Dependence of operational parameters of laccase-based biosensors on structure of photocross-linked polymers as holding matrixes

European Polymer Journal 115 (2019) 391–398

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Dependence of operational parameters of laccase-based biosensors on structure of photocross-linked polymers as holding matrixes

T

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Taras Kavetskyya,b, , Oleh Smutokc, Olha Demkivc, Sigita Kasetaited, Jolita Ostrauskaited, Helena Švajdlenkováe, Ondrej Šaušaf, Khrystyna Zubrytskaa, Nataliia Hoivanovycha, Mykhailo Goncharc a

Drohobych Ivan Franko State Pedagogical University, 82100 Drohobych, Ukraine The John Paul II Catholic University of Lublin, 20950 Lublin, Poland c Institute of Cell Biology, National Academy of Sciences of Ukraine, 79005 Lviv, Ukraine d Kaunas University of Technology, 50254 Kaunas, Lithuania e Polymer Institute, Slovak Academy of Sciences, 84541 Bratislava, Slovakia f Institute of Physics, Slovak Academy of Sciences, 84511 Bratislava, Slovakia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Photocrosslinking Swellability Positron annihilation Amperometric biosensor Laccase

Laccase-based amperometric enzyme biosensors of the third generation for analysis of phenol derivates have been constructed using graphite rods (type RW001) as working electrodes and the photocross-linked polymers as a matrix. Such matrix consisted of epoxidized linseed oil (ELO), bisphenol A diglycidyl ether (RD) as reactive diluent and 50% mixture of triarylsulfonium hexafluorophosphate in propylene carbonate (PI) as photoinitiator. The synthesis was made by the reaction of ELO and 10 mol.% or 30 mol.% of RD, using 3 mol.% of PI (ELO/10RD and ELO/30RD, respectively). The holding matrixes were used for an immobilization of commercial laccase from the fungus Trametes versicolor. The network properties of the polymer matrixes, holding biosensing element, were studied by means of swelling and positron annihilation lifetime spectroscopy measurements. The amperometric enzyme biosensor parameters were evaluated using cyclic voltamperometry and chronoamperometric analysis. A correlation between the constructed biosensor parameters and microscopical free volume of the biosensor holding matrixes was established.

1. Introduction Traditional analytical methods for the determination of the quality of drinking water, as well as pollutants in wastewater, detection of phenolic content, etc. are expensive, time-consuming, not effective in tracing multiple harmful components, and require extensive sample pre-treatment [1,2]. Therefore, due to rapid, sensitive, and selective monitoring features enzyme-based biosensors are attractive alternative [3,4]. The enzyme immobilization procedure in biosensorics has a purpose of allowing the biological element exhibits a maximum activity with the sufficient stability and the reusability of the bioelectrodes. Common methods of e.g. laccase immobilization in biosensing layer are covalent bonding, adsorption, cross-linking and entrapment [5]. Covalent bonding is based on the chemical activation of groups in the support matrix so that they react with functional groups in the biomaterial, which are not essential to catalytic activity [6]. The weak point of this

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method is possible changes in the structure of the enzyme active center [7]. Adsorption is a simple, low cost and fast immobilization method. The enzyme is bound to a support via ionic interactions or weak forces, such as van der Waals and hydrogen bonds. However, the biological elements immobilized through this method are mechanically unstable and can be easily desorbed under operating conditions [8]. Crosslinking uses bifunctional reactives to generate intramolecular bonds between the molecules of the enzyme. However, large quantities of the enzyme are needed, and factors, such as pH and ionic strength, must be controlled [9]. At the entrapment of the biomaterial with a porous solid matrix, the enzyme suffers minimum alteration and this immobilization approach looks most promising in sensor technologies. On the other hand, the method needs pore-size controlling to reduce diffusional limitation of the substrate to the enzyme. Application of polymer materials as holding matrixes of immobilized enzyme is an innovative approach in a construction of the non-mediated enzyme-based biosensors of the third generation [10].

Corresponding author at: Drohobych Ivan Franko State Pedagogical University, 82100 Drohobych, Ukraine. E-mail address: [email protected] (T. Kavetskyy).

https://doi.org/10.1016/j.eurpolymj.2019.03.056 Received 6 March 2019; Received in revised form 27 March 2019; Accepted 28 March 2019 Available online 28 March 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.

