Wassim El Housseini

François Lapicque

Steve Pontvianne

Neus Vilà

Ievgen Mazurenko

Alain Walcarius

Mathieu Etienne
email: mathieu.etienne@cnrs.fr

Dr Etienne Mathieu

Hybrid flow bioreactor with all catalysts immobilized for enzymatic electrosynthesis

Keywords: immobilization, electrochemical regeneration, redox flow, gas diffusion electrode, β-nicotinamide adenine dinucleotide, bioconversion

The electrochemical regeneration of the NADH cofactor was realized in a hybrid flow reactor coupling fuel cell technology and redox flow device, paying attention to the robust immobilization of all catalysts. The rhodium catalyst Rh(Cp * )(bpy)Cl + was covalently immobilized on a MWCNT layer and the association with the gas diffusion electrode was carefully optimized. High stability and activity of the electrochemical system were assessed by cyclic voltammetry and amperometry in the flow reactor. Afterwards, the optimal cofactor regeneration was applied to NADH-dependent biosynthesis using immobilized lactate dehydrogenase for the conversion of pyruvate to lactate in the flow cell in the presence of cofactor concentration as low as 10 µM. 79 % faradaic efficiency was achieved and remarkable total turnover number (TTN) were reached: 2500 and 18000 for the NADH cofactor and the Rh complex, respectively.

Introduction

The catalytic hydrogenation using heterogeneous or homogeneous transition metal catalysts is well set up in industrial fine chemicals synthesis [1] where 10 % to 20 % of chemical steps involves catalytic hydrogenations. [2] Noble metals are highly exploited as catalysts in this area [3,4] . In these processes, high pressures of hydrogen are generally required for the efficient catalytic hydrogenation (>80 bars) [4] . Alternatively, the synthesis of fine chemicals, e.g. the reduction of the "C double bond X" in the ketones, alkenes, and imines, requires the use of biological catalysts, e.g., oxidoreductases (known for their high productivity, enantio-and chemo-selectivity). [5] This particularity is valuable in the chemical and pharmaceutical sectors where 90 % of the chiral molecules were sold as racemic mixture before 1993 [START_REF] Roche | Continuous Regeneration of the NADH Cofactor Catalysed by Formate Dehydrogenase Immobilized in a Filter Press Reactor[END_REF] and 75 % of new drugs in 2002 were single enantiomers. [START_REF] Agranat | [END_REF] However, oxidoreductases applied in bioconversions cannot use molecular hydrogen directly.

Hydrogen can be transferred through enzymatic cofactors such as NADH which plays an important role in biological systems and can be applied as electron carrier in enzymatic systems catalyzing stereo-and regio-selective reactions having a great biotechnological potential. [5,8] The use of enzymatic cofactors in biosynthesis applications (bioconversion) requires their regeneration due to their high costs. [9] The NADH cofactor can be indirectly regenerated in situ by electrochemistry (using suitable redox mediators), which offers an easy separation of products and a high control of the process. [9,10] Other NAD(P)H regeneration techniques can be applied such as the chemical, photochemical, enzymatic, and biological ones. [11][12][13] The application of the electrochemical regeneration of the NAD(P)H cofactor requires the use of an electrochemical mediator in order to lower the cathodic overpotentials associated to the reduction of NAD(P) + , thus preventing the formation of the inactive dimer form (NAD(P))2 occurring at more cathodic potential values. [START_REF] Gorton | Encycl. Electrochem[END_REF] One efficient mediator for the electrochemical regeneration of 1,4-NAD(P)H, the active form of NAD(P)H, is chloro(2,2′-bipyridyl) (pentamethylcyclopentadienyl)-rhodium (III) chloride complex [𝐶𝑝 * 𝑅ℎ(𝑏𝑝𝑦)𝐶𝑙] + . [START_REF] Chenault | [END_REF][16][17] Catalysts applied in the electrochemical or the electroenzymatic regeneration of the NAD(P)H cofactor, either molecular or enzymatic, e.g., the aforementioned rhodium complex or Ferredoxine NADP(+) reductase (FNR), can be immobilized on electrode surfaces, [START_REF] Zhang | Funct. Electrodes Enzym. Microb[END_REF] [START_REF] Cheng | [END_REF] which is important for the easy recovery of the products formed (i.e., no need for separation) and the possible re-use of the electrocatalyst.

There is expanding interest in implementing hydrogenation in continuous flow reactors, where reactants are continuously fed to the reactor and emerge as a continuous stream of products. [START_REF] Irfan | [END_REF]21] Advantages of continuous flow reactors include increased yields of bioconversion due to a high mass transport between reactants and increased selectivity through refined cell design and technology. [22,23] The electrochemical regeneration of the NADH cofactor has been previously reported for biocatalytic hydrogenations and dehydrogenations. [9][24] However, the translation into flow electrochemical reactor has suffered from different constrains such as the reactor design [25] and low conversion efficiencies related to slow kinetics of regeneration of NADH at conventional electrodes [26] . Previously, we have described a new modular route to combine efficiently the oxidation of hydrogen to the regeneration of NADH using a mediator in solution. [27] The idea was to couple the two redox reactions in a hybrid reactor that combines a gas diffusion electrode and a redox flow device with the help of an ion-exchange membrane for protons conduction from the anodic to the cathodic compartment. More precisely, the reactor allows the electrochemical regeneration of the NADH cofactor mediated by a rhodium complex in solution at the cathode with continuous flow reaction, and hydrogen oxidation at the gas diffusion anode. Several parameters were optimized such as hydrogen and solution flow rates (20 mL.min -1 ), pH (7.2), and the molar ratio between NAD + and the rhodium complex. [27] The efficiency of the approach was demonstrated first in H2-driven NADH production, then to supply an NADH-dependent lactate dehydrogenase immobilized by adsorption on multi-walled carbon nanotubes for reduction of pyruvate to lactate. The lactate dehydrogenase is unable to use electrons directly and, therefore, only operated if supplied with reducing equivalents in the form of NADH regenerated by the rhodium complex mediator added in solution. The next challenge is thus the immobilization of all catalysts, compatible with the use of a sustainable flow bioreactor using low amount of enzymatic cofactor.

In the context of immobilized complex for NADH electrochemical regeneration, two essential aspects have been investigated in the present work. First, the method for immobilization of the rhodium complex [𝐶𝑝 * 𝑅ℎ(𝑏𝑝𝑦)𝐶𝑙] + have been optimized using carbon-based supports to obtain Rh-grafted matrices exhibiting high stability and activity in the redox flow reactor.

Carbon materials has been selected because of their low cost, large potential window, relatively inert surface, and active electrochemical properties for redox reactions, [28] . Then, the immobilization of the lactate dehydrogenase has been achieved in order to promote rapid transfer of NAD + /NADH from the molecular catalyst to the enzymatic catalyst. NAD + amounts as low as 10 µM were shown sufficient for the bioconversion of 25 mM pyruvate to lactate in the optimal cell. The original design of the cathode chamber led to a total turnover of NADH larger than the best values ever reported in the literature for electrochemical regeneration. [29]

Results and Discussion

Immobilization of [𝑪𝒑 * 𝑹𝒉(𝒃𝒑𝒚)𝑪𝒍] +

The immobilization of the rhodium complex on a carbon support involves a two-step process as described in the literature. [17] First, 4-amino-2,2-bipyridine reacts quantitatively in solution to produce bipyridyl diazonium cations. Then, the functionalization of the carbon support with 2,2-bipyridine (bpy) moieties is carried out by cyclic voltammetry, involving the electroreduction of the diazonium cations with concomitant release of dinitrogen and generation of aryl radicals prone to be grafted on the carbon support. The rhodium complex is then formed after a complexation reaction of (RhCpCl2)2 with the bipyridyl-functionalized electrode, giving rise to rhodium complexes covalently bonded to the carbon support. (Figure S1)

Selection of the carbon electrode material

Multi-walled carbon nanotubes (MWCNTs) can allow significant improvement of the efficiency of the electroenzymatic synthesis system by tuning some electrochemical properties, e.g., by enhancing the electroactive surface area. [30] On the basis of this opportunity, MWCNTs had been identified as suitable supporting materials for Rh complex immobilization. [29,31] Based on the perspective of combination between fuel cell technology and redox flow technique, a carbon paper (CP) coated with MWCNTs (noted CP-MWCT) was chosen for the construction of the catalytic layer after comparing it with bucky paper and graphite felt electrodes. The electrocatalytic behavior of the so-formed CP-MWCNT-bpy-Rh electrode was evaluated through cyclic voltammetry (Figure 1B), in a configuration where the working electrode has been arranged in the redox flow reactor, at a scan rate of 5 mV.s -1 in 50 mM phosphate buffer solution (PBS) at pH 7.2, with hydrogen and solutions flow rates of 20 mL.min -1 . As shown in scheme S1 in Supporting Information, a graphite felt (16 cm²) was placed on the top of the CP-MWCNT-bpy-Rh electrode in order to ensure a uniform distribution of the solution in the cathodic compartment and to maintain good electrical conduction with the graphite bipolar plate. A gas diffusion electrode, allowing hydrogen oxidation was used in the anodic compartment A Nafion membrane (N-212) was placed between the two electrodes for separation of the two compartments and to permit a uniform proton transport rate from the anode to the cathode. In the absence of NAD + in the medium, a cathodic current was observed at -0.31 V vs ref. (H2) (see curve a in Figure 1B), which was less negative than the potential reported in previous works for the same Rh complex immobilized on a bucky paper (BP) electrode (-0.65 V vs ref. (Ag/AgCl) ≈ -0.45 V vs ref. (H2)) [32] or in solution (-0.4 V vs ref.

