Chronoamperometric determination of d -lactate using screen-printed enzyme electrodes

Analytica Chimica Acta 433 (2001) 81–88

Chronoamperometric determination of d-lactate using screen-printed enzyme electrodes Alina Avramescu a,b , Thierry Noguer b , Vasile Magearu a , Jean-Louis Marty b,∗ a

b

Faculty of Chemistry, University of Bucharest, 90-92 Sos. Panduri, Bucharest, Romania Université de Perpignan, Centre de Phytopharmacie, UMR CNRS 5054, 52 avenue de Villeneuve, 66860 Perpignan Cedex, France Received 21 July 2000; received in revised form 11 December 2000; accepted 11 December 2000

Abstract A reagentless biosensor for d-lactate was developed using the screen-printing technology. In a simple design, d-lactate dehydrogenase and NAD+ were deposited onto the surface of a carbon electrode, modified with an insoluble salt of Meldola’s Blue. The detection of d-lactate was performed by chronoamperometry and took advantage of the constant potential oxidation of NADH, formed in the reaction catalysed by d-lactate dehydrogenase. The total of 150 s were necessary for a measurement and linear detection of d-lactate was achieved over the range 0.1–1 mM. Twenty-five microliter sample volumes were required for the assays. The choice of a properly applied potential together with the use of polyethyleneimine–Nafion in the sensing layer allowed to minimise the interferences for the detection of d-lactate in real samples of wine. © 2001 Elsevier Science B.V. All rights reserved. Keywords: NADH oxidation; Meldola’s Blue; Screen-printed electrodes; d-Lactate; Wine

1. Introduction d-Lactate concentration has been shown to be a very important parameter to control in food and wine production, because it is an indicator of a bacterial activity, which may conduct to a drastic alteration of the quality of the final product. In wines, after the alcoholic and during the malo-lactic fermentation, some heterolactic bacteria are able to transform the remaining sugars into acetic acid, d-lactic acid and mannitol [1]. These irreversible transformations, designated by the French term “piqûre lactique”, lead to a bad sour taste of the wine. ∗ Corresponding author. Tel.: +33-468662254; fax: +33-468662223. E-mail address: [email protected] (J.-L. Marty).

The analysis of the d-optical isomer of lactate is seldom performed because it must be carried out in a very specific manner. Amperometric enzyme sensors based on the NAD+ -dependent d-lactate dehydrogenase represent promising tools for the specific and sensitive analysis of d-lactate due to their low cost and the need of simple equipment [2–8]. The main difficulties in the development of NAD+ -dependent dehydrogenase sensors are related to the mandatory addition of the expensive cofactor and to the oxidation of NADH, which requires high overpotentials. In addition, the equilibrium constant for the enzymatic oxidation of lactate at pH 7 is Keq. = 2.7 × 10−6 M [9]. Many attempts to shift the equilibrium towards the product side have been reported based on the use of additional coupled reactions

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 3 8 6 - 6

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(chemical or enzymatic) combined or not with the use of large amounts of NAD+ and alkaline pH [2,5,7,8,10]. Some works have reported the use of extremely expensive NAD+ -analogues, which show a higher oxidising power [11]. We have chosen an alternative solution based on the use of an electron transfer mediator. This approach allows not only to shift the equilibrium of the enzymatic reaction to the product side but also to reduce the overpotential for the oxidation of NADH [12–15]. This work presents the modification of a screenprinted carbon electrode with an insoluble salt of Meldola’s Blue (MBRS), a phenoxazine dye having a fast rate of electron transfer with NADH [16–23]. The sensor was characterised and further developed to produce a low cost biosensor for d-lactate based on the association of screen-printing technology and chronoamperometry. As the construction of reusable biosensors by immobilising the low-weight cofactor was shown to be a very difficult task [24,25], the problems related to NAD+ were overcome in this work by the simple design of the sensors: disposable biosensors for d-lactate were obtained by direct deposition of small amounts of d-lactate dehydrogenase and NAD+ on the planar surface of a mediator-incorporating carbon electrode. The final goal was to apply the biosensor for the detection of d-lactate in wines, the operating conditions being optimised in order to minimise interferences due to easily oxidable compounds, present in wines.