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swelling and PALS measurements. The amperometric enzyme biosensor parameters were evaluated using cyclic voltamperometric and chronoamperometric analysis. A correlation between the constructed biosensor parameters and hole volume of the biosensor holding matrixes was established. This finding demonstrates a way to control the functionality of amperometric enzyme biosensor of the third generation using the knowledge about the microstructure of a holding polymer matrix.

The use of vegetable oils as starting materials for the preparation of polymers is widely spread due to their inherent biodegradability, low toxicity, and ability to modify functional groups [11]. Curing reactions of raw oils take a long time due to their low reactivity. However, the curing time can be shortened by using oils with more reactive functional groups, for example, epoxidized oils [11]. Vegetable oil-based photopolymers could be used as a holding matrix in biosensors. Photopolymerization shortens the reaction time from hours to minutes compared to the thermal polymerization [12]. The photocurable formulation consists of monomer, reactive diluent, which reduces the viscosity and participates in the reaction, and photoinitiator, which absorbs the light and generates the reactive species [13]. A swelling test provides information about a crosslink density and flexibility of polymer network and is commonly used to characterize the structure of a cross-linked polymer. The knowledge of the polymer microstructure, especially from the view of a free volume behavior over a wide temperature range, allows better understanding of the properties of composite biosensor made on its basis. There are many experimental techniques for the research of free volumes in polymers such as photoisomerization, photochromic probe, inverse gas chromatography, small angle X-ray diffraction [14–18] and positron annihilation spectroscopy [18–20]. Annihilation of a positron is a very sensitive tool for the examination of local regions with low electron density, local freevolumes, i.e. intermolecular spaces, voids or pores with different size. In the organic materials the bound state of positron-electron, positronium (Ps), can be created. This atomic probe in triplet spin state (ortho-Positronium) annihilates in the vacuum with characteristic lifetime of 142 ns. The typical annihilation of o-Ps in organic materials is the pick-off process at which e+ from o-Ps annihilates with e− from the void surface which is resulting in a reduced lifetime τo-Ps compared to the vacuum value. The value of that reduced lifetime depends on hole size. The advantage of this technique – positron annihilation lifetime spectroscopy (PALS) – is a relative simple arrangement, ability to measure of the lifetime at different external conditions (temperature, pressure, etc.) and sensitivity of lifetime to the microstructural freevolume size. Thus, lifetime measurements provide the important information about the local free volume and arrangement of the polymeric structure on the range of 0.2–50 nm [21]. For the simple explanation, we can define total specific volume V as a sum of specific occupied volume (by atoms) Vocc and the specific free volume Vf. This specific free volume can be written as Vf = VhNh, where Vh is the hole volume (local free volume) determined by PALS and Nh is the number of free-volume holes per unit mass. The aim of this study was a usage of epoxidized linseed oil (ELO), bisphenol A diglycidyl ether (RD) as reactive diluent, and 50% mixture of triarylsulfonium hexafluorophosphate in propylene carbonate (PI) as photoinitiator for the formation of biosensor holding matrix on the base of previous results [22]. The commercial laccase was used as a catalytic bioselective element of amperometric enzyme biosensor sensitive to different aromatic phenols and amines. Laccase (polyphenoloxidase from Trametes versicolor; EC 1.10.3.2) is a member of the blue multicopper-oxidase family. This enzyme has been studied for a long time, due to their ability to oxidize a variety of organic substrates and to reduce molecular oxygen to water [23]. Use of nanoimmobilized laccases to remove micropollutants from wastewater has also been recently demonstrated [24]. Laccase is made up of a cluster of four copper atoms (type I copper; type II copper and two type III copper atoms) that form the active site of the enzyme [25]. The copper consisting cluster provides the ability of laccase to direct electron transfer from reduced enzyme to a surface of transducer. We have used these enzymatic properties for construction of new non-mediated laccase biosensor of the third generation. In the present study, we report the results on construction and characterization of biosensor based on laccase and two photocrosslinked polymers ELO/10RD and ELO/30RD as holding matrixes. The network properties of the polymer matrixes were studied by means of

