring, Anal. Chim. Acta 281 (1993) 503–511. [37] G. Urban, G. Jobst, E. Aschauer, O. Tilado, P. Svasek, M. Varahram, Performance of integrated glucose and lactate thin-film microbiosensors for clinical analysers, Sens. Actuators B 19 (1994) 592–596. [38] S. Ghisla, V. Massey, Y.S. Choongs, Covalent adducts of lactate oxidase, J. Biol. Chem. 254 (21) (1979) 10662–10669.

Biographies Marcelo Ricardo Romero, Biochemist (Universidad Nacional de C´ordoba, 2001). Currently he is a PhD student at the Department of Physical Chemistry, School of Chemical Science, Universidad Nacional de C´ordoba. His fields of interest include electrochemistry, analytical chemistry, polymer science, electronic devices and biosensors. Fernando Sebasti´an Garay, Doctor (Universidad Nacional de C´ordoba, 2002). Currently he is an assistant professor at the Department of Physical Chemistry, School of Chemical Science, Universidad Nacional de C´ordoba and research fellow of CONICET, Argentina. His fields of interest include electrochemistry, numerical and digital simulations, analytical chemistry, in situ techniques, polymer science and biosensors. Ana Mar´ıa Baruzzi, Doctor (Universidad Nacional de C´ordoba, 1981). Currently he is a professor at the Department of Physical Chemistry, School of Chemical Science, Universidad Nacional de C´ordoba and Research Fellow of CONICET, Argentina. His fields of interest include electrochemistry, analytical chemistry, polymer science and biosensors.