2. General principles The detection of d-lactate is based on a three-step sequence: in a first step, d-lactate is transformed in pyruvate by d-lactate dehydrogenase, with concomitant reduction of the cofactor NAD+ : + D-lactate + NAD

 pyruvate + NADH + H+

The oxidised form of the electron transfer mediator reacts with NADH as follows: Medox + NADH → NAD+ + H+ Medred + 2e− In the final step, the mediator is electrochemically reoxidised at a proper potential E>E

◦0

Medred → Medox + 2e− + H+

A simple two-electrode system was used in the assays with the MBRS-modified carbon electrode being the working electrode and the Ag/AgCl electrode playing the role of both reference and counter electrode. This simplification of the classic three-electrode configuration required in amperometry experiments was made possible by the negligible ohmic drop expected for the described screen-printed two-electrode system. Additionally, the screen-printed Ag/AgCl electrode is behaving as a reliable “reference” electrode, providing the concentration of Cl− is kept constant during the measurements. The zero current potential of the screen-printed Ag/AgCl electrode in 0.05 M phosphate buffer pH 7 containing 0.1 M KCl was established as 42 ± 3 mV versus SCE (n = 5), in accordance with other values reported in the literature [23,26]. 3. Experimental 3.1. Materials and reagents Meldola’s Blue, polyethyleneimine, d-lactic acid, d-lactate dehydrogenase (E.C. 1.1.1.28) from Lactobacillus leichmanii (92 IU/mg solid) and ␤-NADH were obtained from Sigma. ␤-NAD+ and the d/llactic acid kit were obtained from Böhringer Mannheim. Reinecke’s salt (ammonium tetrarhodanatodiaminechromate III) and hydroxyethylcellulose were supplied by Fluka. Nafion (perfluorinated ion-exchange resin, 5% solution in lower alcohols/ water) was obtained from Aldrich. The inks for the fabrication of screen-printed electrodes were provided by Acheson France: silver ink (Electrodag PF 410); graphite ink (Electrodag 423 SS); silver/silver chloride ink (Electrodag 6037 SS). A chemically modified ink was prepared in the laboratory by mixing 23 ml of hydroxyethylcellulose (3% w/v) with 3.5 g of graphite (Timrex T15 graphite, provided by Timcal, Switzerland) and 140 mg of an insolubilized salt of the mediator Meldola’s Blue. This salt was obtained by precipitating Meldola’s Blue with Reinecke’s salt (by mixing equimolar solutions in the ratio 1:1 v/v), according to the method reported by Schuhmann et al. [22]. Clear PVC sheets (200 mm × 100 mm, 0.5 mm thick, supplied by SKK, Germany) were used as the

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support for the screen-printing electrodes. A commercial acrylic paint was used as insulating ink. The supporting electrolytes used in this work were 0.1 M pyrophosphate buffer pH 8.5 containing 0.1 M KCl and a Sörensen phosphate buffer 0.05 M pH 7 with 0.1 M KCl. All lactate and NADH solutions were prepared immediately before use and stored on ice. 3.2. Apparatus A DEK 248 screen-printing system (produced by DEK, UK) was used to fabricate the electrodes. Cyclic voltammetry experiments were carried out using a scanning potentiostat model 362 provided by EG&G Princeton Applied Research, USA, connected to a LY 1400 x–y plotter. Chronoamperometry studies were performed using a Metrohm potentiostat (641 VA Detector). A PC equipped with a data acquisition and treatment software (Pico Solutions, USA) served to record the signal generated in the electrochemical cell.

83

Fig. 1. Schematic representation of the screen-printed twoelectrode system.