2. Experimental 2.1. Materials Epoxidized linseed oil (ELO, having an average number of 6 epoxy groups per molecule) was purchased from Chemical Point, Germany. Bisphenol A diglycidyl ether (RD), 50% mixture of triarylsulfonium hexafluorophosphate in propylene carbonate (PI), laccase from Trametes versicolor with activity of ≥10 U mg−1, 2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) (ABTS), anhydrous ethanol (EtOH), sodium acetate and acetic acid were purchased from Sigma Aldrich. Tetrahydrofuran (THF) (99.9%) was purchased from Eurochemicals. All chemicals were of analytical reagent grade and all solutions were prepared using HPLC-grade water. 2.2. Synthesis and characterization of photocross-linked polymer matrixes 2.2.1. Sample preparation The photocross-linked polymers ELO/10RD and ELO/30RD were synthesized by the reaction of ELO and 10 mol.% or 30 mol.% of RD, using 3 mol.% of PI. Such concentrations of RD and PI were chosen according to previous results [22]. Chemical structures of ELO, RD and PI are shown in Fig. 1. The minimal amount of THF was used for the preparation of compositions. The mixtures of starting materials were poured in a Teflon mold with the diameter of 15 mm, the height of 3 mm and cured under UV/Vis light using a 500 W Helios Italquartz, model GR.E UV lamp with a wavelength range of (250–450) nm at an intensity of 310 mW cm−2. The obtained photocross-linked polymers ELO/10RD and ELO/30RD were transparent, smooth, and had a yellowish color. 2.2.2. Swelling experiment The cured samples (not identical with the samples used for PALS experiment) with the geometry 15 mm in diameter, 3 mm of thickness and known weight (m0) were swollen in the distilled water (H2O) and in the anhydrous ethanol (EtOH). The swelling tests were performed at room temperature (25 °C) for 5 days in H2O and for 2 days in EtOH until the equilibrium state [26]. The swollen samples were weighed (mswollen) and dried at room temperature to the constant weight of samples (mdry) during 10 days for H2O and 13 days for EtOH. The accuracy of mass determination was ± 0.1%. The swelling capacity S in % is defined here as:

m − m0 ⎤ S = ⎡ swollen × 100 ⎥ ⎢ m0 ⎦ ⎣

(1)

The measure of disintegration, Dis in %, is defined as

m 0 − mdry ⎤ Dis = ⎡ × 100 ⎢ ⎥ m0 ⎣ ⎦

(2)

2.2.3. PALS experiment The positron lifetime spectra were measured by the fast-fast coincidence spectrometer with a time resolution of 320 ps (the full width at half of maximum of the time resolution curve). The instrumental resolution function was calculated from lifetime spectra of an Al defectfree sample. The sandwich arrangement of sample-source was used with 392

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Fig. 1. Chemical structures of epoxidized linseed oil (ELO), bisphenol A diglycidyl ether (RD), and triarylsulfonium hexafluorophosphates (used as PI).

Fig. 2. The absorption and desorption of EtOH (left) and H2O (right) of the polymers ELO/10RD and ELO/30RD during 15 days.

the radioactive positron 22Na source with an activity of 0.8 MBq between two identical samples. The annihilation of positrons in the source was taken into account at the calculation. The PALS spectra were analyzed by the LT program for the discrete term-analysis [27]. Temperature measurements of samples were made in a helium closed-cycle refrigerator Janis CCS-450 system with a temperature stability of 0.2 K. The average spherical hole size R was determined from the measured o-Ps lifetime by a semiempirical relation Eq. (3), where ΔR = 0.166 nm is an empirical constant [28,29]. The corresponding mean hole volume Vh is given by Eq. (4):

Amperometric measurements were carried out using a potentiostat CHI 1200A (IJ Cambria Scientific, Burry Port, UK) connected to a personal computer and performed in a batch mode under continuous stirring in a standard 40 ml electrochemical cell at room temperature. Graphite rods (type RW001, 3.05 mm diameter, area 7.3 mm2, Ringsdorff Werke, Bonn, Germany) were used as working electrodes. They were sealed in glass tubes using epoxy glue thus forming disk electrodes. Before sensor preparation, the graphite electrodes were polished with emery paper and cleaned with distilled water.

