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Development of a New Two-Enzyme Biosensor Based on Poly(Pyrrole-Co-3,4-Ethylenedioxythiophene) for Lactose Determination in Milk.

INTRODUCTION

Lactose (or milk sugar), a significant component of milk and whey, is a disaccharide composed of d-glucose and d-galactose bonded by a [beta]-1,4 glycosidic linkage [1]. Lactose concentration in milk is a basic indicator for the evaluation of raw and processed milk quality and detection of abnormal milk (having a low lactose level) from cows suffering with mastitis [2, 3], Effective control of lactose concentrations is also important for fermented dairy products to control the fermentation processes.

Several methods have been reported for the determination of lactose content, including gravimetric analysis [4], the colorimetric method [5], polarimetry [6], enzymatic assay [7], spectrophotometry [8], high-pressure liquid chromatography [9, 10], ultra-high-pressure liquid chromatography-tandem mass spectrometry [11], and Fourier transform infrared spectrometry [12]. However, many of these methods are generally time-consuming, expensive, complex, and labor-intensive for routine quality control applications.

Electrochemically modified electrodes have a great importance for the analysis of the biological and pharmaceutical samples [13-17] owing to their unique characteristic of specificity. One of the most important reasons for designing and developing biological and pharmaceutical elements immobilized modified electrodes is efficient and reproducible immobilization of these materials on the electrode surface. Besides, the amount and homogen distribution of these elements within the conducting polymer synthesized on the electrode with a small and limited area can be easily monitored by electrochemical immobilization technique [18, 19],

Biosensors have attracted a great deal of interest throughout the last decade. A key search--as a title--for "biosensor" in the Web of Science database reveals more than 9000 papers during the last 10 years (from 2006 to 2016). Biosensors offer a cheap, quick, and reliable alternative for the determination of lactose. Several types of lactose biosensors have been reported in literature [2, 3, 20-29].

Conducting polymers (CPs) are suitable matrixes for the entrapment of enzymes [30], and they are promising candidates for the enzyme electrode component because they are readily synthesized in the form of thin films by electrochemical polymerization of heterocyclic compounds such as pyrroles and thiophenes [31-33]. Generally, the immobilization of enzymes is achieved either by in situ entrapment during electrochemical polymerization in an enzyme-containing solution or by postimmobilization, such as the covalent binding of enzymes to conducting polymer films and their adsorption onto conducting polymer films [34]. The applications of CPs to biosensors have been reviewed in literature [30, 35, 36]. Among these CPs, polypyrrole (PPy) is one of the most extensively studied conducting polymers [37] because of its high electrical conductivity, low cost, considerable environmental stability, and interesting technological applications in the fabrication of biosensors [36]. Compared with other CPs, PPy can be grown on different electrodes from aqueous solutions, which are compatible with many biomolecules [38]. However, lactose amount can be found by the sensor output as a function of the oxygen concentration observed during measurements [39]. The conductivity and the sensitivity of polypyrrole can be decreased under these conditions [40]. On the other hand, poly(3,4-ethylenedioxythiophene) (PEDOT) is a relatively new polymer and a new class of polythiophenes with very high electrochemical stability in oxidized states and a low band-gap [41]. In our study, we immobilized the enzymes by entrapment method simultaneously with electrochemical copolymerization of PPy and PEDOT in buffer solution (pH 6.7) at 25[degrees]C. These immobilization conditions are quite soft for not to denaturated of enzymes. But, at these conditions, PEDOT cannot be polymerized alone. At this point, poly(3,4-ethylenedioxythiophene) (PEDOT) was used as an electroactive component to form a copolymer structure for our lactose biosensor to enhance the electrochemical stability of PPy [42] and enables the long-term measurements of modified electrode due to its high stability [43]. Moreover, the addition of PEDOT to PPy structure could enhance the entrapment of enzymes due to molecular structure of PEDOT and improve the analytical performance of the electrochemical biosensor. PEDOT has exhibited considerable electrocatalytic activity toward detection of various analytes such as glucose [44], pesticide [45], and dopamine [46].

