sensors Article

Reagent-Less and Robust Biosensor for Direct Determination of Lactate in Food Samples Iria Bravo 1,2 , Mónica Revenga-Parra 1,2,3 , Félix Pariente 1,3 and Encarnación Lorenzo 1,2,3, * 1

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Departamento de Química Analítica y Análisis Instrumental, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain; [email protected] (I.B.); [email protected] (M.R.-P.); [email protected] (F.P.) Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia), Faraday, 9, Campus UAM, Cantoblanco, 28049 Madrid, Spain Institute for Advanced Research in Chemical Sciences (IAdChem), Universidad Autónoma de Madrid, 28049 Madrid, Spain Correspondence: [email protected]; Tel.: +34-91-497-4488

Academic Editors: Jesus Iniesta Valcarcel and Craig E. Banks Received: 28 September 2016; Accepted: 11 January 2017; Published: 13 January 2017

Abstract: Lactic acid is a relevant analyte in the food industry, since it affects the flavor, freshness, and storage quality of several products, such as milk and dairy products, juices, or wines. It is the product of lactose or malo-lactic fermentation. In this work, we developed a lactate biosensor based on the immobilization of lactate oxidase (LOx) onto N,N 0 -Bis(3,4-dihydroxybenzylidene)1,2-diaminobenzene Schiff base tetradentate ligand-modified gold nanoparticles (3,4DHS–AuNPs) deposited onto screen-printed carbon electrodes, which exhibit a potent electrocatalytic effect towards hydrogen peroxide oxidation/reduction. 3,4DHS–AuNPs were synthesized within a unique reaction step, in which 3,4DHS acts as reducing/capping/modifier agent for the generation of stable colloidal suspensions of Schiff base ligand–AuNPs assemblies of controlled size. The ligand—in addition to its reduction action—provides a robust coating to gold nanoparticles and a catalytic function. Lactate oxidase (LOx) catalyzes the conversion of L-lactate to pyruvate in the presence of oxygen, producing hydrogen peroxide, which is catalytically oxidized at 3,4DHS–AuNPs modified screen-printed carbon electrodes at +0.2 V. The measured electrocatalytic current is directly proportional to the concentration of peroxide, which is related to the amount of lactate present in the sample. The developed biosensor shows a detection limit of 2.6 µM lactate and a sensitivity of 5.1 ± 0.1 µA·mM−1 . The utility of the device has been demonstrated by the determination of the lactate content in different matrixes (white wine, beer, and yogurt). The obtained results compare well to those obtained using a standard enzymatic-spectrophotometric assay kit. Keywords: Schiff base tetradentate ligand modified gold nanoparticles; lactate determination; enzymatic biosensor; gold nanoparticles; lactate oxidase biosensor

1. Introduction L-lactate is a metabolite generated during anaerobic glucose metabolism. Its production involves an increase in the proton concentration inside the cells. L-lactate levels in blood range from 0.5 to 1.5 mM in normal people at rest, and can increase to 12.0 mM during exercise [1]. Under extreme conditions of excessive exercise, lactate levels can become as high as 25 mM. In this case, the cellular proton buffering may be exceeded. Hence, the cell pH decreases, and this may result in cell lactic acidosis, which disrupts the performance of the muscles [2]. Therefore, knowledge of personal lactic threshold is very important to perform any physical activity, and this is the reason why sport medicine requires the monitoring of L-lactic levels in order to evaluate the so-called lactic threshold [3], which