For the chronoamperometric studies the sensing layer of the working electrodes was prepared by simply depositing 2 ␮l of a mixture containing d-lactate dehydrogenase (2.7 mg/ml), ␤-NAD+ (50 mg/ml), polyethyleneimine (0.5%) and Nafion (0.05%) on the working area of the electrodes. The sensors were left to dry overnight. The two-electrode screen-printed systems were cut from the sheet and inserted in a laboratory-made electrode holder. 3.4. Voltammetric studies

3.3. Fabrication of screen-printed electrodes 3.4.1. Cyclic voltammetry The electrodes were prepared in five steps, by screen-printing different consecutive layers on clear PVC sheets (64-electrodes/sheet, corresponding to 32 working and 32 reference electrodes). The fabrication procedure and the design of the electrodes were similar to those described by Kulys and Costa [26]. Stainless steel screens with a screen mesh size of 200 counts per inch were used to print the electrodes. A first conducting track was deposed using silverloaded ink. A graphite layer was deposed to cover the bottom part of the silver track. The role of this layer is to avoid that electrochemical reactions take place on the silver-conducting track. The third layer consisted in silver/silver chloride ink for the reference/counter electrode and Meldola’s Blue modified graphite ink for the working electrode. This layer allowed to define a geometric working area of 8.5 mm × 2 mm. Finally, an insulating ink was deposited by screenprinting a commercial paint in order to leave uncovered a 9 mm long portion of the silver track, necessary for the electrical contact (Fig. 1). After each screen-printing step, the strips were cured for 20 min in an oven at 50◦ C.

3.4.1.1. Electrochemical oxidation of NADH. The possibility of using MBRS-modified electrodes as sensors for NADH was investigated by cyclic voltammetry. In this experiment an MBRS-modified screen-printed electrode served as the working electrode, a platinum electrode was the counter electrode and the saturated calomel electrode (SCE) was used as reference. The potential was scanned between −0.4 and 0.4 V at a scan rate of 20 mV/s. Voltammograms were recorded in 50 mM phosphate buffer pH 7 containing 0.1 M KCl and in a 5 mM NADH solution prepared in the same buffer, respectively. 3.4.1.2. Oxidation of interfering compounds from wine samples. Cyclic voltammetry was also used to observe the electrochemical interferences on the proposed sensor, which may be originating from wine samples. For this purpose, voltammograms were recorded for three different wine solutions using the same three-electrode configuration as described above. These solutions were 10-fold dilutions in 0.2 M pyrophosphate buffer pH 8.5 of either model

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wine solution [27], untreated wine or wine previously treated with polyvinylpolypyrolidone (PVPP). In these series of experiments the potential was scanned between −0.5 and 0.5 V versus SCE at a scan rate of 2 mV/s. The obtained voltammograms were compared to those registered in plain buffer. 3.4.2. Chronoamperometry 3.4.2.1. NADH sensor. In a first approach, chronoamperometry was used to evaluate the potential of MBRS-modified screen-printed electrodes as sensors for NADH. The two-electrode screen-printed strips were supported horizontally during the measurements. At time zero, 25 ␮l of sample were deposited on the electrodes in such a manner that the liquid area was covering both the electrodes. After 30 s, a difference of 100 mV was imposed between the working and the Ag/AgCl electrode. Measurements of currents were made after another 30 s. 3.4.2.2. d-Lactate biosensor. Prior to experiments the biosensors were optimised with respect of operating pH, buffer composition and enzyme/NAD+ loading. A 120 s delay was observed between depositing the samples and imposing the potential in order to allow the enzymatic conversion of d-lactate. The working electrode was poised at −50 mV versus screen-printed Ag/AgCl electrode. Polyethyleneimine and Nafion

were included in the sensing layer of d-lactate biosensor to prevent interfering compounds present in wine samples from reaching the electrode. The intensity of the current was registered 30 s after setting the potential. Finally, the developed biosensor was applied to the analysis of d-lactate in real samples of wine. The results were compared to those obtained using the Böhringer enzyme-spectrophotometric kit, which is based on the official method of the International Organisation of Wine (OIV, [28]). The ambient temperature during all experiments was 25◦ C. The results were expressed as mean of triplicate assays.