To the best of our knowledge, lactose biosensor designed by PEDOT is not available in the literature. Although several articles relating to the designation of biosensors using conductive polymers have been published [47-50], articles on the development of lactose biosensors using conductive polymers are rather scarce. In this study, for the first time, we present the preparation of a lactose biosensor using a copolymer of polypyrrole and poly(3,4-ethylenedioxythiophene), synthesized using an electropolymerization method.

EXPERIMENTAL

Materials

Pyrrole (Py), 3,4-ethylenedioxythiophene (EDOT), [beta]-galactosidase ([beta]-Gal, EC 3.23.1.23, [greater than or equal to] 8000 units/g, from Aspergillus oryzae) and galactose oxidase (GaOx, EC 1.1.3.4, 179000 units/g, type VII-S from Dactylium dendroides) were purchased from Sigma-Aldrich. Sodium dodecylbenzene sulfonate (NaDBS) was obtained from Fluka, and sodium dihydrogen phosphate dihydrate (Na[H.sub.2]P[O.sub.4] x 2[H.sub.2]O) and lactose were purchased from Riedel De Haen. An alumina polishing suspension agglomerate (0.05CR micron) (Baikowski) was used as an electrode polisher. All chemicals were of analytical grade and used without further purification. Whole, low-fat, and skimmed milk were obtained from local markets.

Instrumental

FTIR spectra of the polymers were recorded between 4000 and 400 [cm.sup.-1] with a resolution of 4 cm1 on a Perkin-Elmer spectrometer (Beaconsfield, HP91QA). SEM was performed using Phillips scanning electron microscope (XL-30S FEG). The electrochemical experiments were performed with a potentiostat/ galvanostat (CompactStat, Ivium Technologies, the Netherlands). The platinum bare and copolymer-modified platinum electrodes were used as working electrodes, platinum wire as counter, and Ag/AgCl as a reference electrode. Indium thin oxide (ITO) electrode was used for the deposition of polymers for the analysis of FTIR and SEM.

Methods

Preparation of PPy and PEDOT Homopolymers and (Py-coEDOT) Copolymer. The homopolymers and copolymer were synthesized using an electrochemical polymerization method. The experiments were performed in a phosphate buffer solution adjusted to a pH of 6.7. The Pt disc was polished by using an agglomerate alumina polishing suspension. Then, the working electrode was washed with the phosphate buffer solution before any of the electrochemical measurements.

The synthesis of homopolymers was carried out in a phosphate buffer solution (pH 6.7) containing 0.5 M monomer and 0.05 M sodium dodecylbenzene sulfonate (NaDBS) as a surfactant. First, cyclic voltammetry (CV) was employed to determine the oxidation potentials of monomers and their mixture. Then, a chronoamperometric method was used to deposit both homopolymers and copolymer onto the Pt disc electrode at 1.0 V at room temperature. Electropolymerization of PEDOT was not achieved onto Pt disc electrode in this condition. But we could polymerize PEDOT onto ITO electrode for FTIR and SEM characterizations. So, we could not make the enzyme electrode study with PEDOT.

Enzyme-Immobilized Electrode Fabrication. The enzymeen-trapped copolymer was electrochemically formed using a mixture of 0.375 M of Py and 0.125 M of EDOT monomers in 0.05 M of NaDBSA, 1 mL 0.1 M of phosphate buffer solution (at pH 6.7) containing 2.94 mg/mL GaOx (40 units), and 2.0 mg [beta]-galactosidase (20 units). A chronoamperometric method was used to deposit an enzyme-immobilized Py-coEDOT copolymer onto the Pt disc electrode at 1.0 V for 30 min at room temperature. Then, the modified working electrode was washed to remove both the nonentrapped enzyme and the remaining monomer with buffer solution and distilled water, respectively. The prepared working electrode was stored at 4[degrees]C in buffer solution (pH 6.7). Figure 1 represents the preparation of the enzyme-entrapped, copolymer-modified electrode.

