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Materials Science and Engineering C 28 (2008) 872 875 www.elsevier.com/locate/msec

Conductometric biosensor based on glucose oxidase and beta-galactosidase for specific lactose determination in milk
Mouna Marrakchi a , Sergei V. Dzyadevych c , Florence Lagarde b , Claude Martelet a , Nicole Jaffrezic-Renault b,
a CEGELY, UMR-CNRS 5005, Ecole Centrale de Lyon, 36, Avenue Guy de Collongue, 69134 Ecully Cedex, France Universit de Lyon, Laboratoire de Sciences Analytiques, UMR CNRS 5180, CNRS-Universit Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, 150 Zabolotnogo Street, Kiev 03143, Ukraine b

Available online 13 October 2007

Abstract This paper investigates the development of a biosensor associating two distinct enzymatic activities, that of the beta-galactosidase and that of the glucose oxidase, in order to apply it for the quantitative detection of lactose in milk. To eliminate interferences with glucose, a differential mode of measurement was used. Results show a linear calibration curve for lactose concentration between 60 and 800 M (0.03 to 0.3 g/L). Tests with real commercial milk samples were carried out to validate the conductometric biosensor. 2007 Elsevier B.V. All rights reserved.
Keywords: Lactose; Biosensor; Conductometric electrodes; Milk; Glucose oxidase; Beta-galactosidase

1. Introduction Lactose is not only the carbohydrate constituting an important source of energy but it also helps the body to absorb rock salt. Unfortunately, some people have intolerance to lactose. This lactose intolerance is caused by a shortage of the enzyme lactase (beta-galactosidase), which is produced by the cells that line the small intestine [1]. Thus, it is interesting to have powerful tools to real-time monitoring of lactose concentration in milk during the production process (in particular for milk with a low content in lactose intended for people intolerant to this sugar). Several methods have been described before, for lactose determination, using different classical analytical methods as enzymatic [2], titrimetric [3], chromatographic [4,5] and spectrometric methods [6]. Many of these methods are expensive, timeconsuming and/or are not in adequacy with real-time measurement in industries. In opposition, biosensors offer considerable
Corresponding author. Tel.: +33 472431182; fax: +33 472431206. E-mail addresses: mounamarrakchi@yahoo.fr (M. Marrakchi), nicole.jaffrezic@univ-lyon1.fr (N. Jaffrezic-Renault). 0928-4931/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2007.10.046

promises for attaining the analytic information in a faster, simpler and cheaper manner compared to conventional assays [7]. Different kinds of lactose biosensors have been previously described. Most of them are based on amperometric electrodes with different immobilized enzymes: galactose oxidase and galactosidase in Langmuir-Blodgett films [8]; horseradish peroxidase, glucose oxidase and -galactosidase in Nafion films [9]; glucose oxidase, -galactosidase and mutarotase in cyclodextrin [10]. However, in spite of the advantages they offer [11] there is no biosensor based on conductometric electrodes for lactose determination. Thus, in this work we were interested in the development of a bi-enzymatic biosensor using glucose oxydase (GOD) and beta-galactosidase activities for lactose determination. 2. Experimental 2.1. Materials Glucose oxidase (GOD) (130 U/mg) is a generous gift of the Laboratory of Biomolecular Electronics, IMBG, Kiev, Ukraine. Bovine serum albumin (BSA), lactose, glucose and

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glutaraldehyde (grade II, 25% in aqueous solution) were purchased from Sigma. 2.2. Sensor design The lactose biosensor is developed on the basis of a thin-film conductometric sensor. This device is a miniature two-electrode cell for the measurement of the resistance of the thin layer immediately near the electrode surface [12]. These two identical pairs of gold interdigitated electrodes (thickness 0.5 mm, dimensions 5 30 mm) were produced by vacuum deposition on a ceramic substrate at the Institute of Semiconductors Physics, Kiev, Ukraine [13]. 2.3. Immobilization of the enzymes The immobilisation of glucose oxidase (GOD) and galactosidase (-gal) was carried out in two steps (see Fig. 1): 1. 0.2 L of a mixture A (5% GOD (w/w), 5% BSA and 10% glycerol in 20 mM phosphate buffer pH 7.4) was deposited on the sensitive part of both work and reference electrodes. Then, the sensor was exposed to glutaraldehyde vapour for 30 min to co-reticulate enzyme with BSA. 2. 0.2 L of a mixture B (5% -gal (w/w), 5% BSA and 10% glycerol in 20 mM phosphate buffer pH 7.4) was deposited on the sensitive part of the work electrode (already containing dried GOD membrane). On reference electrode, 0.2 L of mixture C (10% BSA (w/w) and 10% glycerol in 20 mM phosphate buffer pH 7.4) was deposited. As for the first step, electrodes were then exposed to glutaraldehyde vapour for 30 min. Biosensors were stored, between experiments, at 4 C, in 5 mM phosphate buffer solution, pH 7.4. 2.4. Preparation of milk samples Different aliquots of commercial milks (1 mL) were distributed in microcentrifuge tubes and centrifuged. Both tube
Fig. 2. Time evolution of biosensor response (conductance variation) after 0.4 mM lactose addition in measurement cell.