(H2)) [27] . This potential shift can be explained by the influence of the electrode material on the electron density of the rhodium complex center [33] or due to the minimized distance between the anodic and the cathodic electrodes separated by the Nafion membrane. The addition of increased amounts of NAD + , from 1 mM to 4 mM (see curves b, c, d and e, in Figure 1B), led to the increase in the cathodic current showing the mediated electrocatalytic activity of the Rh complex towards NAD + reduction. A maximum cathodic current of 2.6 mA was observed at the concentration of 3 mM NAD + . Alternatively, for comparison with a previous work where [𝑪𝒑 * 𝑹𝒉(𝒃𝒑𝒚)𝑪𝒍] + was reacting on graphite felt, [27] the direct immobilization of the Rh complex on a graphite felt electrode (GF-bpy-Rh) was evaluated. The cathodic current peak observed at -0.35 V vs ref (H2) decreased with increasing NAD + concentrations, expressing a lower electroactive surface of the GF than with MWCNT [34] and indicating the poor stability of the support-Rh complex system under high flow rate conditions (Figure S2A&B), confirming the advantage of CP-MWCNT-bpy-Rh electrode for the present study.

In addition, the catalytic activity of CP-MWCNT-bpy-Rh towards NADH regeneration was also confirmed by amperometry at constant potential (-0.3 V vs ref (H2)) and the corresponding current variation for the conversion of 1 mM NAD + in solution (V=20 mL) is shown in Figure S3A (curve a). Complete conversion of NAD + into NADH was observed within 50 min electrolysis (Figure S3B, curve a) with an averaged faradaic efficiency of 71 % (Figure S3C, experiment a) using the CP-MWCNT-bpy-Rh cathode. The amount of rhodium complex on the CP-MWCNT electrode, 4.125 nmol.cm -2 , was estimated from cyclic voltammetry by integrating the cathodic peak. From that amount, a turnover frequency of 1.7 s -1 was calculated during the first 5 min of NADH electrochemical regeneration. This value of turnover frequency is very close to the highest value reported in the literature (3.6 s -1 ) for an immobilized Rh complex on MWCNTs. [31] Furthermore, total conversion of NAD + into NADH was obtained with a ratio of NAD + over rhodium complex concentrations in the order of 300, which is much larger than the maximal value achievable with dissolved Rh complex (ratio between NAD + and Rh complex not exceeding 40). [27] Such improvement in terms of performance came in addition to the advantage of CP-MWCNT-bpy-Rh leading to effective NADH regeneration while avoiding the use of dissolved rhodium complex.

To complete this study, bucky paper functionalized by the rhodium complex was also evaluated for the conversion of 1 mM NAD + into NADH in the flow bioreactor. Its catalytic activity for NAD + reduction was confirmed by cyclic voltammetry (Figure S2D), and the conversion reached 92 % after 50 min (Figure S3B, curve b) with a faradaic efficiency at only 56 % (Figure S3C, experiment b). In addition, with the BP-bpy-Rh system, residual amounts of the dissolved (RhCpCl2)2 could not be removed by dissolution in the solvent (DCM) which renders the technique poorly attractive.

Among the three functional electrodes that were tested, a carbon paper modified with a layer of MWCNT and functionalized with [𝑪𝒑 * 𝑹𝒉(𝒃𝒑𝒚)𝑪𝒍] + (CP-MWCNT-bpy-Rh) was thus selected here as the best one for regeneration of the NADH cofactor.

Stability and sustained activity

In order to ensure the robustness of the CP-MWCNT-bpy-Rh electrode, a cell assembly was prepared by pressing the GDE, the Nafion membrane (N212), and the CP-MWCNT-bpy-Rh together at 40 bars at room temperature, as illustrated in Figure 2A. The technique was expected to improve mechanical and electrical contacts in the whole flow cell (anode, membrane and cathodic chamber). CP-MWCNT-bpy-Rh system was characterized by cyclic voltammetry (Figure 2B) for different times under flow conditions after the electrode preparation: 4 h, 12 h, 24 h, and 120 h. The electrode was left inside the reactor and was tested each time with a new solution of phosphate buffer (50 mM; pH = 7.2). The very similar cathodic currents observed revealed that the electrode has maintained its activity for more than five days, and more precisely that the rhodium complex remained immobilized under flow conditions ensuring a long-term operational stability of the electrode. After confirming the electrode activity towards NADH regeneration by mediated NAD + electroreduction, its application in electroenzymatic biosynthesis was further explored. and hydrogen and solution flow rates were at 20 mL.min -1 .

Application of regenerated NADH in an enzymatic reaction

Parameters optimization for the efficient bioconversion of pyruvate

Coupling an enzymatic reaction to NADH regeneration should allow demonstrating that the active form of NADH (1,4-NADH) [35] was really produced by the redox flow reactor, and would be compatible with the production of fine chemicals with electrochemically regenerated NADH as a reactant involved in the biocatalytic reduction of the organic substrate. NAD-dependent lactate dehydrogenase LDH was used here as a convenient example (robust enzyme, commercially available, with reliable analytical methods for lactate and pyruvate). While the cofactor in its oxidized or reduced form is used as an active mediator leading to the bioconversion, we worked with low amounts of the cofactor (10 µM) which is economically desired and proves the stability of the cofactor with time. In addition, low amounts of cofactor are targeted to reach high TTNs. [36] Effect of the arrangement of supported Rh complex and LDH in the cell

The bioconversion of 12.5 mM pyruvate (in 100 mL solution containing 10 µM NAD + ) applying the process described in Scheme 1 was carried out potentiostatically, at -0.3 V vs ref.

(H2) for 72 hours, with the two electrodes CP-MWCNT-bpy-Rh and CP-MWCNT-LDH onto which 250 units of LDH were immobilized (≈ 6.6 nmol). The current near -4 mA in the first instants, decreased gradually to reach -1 mA after 72 hours (Figure 3A) whereas 100 % of pyruvate introduced was converted to lactate within this period (Figure 3B). The sum of the two species concentrations was nearly constant at 12.5 mM (Figure 3B) indicating the high selectivity of the enzymatic reaction. The faradaic efficiency was equal to 73 %, a value comparable to that of NADH regeneration without enzymatic reaction (Figure S2C). TTNs of Rh complex, NAD + , and LDH were respectively larger than 1.8×10 4 , 1250, and 1.8×10 5 . TTN for the Rh complex isto our state of knowledgethe highest value with rhodium complex applied in electroenzymatic synthesis. [32,37] The turnover frequency was 0.28 s -1 after 3 hours of the bioconversion. This value is higher than the value reported in our previous work, [17] thanks to the flowing system allowing of the performance cofactor regeneration coupled to the bioconversion of pyruvate.

On the other hand, setting the CP-MWCNT-LDH layer in another reactor downstream of the "electrochemical cell" provided with the CP-MWCNT-bpy-Rh layer for NADH regeneration (see Scheme S2 in Part 5 of supporting information) led to a lower bioconversion of 12.9 % with only 49% faradaic efficiency (Figure S4), far below the performances obtained with the two modified CP layers (CP-MWCNT-Rh and CP-MWCNT-LDH) stacked in the same cell. This reported experiment leads to the conclusion that the bioconversion of pyruvate is facilitated by close interaction between the Rh complex and LDH, under flow conditions for efficient mass transport rates, leading to increased overall conversion kinetics.

As a control experiment and following reported works in the literature where both catalysts for the regeneration of the cofactor and the production of fine chemicals were placed on the same electrode, [38] LDH enzymes were adsorbed directly on the Rh-immobilized CP-MWCNT layer forming the cathode. The efficiency of the single layer system, where both the Rh complex and LDH were immobilized on the same CP-MWCNT, was tested by studying the conversion of 20 mM pyruvate (50 mL at pH=7.2) with 500 units LDH adsorbed, in the presence of 0.5 mM NAD + , at -0.3 V vs ref (H2). The recorded currents decreased rapidly (in absolute value) from -1.5 mA to -0.25 mA after 5 hours (Figure S5A). Chemical analysis of the solution showed that the bioconversion had not occurred (Figure S5B), whereas NAD + was entirely reduced to NADH as revealed by UV-visible measurement at 340 nm. This result suggests that LDH was totally deactivated while being placed in a direct contact with rhodium complex, in agreement with former works reporting deactivation of the rhodium complex or lactate dehydrogenase. [16] In conclusion, the best strategy for optimal activity of these two catalysts, the molecular one and the biological one, is to associate them closely while keeping a separation, as it is illustrated here with the two catalytic layers.

Towards higher production rate of lactate

Production rate of the designed system (scheme 1) is a quite important criterion to address. For this purpose, four experiments have been designed and carried out, in particular for optimization of the amount of Rh complex in relation to LDH immobilized on the separated CP-MWCNT material. Conditions and results of these tests are reported in Table 1. First, in order to evaluate the effect of LDH amount on the productivity, Experiment (1) and Experiment (2) were performed with the same amount of immobilized Rh complex and different amounts of LDH (Table 1). The current obtained with the system in Experiment (2),

where the ratio between the Rh complex and LDH is equal to 10, led to a total (100 %) bioconversion (Figure 3B) approximately 1.5 times higher than the one obtained in Experiment (1) where the ratio between the Rh complex and LDH is equal to 5 for which the bioconversion was restricted to 59 % (see Figure S6 in Part 7 of supporting information). Faradaic efficiencies for both systems were in the same order of magnitude. This result leads to the consequence that an equilibrium between the regeneration of NADH by the Rh complex and its oxidation by LDH is important in order to reach a total bioconversion within an optimal time.

Later, for the sake of higher lactate production within an optimal time and to obtain a system for the electrochemical regeneration of NADH comparable with the electroenzymatic one involving enzymatic mediators, e.g. FNR, [37,38,39] additional tests (not reported in Table 1) were carried out by inserting two CP-MWCNT-bpy-Rh layers in the electrochemical bioreactor instead of one. As a matter of fact, the immobilization of the Rh complex on the same CP-MWCNT with a larger amount of Rh complex allowed by a larger concentration of 2,2bipyridine moieties led only to the blockage of the electrode.