Lactose Determination in Standard Solution. Oxygen is passed through the cell at a constant speed for 10 min to obtain the desired measuring medium, saturated with oxygen. Oxygen gas flow was continued over the solution to keep a constant oxygen concentration during measurement. The response of the enzyme electrode to lactose was investigated using the CV method based on electrooxidation of [H.sub.2][O.sub.2] produced enzymatically in a potential range from--0.1 to 1.0 V. A standard solution of 0.02 M of lactose solution was used. The measurements were recorded after the addition of 100 [micro]L of lactose to the buffer solution (pH 7.4) at 0.40 V, representing the denaturing potential for the pair of enzymes ([beta]-gal/GaOx) [24].

Lactose Determination in Milk Samples. Whole, low-fat, and skimmed milk provided from different markets were diluted with a phosphate buffer solution (1:10 v/v) and used straight away. The experimental procedure for lactose determination in milk samples is similar to that for the standard solution. The CV measurements were recorded after the addition of 100 [micro]L of 10-times diluted milk to the buffer solution (pH 7.4) at 0.40 V, representing the denaturing potential for the pair of enzymes: [beta]-gal/GaOx. Lactose concentrations of milk samples were also determined using the International Dairy Federation (IDF)approved, mid-infrared absorbance method [51] using a Bentley 150 instrument (Bentley Instruments, Inc., MN, USA).

RESULTS AND DISCUSSION

Cyclic voltammetry (CV) experiments were carried out on unstirred solutions at a scan rate of 25 mV [s.sup.-1]. The electrochemical properties of monomers and the monomer mixture were investigated in a phosphate buffer solution containing 0.05 M NaDBS. Figure 2a shows the first anodic scan of Py and EDOT monomers and their mixture. Py and EDOT samples exhibited onset oxidation potentials of 526 and 806 mV, respectively. Mixture of Py and EDOT exhibited moderately different electrochemical behaviors compared to Py and EDOT alone. The voltammogram of the monomer mixture displays the oxidation peak at 628 mV, which is between the oxidation potential of the monomers. The CV of the monomer mixture shows different redox behavior and oxidation potentials compared to individual monomers. Additionally, the CV of the copolymer indicates the characteristic electrochemical properties of both monomers, according to their CV curves. This electrochemical behavior is attributed to the formation of a copolymer film onto the Pt electrode.

The electrochemical behaviors of the copolymer-coated Pt, and enzyme-entrapped copolymer-coated Pt disc electrodes were investigated using cyclic voltammetry (CV). The CVs of the electrodes using a scanning rate of 50 mV [s.sup.-1] are shown in Figure 2b. First, the P(Py-co-EDOT)-modified Pt disc electrode was dipped into a buffer solution and scanned between--0.1 and 1.0 V, at a scan rate of 50 mV [s.sup.-1]. A similar procedure was performed on the electrode modified with the enzyme-entrapped copolymer. For comparison, the CV of the bare Pt electrode was also added to the figure. Over the same potential range, the enzyme-entrapped, copolymer-modified Pt electrode showed higher current compared to that of the copolymer-modified electrode without enzymes. The bare Pt electrode exhibited very little increase in current due to its uncoated surface.

The results obtained above strongly suggest that the electrochemical oxidation of the pyrrole and 3,4-ethylenedioxythiophene monomers form their corresponding copolymers on the electrode surface. At this point, the adherence of copolymers with and without enzyme on the electrode surface, and their electrochemical response, were both investigated. Once the Pt electrodes were coated with the copolymers, as described above, they were immersed into a monomer-free buffer solution. Figure 3 shows the CVs of the copolymer films recorded with different scan rates (from 25 to 200 mV [s.sup.-1]), and the corresponding anodic peak currents. Anodic peak currents for enzyme-free and enzyme-entrapped copolymers increase linearly with increasing scan rate, indicating that the copolymer films are well adsorbed onto the working electrode surface. In cyclic voltammetry, the Randles-Sevcik equation describes how the effect of scan rate on the peak current, [i.sub.p], depends not just on the concentration and diffusional properties of the electroactive species, but also on the scan rate [52].