−1

R 1 2π R ⎤⎫ + τo − Ps = 0.5 ⎧1 − sin ⎡ ⎨ + R Δ R 2 π R ⎣ + ΔR ⎦ ⎬ ⎭ ⎩ Vh =

4 π R3 3

2.3.2. Immobilization of laccase The laccase immobilisation includes the following stages: (1) dropping of 5 µl of laccase solution (1 mg ml−1 with an activity 13 U mg−1) on the working electrode surface and drying for 5 min at 15 °C; (2) covering the dried enzyme by 0.3 mm thick layers of photocross-linked polymers ELO/10RD or ELO/30RD; and (3) fixation of the formed enzyme-polymer matrix by net cap using plastic “O”-ring. The formed bioelectrode was washed by 50 mM acetic buffer, pH 4.5 and stored at 8 °C till using.

(3) (4)

2.3. Biosensors’ preparation and evaluation 2.3.1. Apparatus and techniques Amperometric biosensors were evaluated using constant-potential amperometry in a three-electrode configuration with an Ag/AgCl/KCl (3 M) reference electrode and a Pt-wire counter electrode. 393

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dependences change at different cycles. The value of Tg is about 230 K for both samples. If we suppose the “relaxed structure” with lower Vh values at slow heating cycles then Tg should be 233 K and 245 K for the polymers ELO/10RD and ELO/30RD, respectively. It can be noted that the average sizes of free-volume holes in the polymer ELO/10RD are larger than that of ELO/30RD. Lifetime measurements showed the reproducible lifetime values at the room temperature after heating over Tg. Values of Vh in the third heating cycle (after first heating and second cooling run) are very similar to the first heating cycle. These facts indicate the chemical network stability during the PALS measurement. Temperature dependences of the positronium formation for polymers ELO/10RD and ELO/30RD represented by the o-Ps intensity Io-Ps are shown in Fig. S2 (Supplementary data). Assuming that this quantity is related only to the concentration of free-volume holes in the material, the polymer ELO/10RD has lower hole concentration compared to ELO/30RD.

Table 1 Weights of initial, swollen and dry states m0, mswollen, mdry for the polymers ELO/10RD and ELO/30RD in EtOH and H2O. The swellability S and structural disintegration Dis are calculated on the base of Eqs. (1) and (2). Polymers ELO/10RD ELO/30RD ELO/10RD ELO/30RD

(EtOH) (EtOH) (H2O) (H2O)

m0 (g)

mswollen (g)

mdry (g)

S (%)

Dis (%)

0.64411 0.65258 0.66329 0.66585

0.79931 0.81451 0.67004 0.67274

0.54702 0.56381 0.66263 0.66489

24.09 24.81 1.018 1.035

15.1 13.6 0.10 0.14

3. Results and discussion 3.1. Swelling data for the photocross-linked polymers The results of swelling experiment for the polymers ELO/10RD and ELO/30RD are shown in Fig. 2. The maximal swollen state of both samples in EtOH and water was reached after 2 days or 5 days, respectively. The swellability of the polymer ELO/10RD in the both solvents (EtOH, H2O) is lower compared to the polymer ELO/30RD. It suggests that the polymer ELO/10RD has a higher crosslinking density. In addition, differences between an initial mass m0 and mswollen (average of three values for a swollen saturated stay in H2O and a maximal value for swollen stay in EtOH after 40 h), expressed by the swellability S in the Table 1, confirm that the polymer ELO/10RD has a higher cross-linked structure than the polymer ELO/30RD. Then, in the desorption process, the weights of both samples began to decrease under the started weight (m0) in the both solvents. The structural disintegration is supposed with their more visible destabilization in EtOH than in H2O (Table 1). The destabilization of structure was reflected on the weights of dried samples mdry, which are lower than the initial ones (m0).