Sensors 2017, 17, 144; doi:10.3390/s17010144

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indicates the physical training level of an athlete. Lactate levels in plasma depend on the balance between factors that produce lactate and lactate clearance factors [4]. Any imbalance between the production and clearance of lactate may lead to the lactic acidosis. The increase of lactate levels in blood may be due to the presence of very diverse pathologies, such as haemorrhagic shock or pulmonary embolism [4], cardiogenic [5] or endotoxic shocks [6], respiratory failure [7], liver diseases [8], and others. High levels of lactate are also the main cause of acidification in the microenvironment of cancer cells and tumors [9], due to the imbalance between the dynamics of the production and consumption of lactate, characteristic for these types of cells and tissues [10]. Thus, the importance of determining lactate level in medical care is evident because the blood lactate level is a valuable indicator of both fitness and clinical states. Lactic acid fermentation is an important process in the food industry. Lactate levels may indicate the stability, freshness, and quality of many fermented dairy products such as milk, yogurt, and creams, as well as raw meat, fruits, and vegetables [11]. In wine production, the malolactic fermentation carried out by bacteria present in the grapes and the must is very important. This fermentation takes place after the alcoholic fermentation, and converts the L-malic acid to L-lactic reducing the wine acidity, and leading to an improvement of the taste and flavor of the final product. The control of this secondary fermentation is very important in the quality of wines, and requires a continuous monitoring of the levels of lactic and malic acids present in the young wine [11]. The examples described above show the great interest that exists in monitoring lactate concentration for clinical diagnosis, intensive care, in the food industry, and in food control, among others. To achieve these goals, there is a great demand for devices capable of determining lactate in a simple and direct way with rapid response, high specificity, low cost, and minimal or no sample preparation. In this regard, a variety of methods have been described, using techniques such as amperometry [12], potentiometry [13], high-performance liquid chromatography [14], fluorometry [15], chemiluminescence [16], microwave sensing [17], holography [18], and magnetic resonance spectroscopy [19]. Because of their simplicity, portability, and easy integration in different devices, amperometric biosensors have reached the most significant development—in particular when reagent-less biosensors based on disposable electrodes are employed. Different biosensors for lactate monitoring have been reported based on immobilized lactate oxidase [20–23]. Bienzyme systems such as lactate oxidase/lactate dehydrogenase (LOx/LDH) have also been described [24]. In these configurations, the enzyme was immobilized in a polymeric matrix inside the electrode material. Recently, in order to improve the biosensor response, lactate biosensors using nanoparticles have been described [25–29]. In general, these devices showed linearity in the range of µM to mM lactate concentration. L-lactate oxidase and lactate dehydrogenase have been the enzymes commonly used in the design of L-lactate biosensors due to the favorable kinetics of the enzymatic reactions, the stability of the immobilized enzymes, and the simple design of the biosensors. In fact, LOx is usually preferred over LDH because its reaction involves O2 and H2 O2 that can be amperometrically detected. However, the oxidation of H2 O2 requires a high overpotential. A strategy employed to overcome this problem is the addition of redox mediators to the solution, and in order to simplify the systems, an improved strategy is to include the mediator in the device development. L -lactate oxidase is a globular flavoprotein with a mean diameter of 5 nm that can be obtained with an acceptable degree of purity from various microorganisms, such as Pediococcus and Aerococcus viridians [30]. In the presence of molecular oxygen, the enzyme catalyzes the oxidation of L-lactate to pyruvate, yielding hydrogen peroxide, which is electrochemically active and can be oxidized or reduced on the electrode surface giving rise to a current which is proportional to the concentration of lactate present in the sample [31,32]. As can be seen in Scheme 1, the electrons involved in the reaction are transferred from the substrate (L-lactate) to the oxidized form of the enzyme cofactor (Flavin Adenine Dinucleotide, FAD) present in the enzyme structure. To regenerate the enzyme catalytic site, molecular oxygen takes the electrons from the reduced cofactor, turning

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Sensors 2017, 17, 144 3 of can 11 be into hydrogen peroxide. The flow of electrons from the substrate to the electrode surface enhanced by the presence of highly conductive nanomaterials in the sensing interface, which can act enhanced by the presence of highly conductive nanomaterials in the sensing interface, which can act as tiny conductive centers, allowing kinetic barriers to be eliminated, and yielding more sensitive as tiny conductive centers, allowing kinetic barriers to be eliminated, and yielding more sensitive electrochemical responses [33]. electrochemical responses [33].

Scheme 1. of the Scheme the 3,4DHS: biosensing platform. 3,4DHS: Scheme 1. Scheme biosensingofplatform. N,N 0 -Bis(3,4-dihydroxybenzylidene)N,N′-Bis(3,4-dihydroxybenzylidene)-1,2-diaminobenzene. 1,2-diaminobenzene.

Recently, we reported the use of a multi-tasking N,N′-Bis(3,4-dihydroxybenzylidene)

0 -Bis(3,4-dihydroxybenzylidene)Recently, we reported the tetradentate use of a ligand multi-tasking -1,2-diaminobenzene Schiff base (3,4DHS) asN,N reductant, stabilizer, and catalyst in a new concept Schiff of gold synthesis as[34]. This ligand contains the 1,2-diaminobenzene basenanoparticles tetradentate(AuNPs) ligand (3,4DHS) reductant, stabilizer, and catalyst quinone/hydroquinone functional group and is capable of reducing HAuCl 4 in water, also acting as the in a new concept of gold nanoparticles (AuNPs) synthesis [34]. This ligand contains a capping agent for the generation of stable colloidal suspensions of Schiff base ligand–AuNPs quinone/hydroquinone functional group and is capable of reducing HAuCl4 in water, also acting assemblies of controlled size by providing a robust coating to AuNPs within a unique reaction step. as a capping agent for the generation of stable colloidal suspensions of Schiff base ligand–AuNPs These 3,4DHS–AuNPs assemblies—deposited on carbon electrodes—show a potent electrocatalytic assemblies of controlled size by providing a robust coating to AuNPs within a unique reaction step. effect towards hydrazine oxidation and hydrogen peroxide oxidation/reduction [34]. Following Thesethese 3,4DHS–AuNPs assemblies—deposited on carbon electrodes—show a potent electrocatalytic previous studies, the present work takes the preparation of 3,4DHS–AuNPs assemblies effectone-step towardsfurther hydrazine oxidation and peroxide [34]. Following by using them for thehydrogen construction of a newoxidation/reduction L-lactate biosensor. This is based on thethese previous studies, the present work takes thealong preparation of 3,4DHS–AuNPs one-step further co-immobilization of L-lactate oxidase with 3,4DHS–AuNPs onto aassemblies screen-printed carbon by using them for the rise construction of a new biosensor, L-lactate biosensor. This based on co-immobilization electrode giving to a reagent-less in an effort to issimplify thethe development of point-of-care lactate analysis systems. of L-lactate oxidase along with 3,4DHS–AuNPs onto a screen-printed carbon electrode giving rise to a Thebiosensor, biosensor in response was in terms of enzyme solution pH, and the effect reagent-less an effort to optimized simplify the development ofloading, point-of-care lactate analysis systems. of potentially interfering substances. Finally, the developed biosensor has been applied theeffect The biosensor response was optimized in terms of enzyme loading, solution pH, andtothe determination of lactate in food and beverage samples. The results were validated by comparing of potentially interfering substances. Finally, the developed biosensor has been applied to the with those obtained with a commercial enzymatic kit. determination of lactate in food and beverage samples. The results were validated by comparing with those2.obtained commercial enzymatic kit. Materialswith and aMethods

2. Materials and and Methods 2.1. Reagents Apparatus Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O, ≥99.9% trace metals basis) and 2.1. Reagents and Apparatus L-(+)-lactic

acid lithium salt 97% were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA).