If the solution is at 25[degrees]C; [i.sub.p] = (2.69 x [10.sup.5]) x A x [D.sup.1/2] x [C.sub.o] x [v.sup.1/2], where [i.sub.p] is the current maximum, v is the scan rate, A is the electrode area, D is the diffusion coefficient of the electroactive species, and [C.sub.o] is the concentration of the electroactive species in the solution. Peak current is proportional to [v.sup.1/2] in the range of scan rates where diffusion control applies [53]. The scan rate dependence of the anodic peak currents shows a linear dependence on the scan rate for the enzyme-entrapped copolymer versus the square root of the scan rate ([v.sup.1/2]) (Figure 3a, b). This indicates that the electrochemical process is diffusion controlled and also shows thin-film formation. This shows that the electrochemical process is extremely reversible, even at high scan rates. Additionally, the redox process has no diffusional obstacles and the electroactive polymer is securely attached to the working electrode surface [49]. However, the linearity of the plot belonging to the enzyme-free copolymer was not as good as the enzyme-entrapped copolymer (Figure 3c, d). It can be concluded that the entrapment of enzymes has a positive effect on the properties of the modified working electrode.

Figure 4 represents the FT-IR spectra of homopolymers and copolymers, with and without enzyme entrapment. To obtain the desired quantities of samples for measurement, an indium tin oxide (ITO) working electrode was used for electrodeposition. In the PPy spectrum, the peaks at 1547 and 1458 [cm.sup.-1] were attributed to C-C and C-N vibrations in PPy. The bands around 1635 [cm.sup.-1] are due to the C=C stretching mode of PPy. In the PEDOT homopolymer spectrum, the bands at 1463 and 1459 [cm.sup.-1] are due to the C=C and C-C stretching of the thiophene backbone, respectively. The broad absorption peak around 1380 [cm.sup.-1], and the peaks at 1174 and 1087 [cm.sup.-1] are attributed to the C-O-C vibration mode of the thiophene ring. The peaks at 977, 927, 837, and 687 cnT1 are due to the C-S bond of the thiophene ring [54]. The FT-IR spectrum of the copolymer consists of absorption peaks attributed to the PPy and EDOT homopolymer. The FT-IR spectrum of the enzyme-entrapped copolymer exhibits an appreciable difference compared to the enzyme-free copolymer. The peaks at 900, 1167, 1290, and 1540 [cm.sup.-1] are attributed to the structure of the enzyme, indicating the enzymes [beta]-gal and GaOx are entrapped in the copolymer structure. The absorption bands around 2927 and 2850 [cm.sup.-1] in all samples are related to aromatic C-H stretch vibrations.

A morphological analysis of the homopolymers and copolymers was carried out by scanning electron microscopy (SEM), with the resulting images presented in Figure 5. This analysis technique was performed to confirm the entrapment of [beta]-gal and GaOx enzymes into the copolymer structures. PPy exhibits a granular and tightly stacked structure, while the PEDOT image reveals a randomly distributed porous structure. The surface of the copolymer exhibits cauliflower-like structures consisting of clusters of globules. The enzyme molecules were immobilized into pinholes, both between the globules and between the cauliflower-like structures.

To determine the response of the enzyme electrode to lactose, a lactose solution of a known concentration prepared in a phosphate buffer solution was added to it. Figure 6 shows the typical cyclic voltammetric response (-0.1 to + 1.0 V) of a [beta]-gal/GaOx- immobilized, copolymer-modified electrode to lactose. The response time of the enzyme electrode was found to be from 8 to 10 s from Figure 6. In Figure 6, we used scan rate at 50 mV/s. The variation of current values of the working electrode for potential values scanning from -0.1 to 1.0 V, versus Ag/AgCl, were measured to determine the increasing current values at 0.40 V caused by electrooxidation of [H.sub.2][O.sub.2] propagated enzymatically. Until 400 mV, the electrode response takes 8 s. Sometimes it can shift to left or right. So, we determined the response time as 8-10 s from Figure 6.

After completing a cyclic voltammogram of a blank solution, 100 [micro]L of 0.02 M lactose solution was added and the currents at 0.40 V, for each added concentration of lactose, were recorded. The current values increased with each addition of lactose solution. These current values were used to form calibration plots.