3.3. Amperometric sensors analysis For the immobilization of laccase on the graphite-rods electrodes, 0.3 mm thick layers of the photopolymers ELO/10RD or ELO/30RD were used. The photopolymers’ layer was held on the electrode surface by means of “O”-ring fixed net cap (Fig. 4). The possibility of a direct electron transfer through laccase to the gold planar electrode was confirmed by us recently [10]. To estimate the possibility of effective direct electron transfer between graphite-rod electrode and laccase, a cyclic voltamperometric analysis was performed (Fig. 5). The laccase-based bioelectrode constructed using ELO/10RD without any mediator shows a difference in cyclic voltamperometric responses before adding and after addition of the increasing concentration of ABTS, as a laccase substrate. It is clearly seen a redox peak as a result of laccase-catalyzed reaction at the potential of −100 mV vs Ag/AgCl reference electrode after addition of the increasing concentration of ABTS. A height of the peak testifies brightly to a high efficiency of the direct transfer between the surface of the graphite-rod electrode and laccase when compared with laccase-adsorbed electrode without adding polymers (Fig. 5). In addition, it allows estimating an optimal working potential of this process, which corresponds to −100 mV vs Ag/AgCl. This potential was selected for further work on the study of characteristics of the constructed bioelectrodes. The chronoamperometric current responses and corresponding calibration curves of the bioelectrodes constructed on the basis of laccase immobilized by ELO/10RD or ELO/30RD on the surface of the graphiterod electrode are presented in Fig. 6. The chronoamperogramms in Fig. 6, as well as corresponding calibration curves of the bioelectrodes constructed by means of ELO/10RD or ELO/30RD, have demonstrated a difference in their operational properties. The bioelectrode based on ELO/10RD has shown a 4-fold higher maximal current at substrate saturation (Imax) versus bioelectrode based on ELO/30RD (4.9 µA vs 1.25 µA, respectively). ELO/10RDbased bioelectrode has shown significantly higher apparent MichaelisMenten constant (KMapp) [30] to the ABTS as the substrate (0.36 vs 0.11 mM of ABTS). It is known from literature that the value of KM to the ABTS for laccase from Trametes versicolor in solution is 0.29 mM [31]. Thus, the obtained KMapp values for the constructed bioelectrodes in the range between 0.11 and 0.36 mM indicate a native configuration of laccase in polymeric matrixes and a high efficiency of direct electron transfer between the enzyme and surface of the graphite-rod electrode. For a more detailed characterization of both types of bioelectrodes (based on ELO/10RD and ELO/30RD), their sensitivity and range of linearity to ABTS were analyzed (Fig. 7). The sensitivity of the bioelectrode based on ELO/10RD was 1.3-fold higher compared with ELO/30RD-based bioelectrode: 1.673 A M−1 m−2 vs 1.234 A M−1 m−2 for ELO/30RD matrix,

3.2. PALS data for the photocross-linked polymers The lifetime spectra of both polymer samples were evaluated by three-component analysis. The shortest component comes from the annihilation of short-lived para-positronium and was fixed to the 125 ps value. The second component is connected with the direct annihilation of positrons with electrons of material and the data from the longest component (ns range of lifetimes) were converted to the average volume of the holes Vh by using Eqs. (3) and (4). Temperature measurements of positron lifetimes were made by cooling the sample (2 K/min) from the room temperature to a low temperature and then they were gradually performed by increasing the temperature to room temperature (RT). One measurement at the defined temperature lasted about 2 h in order to achieve a sufficient number of annihilation events in the time spectrum. After reaching the room temperature, the sample was measured in the ‘cooling’ cycle, while the temperature gradually decreasing. The temperature dependences of the hole volume Vh calculated from o-Ps lifetimes for the polymers ELO/10RD and ELO/30RD are presented in Fig. 3. The significant difference in Vh(T) dependences between temperature cycles of heating and cooling for both samples, especially for polymer ELO/10RD was observed. Such hysteresis indicates the unstable structure relaxing over time in different temperature regions. This fact supports the experiment with the fast cooling from 300 to 60 K and then the isothermal measurement of o-Ps lifetime for the photocross-linked polymer ELO/10RD (Fig. S1, Supplementary data). The typical constant of the relaxation time is 5.1 h for this temperature (60 K). This instability of the structure referred here is understood in this case as a slow network reaction at temperature change. Changes in hole volume depend on cooling or heating rate. After heating to room temperature the value of hole volume is reproduced. The determination of the glass transition temperature Tg is complicated due to the different position of points where the slopes of Vh(T) 394

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Fig. 3. Hole volume temperature dependences Vh(T) for the polymers ELO/10RD (left) and ELO/30RD (right) in the heating and cooling cycles.

Fig. 4. The scheme of biorecognition layer formation on the graphite-rods electrodes by means of photopolymers holding via “O”-ring fixing net cap.