Hydrogen tetrachloroaurate (III)from trihydrate (HAuCl 3H2 O, ≥99.9% trace Lactate oxidase (LOx, EC 232-841-6 Pediococcus species) powder wasmetals obtainedbasis) from and 4 ·lyophilized L -(+)-lactic lithium were fromsolutions Aldrichwere Chemical Co. 200 (Milwaukee, WI,MUSA). Sigma acid Chemical Co. salt (St. 97% Louis, MO,obtained USA). Stock prepared U·mL−1 in 0.1 phosphate 7.0) and stored −30 °C. Under these conditions, powder the enzymatic activitiesfrom Lactate oxidase buffer (LOx, (pH EC 232-841-6 from at Pediococcus species) lyophilized was obtained stableCo. for several weeks.MO, The USA). enzymatic assay kit for lactate Sigmaremain Chemical (St. Louis, Stock solutions were determination prepared 200(K-LATE U·mL−107/14) in 0.1 M was purchased from Megazyme (Ireland). Other chemicals used in this work were reagent grade ◦ phosphate buffer (pH 7.0) and stored at −30 C. Under these conditions, the enzymatic activities remain stable for several weeks. The enzymatic assay kit for lactate determination (K-LATE 07/14) was purchased from Megazyme (Ireland). Other chemicals used in this work were reagent grade quality

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and used as received without further purification. MilliQ water (Millipore® , Darmstadt, Germany) was used throughout. N,N 0 -Bis(3,4-dihydroxybenzylidene)-1,2-diaminobenzene (3,4DHS) was prepared as previously reported [35] by condensation of 1,2-phenylendiamine with the 3,4-dihydroxybenzaldehyde isomer in a 1:2 molar stoichiometric ratio. The resulting compound was recrystallized from hot methanol. Stock solutions of 3,4DHS (typically 2.0 mM) were prepared just prior to use by dissolving the solid in dimethylformamide. All electrochemical measurements were carried out using an Autolab potentiostat/galvanostat type PGSTAT 302N (Eco Chemie, Utrecht, The Netherlands) using the software package GPES 4.9. Integrated screen-printed carbon electrodes (4 mm diameter, SPCEs) from DropSens were used. They include a silver pseudoreference electrode and a carbon counter electrode. A 1.5 mL home-made glass electrochemical cell was employed to carry out the measurements. All experiments were carried out in 0.1 M phosphate buffer (pH 7.0) at 25 ◦ C. 2.2. Procedures 2.2.1. Synthesis of 3,4DHS Capped Gold Nanoparticles (3,4DHS–AuNPs) 3,4DHS–AuNPs were synthesized as previously reported [34] by mixing aqueous HAuCl4 with 3,4DHS solution, and chemical reduction was left to proceed for 24 h at room temperature. The 3,4DHS capped AuNPs thus formed were separated by centrifugation at 9000 rpm (7700 g) and washed with water to remove excess 3,4DHS. The size and concentration of 3,4DHS–AuNPs were estimated from the ratio of absorbance measured at the surface plasmon band (~520 nm) and the absorbance at 450 nm, as described by Haiss et al. [36]. The particle size value was confirmed by transmission electron microscopy [34]. Thus, a stock solution of 17.7 nM (33 ± 3 nm) 3,4DHS–AuNPs was employed. 2.2.2. Biosensor Preparation Screen-printed carbon electrodes (SPCEs) were modified by transferring of 4 µL of the 3,4DHS–AuNPs solution onto the working electrode surface (3,4DHS–AuNP/SPCE) followed by air-drying at room temperature. Once dried, 5 µL of LOx (1 U) were deposited on the 3,4DHS–AuNP/SPCE surface and placed for 1 h at 4 ◦ C to assemble the protein on the electrode surface (LOx/3,4DHS–AuNP/SPCE). 2.2.3. Determination of Lactate in Food Samples Lactate was determined in food samples using the developed amperometric biosensor. Results were compared with those obtained using a commercial enzymatic assay kit following the procedure described by the manufacturer. White wine, beer, and yogurt were purchased in a local store and were analyzed by standard addition method without any pre-treatment other than dilution in 0.1 M phosphate buffer (pH 7.0). 3. Results and Discussion 3.1. Lactate Oxidase Biosensor Development The modification of AuNPs with electroactive substances can be of great interest in designing new nanostructured platforms with applications in catalytic processes, and in the construction of biosensors [37]. In a previous work, we reported the multi-tasking N,N 0 -Bis(3,4-dihydroxybenzylidene)1,2-diaminobenzene Schiff base tetradentate ligand (3,4DHS) as reductant, stabilizer, and catalyst in a new concept of AuNPs synthesis. Once these assemblies were deposited on screen-printed carbon electrodes, the resulting nanostructured platforms exhibited excellent electrocatalytic activity towards the oxidation and the reduction of peroxide in alkaline medium [34]. In this work, we have gone