The lactose biosensor works based on the following sequence of enzymatic reactions. The first two biochemical reactions are the hydrolysis of lactose by [beta]-galactosidase, and the catalytic oxidation of galactose by galactose oxidase, respectively (Eqs. 2 and 3). The [H.sub.2][O.sub.2] produced at reaction (Eq. 2) was oxidized electrochemically as shown by the reaction in Eq. 3 [27].

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[mathematical expression not reproducible] (2)

[mathematical expression not reproducible] (3)

Optimization of some parameters (copolymer ratio, electrode-position time, and enzyme concentration) on the response of enzyme electrode was performed before starting the biosensor measurements. First, we optimized the electrodeposition time of copolymer onto electrode surface. We tried 15, 30, 45, and 60 min for electrodeposition. The maximum current response was obtained with 30 min. After that, we optimized the copolymer ratio. We used (0.50M Py), (0.45M pyrrole-0.05M EDOT), (0.375M pyrrole-0.125M EDOT), (0.25M pyrrole-0.25M EDOT), and (0.50M EDOT) containing solutions, respectively. The 0.50M EDOT modified enzyme electrode could not be obtained due to nonpolymerization of EDOT in our conditions. After preparation of polymer-modified enzyme electrodes, response of electrodes on the lactose was examined. Current response-to-copolymer ratio was plotted (not shown here) by applying the cyclic potential between the range of -0.10 to 1.0 V versus Ag/AgCl. It has been observed that the solution, which includes (0.375M pyrrole-0.125M EDOT), gives the best response to lactose concentrations. Then, we decided to use (0.375M pyrrole-0.125M EDOT)-modified enzyme electrode for our further studies.

The enzyme concentrations used in our study was selected from literature [24]. However, we optimized the enzyme concentration using (5.88 mg/mL GaOx and 4.0 mg [beta]-galactosidase), (2.94 mg/mL GaOx and 2.0 mg [beta]-galactosidase) and (1.47 mg/mL GaOx and 1.0 mg [beta]-galactosidase), respectively, against enzyme electrode response to lactose concentrations. The maximum current response was observed with the enzyme electrode prepared with 2.94 mg/mL GaOx (40 units), and 2.0 mg [beta]-galactosidase (20 units).

Figure 7 indicates the correlation between the reaction rate of the [beta]-gal/GaOx-immobilized, copolymer-modified electrode with the lactose concentration in the buffer solution. As can be seen from Figure 7, the linear working range of the enzyme electrode is across a lactose concentration of 0.2-2.3 mM, where the detection limit of the enzyme electrode was found to be 1.4 z [10.sup.-5] M for lactose.

The characteristics (linear working range, detection limit, and sensitivity of enzyme electrode) of some lactose biosensors were given in Table 1 to make comparison with the characteristics of lactose biosensor developed in this study. The sensitivity of enzyme electrode was determined as 1.08 A [M.sup.-1] [cm.sup.-2] from the plot of current density (current/area) against concentration. The upper limit of linear working range of developed biosensor is better than that of some studies in literature [2, 50, 55]. Limit of detection is lower than that of [20, 25, 27, 56]. The high sensitivity of the biosensor to hydrogen peroxide produced from enzymatic reactions is due to the high efficiency of the electron transfer between enzymes and a Pt electrode by the way of P(Pyco-EDOT) film. GaOx and [beta]-galactosidase based lactose biosensor showed a much higher sensitivity for lactose compared to other biosensors [2, 20]. Also the sensitivity value of biosensor is in comparable level with the study made by Tasca et al. [55].