Further, the electrocatalytic activity of the two coupled CP-MWCNT-bpy-Rh electrodes was evaluated by cyclic voltammetry (Figure S7A). Without NAD + in the flow cell, the cathodic peak was located at -0.3 V vs ref.

(H2) and a cathodic current near 3.5 mA was measured, as compared to 1.6 mA with a single CP-MWCNT-bpy-Rh layer (Figure S7B), in agreement with the higher amount of rhodium complex immobilized in the former case. The calculated surface coverage with the rhodium complex estimated by charge balance and Faraday's law from voltammograms was found equal to 138 nmol. The surface concentration near 4.3 nmol.cm -2 was very close to that exhibited by a single CP-MWCNT-bpy-Rh layer. Then, when adding increasing amounts of NAD + in the flow cell, significant increases in the cathodic currents were observed, much larger than for the single CP-MWCNT-bpy-Rh layer (Figure S7B). In the NAD + concentration range investigated, the cathodic current measured at -0.3 V increased gradually with the increase of NAD + until levelling off with NAD + concentration higher than 3 mM. The improved activity of the bilayer electrochemical system appears promising for more efficient regeneration of NADH coupled to pyruvate bioconversion.

Two experiments of lactate biosynthesis were performed in the presence of the two layer CP-MWCNT-bpy-Rh electrode associated to one CP-MWCNT-LDH on which distinct amounts of LDH were immobilized (Table 1, Experiment 3 and Experiment 4).

Let's first consider the case where 500 units (approximately 13.8 nmol) of enzyme were adsorbed on a CP-MWCNT, corresponding to Rh-over-LDH molar ratio near 10, for which the bioconversion of 100 mL solution of 25 mM pyruvate was carried out in the presence of 10 µM NAD + , under usual flow and potential conditions (Experiment 3 in Table 1). The current monitored at -0.3 V vs ref.

(H2) decreased regularly from -5.5 mA to reach a fairly stable current value at -1.8 mA after 72 hours (Figure 4A). This current was larger than that measured with a single Rh electrode (Figure 3A) in correlation with the two-time larger amount of immobilized rhodium complex. Pyruvate was continuously converted along the run, until its disappearance after 72 hours. In the meanwhile, lactate was formed quantitatively (Figure 4B). These results

show the positive impact of increasing the amount of immobilized Rh complex on the overall performance of the regeneration of the cofactor coupled to the enzymatic reaction. Furthermore, these results showed than the regeneration system of NADH coupled to pyruvate bioconversion can be scaled up and can lead to a high bioconversion yield following a ratio of 10 between the rhodium complex and lactate dehydrogenase both immobilized on separated CP-MWCNT.

TTNs obtained with the bilayer system for the Rh complex, NAD + , LDH were respectively calculated at 1.81×10 4 , 2.5×10 3 and 1.8×10 5 . The TTN for the rhodium complex is in the same order of magnitude as the one reported in the literature for the FNR-mediated regeneration of NADPH. [START_REF] Cheng | [END_REF] In addition, the TTN obtained for NAD + isto our state of knowledgethe highest value among those reported in the literature. [START_REF] Cheng | [END_REF]38] On the other hand, the bioconversion of the 25 mM lactate in the presence of two CP-MWCNTbpy-Rh layers, corresponding to 13.8 nmol of immobilized Rh complex, and one CP-MWCNT-LDH layer with only 100 LDH units (2.76 nmol) showed a bioconversion current (Experiment 4 in Table 1) starting near -5 mA and decreasing to -1 mA after 72 h (Figure S8A). This bioconversion current is lower in absolute value than the one obtained in Experiment 3 with 500 units of LDH. As a consequence, 16.5 mM of lactate were synthetized after 72 h (Figure S8B) with a faradaic efficiency of 79 %. Moreover, extending the electrosynthesis up to 120 h resulted in the bioconversion of 21.5 mM of pyruvate (Figure S8B).

To conclude, an optimal ratio between the Rh complex and LDH of 10 allows a total bioconversion of pyruvate within an optimal time (72 hours).

Conclusion

Thanks to the robust immobilization of all catalysts and fine optimizations, the presented continuous flow electrochemical reactor shows remarkable performances for catalytic hydrogenation of pure chemicals through NADH regeneration. The work deals with different challenges such as the choice of the cathode material, the immobilization of both the redox catalyst and the enzymes, their association or separation, the molar ratio between each of them, and the use of low amounts of the enzymatic cofactor (10 µM).

Different aspects have to be taken into consideration when coupling efficient electrochemical regeneration of the NADH cofactor to enzymatic bioconversion of chemicals:

1) Immobilizing the Rh complex on carbon paper with MWCNT, and pressing at 40 bars this catalytic layer with a GDE and Nafion membrane led to an active system for the efficient electrochemical regeneration of the NADH cofactor system with a stability of more than 5 days under continuous flow.

2) Reducing the distance between the Rh complex and LDH by immobilizing each of them on separated, stacked layers led to an active loop for the continuous regeneration of the cofactor and pyruvate bioconversion.

3) The ratio between the immobilized Rh complex and enzymes units has to be at least equal to 10 in order to reach the highest faradaic efficiency (79 %), productivity (1.08 µmol.cm -2 .h -1 ) and TTN for cofactor (2500), rhodium catalyst (18000) and enzyme (180000).

4) Finally, hydrogen and solution flow rates can be fixed at 20 mL.min -1 in the bench cell as described previously [27] and the applied electrolysis potential at -0.3 V ref. (H2). In addition, the flow cell was built advantageously using a graphite felt electrode allowing uniform distribution of the solution in the cathodic compartment and ensuring electrical contact between the functional layers and the current collector of the cell.

Lactate produced from the bioconversion of pyruvate can have a wide range of applications in in cosmetics, food, and pharmaceutical industries. [41] Although we have focused on the hydrogenation of a ketone catalyzed by an alcohol dehydrogenase, the approach presented here should be applicable to many other NADH-dependent bioconversions.

Materials and methods

Chemical and reagents

All chemicals and LDH from porcine heart (250 units.mg -1 protein) used were from Sigma-Aldrich. Multi-walled carbon nanotubes (MWCNT, NC7000™ series) were from Nanocyl (Belgium) and carbon papers (SpectraCarb 2050L-0550 Carbon Paper) were acquired from Fuel Cell Store (USA). MWCNT were dispersed in a solution of ethanol (96 %). High purity water (18 MΩ.cm) was used to prepare all solutions.

Immobilization of [𝑪𝒑 * 𝑹𝒉(𝒃𝒑𝒚)𝑪𝒍]

+ on a carbon support electrode

Graphite felt electrode pretreatment

A piece of graphite felt (GFD4.6EA, SGL, Germany) being 4 cm x 4 cm x 0.4 cm in dimensions was used as cathode in the first part of the study. In order to reduce its hydrophobicity, the graphite felt electrode was washed and ultrasonicated with different proportions of ethanol and water at a temperature of 25 °C following the adopted protocol in our previous work. [27]

Carbon paper with MWCNT (CP-MWCNT) preparation

A piece of SpectraCarb carbon paper (CP) (4 cm x 4 cm x 0.05 cm) covered with MWCNT was prepared following a reported work in the literature [42] where 10 mg of MWCNT were ultrasonicated in a solution of ethanol (50 mL) for 5 hours. This suspension was then vacuum filtered on the CP and the resulting material was dried overnight. The CP coated with MWCNT was then denoted CP-MWCNT and was used as cathode for the rhodium complex covalent immobilization and as a support material for the immobilization of L-lactate dehydrogenase.

Carbon support electrode functionalization with [𝑪𝒑 * 𝑹𝒉(𝒃𝒑𝒚)𝑪𝒍] +

The carbon support was functionalized following a protocol from the literature. [17] 1 mM 4amino-2,2-bipyridine (synthetized following a reported procedure [17] ) was mixed with 2 mM sodium nitrite in 0.5 M HCl under gentle stirring for 5 min in order to generate 2,2-bipyridyl diazonium cations. A negative potential was then applied by running 2 cyclic voltammograms between 0.4 V and -0.8 V (vs Ag/AgCl (3M)) at a scan rate of 20 mV.s -1 . The electrode was then rinsed with ultra-pure water and left to dry.

The functionalized electrode with 2,2-bipyridine moieties was then placed into a solution of DCM containing 0.15 mM of ((RhCp*Cl2)2 for 4 h with a gentle stirring. The functionalized electrode with the rhodium complex was then rinsed with DCM for 5 min in order to remove the unreacted residues.

Enzymatic carbon paper with MWCNT (CP-MWCNT-LDH) preparation

CP-MWCNT was prepared following the same protocol in 4.2.2. LDH enzyme was adsorbed on the MWCNT surface by depositing 50 µL suspension of LDH and keeping for 2 hours at a temperature of 3°C.

Electrochemical reactor

Scheme S1 shows the bioelectrochemical bioreactor used in this study. The bioreactor has been presented in a previous work. [27] A gas diffusion electrode (4 cm * 4 cm), used as anode, was placed on a grooved graphite bipolar plate allowing the introduction and the distribution of hydrogen in the anodic compartment at flow rate of 20 mL.min -1 . [27] At the same time, the reaction solution containing 50 mM phosphate buffer (pH = 7.2), NAD + , and pyruvate was flowed continuously to the cathodic compartment using peristaltic pump at flow rate of 20 mL.min -1 . [27] A CP-MWCNT on which is immobilized the rhodium complex was used as cathode. On the top of the CP-MWCNT-Rh was placed a graphite felt electrode known as a good electrical conductor and acting as a three-dimensional cathode, with homogeneous distribution of the solution. For pyruvate bioconversion tests, a CP-MWCNT-LDH was placed on the top of the CP-MWCNT-Rh electrode in addition to the graphite felt electrode.