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a stepSensors further and have used these platforms to build a portable lactate biosensor based on lactate 2017, 17, 144 5 of 11 oxidase in which the lactate is oxidized to pyruvate and hydrogen peroxide in presence of oxygen. Sensors 2017, 17, 144 5 of 11 portable lactategenerated biosensor based oxidase in which the lactate is oxidized to pyruvatemodified and The enzymatically H2 O2onislactate electrocatalytically oxidized at the 3,4DHS–AuNPs portable lactate biosensor based on in which thegenerated lactate is oxidized to pyruvate and hydrogen peroxide presence of lactate oxygen. The enzymatically H2O2 is electrocatalytically electrode, giving rise toin the analytical signaloxidase (Scheme 1). hydrogen peroxide in presence ofmodified oxygen. electrode, The enzymatically generated H2O2 is signal electrocatalytically oxidized the 3,4DHS–AuNPs giving rise to the analytical (Scheme 1). with At a first at step, the electrochemical behavior of the screen-printed carbon electrode modified oxidized at the 3,4DHS–AuNPs modified electrode, giving rise to the analytical signal (Scheme 1). At a first step, the electrochemical behavior of the screen-printed carbon electrode modified 3,4DHS capped gold nanoparticles (3,4DHS–AuNP/SPCE) was studied using cyclic voltammetry in a first step, the electrochemical behavior of the screen-printed was carbonstudied electrode modified withAt 3,4DHS capped gold nanoparticles (3,4DHS–AuNP/SPCE) using cyclic 0.1 M phosphate buffer (pH gold 7). Asnanoparticles can be seen in(3,4DHS–AuNP/SPCE) Figure 1a, a well-defined redox couple with cyclic anodic and with 3,4DHS capped was studied using voltammetry in 0.1 M phosphate buffer (pH 7). As can be seen in Figure 1a, a well-defined redox cathodic peak potentials centered at buffer +0.13 V and V, be respectively, was1a, observed. This redox couple voltammetry in 0.1 Mand phosphate (pH 7).+0.02 Ascentered can seen in Figure a well-defined redox couple with anodic cathodic peak potentials at +0.13 V and +0.02 V, respectively, was is ascribed to the quinone/hydroquinone functional groups present in the 3,4DHS–AuNPs. The couple with anodic and cathodic peak potentials centered at +0.13 V and +0.02 V, respectively, was observed. This redox couple is ascribed to the quinone/hydroquinone functional groups present formal in potential (E =This +0.08 V) was constant lowV)scan and the anodic cathodic observed. redox couple is ascribed to the functional groups in peak the 3,4DHS–AuNPs. Thepractically formal potential (E quinone/hydroquinone = at +0.08 wasrates, practically constant at and lowpresent scan rates, the 3,4DHS–AuNPs. formal potential (E =showed +0.08 V) was constant at low currents showed a linear dependence with the scan rate thepractically range studied, asthe anticipated for a redox and the anodic and The cathodic peak currents a in linear dependence with scanscan rate rates, in the and the anodic and cathodic peak currents showed a linear dependence with the scan rate in the range studied, anticipated a redox couple1b). confined on the electrode surface (Figure The couple confined onasthe electrodefor surface (Figure The peak-to-peak separation (∆Ep)1b). was 0.11 V range studied, as anticipated for a aredox confined on the electrode 1b). The and peak-to-peak separation (ΔEp) was 0.11 couple V (farredox from the 0 value anticipated forelectrode a(Figure reversible redox (far from the 0 value anticipated for reversible process confined onsurface the surface), peak-to-peak separation (ΔEp) wassurface), 0.11 V (far the 0 value anticipated forrates a reversiblethan redox process confined onfor thescan electrode andfrom increased scan increased significantly rates higher than 1.0 V·s−1 ,significantly suggesting for severe kinetichigher limitations1.0in the process confined onsevere the electrode andin increased significantly scan rates higher than 1.0 V·s−1, suggesting kinetic surface), limitations the charge transfer for (Figure 2a). The anodic and charge −1transfer (Figure 2a). The anodic and cathodic peak potentials were plotted vs. log (v; scan rate) V·s , suggesting severe kinetic limitations 2a).plot The anodic and cathodic peak potentials were plotted vs. login (v;the scancharge rate) intransfer a typical(Figure Laviron’s [38] (Figure 2b). in a typical Laviron’s plot [38] (Figure 2b). log At scan rates higher than 5 V·s−1 , the potential depicts −1, thevs. cathodic were plotted (v; scan rate) in aatypical plotpeak [38]log (Figure 2b). At scan peak ratespotentials higher than 5 V·s peak potential depicts linear Laviron’s dependence with (v). From a linear dependence with log (v). From the ratio of the slopes of these straight lines, according to −1 At scan rates higher thanof5 these V·s , straight the peaklines, potential depictstoaLaviron’s linear dependence From the ratio of the slopes according equation with Ep = log E° +(v). (RT/αnF) ◦ + (RT/αnF) [ln(RT·k /αnF) − lnv], the electron-transfer coefficient (α) and Laviron’s equation Ep = E the ratio of the −slopes of these straight lines, according Laviron’s equation Ep =electron-transfer E° + (RT/αnF) s [ln(RT·k s/αnF) lnv], the electron-transfer coefficient (α)toand the apparent surface [ln(RT·k s/αnF) − slnv], thefound electron-transfer coefficient and the apparent surface electron-transfer the apparent surface rateand constant (k(α) found to be 0.50 and 47 s−1 , respectively. s ) were rate constant (k ) electron-transfer were to be 0.50 47 s−1, respectively. rate constant (ks) were found to be 0.50 and 47 s−1, respectively. 2.0