For the purpose of determining the thermal stability and activation energy of the enzyme system, the response of the co-immobilized enzyme electrode was measured in a temperature range from 20 to 50[degrees]C. The current response increases linearly with temperature from 20 to 50[degrees]C (not shown here). The activity of the immobilized enzymes reached a maximum value at about 50[degrees]C, compared to around 40 and 50[degrees]C for the native [beta]-galactosidase and galactose oxidase, respectively, reported previously [57, 58]. This result indicates that the immobilized enzyme has a little increased temperature tolerance. The increase in optimum temperature may be due to diffusional effects: the immobilized enzyme can be more easily contacted by the substrates with the increase in the bulk temperature [59]. Accordingly, we can conclude that P(Py-co-EDOT) can offer a good environment for the enzyme system, which make the sensor more stable at high temperature. The temperature tolerance of the immobilized enzymes mainly not only depends on the nature of enzymes but also keeping enzyme in a safety environment. The temperature has negative effect on the electrical conductivity of conducting copolymers. But the physical conditions are more effective on the response of electrode [34]. Besides, the maximum activity of immobilized [beta]-galactosidase and (GaOx) with temperature was found to be at 55[degrees]C [60] and 45[degrees]C [56], respectively. Consequently, it was found that the activity of lactose enzyme electrode with temperature developed in this study is consistent with literature. Owing to the denaturation of enzymes at higher temperatures, we have not worked at temperatures higher than 50[degrees]C [21, 56]. However, for practical applications, experimental temperature was controlled at 25[degrees]C.

The activation energy of the co-immobilized enzyme system can be calculated based on the Arrhenius Formula [In k = In A -(Ea/RT), where k is the rate constant and Ea is the apparent activation energy]. The response current is proportional to the rate constant k [61]. The natural logarithm of I can be used instead of In k in the formula. The relationship between In I and 1/7* was plotted, and a straight line was obtained. The activation energy of this co-immobilized enzyme system was calculated to be 21.24 kJ [mol.sup.-1] from the Arrhenius plot, which is lower than free enzymes [62, 63] (Figure 8). The comparison of Ea values of the immobilized and free enzymes also confirms the fact that the immobilization of the enzymes causes no deformations in the structures of the enzymes in the immobilized state.

The relationship between the reciprocal of the response current ([i.sup.-1]) and the reciprocal of the lactose concentration ([C.sup.-1]) was obtained according to the Lineweaver-Burk form of the Michaelis-Menten equation (Figure 9) [64]. The Michaelis-Menten constant is important to characterize the enzyme electrode due to the character of the aforementioned constant (KM). In the Michaelis-Menten assumption, the transformation rate of the substrate due to the products depends on the degradation rate of the enzyme-substrate complex. When the value of the [K.sub.m] constant decreases, the affinity between the enzyme and the substrate is strengthened, according to the Michaelis-Menten equation [65]. In this study, the apparent Michaelis-Menten constant ([K.sub.Mapp] = 0.65 mM) for lactose was computed by using the slope and intercept values of the Lineweaver-Burk equation. This value is lower than the value found in other literature for the free enzyme system (2.30 mM) [66] and may be due to a stronger affinity between the enzyme and the electrode, and also the electronic attraction of P(Py-co-EDOT)-NaDBS. KM values of both entrapped- and free-enzyme systems show that there were no diffusional limitations or denaturing character in the entrapment procedure of enzymes [67].

The operational stability of the enzyme-immobilized P(Pyco-EDOT)-NaDBS, film-modified biosensor for different lactose concentrations was investigated (Figure 10). The working electrode was measured over 11 days. The enzyme electrodes were stored at 4[degrees]C when not in use. It was found that during the first day, the electrochemical activity decreased by more than 60% and remained stable afterwards. This large initial fall in activity is presumably caused by oxidation (doping of the polymer) or rearrangement of the pristine polymer [68]. The amperometric current was found to decrease from 378 to 109 [micro]A over 11 days. This may be due to partial deactivation of the enzyme [24]. According to these results, the electrode has lost some of the maximum current that lactose has shown during these measurements, but it retains its stability for a long time at this value. The reproducibility of the biosensor was performed by the five different biosensors prepared under the same conditions in a day. The results showed that the biosensor had a good reproducibility with a standard deviation of 0.013 [micro]A and a variation coefficient of 4.1%.

Biosensors based on electrochemical oxidation of hydrogen peroxide at a Pt electrode are inclined to interference from other electroactive species such as ascorbic acid, uric acid, and glucose. However, these species are the major interferents in the milk and may affect the response of the lactose biosensor. For this reason, the response current in the presence of these substances was studied at their normal physiological levels ascorbic acid (2.21 mg [dL.sup.-1]) [69], uric acid (1.5 mg [dL.sup.-1]) [70], and glucose (max. 6 mg [dL.sup.-1]) [71] with 6.8 g [dL.sup.-1] lactose. Ascorbic acid, uric acid, and glucose were found to have no significant interfering effect on the biosensor response.