Finally, a Nafion membrane (N-212) was placed between the two compartments to separate them and to transport protons from the anodic to the cathodic compartment.

Cyclic voltammograms and batch electrolysis were performed in a two-electrode configuration, the hydrogen anode acting also as reference electrode. Potential scan rate and electrolysis potential were fixed at 5 mV.s -1 and -0.3 V vs ref (H2).

Quantitative analysis of pyruvate bioconversion was performed by High Performance Liquid Chromatography (HPLC), using the Aminex HPX-87H HPLC column (300 mm x 7.8 mm, Phenomenex) thermostated at 45 °C, and a refractive index detector. The aqueous phase was flowed at 0.7 mL.min -1 . 200 µL aliquots were taken at different intervals from the reaction mixture. These aliquots were further analyzed by HPLC to determine the pyruvate and lactate concentrations.

Diazonium electrografting on a 16

 16 cm² CP-MWCNT electrode was performed by cyclic voltammetry from 0.4 V to -0.8 V vs Ag/AgCl, as shown in Figure 1A. During the first scan, an irreversible wave was observed at -0.35 V vs Ag/AgCl (curve a) corresponding to the grafting of 2,2-bipyridine and the release of N2. A cathodic reduction peak of lower intensity was observed in the second scan (curve b) due to the partial blockage of the CP-MWCNT electrode surface, indicating successful electrografting. The CP-MWCNT electrografted with 2,2-bipyridine moieties was then immersed in a solution of (RhCpCl2)2 for the formation of rhodium complexes covalently immobilized onto the electrode surface.

Figure 1 .

 1 Figure 1. (A) (a) first and (b) second cyclic voltammograms for the reduction of diazonium cations generated

Figure 2 .

 2 Figure 2. (A) Schematic representation of GDE-N212-(Cp-MWCNT-bpy-Rh) system pressed together at 40 bars. (B) Cyclic voltammograms recorded at a potential scan rate of 5 mV.s -1 using a [Cp*Rh(bpy)Cl] + functionalized CP-MWCNT (using the pressed system in Figure 2A) electrode in 50 mM PBS buffer at pH 7.2 under nitrogen after 4 h, 12h, 24 h, and 120 h of electrode preparation. The geometric surface area of the electrode was 16 cm 2

Scheme 1 .

 1 Scheme 1. Schematic representation of the electroenzymatic reactor including CP-MWCNT-bpy-Rh electrode and CP-MWCNT-LDH enzymatic system reactions with the LDH-MWCNT-CP system.

Figure 3 .

 3 Figure 3. (A) Variation of sampled currents with time during the electroenzymatic synthesis of lactate from 12.5 mM of pyruvate in 100 mL solution containing 10 µM NAD + (and 50 mM PBS, pH 7.2) in the flow reactor provided with one CP-MWCNT-bpy-Rh electrode and one CP-MWCNT-LDH (250 units) layer. (B) Variation of concentrations with time of (a)Pyruvate, (b) Lactate, and (c) the sum pyruvate and lactate, as determined by HPLC.

Figure 4 .

 4 Figure 4. Electroenzymatic synthesis of lactate from 25 mM pyruvate in 100 mL solution containing 10 µM NAD + (and 50 mM PBS, pH 7.2) in the hybrid flow cell provided with two CP-MWCNT-bpy-Rh electrodes and one enzymatic CP-MWCNT-LDH (500 units) layer (Experiment 3). (A) Variations of the current in the run. (B) Time variations of the concentrations of (a) pyruvate, (b) lactate, and (c) the sum pyruvate and lactate, as determined by HPLC.

Table 1 .

 1 Ratio optimization between Rh complex and LDH for the optimal bioconversion of pyruvate (V = 100 mL). (*) Current efficiency is for lactate production.

		[Pyruvate]	LDH	Rh	nRh/nLDH	[Lactate]	(*) Current
	Experiment	at t=0 (mM)	(nmol)	(nmol)	ratio	after 72 h (mM)	efficiency (%)
	1	20	13.8	66	5	7.2	76.7
	2	12.5	6.6	66	10	12.5	73
	3	25	13.8	138	10	25	78
	4	25	2.76	138	50	16.5	79

ACKNOWLEDGMENTS

We gratefully acknowledge CNRS for funding of the project NADP/H2. We also thank Xavier Framboisier (LRGP) in the development of the HPLC analytical method.

Graphical Abstract

High total turnover of NADH cofactor can be achieved with using all-immobilized catalysts in a hybrid flow bioreactor allowing efficient transfer of electrons and protons from H2 reacting in a gas diffusion electrode to the enzymatic reaction with only 10 µM enzymatic cofactor


Július Gajdár

Grégoire Herzog

Mathieu Etienne
email: mathieu.etienne@cnrs.fr

Amperometric Sensor for Selective On-Site Analysis of Free Sulfite in Wines

Keywords: Amperometry, wine, sulphur dioxide reduction, electrochemical oxygen filter, screen printed electrodes

Accurate and rapid on-site analysis of free SO2 content is crucial in the process of winemaking from a producer and consumer perspective. Herein, we present an amperometric sensor based on commercially available screen-printed electrodes coupled with an electrochemical oxygen filter. The developed amperometric method gave a linear response in a concentration range up to 200 mg L -1 with a limit of quantification of 7.5 mg L -1 . The applicability of the developed sensor was successfully tested on 27 white and red wine samples and compared to the Ripper method (iodometry) which is a standard procedure for free SO2 determination. The sensor exhibits similar precision and accuracy but shows no interference from oxidizable species such as ascorbic acid, which is a major advantage over iodometric titration. Performance of the sensor was in addition positively evaluated during on-site analysis in a winery.

Sulphites are widely used as food preservatives, antioxidants, and anti-browning agents. They are usually added to foods such as dried fruit or vegetables, bottled soft drinks, juices, wine or beer. Sulphites establish an equilibrium between 'free' sulphites (which can be found as a dissolved salt HSO3 -; SO3 2-, or as molecular SO2 depending on pH) and 'bound' sulphites that are complexed with carbohydrates, lipids, or proteins in foods. This equilibrium varies in different kinds of foods, and depends on the temperature, pH and macromolecular composition of food. [START_REF] Vally | Clinical Effects of Sulphite Additives[END_REF][START_REF] Garcia-Fuentes | Short Review of Sulphites as Food Additives[END_REF] In winemaking, sulphites also provide microbial stabilization, regulation of offflavours and preservation of aromas and colour. [START_REF] Oiv | Resolution OIV-OENO 631-2020: Review of Practices for the Reduction of SO2 Doses Used in Winemaking[END_REF] Many studies have confirmed a sensitivity of some individuals to sulphite additives. Adverse reactions include asthmatic and anaphylactic episodes. [START_REF] Garcia-Fuentes | Short Review of Sulphites as Food Additives[END_REF] However, there is minimal risk involved for the general population following the acceptable daily intake of 0 -0.7 mg / kg of body weight. [START_REF] Vally | Clinical Effects of Sulphite Additives[END_REF][START_REF] Garcia-Fuentes | Short Review of Sulphites as Food Additives[END_REF] During the winemaking process, sulphite can be added at harvesting, before and after the fermentation process, at the start of the ageing process, and at the bottling stage. A free sulphite concentration between 20 and 40 mg L -1 is advised to guarantee a protection against the oxidation of the wine. [START_REF] Oiv | Resolution OIV-OENO 631-2020: Review of Practices for the Reduction of SO2 Doses Used in Winemaking[END_REF] Total sulphite content in wines must not exceed 150 mg L -1 in red wines and 200 mg L -1 in white / rosé wines (with higher limit for some sweet wines) according to the EU legislative [START_REF]Commission Regulation (EC) No 118/2006 of 19 December 2006 Setting Maximum Levels for Certain Contaminants in Foodstuffs[END_REF] and OIV (International organisation of vine and wine) recommendations. [START_REF]OIV Resolution OENO 09[END_REF] Wines containing more than 10 mg L -1 of sulphites must include a statement "contains sulphites" on their label. Sulphites (total content of up to 100 mg L -1 ) may also be formed by the yeast during the fermentation. [START_REF] Stockley | OIV Collective Expertise Document SO2 and Wine: A Review[END_REF] In recent years, following the consumer demand, there is also a tendency to reduce (or completely eliminate) the amount of additives like SO2 in wines. Therefore, it is important for winemakers to accurately determine the amount of free SO2 in the different stages of winemaking process to guarantee the quality of the final product for consumers.