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0.4 / V 0.2 E/V Figure (a) Cyclic voltammogram screen-printed carbon modified with 3,4DHS capped Figure 1. (a)1.Cyclic voltammogram ofofscreen-printed carbonelectrode electrode modified with 3,4DHS capped −1; Thecapped Figure 1. (a) Cyclic voltammogram of screen-printed carbon electrode modified withV·s 3,4DHS inset (b) gold nanoparticles (3,4DHS–AuNP/SPCE) in 0.1 M phosphate buffer (pH 7) at 0.04 − 1 gold nanoparticles (3,4DHS–AuNP/SPCE) in 0.1 M phosphate buffer (pH 7) at 0.04 V·s ; The inset gold nanoparticles (3,4DHS–AuNP/SPCE) 0.1 M phosphate buffer (pHscan 7) atrate. 0.04 V·s−1; The inset (b) shows the dependence of the anodic and in cathodic peak current on the (b) shows the dependence of the anodic and cathodic peak current on the scan rate. shows the dependence of the anodic and cathodic peak current on the scan rate. 40 40 30

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Figure (a) voltammetric response ofof3,4DHS–AuNP/SPCE M phosphate buffer Figure 2. Cyclic (a) Cyclic voltammetric response 3,4DHS–AuNP/SPCE inin 0.10.1 phosphate buffer (pH 7) at 2.different scan rates; (b) Laviron’s plot showing the dependence ofMthe peak potential on(pH the 7) at atlogarithm different scan rates; (b) Laviron’s plot showing the dependence the peak potential onlogarithm the different scan rates; (b) Laviron’s plot showing the dependence of theofpeak potential on the of scan rate. logarithm of scan rate. of scan rate.

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The electrocatalytic behavior of the 3,4DHS–AuNP/SPC modified electrodes toward the oxidation behavior of the 3,4DHS–AuNP/SPC modified electrodes toward the of H2 O2 atThe pHelectrocatalytic 7—the optimal for lactate oxidase enzyme function—was studied. Figure 3a oxidation of H2O2 at pH 7—the optimal for lactate oxidase enzyme function—was studied. Figure 3a shows the cyclic voltammograms of 3,4DHS–AuNP/SPCE in 0.1 M phosphate buffer (pH 7) in shows the cyclic voltammograms of 3,4DHS–AuNP/SPCE in 0.1 M phosphate buffer (pH 7) in the the absence and presence of 1.0 mM of H2 O2 recorded at 0.01 V·s−1 . In the absence of H O , absence and presence of 1.0 mM of H2O2 recorded at 0.01 V·s−1. In the absence of H2O2, a well-defined2 2 a well-defined electrochemical (Figure grey line) ascribed the oxidation/reduction electrochemical response response (Figure 3a, grey 3a, line) ascribed to the to oxidation/reduction of the of the hydroquinone/quinone was observed. observed.However, However, the presence hydroquinone/quinone moieties moieties of of the the 3,4DHS 3,4DHS was inin the presence of of H2H O22, O2 , there was dramatic increase in the anodic andpractically practically current observed therea was a dramatic increase in the anodicpeak peakcurrent, current, and no no current was was observed in the reverse (cathodic) scan (Figure 3a, black line). This behavior is consistent with a very strong in the reverse (cathodic) scan (Figure 3a, black line). This behavior is consistent with a very strong electrocatalytic 3,4DHS–AuNP/SPCEhas hasan anexcellent excellent electroacatalytic towards electrocatalytic effect.effect. Thus,Thus, 3,4DHS–AuNP/SPCE electroacatalyticactivity activity towards the oxidation of H 2O2, and facilitates electrochemical detection of this compound at lower potential. the oxidation of H2 O , and facilitates electrochemical detection of this compound at lower potential. 2 24

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3. (a) Cyclic voltammogram of 3,4DHS–AuNP/SPCEin in 0.1 0.1 M M phosphate (pH 7) 7) in the Figure Figure 3. (a) Cyclic voltammogram of 3,4DHS–AuNP/SPCE phosphatebuffer buffer (pH in the (b) Cyclic Cyclic voltammogram of of lactate (grey or line) or presence (black of 1.0 mM absenceabsence (grey line) presence (black line)line) of 1.0 mM of of H2HO2O2 ;2;(b) voltammogram lactate (LOx)/3,4DHS–AuNP/SPCE in 0.1 M phosphate buffer (pH 7) in the absence (dotted line) or oxidaseoxidase (LOx)/3,4DHS–AuNP/SPCE in 0.1 M phosphate buffer (pH 7) in the absence (dotted line) or −1. presence (solid line) of 0.5 mM lactate. Scan rate: 0.01 V·s presence (solid line) of 0.5 mM lactate. Scan rate: 0.01 V·s−1 .