Milk samples with different fat contents (whole, low-fat, and skimmed) were analyzed to demonstrate the applicability of the developed biosensor for determining lactose using [beta]-galactosidase and galactose oxidase to determine lactose. The analyses were performed on diluted (1/10 v/v) milk samples without any pretreatment. The results were compared with those obtained by the reference method [51]. Lactose concentrations of whole milk samples determined by the reference method and by the biosensor are shown in Table 2. The p value of 0.98 obtained by the Student's t test showed that the mean lactose concentrations determined by the two methods were statistically similar (p > 0.05). Our results are in agreement with the findings of Conzuelo et al. [3] ([t.sub.tabulated] = 3.812 > i = 0.774, for 95% confidence) and Eshkenazi et al. [22] (p = 0.15). Analysis of variance showed that lactose concentrations of the milk samples were not affected by fat content (p > 0.05).

CONCLUSIONS

It has been demonstrated that a useful biosensor can be developed for lactose determination, using the influence of the relationship between the enzymatic activities of [beta]-galactosidase and galactose oxidase immobilized in P(Py-co-EDOT)-NaDBS, to deliver a useful electrochemical signal. The formation of a copolymer film was confirmed by CV, FTIR, and SEM measurements. The optimized lactose biosensor exhibited a linear response range from 0.20 to 2.30 mM, and a detection limit of 1.4 x [10.sup.-5] M for lactose. This enzyme-immobilized, P(Py-coEDOT)-NaDBS film can be used for the estimation of lactose. The maximum reaction rate (Vmax) was estimated at 233 [micro]A. Km values of both entrapped (0.65 mM) and free enzyme (2.30 mM) systems show that there were no diffusional limitations or denaturing characteristics in the entrapment procedure of the enzymes. The enzyme electrode is stable up to 50[degrees]C. The major interferents in the milk (ascorbic acid, uric acid, and glucose) were found to have no significant interfering effect on the biosensor response. The response time of the enzyme electrode was found to be from 8 to 10 s. Sensitivity of enzyme electrode was determined as 1.08 A [M.sup.-1] [cm.sup.-2]. Designed biosensor had a good reproducibility with a standard deviation of 0.013 [micro]A and a variation coefficient of 4.1 %.

This study shows that synthesized P(Py-co-EDOT)-NaDBS film can be used to construct stable and sensitive lactose biosensors. The biosensors based on P(Py-co-EDOT)-NaDBS, film-modified enzyme electrodes have several advantages with respect to wide linear range with a low detection limit, temperature stability, short response time, a strong relationship between enzyme and substrate (low KM), and simplicity, which allows for the rapid detection of lactose. P(Py-co-EDOT)-NaDBS, film-modified enzyme electrodes have additional advantages such as cost effectiveness, and a simple method of fabrication when compared with conventional electrodes. The applicability of the biosensor to determine lactose concentration in milk was demonstrated. Agreement between the biosensor and the reference method results has shown that the developed sensor has important potential for measuring lactose in real milk samples.

ACKNOWLEDGMENT

The authors thank Ozge Gokce for her technical support during milk analysis.

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Oguz Gursoy, (1) Songul Sen Gursoy [iD], (2) Sadik Cogal, (3) Gamze Celik Cogal (4)

(1) Department of Food Engineering, Faculty of Engineering and Architecture, Mehmet Akif Ersoy University, Burdur, Turkey

(2) Department of Chemistry, Faculty of Arts and Sciences, Mehmet Akif Ersoy University, Burdur, Turkey

(3) Department of Polymer Engineering, Faculty of Engineering and Architecture, Mehmet Akif Ersoy University, Burdur, Turkey

(4) Department of Chemistry, Institute of Natural and Applied Sciences, Suleyman Demirel University, Isparta, Turkey

Correspondence to: S. Sen Gursoy; e-mail: ssen@mehmetakif.edu.tr

Contract grant sponsor: Scientific and Technological Research Council of Turkey (TUBITAK); contract grant number: 1140904.