There are currently two methods recommended by OIV for determination of free or total SO2 content in wines. The Ripper method is an iodometric titration based on the oxidation of SO2 in an acidified sample with starch as an indicator. To get a total SO2 content, the wine sample must be pre-treated with a basic solution to release all bound SO2. [START_REF]Compendium of international methods of wine and must analysis[END_REF] The other method is based on Monier-Williams procedure in which a sample is distilled under inert atmosphere and all SO2 is then captured in a peroxide solution where it reacts to sulphuric acid. The acid is then determined by acido-basic titration. This method is more accurate with less interfering species, but it is more complicated to set up and the time of analysis is consequently longer. [START_REF]Method OIV-MA-AS323-04A[END_REF] Determination of sulphites is usually done with automatic titrators [START_REF][END_REF] based on iodometry for its simplicity compared to the Monier-Williams procedure. Commercially available and affordable titration kits based on the Ripper method are currently available to winemakers for on-site analysis. [START_REF]Vinmetrica SC-100A Sulfite (SO2) Analyzer Kit[END_REF] However, this method is prone to interferences from any oxidizable compounds such as polyphenols or ascorbates. [START_REF]Compendium of international methods of wine and must analysis[END_REF] Additionally, cheap colorimetric paper strip tests are also available but they obviously offer lower precision and difficult interpretation especially for red wines. [START_REF]Free Sulfite Test Strips[END_REF] Recently, a commercial sensor based on electrochemical detection of free SO2 was also introduced for winemakers. [START_REF]Sentia Free SO₂ Test Strips -Universal Biosensors[END_REF] Many recent analytical studies have been dedicated to their determination. SO2 content in wines was determined by GC-MS, [START_REF] Carrascon | Gas Chromatography-Mass Spectrometry Strategies for the Accurate and Sensitive Speciation of Sulfur Dioxide in Wine[END_REF] ICP-MS, [START_REF] Wang | Direct Quantification of Total Sulfur Dioxide in Wine Using Triple Quadrupole ICP-MS[END_REF] microextraction coupled with Surface-Enhanced Raman Scattering, [START_REF] Deng | Headspace Thin-Film Microextraction Coupled with Surface-Enhanced Raman Scattering as a Facile Method for Reproducible and Specific Detection of Sulfur Dioxide in Wine[END_REF] or UV-vis spectrophotometry, [START_REF] Gómez-Otero | Ultrasensitive, Simple and Solvent-Free Micro-Assay for Determining Sulphite Preservatives (E220-228) in Foods by HS-SDME and UV-Vis Micro-Spectrophotometry Microextraction Techniques[END_REF] conductometry in a flow system based on a gas-diffusion cell, 17 by using solid polymer electrolyte electrodes in a flow system with amperometric detection, [START_REF] Toniolo | A Sensor Based on Electrodes Supported on Ion-Exchange Membranes for the Flow-Injection Monitoring of Sulphur Dioxide in Wines and Grape Juices[END_REF] and by a device based on a gas-diffusion membrane and pH-ISFET (Ion Selective Field Effect Transistors) system. [START_REF] Giménez-Gómez | Microanalytical Flow System for the Simultaneous Determination of Acetic Acid and Free Sulfur Dioxide in Wines[END_REF] All these examples are based on methods that are either not suitable for on-site analysis due to the size and the price of the instrumentation, or require some complex sample pre-treatment (extraction, flow systems, gas-diffusion cells, etc.). Both these reasons are a hindrance for a development of a robust, portable and costeffective sensor. Recent study based on colorimetric assay on a microfluidic paper-based device could be considered as an example of sensor fulfilling these requirements. [START_REF] Arroyo | Capillary Microfluidic Platform for Sulfite Determination in Wines[END_REF] Electrochemical methods of analysis on disposable screen-printed electrodes (SPE) provide a rapid, reliable, and affordable way of in-situ analysis in complex matrices at portable devices. [START_REF] Couto | Recent Developments, Characteristics and Potential Applications of Screen-Printed Electrodes in Pharmaceutical and Biological Analysis[END_REF] The analysis of sulphites by electrochemical methods has been extensively studied before. Sulphites are more often determined by oxidation. [START_REF] Isaac | Electroanalytical Methods for the Determination of Sulfite in Food and Beverages[END_REF][START_REF] Pisoschi | Analytical Methods Applied to the Assay of Sulfur-Containing Preserving Agents[END_REF] However, in the case of wines, this approach is sensitive to interference from various molecules: e.g. polyphenols or ascorbates. These interfere with the analysis itself, and also significantly contribute to the electrode passivation, further complicating the analysis. [START_REF] Isaac | Electroanalytical Methods for the Determination of Sulfite in Food and Beverages[END_REF] On the other hand, reduction does not have interferents in the wine matrix apart from oxygen. [START_REF] Isaac | A Novel Electroreduction Strategy for the Determination of Sulfite[END_REF] SO2 is reduced under acidic conditions (0 < pH < 2) by 2 electrons to sulfoxylic acid H2SO2:

SO2 + 2H + + 2e -↔ H2SO2
At more positive pH (2 < pH < 5) when HSO3 -species is prevalent, reduction occurs by one electron to SO2 •-species, which then dimerizes to S2O4 2-: [START_REF] Isaac | A Novel Electroreduction Strategy for the Determination of Sulfite[END_REF][START_REF] Streeter | Cathodic Reduction of Bisulfite and Sulfur Dioxide in Aqueous Solutions on Copper Electrodes: An Electrochemical ESR Study[END_REF] HSO3 -+ e -+ H + → SO2

•-+ H2O 2SO2 •-↔ S2O4 2-
Reduction of SO2 was used for its determination only in limited number of works. [START_REF] Isaac | Electroanalytical Methods for the Determination of Sulfite in Food and Beverages[END_REF][START_REF] Silva | Carbon Nanotubes for Voltammetric Determination of Sulphite in Some Beverages[END_REF][START_REF] Ramos | Free Sulphite Determination in Wine Using Screen-Printed Carbon Electrodes with Prior Gas-Diffusion Microextraction[END_REF][START_REF] Gonçalves | Determination of Free and Total Sulfites in Wine Using an Automatic Flow Injection Analysis System with Voltammetric Detection[END_REF][START_REF] Ramos | Electrochemical Sensing of Total Sulphites in Beer Using Non-Modified Screen-Printed Carbon Electrodes[END_REF][START_REF] Martins | Laser-Pyrolyzed Electrochemical Paper-Based Analytical Sensor for Sulphite Analysis[END_REF][START_REF] Yilmaz | Determination of Trace Sulfite by Direct and Indirect Methods Using Differential Pulse Polarography[END_REF] SO2 was determined on carbon nanotubes modified electrode in various beverages by square wave voltammetry. [START_REF] Silva | Carbon Nanotubes for Voltammetric Determination of Sulphite in Some Beverages[END_REF] Gas diffusion extraction was used for sulphite determination by electrochemical methods in several publications by the same group of authors for detection of free SO2 in matrices of wine and beer. Free SO2 was selectively extracted from samples and determined by voltammetry in a solution free of any matrix effects. However, experimental procedure requires several steps and determination was only done by standard additions which are not applicable for on-site analysis due to the instability of standard sulphite solutions. [START_REF] Ramos | Free Sulphite Determination in Wine Using Screen-Printed Carbon Electrodes with Prior Gas-Diffusion Microextraction[END_REF][START_REF] Gonçalves | Determination of Free and Total Sulfites in Wine Using an Automatic Flow Injection Analysis System with Voltammetric Detection[END_REF][START_REF] Ramos | Electrochemical Sensing of Total Sulphites in Beer Using Non-Modified Screen-Printed Carbon Electrodes[END_REF][START_REF] Martins | Laser-Pyrolyzed Electrochemical Paper-Based Analytical Sensor for Sulphite Analysis[END_REF] A similar approach based on gas-diffusion membrane and electrochemical detection was applied for determination of total SO2 content in wines. [START_REF] Prasertying | Disposable Microchamber with a Microfluidic Paper-Based Lid for Generation and Membrane Separation of SO2 Gas Employing an In Situ Electrochemical Gas Sensor for Quantifying Sulfite in Wine[END_REF] Sulphites were also determined by polarography in dried fruit samples. Sample preparation included deoxygenation of buffers (pH 4) and was reported to be difficult considering the volatility of SO2. [START_REF] Yilmaz | Determination of Trace Sulfite by Direct and Indirect Methods Using Differential Pulse Polarography[END_REF] The removal of oxygen proved to be a tricky problem considering the volatility of molecular SO2 and the necessity to purge the solution before electrochemical analysis with inert gas to remove dissolved oxygen. Our solution of localised oxygen removal using a porous second working electrode (the so-called "oxygen filter") positioned in the vicinity of a sensing electrode was presented in previous publications. [START_REF] Le | Local Removal of Oxygen for NAD(P)+ Detection in Aerated Solutions[END_REF][START_REF] Etienne | Electrochemical Filter to Remove Oxygen Interference Locally, Rapidly, and Temporarily for Sensing Applications[END_REF][START_REF] Rotureau | Electroanalytical Metal Sensor with Built-in Oxygen Filter[END_REF][START_REF] Le | Improved Productivity of NAD+ Reduction under Forced Convection in Aerated Solutions[END_REF] The oxygen filter has been so far used in an applied electrochemical sensor only for sensing of heavy metals (zinc, lead, cadmium) by stripping chronopotentiometric technique [START_REF] Rotureau | Electroanalytical Metal Sensor with Built-in Oxygen Filter[END_REF] and for a fructose biosensor based on NAD + /NADH regeneration which is sensitive to the oxygen presence. [START_REF] Le | Local Removal of Oxygen for NAD(P)+ Detection in Aerated Solutions[END_REF] Here we present a novel use of the oxygen filter fully integrated into an electrochemical sensor designed specifically for on-site amperometric sensing of free SO2 in complex wine samples. The oxygen filter offers an analysis without sample purging with an inert gas significantly facilitating the use of the method outside of the laboratory. Idea of a simple on-site analysis is further supported by the use of low-cost commercially available unmodified disposable SPEs, minimal sample pre-treatment, and a robust method with facile automatic evaluation.

Experimental Chemicals and materials

A stock solution of SO2 (1 g L -1 ) was prepared daily from anhydrous Na2SO3 (98 %, Fluka) in 10 mL of deionized water purged with nitrogen to obtain a stable solution. [START_REF] Gómez-Otero | Ultrasensitive, Simple and Solvent-Free Micro-Assay for Determining Sulphite Preservatives (E220-228) in Foods by HS-SDME and UV-Vis Micro-Spectrophotometry Microextraction Techniques[END_REF] Concentration of the solution was checked using iodometry. 0.1 mol L -1 HCl-KCl solutions and 0.1 mol L -1 Britton-Robinson buffers with given pH were prepared as previously reported for the electrochemical characterisation of the set-up. [START_REF] Fernández | Buffer Solutions[END_REF] 37 % hydrochloric acid (Fluka) was diluted to 10 mol L -1 concentration. pH of buffers and wines was checked using pH meter Meterlab PHM210 (Radiometer Copenhagen). All solutions were prepared using deionized water. Lowsulphite white and red wines were used as model samples for the optimization of the amperometric method and construction of the calibration curves. These wines were identical to wines R1 and W1, but they were opened and then stored in a fridge and used for a month. All experiments with wines were carried out in these matrices unless stated otherwise.