pellet (insoluble proteins) and supernatant (milk fats) were eliminated. The clarified part of milk is diluted twice and preserved for lactose determination using the biosensor. 2.5. Measurements For measurements, sinusoidal wave of 100 KHz frequency with small-amplitude alternating voltage (10 mV peak-to-peak about 0 V) was generated to reduce Faradaic processes, doublelayer charging and concentration polarization at the sensor surface. Conductometric measurements were conducted in magneticstirred cell filled with 5 mM phosphate buffer pH 7.4 at room temperature. After stabilisation of the output signal, defined volumes of a lactose concentrated solution were added into the measurement cell to reach the final substrate concentration. The differential output signal (between reference electrode and working electrode) was recorded using a Stanford Research Systems Lock-in amplifier SR510. Thus, the output of the lock-in amplifier was directly proportional to the cell conductance and

Fig. 1. Schematic representation of the different steps of the enzymes immobilization on the gold interdigitated microelectrodes.

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Fig. 3. Dependence of biosensor response (S) on lactose concentration (in 5 mM PBS buffer, pH 7.4). a: curve of response with saturation effect beginning for 1 mM lactose. b: linear calibration curve (R2 = 0.988) for lactose concentration between 60 and 800 M (0.03 to 0.3 g/L).

the responses of the biosensor were recorded as a function of the final lactose concentration in the cell. 3. Results and discussion 3.1. Lactose determination Biosensor response as a function of time, after 0.4 mM lactose addition is shown in Fig. 2. The steady-state response (after signal stabilisation) is reached after at least 4 min. This response time is in adequacy with real-time measurements, in comparison with other works describing time consuming biosensors for lactose determination (construction of biosensor needing about 35 h [9], time-response about 1 h and baseline recovered after 2 h [14]). Calibration curve is presented in Fig. 3. The biosensor showed a linear response for an interval of lactose concentration between 30 and 600 M (0.01 and 0.6 g/L). The detection

Fig. 5. Comparison of signal evolution after addition of 0.4 mM lactose and 0.4 mM glucose with: a: biosensor without GOD on reference electrode. b: biosensor with GOD on reference electrode.

limit of this conductometric biosensor is much lower than those obtained with biosensors developed by Sharma et al. [8] and Amarita et al. [14]. 3.2. Study of biosensor operational stability The operational stability of the conductometric biosensor for different lactose concentrations was studied (Fig. 4). We observed a significant response decreasing from the fifth day of biosensor exploitation for the highest lactose concentrations (28.5% loss of signal between the first day and the fifth day of measurements). However for the lowest lactose concentrations (in the linear range of response), there is a negligible variation of the biosensor response. 3.3. Interferences with glucose As presented in Section 3.3, glucose oxidase was immobilized on both work and reference electrodes to select only the effect of conductance variation due to lactose hydrolysis. Thus, glucose effect must be normally cancelled by the use of the

Fig. 4. Results of the study of the operational stability of the biosensor during 5 days (in 5 mM PBS buffer, pH 7.4, for lactose concentrations from 0 to 2 mM).