The 3,4DHS–AuNP/SPCE platform was used to develop an amperometric lactate biosensor based on the quantification of the H2O2was liberated the enzymatic reaction. Thelactate biosensor was The 3,4DHS–AuNP/SPCE platform usedduring to develop an amperometric biosensor developed by coupling the lactate oxidase enzyme to the 3,4DHS–AuNP/SPCE platform, based on the quantification of the H2 O2 liberated during the enzymatic reaction. The biosensoraswas described in the experimental section.enzyme The cyclic voltammetric biosensor response (LOx/3,4DHS– developed by coupling the lactate oxidase to the 3,4DHS–AuNP/SPCE platform, as described AuNP/SPCE) in the presence and absence of the substrate (lactate) was used to assess its catalytic in the experimental section. The cyclic voltammetric biosensor response (LOx/3,4DHS–AuNP/SPCE) activity (Figure 3b). In the absence of lactate (grey line), the well-behaved redox response of 3,4DHS– in the presence and absence of the substrate (lactate) was used to assess its catalytic activity (Figure 3b). AuNP was readily apparent. Upon the addition of lactate (to a final concentration of 0.5 mM), there In the absence of lactate (grey line), the well-behaved redox response of 3,4DHS–AuNP was readily was an increase in the anodic peak current due to electrocatalytic oxidation of the H2O2 generated in apparent. the addition (to aa decrease final concentration of 0.5 mM), therewas wasobserved an increase in the the Upon enzymatic reaction. of Inlactate addition, in the cathodic peak current (black anodic line). peak To current due torole electrocatalytic oxidation of the response H2 O2 generated in the enzymatic reaction. confirm the of the enzyme in the catalytic to substrate, 3,4DHS–AuNP/SPC In addition, a decrease in without the cathodic peak current wasimmersed observedin(black To confirm the role of modified electrodes immobilized LOx were 0.1 M line). phosphate buffer solution (pH 7.0). As one wouldresponse expect, upon addition of3,4DHS–AuNP/SPC lactate, no catalytic waves were observed. the enzyme in the catalytic to substrate, modified electrodes without The amount both LOx and 3,4DHS–AuNPs the biosensor development was optimized. For immobilized LOx wereofimmersed in 0.1 M phosphateinbuffer solution (pH 7.0). As one would expect, this purpose, different with increasing amounts of both components were prepared, and upon addition of lactate, no biosensors catalytic waves were observed. their responses to lactate The biosensor increased as the units of optimized. enzyme The amount of both LOxwere and obtained. 3,4DHS–AuNPs in theresponse biosensor development was included in the biosensing layer increased from 0.3 U to 1.0 U (Figure 4a). At higher enzyme loading, For this purpose, different biosensors with increasing amounts of both components were prepared, there was a decrease in the biosensor response. This is probably due to an excess of protein on the and their responses to lactate were obtained. The biosensor response increased as the units of enzyme biosensing layer that could prevent the charge transfer towards the electrode surface. In accordance included in these the biosensing layer increased fromas0.3 U to 1.0 U (Figure 4a). At higher enzyme loading, with results, 1.0 U of LOx was chosen optimal. there was aAs decrease in the biosensor response. This is probably to an excess of protein one would expect, the LOx biosensor response showeddue a significant dependence on on thethe biosensing layer that could prevent the charge the electrode surface. In 20, accordance amount of 3,4DHS–AuNPs deposited on thetransfer electrodetowards surface. Biosensors prepared using 70, or with these results, 1.0 U of LOx was chosen as optimal. As one would expect, the LOx biosensor response showed a significant dependence on the amount of 3,4DHS–AuNPs deposited on the electrode surface. Biosensors prepared using 20, 70, or 180 fmol of

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180 fmol of 3,4DHS–AuNPs showed different responses. Although the highest catalytic response 7 of 11 was obtained when 180 fmol were employed (Figure 4b), the catalytic efficiency—defined as the ratio between the catalytic current and the current measured for the LOx/3,4DHS–AuNP/SPCE in the absence showed of lactatedifferent (ICAT/IMEDresponses. )—was higher when 70 3,4DHS–AuNPs were employed. 3,4DHS–AuNPs Although thefmol highest catalytic response was obtained Therefore, this amount was chosen for further studies. when 180 fmol were employed (Figure 4b), the catalytic efficiency—defined as the ratio between the The effect of the buffer solution pH on the biosensor response was investigated over the range Sensors 2017, 17,and 144 the current measured for the LOx/3,4DHS–AuNP/SPCE in the absence7of of 11 catalytic current lactate 5.0–8.0. The results showed that the response increased upon increasing the pH, reaching a (ICATmaximum /IMED )—was higher when 70 fmol 3,4DHS–AuNPs were employed. Therefore, this amount was at 7.0, and then the response (Figure 4c). Hence, 0.1 Mcatalytic phosphate buffer 180 fmol ofvalue 3,4DHS–AuNPs showed differentdecreased responses. Although the highest response chosen for further studies. (pH was selected determination of lactate. was 7) obtained whenfor 180the fmol were employed (Figure 4b), the catalytic efficiency—defined as the Sensors 2017, 17, 144

ICAT / µA

ICAT / µA

ICAT / µA

ratio between the catalytic current and the current measured for the6LOx/3,4DHS–AuNP/SPCE in 6.0 the absencea of lactate (ICAT/IMED)—was7 higher when 70 fmol 3,4DHS–AuNPs were employed. c b 5.5 Therefore, this amount was chosen for further studies. 4 6 5.0effect of the buffer solution pH on the biosensor response was investigated over the range The 5.0–8.0. 4.5The results showed that the 5response increased upon increasing the pH, reaching a 2 maximum value at 7.0, and then the response decreased (Figure 4c). Hence, 0.1 M phosphate buffer 4 4.0 (pH 7) was selected for the determination of lactate. 0 0.3