Electrochemical apparatus and methods

Screen-printed electrodes (DRP-110) containing carbon ink working and counter electrodes, and silver ink quasi-reference electrode were obtained from Dropsens / Metrohm France. Electrodes were connected to a bipotentiostat Dropsens µStat ECL controlled by Dropview 8400 software. A platinum mesh (aperture: 0.12 mm, wire diameter: 0.04 mm) for the construction of the oxygen filter was obtained from Goodfellow France. 3D printer Prusa I3 MK3S+ (Prusa Research, Czech Republic) was used for 3D printing. The software Blender (Blender studio) was used for the creation of the 3D model of the electrochemical cell. Polyethylene terephthalate glycol (PETG) and polylactic acid (PLA) polymers (both Prusa Research) were used for the prints. Nitrile rubber coated nylon diaphragm sheeting of 0.25 mm thickness (Coruba, UK) was cut out to the appropriate size to act as an O-ring.

Cyclic voltammetry and linear sweep voltammetry were usually carried out at 50 mV s -1 scan rate. Amperometric detection was carried out at the SPE at the potential -0.65 V with 1 s sampling rate. The oxygen filter was connected as a second working electrode. Potential of -0.3 V was applied at the oxygen filter for 60 s before the start of the scan at the SPE and was kept on for the whole experiment. Current value was evaluated after 240 s as an average value of the current from the last 10 points. SPEs could be used throughout the day for several experiments (maximum was 15 wine samples in a day) with no observable degradation of SO2 signal. All potential values were reported versus silver quasi reference electrode.

Analytical procedures

At first, the cell was assembled by placing first SPE (used as a working electrode only) in the designed place in a cell. The cell was then enclosed using a sealing and all 3D printed parts as shown in Fig. 1A. After the introduction of a sample, the cell was closed using a cap with a second SPE (used as a reference and a counter electrode). The fully assembled cell could be used for several experiments with various wine samples.

For the construction of a standard calibration and first experiments with low sulphite wines the procedure was the following: (i) 10 mL of the sample solution (wine sample) was added to the cell and then purged with nitrogen (to limit the reaction of SO2 with oxygen); (ii) 100 µL of 10 mol L -1 HCl was added to modify the pH to the optimal conditions. HCl was used as it also provided a constant high concentration of Cl -ions necessary to stabilize the potential of silver quasi-reference electrode; (iii) Given amounts of SO2 standard were then pipetted to the sample in the cell.

For the determination in wine samples: (i) 10 mL of wine was pipetted into the cell (List of wines is given in Table S1); (ii) 100 µL of 10 mol L -1 HCl was added followed by a gentle stir of a solution; (iii) Amperometric scan was then immediately started; (iv) Concentration of free SO2 was then calculated by dividing the current with the obtained standard slope separately for red and white wines.

Iodometric titration

Method for iodometric titration of free SO2 was taken from OIV method [START_REF]OIV Resolution OENO 09[END_REF] . Metrohm DMS Titrino716 automatic titrator controlled by Tiamo 1.3 software was utilized. End point detection was carried out potentiometrically at the inflex point of the titration curve. 25 mL of wine sample was diluted with 35 mL of deionized water and after the addition of 5 mL of 25% H2SO4, the titration was immediately started. Titration solution was 0.01 M solution of I2 prepared from 0.1 N Iodine solution (SDS France) that was standardized against As2O3. [START_REF] Kar | Iodometric Assays[END_REF] All measurements were repeated in duplicate for every wine sample. In a case of high interferent (ascorbic acid) concentration, 5 mL of 7 g L -1 acetaldehyde solution in ethanol was added to the wine sample and let rest for at least 30 mins. Free SO2 concentration was then obtained by calculating the difference between the first run (SO2 + interferents) and the second run (interferents only). This was done for wine samples that showed at least twice higher free SO2 concentration measured by iodometry than by amperometry.

Statistical evaluation

Statistics were calculated with the help of Origin software and Microsoft Excel. All errors are given at 95% confidence limit. Errors in standard slope are calculated at 95% confidence limit as 1.96-times standard deviation of the fitted curve and converted to relative error which is then applied to every result. [START_REF] Miller | Statistics and Chemometrics for Analytical Chemistry[END_REF] Limit of quantification was calculated as a concentration corresponding to a 10-fold standard deviation of a slope of a fitted concentration curve. [START_REF] Miller | Statistics and Chemometrics for Analytical Chemistry[END_REF] Ttest was used to evaluate the accuracy of the results obtained by two analytical methods. Critical value ts for two-tailed test (P = 0.05, 4 degrees of freedom) was 2.78. [START_REF] Miller | Statistics and Chemometrics for Analytical Chemistry[END_REF]

Elimination of false positive results

In the cases of wines with low free SO2 concentration (below 15 mg L -1 ), a procedure was devised to eliminate current readings that did not correspond to SO2 signal and only represented the charging current. This was done by evaluating the average current value of 10 data points and comparing them to the average current value of previous 10 data points. This constraint can be expressed by an equation:

( 𝑖̅ (𝑛-10;𝑛-20) 𝑖̅ (𝑛;𝑛-10) -1) × 100% < 2%
where 𝑖̅ is an average current of n th to (n-m) th data points. This evaluation was done for every data point in the exported data file of the amperometric curve. Two amperometric recordings (false positive and correct reading) and the calculation of their decrease is shown in Table S4.

Only data points at the end of an amperometric recording were considered (from 200 to 240 s).

If the equation was valid for data points in this range (decrease less than 2%), this result is correct and the obtained concentration reading corresponds to free SO2 concentration. If the current decrease is more than 2% in the evaluated range, this sample is a false positive and the concentration of free SO2 was labelled to be below LOQ of the method. This principle is also graphically illustrated in Fig. S6B with total of 10 amperometric recordings from 8 wine samples. The critical value of 2% clearly divides false positive and correct readings.

Results

Sensor cell design with built-in oxygen filter

This work started with a careful design and optimization of the electrochemical cell that would incorporate an oxygen filter and could be used with SPEs. The previous cell design presented in a previous publication for analysis of metals was a starting point. [START_REF] Rotureau | Electroanalytical Metal Sensor with Built-in Oxygen Filter[END_REF] In that work, oxygen filter was a single part of a cell consisting of several pieces and quite complicated assembly. The aim was to create and design a more robust and more user-friendly electrochemical cell. 3D printing with PETG thermoplastic polymer was used for this purpose. The printed cell was used for the period of several months with no obvious leaking or degradation of plastic while working with wine samples. At the start of the experimental work, a set-up using only one single SPE ((3) in Fig 1A ) was at first used. However, this configuration was found to be unsuitable for a robust sensor design. It was found that the proximity of reference and counter electrodes in a thin layer with the working electrode and the oxygen filter causes a rapid increase of observed current on both working electrodes. This can be explained by unfavourable feedback loop caused by the products of a reaction at a counter electrode that are then reduced at working electrodes. Therefore, a second SPE (acting only as a reference and a counter electrode) had to be placed further from the working electrode to prevent this issue. The second SPE was inserted into the cap of the cell (( 6) in Fig 1A). There was no significant change in parameters of determination of SO2 when using one or two SPEs.

The cell was at first optimized in order to maximize the efficiency of oxygen removal. It was proven that with the increasing number of layers (1, 2 or 4), the effectiveness of the oxygen filter was improved (the time after no O2 response was observed by CV was decreased). We used several commercially available sealing rubber-based materials of various thickness to observe the effect of a distance between the oxygen filter and the sensing electrode. As expected, the speed of oxygen removal increased with shorter distance between the filter and SPE. However, a seal with a thickness, d, lower than 250 µm did not keep the cell properly tight and the solution leaked. Therefore, a seal with 250 µm thickness and the oxygen filter consisting of 4 layers of platinum mesh were used for this work (Fig S1). In the later parts of the work, after obtaining the optimal conditions for SO2 determination, the parameters such as the applied potential and time of operation of the filter before the detection at the sensing electrode were further optimized.

Voltammetric detection of SO2

The investigation into SO2 sensor was started by confirming the reported pH dependency of SO2 reduction on a carbon SPE. [START_REF] Isaac | A Novel Electroreduction Strategy for the Determination of Sulfite[END_REF][START_REF] Streeter | Cathodic Reduction of Bisulfite and Sulfur Dioxide in Aqueous Solutions on Copper Electrodes: An Electrochemical ESR Study[END_REF] This reaction was studied in solutions of pH 0-6 (Fig S2).

The highest peak currents were observed for pH 0 and 1 and a continuous decrease of the peak current was then observed from pH 2 until 6 in accordance with previously reported results. [START_REF] Isaac | A Novel Electroreduction Strategy for the Determination of Sulfite[END_REF][START_REF] Streeter | Cathodic Reduction of Bisulfite and Sulfur Dioxide in Aqueous Solutions on Copper Electrodes: An Electrochemical ESR Study[END_REF] The potential applied at the oxygen filter was varied with pH to avoid hydrogen evolution and is summarized in Table S2.