4. Results and discussion 4.1. Electrochemical oxidation of NADH Cyclic voltammograms show that the peak for the oxidation of MBRS on the screen-printed electrodes corresponds to −125 mV versus SCE (Fig. 2a). In the presence of NADH, an important increase in the current was observed starting at the peak potential, which is specific for the oxidation of MBRS (Fig. 2b). This emphasises the electrocatalytic current due to electron shuttling by MBRS from NADH to the electrode surface. The oxidation of NADH mediated by MBRS takes place at a very low potential (the decrease in the overpotential for the oxidation of NADH being about

Fig. 2. Cyclic voltammetry of MBRS-modified screen-printed electrodes in 0.05 M phosphate buffer pH 7 containing 0.1 M KCl (a) and in a 5 mM NADH solution prepared with the same buffer (b). The potential was scanned between −400 and +400 mV vs. SCE at a scan rate of 20 mV/s.

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0.7 V) and became diffusion-limited at about −50 mV versus SCE (Fig. 2b).

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The overall coefficient of variation for the determination of d-lactate using the biosensor was 10.8% (n = 30). This coefficient was found to be mainly induced by the screen-printing procedure, differences in the responses of the sensors being caused by small differences in area or width of the modified graphite layer. Reports concerning biosensors for d-lactate are rather rare in the literature (Table 1) and most of the procedures rely on the use of two-enzyme systems [2,5,7,8,10]. To the author’s knowledge, this is the first description of a biosensor for d-lactate based on a screen-printed carbon electrode. The sensitivity of the biosensor described in this work is better compared to other d-lactate mono-enzyme methods [3,4,6]. Although the use of electron transfer mediators providing fast kinetics with NADH is expected to displace the equilibrium of the enzymatic reaction towards the production of pyruvate, this effect is not so pronounced as in the case of two-enzyme methods or for enzyme reactors integrated in FIA systems [2–5,7,8,10]. These latter approaches allow a more efficient conversion of d-lactate and thus, the achievement of lower detection limits, up to 5 × 10−6 M (Table 1). Over these two methods, the described biosensor has the major advantages of the simplicity and low cost. When comparing the calibration data for the d-lactate biosensor with the characteristics of the basic NADH sensor it becomes obvious that the linear range

4.2. NADH sensor The detection limit for NADH using the MBRSmodified sensor was 0.01 mM (signal/noise = 5/2). Sensitive detection of NADH was achieved for concentrations ranging from 0.02 to 1 mM: I (nA) = 36.02 + 2026.3 [NADH](mM) (r 2 = 0.9955). The coefficient of variation for the determination of NADH in these conditions was 6.5% (n = 30). Incorporation of an insoluble salt of Meldola’s Blue in the screen-printed carbon electrode conducted to NADH sensors, which are suitable for repetitive assays with negligible contamination of the sample. However, the aim of our work being the construction of reagentless and easy-to-use d-lactate sensors and by considering their low price and good reproducibility, the sensors were developed in this work as disposable devices. 4.3. d-Lactate biosensor The calibration plot for d-lactate in 0.1 M pyrophosphate buffer pH 8.5 containing 0.1 M KCl shows linearity in the range from 0.1 to 1 mM (r 2 = 0.9955), with a detection limit of 0.05 mM (signal/noise = 5/2): I (nA) = 39.94 + 207.02 [d-lactate] (mM).

Table 1 Characteristics of some enzyme-based methods for the detection of d-lactate reported in the literature Reference

Detection limit (mM)

Linear range (mM)

Sensitivity (nA/mM)

Response time (s)

Biocatalyst

Observations

[26]

3 × 10−3

3 × 10−3 –3.6

–

∼1200

d-LDH + GPTb in solution

[3,4]

5 × 10−3

1 × 10−3 –4 × 10−1

70a

–

[2]

5 × 10−3

5 × 10−3 –1.5 × 10−1

200, 900

60, 180

[27] [7]