Fig. 5. Cyclic voltamperograms of bioelectrode based on the use of ELO/10RD as a polymer matrix for laccase (left) and the laccase-adsorbed bioelectrode without adding polymers (right); without ABTS (black line) and upon subsequent additions of ABTS (red and green lines). Conditions: scan rate 10 mV s−1 vs Ag/AgCl (reference electrode), 50 mM acetate buffer, pH 4.5 at 23 °C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Chronoamperogramms (left) and calibration curves (right) of response on the increasing concentrations of ABTS for the graphite-rod electrode modified by laccase-containing photopolymers’ matrix. Conditions: working potential −100 mV vs Ag/AgCl (reference electrode), 50 mM acetate buffer, pH 4.5 at 23 °C and constant stirring using a magnetic mixer.

respectively (Fig. 7). On the other hand, the linearity of the ELO/10RDbased bioelectrode was 1.5-fold wider compared with the usage of ELO/ 30RD (from 0.006 to 0.15 mM vs 0.025–0.1 mM toward ABTS). The wider linear frames and higher sensitivity of bioelectrodes based on ELO/10RD make them more perspective in the analysis of the real samples of drinking water or wastewater due to outhitting the dilution step. The reproducibility of the best type of the sensor (ELO/10RD-based) was analyzed using three bioelectrodes in three independent chronoamperometric analyses (Fig. 8). The sensitivities for three independent chronoamperometric studies were between 1.5 and 1.7 A M−1 m−2 which suggest a good reproducibility of the constructed biosensor. Thus, the developed prototype of laccase-based amperometric biosensor can be used for practical application in industry and research, as well as for the environmental control of phenols pollution.

Fig. 7. Analysis of linearity range and sensitivity of the bioelectrodes based on ELO/RD-polymers. The diameter of the graphite electrode is 3.06 mm with the area 7.35 mm2. Abbreviation: B is the slope of the calibration curve in the linear range.

3.4. Properties of biosensors parameters vs structure of holding polymer matrixes The network properties of the biosensor holding polymer matrixes and biosensor’s parameters obtained in this work are gathered in Tables 2 and 3, respectively. The results of the hole volume thermal expansion experiments show a higher TgPALS value, a lower slope of Vh(T) dependence αF2 above Tg as well as a smaller change in slopes, expressed through αF2 − αF1, for the polymer ELO/30RD (Table 2). This would indicate a more rigid structure typical for dense cross-linked systems [32,33]. This would be in conflict with the finding of swelling that indicates that a denser crosslinked system is ELO/10RD (Table 2). In fact, however, other factors may also play a significant role at network behavior, causing a more packed structure and reducing the mobility of the polymer network above Tg. It is, for example, the presence of weak physical bonds that can reduce the network flexibility and increase Tg, although the system may have a low crosslink density. Likewise, the presence of aromatic groups causes a suppression of the network mobility due to steric hindrance. Thus, for example, the 2 M/EDDT and 2 M/DAS photopolymer networks [21,34] behaved similar, where the first photopolymer had lower Tg and higher crosslink density in comparison with the second one. In this work, the higher amount of polar sites and aromatic groups in the polymer ELO/30RD due to higher concentration of RD in this sample can play an important role. Therefore, the PALS results of the present study confirm that the coefficients for the thermal expansion of free-volume holes αF1, αF2 as well as their difference (αF2 – αF1) are

Fig. 8. The calibration curves of three independent chronoamperometric experiments used for estimation of the reproducibility of the ELO/10RD-based bioelectrodes. Conditions: working potential −100 mV vs Ag/AgCl (reference electrode), 50 mM acetate buffer, pH 4.5 at 23 °C and constant stirring using a magnetic mixer.

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Table 2 Hole volume Vh at glass transition temperature Tg, swellability S in EtOH, and slopes αF1, αF2 of the Vh(T) dependences in the regions below and above Tg, respectively, as well as their differences. Values for heating and cooling cycles are in the top and bottom part of the boxes, respectively. Polymers

Vh (nm3)

Tg (K)

S (%)

αF1 (10−4 K−1)

αF2 (10−4 K−1)

αF2 − αF1 (10−4 K−1)