To be selective, the oxygen filter must not react with SO2. This was confirmed by cyclic voltammetry showing that no current increase was observed on a platinum mesh used as the oxygen filter when 200 mg L -1 of SO2 was added in the acidified low-sulphite white wine (Fig 2A). The only variation observed after an addition of this high concentration of sulphite in a solution was a decrease of cathodic current at the platinum mesh, possibly because of the removal of trace oxygen in the solution or at the electrode surface by the reaction with sulphite. When the oxygen filter was active, the height of the peak current measured on SPE by CV increased linearly with SO2 concentration in the range of 10-100 mg L -1 in the optimal conditions in HCl-KCl buffer pH 1 (Fig S3).

During the next part of the investigation, we were focused on the application directly in wine samples. Therefore, only the addition of the HCl acid was carried out to adjust the pH of the wine close to 1. The attempt at the determination of SO2 in the wine matrix gave about 3-times lower peak height (compared to the buffer solution) and more pronounced distortion of the SO2 peak, giving two maxima (Fig 2B). This complicated the evaluation as only the first maximum could give a linear response with increasing concentration. These phenomena caused by the wine matrix rendered a voltammetric sensor based on carbon SPEs hardly applicable. The design of the voltammetric sensor for the eventual commercial use is in general more complex due to the complexity of the proper peak evaluation. On the other hand, amperometric sensors are much easier to design since the obtained current reading can be directly converted into the concentration value, bringing much less complexity into the design of a commercial sensor. One of obvious examples are commercial glucometers, which mostly work on the principle of amperometry. [START_REF] Yoo | Glucose Biosensors: An Overview of Use in Clinical Practice[END_REF] Amperometric response is less prone to signal distortion than voltammetric response and it allows easier signal evaluation. Therefore, a possibility of amperometric sensor was closely investigated next.

Amperometry of SO2

For amperometric sensing of SO2, the use of the oxygen filter is crucial. Amperometric responses of oxygen and SO2 cannot be separated, therefore, if there was some oxygen present in the wine, the sensor would overestimate the amount of SO2. The use of the oxygen filter allows the separation of oxygen and SO2 signals. If we consider an experiment with a red wine sample that has been opened for some time, we can assume that it is saturated with dissolved oxygen. If no oxygen filter was used, the recorded current after an addition of 500 µg of SO2 to 10 mL of sample was lower than in the blank wine sample (see curves 1, 2 in Fig 3). This was caused by the reaction of added SO2 and oxygen which decreased oxygen concentration in a solution. If the oxygen filter was used for an analysis of the same sample (see curves 3, 4 in Fig 3 ), the current value in blank was significantly lower as no oxygen was present near the SPE surface and the same addition of 500 µg of SO2 correctly increased the current value. This experiment proves that the accurate determination of SO2 is impossible without the electrochemical removal of oxygen.

The parameters of the method related to the oxygen filter were optimized next. The optimal potential for oxygen filtering was -0.3 V (Fig S4A). Potentials closer to 0 V did not provide effective oxygen removal and evolution of hydrogen bubbles was observed at potentials more negative than -0.3 V, which could block the contact of electrodes with the solution during analysis. Moreover, it was determined that by starting the oxygen filter 60 s before the amperometric detection scan, it was possible to rapidly achieve the steady state on SPE (Fig S4B ) with no significant change using a longer time of oxygen filtration. The amount of HCl added (pH) was additionally optimized in a narrower range of pH from 0.7 to 2 for amperometric detection (Fig S5A). It was found out that at pH 0.7 the signal of SO2 started to significantly decrease. This decrease can be probably attributed to too rapid SO2 evaporation and its escape from the solution. Therefore, the addition of 100 µL of 10 M HCl was chosen as optimal for several reasons. The final pH of wine samples (see Tab S3) was always noted after acidification to check that it was in the range of 1.3 to 1.6. This is due to the wine acting as a buffer, and we can assume that depending on a type of wine, the buffering capacity would differ. The current variation for SO2 reduction between pH 1.1 to 2 (which corresponds to 200 and 50 µL of added acid) is less than 10 %, (see Fig S5A) therefore we can assume that the addition of 100 µL is robust and that a slight change of pH (within 0.3 units) does not significantly induce error in measurements.

Based on previous voltammetric experiments, the detection potential was optimized (Fig S5B).

The viable range was determined between -0.6 V and -0.7 V. Reduction of SO2 would not occur at less negative potentials and at more negative potentials hydrogen evolution took place at the SPE. The optimal potential was therefore determined as -0.65 V. Detection time was kept at 240 s in order to decrease the capacitive current as much as possible and to keep a total analysis time within 5 mins. The optimal technique for SO2 determination in wine was: the oxygen filter was biased at a potential of -0.3 V for 60 s before the start of the amperometric scan at SPE biased at -0.65 V for 240 s (with the oxygen filter still biased at -0.3 V).

Using this protocol, we followed the increase of an amperometric response with the concentration of sulphite in a model red wine matrix (Fig 4A). The reduction current increased linearly in the concentration range from 6 -200 mg L -1 SO2 (Fig 4B ). The calculated limit of quantification was 7.5 mg L -1 in the case of this low-sulphite red wine sample from Fig 4B .   Next step of the investigation was to obtain a standard calibration curve for SO2 in model matrices for both red and white wines. We investigated the reliability and repeatability of the amperometric response at different carbon SPEs. We obtained 4 similar calibration curves for the low-sulphite red wine samples measured within two days with 4 different SPEs (as working electrodes). The calculated average slope value was -0.0609 µA L mg -1 with an error of 9.7 % (see Fig 4C). The same was repeated with low-sulphite white wine. The average slope in this case was -0.0789 µA L mg -1 with an error of 4.0 % (see Fig 4D) and slightly better repeatability, probably due to the less complex matrix of white wines. These values were then used to calculate the unknown concentration of free SO2 in 27 samples of white and red wines. In both cases, intercept values were not statistically different from 0 and therefore, only slope value was used for the following calculations.

Determination of free SO2 in red and white wines

The optimized amperometric method was used for the determination of free SO2 in a total of 27 different wine samples (14 white and 13 red) produced in different regions of France (see Table S1). To note, standard addition method is not suitable for any on-site (or commercial) sensor due to the inherent instability of stock solutions of SO2. The second method used for determination of free SO2 is already established iodometric titration method (using the protocols published by OIV [START_REF]Compendium of international methods of wine and must analysis[END_REF] ).

The content of free SO2 measured by the two methods is summarized in Table 1. Most of the wines (with three exceptions) contained between 10 to 50 mg L -1 of free SO2. This was well within the range of the standard calibration curves built in Fig 4C and4D. The comparison of free SO2 content obtained by amperometry and iodometry gave an excellent correlation (Fig 5).

A perfect agreement between the two methods would give a regression equation of 𝑦 = 1.0 𝑥. The obtained regression slopes were 0.982 for red wines and 1.022 for white wines. Correlation coefficients were greater than 0.95 in both cases. The t-test values (listed in Table 1) were all

Commenté [ME2]: Ajouter potentil appliqué au capteur lower than the critical value, 𝑇 𝑐𝑟𝑖𝑡 = 2.78, indicating that there were no significant differences between the two analytical methods in all cases where the value could be calculated.

As can be seen on three examples of low sulphite wines, i.e. wines R1, R11, and R12, results obtained by amperometry were discarded by the rule related to the mathematical elimination of false positive, as described in Experimental Section. Values obtained by iodometry confirmed that the free SO2 concentration is below LOQ of the amperometric method (see Table 1). The concentration dependency has a U-shape in the range of 0 -12 mg L -1 SO2. The higher current values for blank (c = 0 mg L -1 ) probably corresponded to the charging current whose value was decreasing more rapidly compared to faradaic current corresponding to SO2 reduction. Therefore, one current value only in this range could correspond to two concentrations and a procedure of eliminating false positive results was devised. The decrease of the current value during the amperometric scan of wines R1, R11, and R12 is greater compared to other wines (R4, R7, R9, R10, and W8) with SO2 concentration around 10 mg L -1 . The critical value of 2% was established empirically comparing in total 8 wine samples with low sulphite content. The elimination of false positives this way is graphically illustrated in Fig. S6B (and Table S4) where 2% decrease clearly eliminates false positive readings.

During the tests with different wine samples, one significant advantage of the method presented here over iodometry was demonstrated. Presence of ascorbic acid as an interferent of iodometry can be seen on three white wines (W8, W9, and W11) and two red wines (R7 and R12). The concentration obtained after the first titration (in brackets in Table 1) was several times higher than the actual free SO2 concentration only obtained after the second titration which estimated the amount of interferents. This was observed because iodine can oxidize numerous compounds that are present in wine such as ascorbic acid. There are some reports of adding sulphites and ascorbic acid together as preservatives into the wine and, therefore, a significant amount of ascorbic acid (30 -400 mg L -1 ) can be present in white wines [START_REF] Barril | Ascorbic Acid and White Wine Production: A Review of Beneficial versus Detrimental Impacts[END_REF][START_REF] Wallington | The Decay of Ascorbic Acid in a Model Wine System at Low Oxygen Concentration[END_REF] . As an illustration, the addition of 36 mg L -1 of ascorbic acid increased the titration volume by about 0.6 mL (from ~1.1 to ~1.7 mL in a prepared model sample) (Fig S7A) but in the same red wine sample the amperometric method was not sensitive to ascorbic acid presence as it is not reducible. Indeed, the addition of the same amount of ascorbic acid (36 mg L -1 ) did not increase the current response. The current before (-5.4 µA) only slightly decreased after the addition of ascorbic acid to -5.2 µA due to the dilution of the sample (Fig S7B). In these cases, a second titration determining the content of interferents is necessary in which free SO2 is masked by the addition of aldehydes (formaldehyde) and the result is then deducted from the first titration. However, this brings more complexity and error into the analysis by iodometry.