– 2 × 10−2 , 1 × 10−2 3 × 10−2

5 × 10−3 –1.5 × 10−3 2 × 10−2 –1 × 10−2 , 1 × 10−2 –1 × 10−2 5 × 10−2 –5 × 10−2

80–320 500

120–300 180, 120

70

180

d-LDH immobilised in solution d-LDH + DPc immobilised in solution d-LDH + DP in solution d-LDH + NADHox d immobilised In solution d-LDH immobilised

Spectrophoto-metric, reference method FIA, enzyme reactor

[6] a

Deducted from the calibration curve. Glutamate-pyruvate-transaminase. c Diaphorase. d NADH oxidase. b

Biosensor Miniaturised biosensor Biosensor FIA, reagentless biosensor

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for the determination of d-lactate is not restricted by NADH detection, but by the enzymatic reaction. Increasing the amount of cofactor/electrode did not lead to a broader linear range. Similar results were reported in the case of llactate dehydrogenase-based screen-printed sensors [23,29,30]. For d-lactate detection, larger linear ranges were obtained only by coupling d-lactate dehydrogenase with pyruvate-glutamate transaminase, NADH oxidase or diaphorase [2,5,7,8,10,28] or by using FIA methods with d-lactate dehydrogenase immobilised in biosensors [6] or enzyme reactors [3,4]. Despite its relatively narrow linear range, the biosensor described in this work appears suitable for applications like the early detection of “piqûre lactique” in wine samples. After 2 weeks of storage at 4◦ C, the d-lactate biosensors showed 75% of their initial activity. Consequently, all the measurements with the biosensors were done within a week from the fabrication.

4.4. Analysis of d-lactate in wine samples Before performing measurements of the concentration of d-lactate in wines, we considered necessary to emphasize the electrochemical interferences in real samples. In fact, significant differences were observed between the cyclic voltammograms obtained using MBRS-modified electrodes in electrolytes based on wine solutions treated or not with polyvinylpolypyrolidone (PVPP, Fig. 3a and b). As PVPP is widely used to remove coloured (phenolic) compounds from wine and fruit juices samples (27), these differences seem to be related to the coloured (phenolic) compounds, which are easily oxidable on the working electrode. This hypothesis is sustained by the voltammogram obtained for a model solution containing all major substances normally present in wine, except the phenolic compounds (Fig. 3c). The voltammogram registered in this solution is practically identical to that recorded in wine previously treated with PVPP.

Fig. 3. Cyclic voltammograms for the MBRS-modified screen-printed electrodes in red wine treated with PVPP (a); red wine (b); “model” wine solution (c). All solutions were 10-fold dilutions in 0.2 M pyrophosphate buffer pH 8.5. The potential was scanned between −500 and 500 mV vs. SCE at a scan rate of 2 mV/s.

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Wines are generally containing up to 3.5 g/l phenolic compounds (expressed as gallic acid). As illustrated by the voltammogram obtained for a red wine diluted 10-fold (Fig. 3b), the oxidation of the phenolic compounds on the MBRS-modified electrode begins at approximately −100 mV versus SCE. A slow increase in the oxidation current is observed from −100 to 0 mV versus SCE, followed by a significant increase in the range 0–120 mV versus SCE. Because of this behaviour, the phenolic compounds are supposed to interfere in NADH detection using the developed MBRS-modified sensor. Gillis et al. [8] reported the same problem, aberrant results being obtained for red wine samples, while white wine matrices caused minor problems. In this work, the problem was solved by the combination between a negative operating potential, a dilution factor for the wine samples higher than 10 and the inclusion of a polymeric membrane in the sensing layer. The validity of this approach for eliminating the electrochemical interferences was verified by chronoamperometry. Unmodified MBRS-sensors were compared to d-lactate biosensors regarding the response towards 0.01 mM NADH, 1 mM NADH and 175 mg/l gallic acid (Fig. 4). The electrolyte was 0.2 M pyrophosphate buffer pH 8.5 containing 0.1 M KCl, the potential was applied 30 s after depositing the sample and the intensity of the current was regis-