ELO/10RD

0.057 ± 0.002 0.068 ± 0.002

233

24.09

3.53 ± 0.30 3.31 ± 0.32

13.02 ± 0.60 11.16 ± 0.55

9.49 ± 0.67 7.85 ± 0.64

ELO/30RD

0.051 ± 0.002 0.049 ± 0.002

245

24.81

3.47 ± 0.33 3.87 ± 0.83

12.42 ± 0.64 8.96 ± 0.48

8.95 ± 0.72 5.09 ± 0.96

Table 3 Biosensor response Imax, apparent Michaelis-Menten constant KMapp toward ABTS as the substrate, the slope of the calibration curve B, the sensitivity of bioelectrodes (working surface area 7.35 mm2) constructed based on laccase immobilized by the polymers ELO/10RD and ELO/30RD, and the range of linearity of the constructed bioelectrodes to ABTS. Polymers

Imax (μA)

KMapp (mM)

B (μA⋅mM−1)

Sensitivity (A⋅M−1⋅m−2)

Range of linearity (mM)

ELO/10RD ELO/30RD

4.9 ± 0.19 1.25 ± 0.17

0.36 ± 0.03 0.11 ± 0.04

12.3 9.07

1.673 1.234

0.006–0.15 0.025–0.10

reliable characteristics of microstructural free volume in the case of photopolymers [21]. According to the comparative analysis of the biosensor’s parameters, it is seen that the laccase-based amperometric biosensor constructed using holding polymer ELO/10RD matrix has better characteristics on the sensitivity of bioelectrodes calculated using the slope of the calibration curve B and area of the electrode used as well as the range of linearity to ABTS, than that constructed using holding polymer ELO/30RD matrix (Table 3). Summarizing the results obtained for the investigated samples one may conclude that the polymer ELO/10RD compared to the polymer ELO/30RD has: (i) the higher crosslink density, (ii) the larger free-volume holes, (iii) the lower concentration of free-volume holes, and (iv) the larger difference in the coefficients for the thermal expansion of free-volume holes in the regions below and above Tg. At the same time, the laccase-based amperometric biosensor constructed using the polymer ELO/10RD as a biosensor holding matrix shows the improved biosensor’s parameters compared to ELO/30RD. It seems that the microstructural network properties of the polymer holding matrix are crucial for the determination of the biofunctionality of constructed amperometric biosensors. Further research is required to prove this correlation for other polymers suitable for immobilization of enzyme and construction of amperometric biosensors with controlled parameters.

compared to the polymer ELO/30RD results in the improvement of bioanalytical characteristics of the laccase-based amperometric biosensor. Further research is needed to prove this correlation for other polymers with more different crosslink density as well as to compare the operational parameters of the constructed biosensors with similar analogues known in literature. Acknowledgements This work was supported in part by the Ministry of Education and Science of Ukraine (projects Nos. 0118U000297 and 0119U100671 to TK, OS, OD, and MG; and project No. 0117U007142 to KZ), National Academy of Sciences of Ukraine in the frame of the Scientific-Technical Program “Smart sensor devices of a new generation based on modern materials and technologies” (project No. 13 to OS, OD, and MG), Slovak Grant Agency VEGA (project No. 2/0127/17 to HŠ and project No. 2/ 0157/17 to OŠ), and Slovak Research and Development Agency (project No. APVV-16-0369 to HŠ and OŠ). TK also acknowledges the SAIA (Slovak Academic Information Agency) for scholarship in the IPSAS within the National Scholarship Programme of the Slovak Republic. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.03.056.

4. Conclusion References We report the results on construction and characterization of the biosensor based on laccase and photocross-linked polymer as a holding matrix to be suitable for analysis of the quality of drinking water, as well as determination of wastewater pollution. The amperometric enzyme biosensors using photocross-linked polymers based on epoxidized linseed oil (ELO), bisphenol A diglycidyl ether (RD) as reactive diluent and 50% mixture of triarylsulfonium hexafluorophosphate in propylene carbonate (PI) as photoinitiator, synthesized by the reaction of ELO and 10 mol.% or 30 mol.% of RD, using 3 mol.% of PI (ELO/10RD and ELO/ 30RD, respectively) as holding matrixes and immobilized commercial laccase from Trametes versicolor have been constructed on the graphite rods as working electrodes. Network properties (crosslink density and local free volume or hole volume characteristics) of the photocrosslinked polymers used have been studied by means of swelling and positron annihilation lifetime spectroscopy. It has been established that the application of the polymer ELO/10RD which is characterized by the higher crosslink density, larger free-volume holes with their lower concentration and larger difference in the coefficients for the thermal expansion of free-volume holes in the regions below and above Tg

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