6.0 5.5

a

0.9

1.5

2.1

0

60

LOx / U

120

180

5

3,4-DHS-AuNPs / fmol 7

6

b

c

6

7

8

pH

ICAT / µA

ICAT / µA

ICAT / µA

Figure (a) Biosensor current response at different loadings LOx enzyme phosphatebuffer Figure 4. (a)4.Biosensor current response at different loadings of of LOx enzyme in in 0.10.1 MM phosphate 4 6 buffer 0.5lactate. mM of lactate. (b) 3,4DHS–AuNP concentrationand and (c) (c) pH 5.0 (pH 7) containing (pH 7) containing 0.5 mM of Effect Effect of (b)of3,4DHS–AuNP concentration pHon on the the response to 0.5 mM lactate of a biosensor prepared with 1.0 U of LOx. 5 response4.5to 0.5 mM lactate of a biosensor prepared with 1.0 U of LOx. 2 4 4.0 the biosensor development and Once work conditions were optimized, its response to lactate 0 The effect0.3 of theby buffer solution the biosensor was investigated over 0.9 1.5 2.1 pH on 0 applying 60 a constant 120 response 180 6 Under 7 the 8 the range was investigated chronoamperometry, potential of5 +0.3 V. optimal LOx / Uthatshowed 3,4-DHS-AuNPs / fmol stable pH conditions, theshowed biosensor a reproducible and response to reaching different alactate 5.0–8.0. The results the response increased upon increasing the pH, maximum concentrations. Figure 5 depicts a typical calibration curve, which follows Michaelis–Menten value at 7.0, and then the response decreased (Figure 4c). Hence, 0.1 M phosphate buffer (pH 7) was Figure 4. (a) Biosensor current response at different loadings of LOx enzyme in 0.1 M phosphate kinetics. This confirms thatof the biosensor response is controlled by the enzymatic reaction. The selected for the determination lactate. buffer (pH 7) containing 0.5 mM of lactate. Effect of (b) 3,4DHS–AuNP concentration and (c) pH on analytical properties of the biosensor were obtained from the linear part (up its to 800 μM) oftothe Oncethe the biosensor development and work conditions optimized, response lactate response to 0.5 mM lactate of a biosensor prepared with 1.0 were U of LOx. calibration plot. The sensitivity—calculated from the slope of the plot—was found to be 5.1 ± 0.1 was investigated by chronoamperometry, applying a constant potential of +0.3 V. Under the −1 μA·mM detection development and quantification limits—calculated as optimized, the concentration of lactate that Once. The the biosensor and a work conditions were its response to lactate optimal conditions, the biosensor showed reproducible and stable response to different lactate gave a signal equal to three and ten times the standard deviation of background current—were was investigated by chronoamperometry, applying a constant potential of +0.3 V. Under the optimal concentrations. 5 depicts a typical calibration curve, which follows Michaelis–Menten kinetics. found to beFigure 2.6 biosensor and 8.6 μM, respectively. The detection with lactate other conditions, the showed a reproducible and limit stablecompares response favorably to different This previously confirms that the biosensor response is controlled by the enzymatic reaction. The analytical described nanostructured on modified screen-printed electrodes concentrations. Figure 5 depicts a lactate typicalbiosensors calibrationbased curve, which follows Michaelis–Menten properties ofThe thereproducibility biosensor were the islinear part (up to 800 µM) of the calibration [28,29]. was evaluatedfrom comparing the analytical obtained using three kinetics. This confirms that the obtained biosensor response controlled by signals the enzymatic reaction. The biosensors in from the same manner. value ofthe lesslinear than 9% was obtained. Finally, plot. different The sensitivity—calculated the slope ofAthe plot—was found to beto5.1 0.1 µA ·mM−1 . analytical propertiesprepared of the biosensor were obtained from part (up 800±μM) of the the stability was examined by measuring the response three biosensors 0.5 The detection and quantification limits—calculated as the concentration of towards lactate that±mM gave a calibration plot. The sensitivity—calculated from theofslope ofdifferent the plot—was found to be 5.1 0.1 for one month. After this period, biosensor retained 85% of its original response. −1.to signallactate equal three and ten times the the standard deviation of current—were found μA·mM The detection and quantification limits—calculated as background the concentration of lactate that

ISS / µA

a signal equalrespectively. to three and ten the standard deviation favorably of background to begave 2.6 and 8.6 µM, Thetimes detection limit compares withcurrent—were other previously 6 found nanostructured to be 2.6 and 8.6 μM, respectively. The detection limit compares favorably with other described lactate biosensors based on modified screen-printed electrodes [28,29]. previously described nanostructured lactate biosensors based on modified screen-printed electrodes The reproducibility was evaluated comparing the analytical signals obtained using three different [28,29]. The reproducibility was evaluated comparing the analytical signals obtained using three 4 biosensors prepared in the same manner. A value of less than 9% was obtained. Finally, the stability different biosensors prepared in the same manner. A value of less than 9% was obtained. Finally, the was examined by measuring the response of three different biosensors towards 0.5 mM lactate for one stability was examined by measuring the response of three different biosensors towards 0.5 mM month. After the biosensor 85% ofretained its original response. 2 retained lactate forthis oneperiod, month. After this period, the biosensor 85% of its original response. 6 0 0.0

0.5

1.0

1.5

2.0

[Lactate] / mM

ISS / µA

4

Figure 5. Calibration curve (n = 3) obtained from chronoamperometric measurements for LOx/3,4DHS–AuNP/SPCE in 0.1 M phosphate buffer (pH 7) in the presence of increasing amounts of 2 lactate. 0 0.0

0.5

1.0

1.5

2.0

[Lactate] / mM

Figure 5. Calibration curve (n = 3) from obtained from chronoamperometric measurements for Figure 5. Calibration curve (n = 3) obtained chronoamperometric measurements for LOx/3,4DHS– LOx/3,4DHS–AuNP/SPCE in 0.1 M phosphate buffer (pH 7) in the presence of increasing amounts AuNP/SPCE in 0.1 M phosphate buffer (pH 7) in the presence of increasing amounts of lactate.of lactate.