87

tered after another 30 s. The operating potential was set at −50 mV versus screen-printed Ag/AgCl for the d-lactate biosensor (working conditions “A”) and at +50 mV versus screen-printed Ag/AgCl for the MBRS-sensor (working conditions “B”). As shown in Fig. 4, an unmodified MBRS-sensor has a very good sensitivity towards NADH but the response for 175 mg/l gallic acid is higher than that for 1 mM NADH. The response of d-lactate biosensor operating at a low negative potential was not influenced by gallic acid even that the sensitivity towards NADH decreased because of the additional diffusion layer. The results obtained for the detection of d-lactate in wine samples emphasise a good agreement with the Böhringer kit for usual concentrations of d-lactate (Table 2). However, for a low concentration tested (cf. red wine 3, Table 2), a significant difference was observed between the results obtained according to the two methods. This discrepancy may be due the fact that such a concentration is close to the limit of the calibration range. The experimental data suggest that other approaches should be considered, for example, by using the standard addition method in order to perform accurate determinations of less than 1 mM d-lactate with the biosensor. Bearing in mind that concentrations greater than 500 mg/l d-lactate (about 5.5 mM) are considered by oenologists as clear signs of the imminence of “piqûre

Table 2 Results obtained for the detection of d-lactate in real samples of wine with the described biosensora Sample

[d-lactate] (mM)b Biosensor

Fig. 4. Quantitative evaluation of electrochemical interferences due to phenolic compounds from wine samples in different working conditions: response of the MBRS: (䊐) sensor for 175 mg/l gallic acid; (䊏) 0.01 mM NADH; ( ) 1 mM NADH (other explanations are given in the text).

Red wine 1 Red wine 2 Red wine 3 Red wine 4 Red wine 5 Red wine 6 Red wine 7 Red wine 8 White wine1

3.7 3.1 0.9 3.0 3.4 2.9 2.2 2.0 1.5

± ± ± ± ± ± ± ± ±

0.4 0.3 0.1 0.4 0.6 0.3 0.1 0.2 0.1

Reference method 3.9 3.5 0.5 3.1 3.3 3.2 2.3 2.0 1.4

± ± ± ± ± ± ± ± ±

0.3 0.2 0.0 0.2 0.5 0.2 0.1 0.5 0.1

a Comparison with values achieved using the reference enzyme-spectrophotometric method. b Mean of n = 3 assays.

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lactique”, the described biosensor seems suitable for the early detection of this microbial accident. The biosensor described in this report has several advantages over the reference spectrophotometric method like the lower price, the simplicity of the procedure and the equipment, as well as the short time required for one assay. It must be also pointed out that the biosensor is a one-enzyme sensor, while the OIV method relies on the use of two coupled enzyme reactions to perform detection of d-lactate. 5. Conclusions Cheap, reliable and sensitive sensors for NADH were produced by screen-printing. These sensors were carbon electrodes incorporating an insoluble salt of Meldola’s Blue, allowing detection of NADH at very low potentials. The NADH sensors have a great potential as the basis for biosensors relying on NADH producing enzymatic reactions. In this work, the development of the sensors into reagentless, disposable biosensors for d-lactate has conducted to linear detection of d-lactate over the range 0.1–1 mM. The price for a d-lactate assay using the biosensor was estimated at 0.6 FF. By its simplicity, the biosensor seems ideal for non-specialist users, for example for a rapid checking of the activity of heterolactic bacteria during wine production. However, further optimisation of the biosensor should be carried out in order to achieve a better sensitivity. For example, a change in the design, by varying the order of deposition of different active elements (mediator, enzyme, cofactor and additives) could appear as very helpful. Additionally, the improvement of the storage stability of the described biosensor is currently under test, short-term studies indicating the potential efficiency of stabilisers like PVA, trehalose, DEAE-dextran or lactitol.

Acknowledgements The authors wish to thank Professor Maurice Comtat (Université Paul Sabatier, Toulouse, France) for helpful discussions.

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