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by the biosensor (after taking into account dilutions) were 59.4 g/L and 64.9 g/L of lactose which is slightly higher than the values given by the provider: about 50 g/L. This can be allotted to the concentration of milk (not taken into account) obtained after the preparation procedure of milk samples described in the Section 2.4. 4. Conclusion The conductometric biosensor developed for lactose determination showed the effectiveness of the association between the enzymatic activities of beta-galactosidase and glucose oxidase to obtain an exploitable conductometric signal. This biosensor presents a linear range of response between 30 and 600 M. Following the injection of lactose, the stabilization of the output signal (conductance) is reached after 4 min. Thus, this biosensor can be used for real time measurements in comparison with other biosensors developed for lactose determination in milk (which requires more than 50 min for analysis [14]). In regard to the stability of the biosensor, it is recommended to change the enzymatic membranes every week. This is not a problem, considering the simplicity of the immobilisation procedure of these enzymatic membranes. The determination of lactose in commercial milk samples using the developed biosensor shows its performance for the measurement of the quantity of this disaccharide. References
[1] Lactose intolerance, national digestive diseases information clearinghouse, NIH Publication No. 062751, 2006 March, http://digestive.niddk.nih. gov/ddiseases/pubs/lactoseintolerance/index.htm. [2] B. Mattiasson, B. Danielsson, Carbohydrate research 102 (1982) 273. [3] D. Amin, K.Y. Saleem, W.A. Bashir, Talanta 29 (1982) 624. [4] C. Brons, C. Olieman, Journal of chromatography 259 (1983) 79. [5] J.M. Beebe, R.K. Gilpin, Analytica Chimica Acta 146 (1983) 255. [6] D. Naresingh, V.A. stoute, G. Davis, D. Persad, Analytica Chimica Acta 258 (1992) 141. [7] V. Anjum, C.S. Pundir, Sensors & Transducers 76 (2007) 935. [8] S.K. Sharma, R. Singhal, B.D. Malhotra, N. Sehgal, Biosensors and Bioelectronics 20 (2004) 651. [9] H. Liu, H. Li, T. Ying, K. Sun, D. Qi, Analytica Chimica Acta 358 (1998) 137. [10] H. Liu, T. Ying, K. Sun, H. Li, D. Qi, Analytica Chimica Acta 344 (1997) 187. [11] S.V. Dzyadevych, V.N. Arkhipova, A.V. El'skaya, C. Martelet, N. Jaffrezic-Renault, Current Topics in Analytical Chemistry 2 (2001) 179. [12] S.V. Dzyadevich, A.A. Shul`ga, S.V. Patskovskii, V.N. Arkhipova, A.P. Soldatkin, V.I. Strikha, Russian Journal of Electrochemistry 30 (1994) 982. [13] M. Marrakchi S.V. Dzyadevych P. Namour C. Martelet N. JaffrezicRenault, Analyticals Letters 40 (2007) 1, in press. [14] F. Amrita, C.R. Fernndez, F. Alkorta, Analytica Chimica Acta 349 (1997) 153.

Fig. 6. Determination of lactose concentration corresponding to biosensor responses after addition of two milk samples (A and B) (dilution factor = 1/1000) using linear calibration curve: Milk A: equivalent lactose concentration= 0.0594 g/L 1000. Milk B: equivalent lactose concentration= 0.0649 g/L 1000.

differential mode for measurements. To verify it, a glucose lactose biosensor was prepared according to the steps described in the Section 3.3 with a single difference in step 1 where, on reference electrode, glucose oxidase was replaced by an inactive membrane (10% BSA). Comparison between the responses of the two types of biosensors is presented in Fig. 5. We notice that the use of a GOD free reference electrode (Fig. 5-a) is resulting into a significant response for the glucose in addition to the one for lactose. On the contrary, for the biosensor developed in this work (with immobilized GOD on the reference electrode), a small increase of the conductance followed by a fast fall of the signal was observed after glucose addition (Fig. 5-b). This phenomenon can be allotted to the time of homogenization of glucose for the obtaining of the same concentration of this sugar in direct contact with both electrodes (work and reference electrodes). Thus, the biosensor developed is selective for lactose. 3.4. Lactose determination in milk samples To validate the conductometric biosensor developed for lactose determination in milk, two commercial milk samples (called A and B) were prepared as described in section 2.4 and added to measurement cell (dilution 1/500) to determine biosensor response. By placing conductance values given by the biosensor, in the presence of milk samples, on the linear calibration curve (see Fig. 6), the corresponding lactose concentrations for the samples A and B respectively are: 0.0594 and 0.0649 g/L. Thus, concentrations of commercial milks given