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3.2. Study of Common Interfering Substances on the Response of Lactate One of the most important aspects to consider for any analytical application of biosensors is the study of the effect of potential interfering substances present in real samples. Therefore, to test the utility of the proposed biosensor in the determination of the lactate present in different real samples (such as white wine, beer and yogurt), a study of the influence of the most usual interfering substances that may be present in these samples was carried out. For this purpose, the biosensor response was obtained under the optimized experimental conditions in the absence and presence of different concentrations of tartaric acid, citric acid, ascorbic acid, acetic acid, glucose, fructose, methanol, and ethanol (Table 1). The presence of the potential interfering compounds—when they were at the same concentration as the analyte—did not affect the response, except in the case of ascorbic acid, where an increase of about 76% of the signal was observed. However, at lower concentrations, the presence of this compound did not show any effect. Therefore, these results suggest that the proposed device can be used to measure lactate concentrations in the presence of a variety of possible interfering substances. Moreover, other important analytical parameters such as sensitivity and stability compare favorably to other previously described lactate biosensors prepared in a similar manner [28,29,39,40]. However, in this work, a conjugate nanomaterial consisting of AuNPs with a reagent that has an electrocatalytic activity towards the oxidation of H2 O2 has been obtained in a unique process for the first time. This conjugate (3,4DHS–AuNP) can be easily immobilized on a screen-printed electrode and combined with the enzyme LOx to prepare a biosensor that could determine lactate in real samples directly without previous treatment and without the need to add reagents to the sample. This is an advantage compared to other lactate biosensors prepared using different nanomaterials or electrocatalysts. Table 1. Interference tests on various compounds for the assay of lactate.

Compound Tartaric acid Citric acid Acetic acid Ascorbic acid Glucose Fructose Methanol Ethanol

Current Ratio 1 1:1

1:0.1

1.05 1.08 1.16 1.76 1.07 1.01 1.12 1.10

0.97 1.00 0.88 1.08 0.96 0.99 0.97 0.94

1

Current ratio = IL+I /IL . IL+I : response of 0.50 mM lactate in the presence of interfering compound; IL : response of 0.50 mM lactate.

3.3. Determination of Lactate in Real Samples Finally, the developed biosensor was applied to the determination of lactate in real food samples—in particular, wine, beer, and yogurt. The importance of lactate in dairy products is well known. Moreover, this analyte plays an important role in the quality of wines, since the amount of lactic, malic, citric or succinic acid in wines is important for the provision of a mild and pleasant acidity. Samples (25 µL of wine, 40 µL of beer, or 10 µL of yogurt) were diluted in 10 mL of 0.1 M phosphate buffer (pH 7.0), and the standard addition method was used in order to minimize matrix effects. The results obtained were compared to those obtained by a commercial enzymatic assay kit based on L-lactate dehydrogenase/glutamate–pyruvate transaminase and photometric measurement at 340 nm of NADH formed during the enzymatic reaction. The results are summarized in Table 2. The average lactate concentration value obtained for three measurements using different biosensors agrees well with that obtained by the commercial enzymatic kit, with the advantage that the experimental procedure is more rapid, direct, and economical when the biosensor is used.

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Table 2. Determination of lactate in different samples with the biosensor and with a commercial enzymatic assay kit. Lactate Content (g/L) ± SD 1

Lactate Content (g/L) ± SD 1

Sample

(LOx Biosensor)

(Commercial Assay Kit)

White wine Beer Yogurt

1.7 ± 0.1 0.10 ± 0.02 5.4 ± 0.6

1.73 ± 0.02 0.100 ± 0.001 5.7 ± 0.4

1

SD: Standard deviation of three determinations.

4. Conclusions Screen-printed carbon electrodes nanostructured with 3,4DHS–AuNPs assemblies are excellent electrocatalytic platforms for the oxidation of H2 O2 . Once these platforms are coupled to lactate oxidase, they result in reagent-less disposable biosensors for direct determination of lactate in different samples (wine, beer, and yogurt) without the need for tedious and time-consuming pretreatment steps, and with excellent sensitivity and selectivity. The resulting biosensors are stable, and retain their activity for more than one month. Acknowledgments: The authors acknowledge Ministerio de Economía y Competitividad (project No. CTQ2014-53334-C2-1-R and CTQ2015-71955-REDT) and Comunidad de Madrid (NANOAVANSENS Program) for financial support. I.B. gratefully acknowledges the FPI-2012 Grant from Ministerio de Economía y Competitividad. E.L. gratefully acknowledges the Fulbright scholarship-Salvador de Madariaga program from Ministerio de Economía y Competitividad. Author Contributions: E.L. and F.P. conceived and designed the experiments; I.B. performed the experiments; I.B. and M.R.-P. analyzed the data; E.L., F.P. and M.R.-P. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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