In all tested wine samples, we have not found any indication of some reducible species that could interfere with the free SO2 analysis. In addition, we do not suspect that any compound usually present in wine [START_REF] Butnariu | Qualitative and Quantitative Chemical Composition of Wine. In Quality Control in the Beverage Industry[END_REF] could significantly interfere with this analysis considering their normal concentrations in wine samples. .01 a -Wines with higher interferent concentration (probably ascorbic acid); value in italics in brackets denotes the concentration value obtained after the first titration. Second titration was carried out after the sample was treated with formaldehyde, see experimental section for details. b -Titration to determine interferents was not carried out. This was the first case where interferent (ascorbic acid) was observed. c -Value was discarded by evaluating the decrease of the current. d -Most likely below LOQ of iodometry. 1. Dashed lines (yellow and red) represent a linear fit for white/red wine samples.

On-site testing

The proposed method was subsequently tested on-site in the winery in Alsace region of France to evaluate the portability, robustness, and easy-to-use aspect of the whole procedure. The analysis was successfully performed by the winemaker himself. Evaluation of the measurement was automated by exporting the current values and using macro in Excell which instantly calculated the concentration of free SO2. Results are summarized in the supplementary material (Tab S5). Sample 1 was later analysed in the laboratory which confirmed the accuracy of the analysis with iodometry and it corresponds to the wine W13 (see Tab 1). The second sample was taken from the wine tank and it is an example of a sample for which an on-site analysis with a portable sensor is highly preferred. This is also an example of a sample for which this sensor is mostly intended to.

Conclusions

In this work we presented an amperometric sensor for the determination of free SO2 in wine. The design of the sensor included the oxygen filter in order to remove oxygen interference in the vicinity of the sensing electrode. This removed a necessity to purge a solution before the analysis which is practically impossible for SO2 solutions considering the fast escape of molecular SO2 during purging.

The proposed method is intended for the on-site analysis of wines. The required sample preparation only includes the addition of the hydrochloric acid (as the only chemical necessary) immediately followed by the detection step. Analysis of one sample including preparation and evaluation of the result can be performed in less than 10 minutes. The amperometric sensing technique is technically simple to construct with straightforward calculation of a concentration from a current reading compared to voltammetric techniques, where an automatic peak evaluation process must be integrated into the sensor. It is built on commercially available and low-cost screen-printed electrodes that proved to give a repeatable performance and provide a standard calibration for the determination of free SO2 in red and white wines. So far, electrochemical sensors based on the reduction of SO2 used only standard additions to determine SO2 content in real wine samples. This is practically impossible to carry out on-site due to the inherent instability of SO2 standard solutions. Using the standard calibration for SO2 determination completely eliminates this issue and greatly improves practical application of the sensor.

The presented sensor provided a linear response up to 200 mg L -1 with a limit of quantification at 7.5 mg L -1 . The application of the sensor for the determination of free SO2 in wine was proven by determining the quantity of free SO2 in 27 red and white wine samples. Results obtained by amperometric sensing method were comparable to the values obtained by the standard method of iodometry recommended by OIV. Additionally, the amperometric method did not suffer from any interferences in all of the measured wines. On the other hand, higher content of ascorbic acid can give a false overestimation of free SO2 content by iodometry. The presented sensor was successfully tested on-site with a winemaker who carried out the analysis correctly. Therefore, we propose this method as an alternative to currently used Ripper method based on iodometry that can be potentially used for the determination of SO2 content in other foods and drinks directly at the point of interest by producers.

Associated content

The following content is available as supporting information: Wine tested in the studies; Optimisation of the electrochemical cell design; Optimisation of the SO2 sensing parameters; Determination of free SO2 in red and white wine.

  3D model of the cell can be seen in Fig 1A. It consists of two 3D-printed plastic parts put together with the help of magnets inserted into the models during 3D-printing process. The lower part (in red on Fig 1A) is designed to host SPE. The upper part (in blue in Fig 1A) has an oxygen filter (shown as (1) on Fig 1A) incorporated in it.The oxygen filter was constructed with 1, 2, or 4 layers of platinum mesh and was inserted into the plastic during 3D printing. The distance between the oxygen filter and SPE was controlled with an O-ring made from nitrile rubber of 250 µm thickness. The 15 mL plastic tube with a cut-out bottom was then attached with epoxy glue into the top part. A sample was placed as a column of liquid in an enclosed space. This prevented a rapid escape of SO2 and created a barrier for oxygen coming into the sample from the air. A scheme illustrating this function of the oxygen filter is in Fig 1B.The cell was finally assembled and ready for measurements with some other 3D printed parts (printed in grey plastic in Fig1A) in order to facilitate the handling of the cell.

Fig 1 :

 1 Fig 1: (A) 3D model of the cell (on the left) and photograph (on the right) of the assembled cell with a red wine sample. Oxygen filter made of a platinum mesh (1) is inserted into the 3D printed upper part. Upper and lower parts are put together and sealed with the rubber O-ring (2) (d = 250 µm) placed on top of the SPE acting as the working electrode only (3). The connection of the oxygen filter to the bipotentiostat as a second working electrode is made with a copper tape (4). Wine sample (5) was pipetted into the cell from the top. SPE acting as a reference and a counter electrode was integrated in the cap(6). (B) Scheme illustrating the removal of the oxygen in the vicinity of the sensing electrode for the determination of SO2 in wines.

Fig 2 :

 2 Fig 2: (A) Cyclic voltammograms ( = 5 mV s -1 ) obtained at the oxygen filter in the acidified low-sulphite white wine sample (pH 1.4). Curve 1 corresponds to the reduction of dissolved oxygen in wine at the oxygen filter. Curves 2 and 3 were recorded after the removal of oxygen by nitrogen purging in the absence (2) and in the presence (3) of 200 mg L -1 SO2 (curve 3). (B) Cyclic voltammograms ( = 50 mV s -1 ) of SO2 (c = 0 (1); 10 (2); 20 (3); 40 (4); 60 (5); 80 (6); 100 (7) mg L -1 ) obtained on SPE in deoxygenated acidified low-sulphite white wine sample (pH 1.4). Oxygen filter was set at a potential -0.3 V. Concentration dependencies are shown in the inset.

Fig 3 :

 3 Fig 3: Amperometric recordings obtained on SPE in 10 mL of the acidified low-sulphite red wine sample (pH 1.6) with the oxygen filter not connected (curves 1, 2) and the filter set at the potential -0.3 V (curves 3, 4). Curves 1 and 3 were measured with dissolved oxygen in the wine sample, curves 2 and 4 were measured after the addition of 500 µg of SO2.

1 Fig. 4 :

 14 Fig.4: (A) Amperometric recordings of SO2 (c = (0) 6 -200 mg L -1 ) obtained on SPE in deoxygenated acidified low-sulphite red wine sample with the oxygen filter set at the potential -0.3 V. (B) Dependency of the current on the concentration of SO2. Typical calibration curves obtained on 4 different SPEs in (C) deoxygenated acidified low-sulphite white wine sample (pH 1.4) and (D) low-sulphite red wine sample (pH 1.6). Shaded areas correspond to 95% confidence interval of the obtained dependencies.

Fig 5 :

 5 Fig 5: Correlation between measured concentration of free SO2 in samples of white wines (yellow circles and error bars) and red wines (red squares and error bars) by amperometric detection and iodometric titration from Table1. Dashed lines (yellow and red) represent a linear fit for white/red wine samples.

Table 1 :

 1 Comparison of determination of free SO2 in 21 different samples of white and red wines using the developed amperometric method and iodometry.

	Wine	Amperometry free SO2, mg L -1	Iodometry free SO2, mg L -1	T-test (Tcrit = 2.78)
	W1	19.7 ±0.8	23.5 ±1.9	2.32
	W2	34.3 ±1.4	30.3 ±2.1	1.36
	W3	20.2 ±0.8	22.2 ±2.3	0.91
	W4	25.5 ±1.0	28.7 ±2.0	1.57
	W5	19.7 ±0.8	22.3 ±1.6	1.93
	W6	44.2 ±1.8	45.7 ±3.9	0.20
	W7	21.2 ±0.8	22.7 ±2.3	0.68
	W8	18.7 ±0.7	13.3 ±3.4 (44) a	1.41
	W9	26.0 ±1.0	24.6 ±3.9 (45) a	0.26
	W10	23.6 ±0.9	24.0 ±1.6	0.24
	W11	23.5 ±0.9	27.9 ±4.1	0.72
	W12	23.3 ±0.9	20.0 ±2.2	1.51
	W13	19.7 ±0.8	17.5 ±1.6	1.74
	W14	25.2 ±1.0	-(64) b	-
	R1	<LOQ c	1.8 ±1.2 d	-
	R2	22.4 ±2.2	23.9 ±2.9	0.24
	R3	26.7 ±2.6	26.5 ±4.8	0.01
	R4	17.8 ±1.7	15.6 ±2.0	0.60
	R5	21.6 ±2.1	16.9 ±2.0	0.94
	R6	44.3 ±4.3	42.8 ±4.0	0.07
	R7	11.7 ±1.1	10.6 ±13.1 (30) a	0.02
	R8	20.9 ±2.0	25.6 ±3.6	0.64
	R9	11.3 ±1.1	15.3 ±3.8	0.77
	R10	16.5 ±1.6	19.3 ±3.2	0.55
	R11	<LOQ c	4.0 ±1.2	-
	R12	<LOQ c	3.3 ±2.5 (10) a, d	-
	R13	17.4 ±1.7	21.2 ±2.2	1

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Acknowledgements

This work was supported by SATT Sayens. The authors thank Laurent Akrour and Pablo Fanjul Bolado (Metrohm / Dropsens) for the discussion, material and technical support. The authors are grateful to Alexandre Arbogast (Dalenheim, France) for his help with on-site analysis and carrying out the analysis of sulphites.