DOI 10.1002/pen.24632

Caption: FIG. 1. Preparation of enzyme-entrapped copolymer-modified Pt electrode. [Color figure can be viewed at wileyonlinelibrary.com]

Caption: FIG. 2. (a) The CVs of Py, EDOT, and Py/EDOT in phosphate buffer solution containing 0.05 M NADBS. Working electrode: Pt disc, scan rate: 25 mV s and reference electrode: Ag/AgCl. (b) Cyclic voltammograms of unmodified Pt, copolymer-coated Pt and enzyme-entrapped copolymer-coated Pt electrodes. [Color figure can be viewed at wileyonlinelibrary.com]

Caption: FIG. 3. Cyclic voitammograms and anodic currents at different square root of scan rates of enzyme entrapped copolymer (a, b) and enzyme free copolymer (c, d) in monomer-free buffer solution. [Color figure can be viewed at wileyonlinelibrary.com]

Caption: FIG. 4. FT-IR spectra of PPy, PEDOT, copolymer, and enzyme-entrapped copolymer.

Caption: FIG. 5. SEM images of PPy (a), PEDOT (b), copolymer (c), and enzyme- entrapped P(Py-co-EDOT) (d).

Caption: FIG. 6. Typical cyclic voltammetric response (-0.1 to 1.0 V) of [beta]- gal/ GaOx-immobilized copolymer to lactose in 0.1 M phosphate buffer solution (pH 6.7). [Color figure can be viewed at wileyonlinelibrary.com]

Caption: FIG. 7. Effect of lactose concentrations on current and calibration curve (inset graph) for the cyclic voltammetric responses of the enzyme modified electrode in the range of -0.1 to 1.0 V applied potentials, in the concentration range 0.3-1.22 mM. [Color figure can be viewed at wileyonlinelibrary. com]

Caption: FIG. 8. Arrhenius plots for immobilized enzyme system. [Color figure can be viewed at wileyonlinelibrary.com]

Caption: FIG. 9. Lineweaver-Burk plots of [beta]-gal/GaOx-immobilized copolymer. [Color figure can be viewed at wileyonlinelibrary.com]

Caption: FIG. 10. Working stability (reusability) of P(Py-co-EDOT)-NaDBS films in the presence of 0.02 M lactose in 0.1 M phosphate buffer, pH 6.7 at 0.40 V (bias voltage). [Color figure can be viewed at wileyonlinelibrary.coml
TABLE 1. Characteristics of some lactose biosensors.

Linear working        Detection        Sensitivity
range (mM)            limit (M)       (A [M.sup.-1]    Reference
                                       [cm.sup.-2])

0.0035-2           3.5 [10.sup.6]          0.10        6
Up to 14           1 x [10.sup.4]          0.01        26
0.05 and 50       5.0 x [10.sup.5]         1.92        19 (a)
0.10 and 40       1.0 x [10.sup.4]         2.15        19 (b)
Up to 1.22        2.6 x [10.sup.6]         2.43        38
0.001-0.150       0.5 x [10.sup.6]         0.48        55
0.198-2.301       1.4 x [10.sup.5]         1.08        This study

(a) Thermometric sensor.

(b) Amperometric sensor.

TABLE 2. Lactose concentrations (mM) of whole milk samples determined
by reference method and by the biosensor.

Sample                        Biosensor    Reference    Relative
                                             method       error

1                             0.0125       0.0123        0.0002
2                             0.0125       0.0128       -0.0004
3                             0.0112       0.0125       -0.0012
4                             0.0135       0.0126        0.0009
5                             0.0124       0.0128       -0.0004
6                             0.0121       0.0127       -0.0006
7                             0.0130       0.0128        0.0002
8                             0.0131       0.0125        0.0007
9                             0.0120       0.0128       -0.0008
10                            0.0141       0.0124        0.0016
Mean                          0.0126       0.0126
Standard deviation            0.0008       0.0002
p value (Student's I test)    0.98
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Author:Gursoy, Oguz; Gursoy, Songul Sen; Cogal, Sadik; Cogal, Gamze Celik
Publication:Polymer Engineering and Science
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Date:Jun 1, 2018
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