Advanced Ga 2 O 3 / Lignin and ZrO 2 / Lignin Hybrid Microplatforms for Glucose Oxidase Immobilization: Evaluation of Biosensing Properties by Catalytic Glucose Oxidation

: In this study, novel Ga 2 O 3 / lignin and ZrO 2 / lignin hybrid materials were obtained and used as supports for the adsorption of the enzyme glucose oxidase (GOx). A biosensor system based on the hybrid supports was then designed to determine the concentration of glucose in various solutions. The obtained bioinspired platforms were analyzed to determine chemical and physical properties of the support structures. A determination was made of the e ﬀ ectiveness of the proposed method of immobilization and the quality of operation of the constructed glucose biosensor in electrochemical tests. To characterize the materials, Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray di ﬀ raction (XRD), thermogravimetric analysis (TGA), electrokinetic (zeta) potential measurements, atomic force microscopy (AFM), particle size measurements (NIBS technique), and elemental analysis (EA) were used. In further research, glucose oxidase (GOx) was immobilized on the surface of the obtained functional Ga 2 O 3 / lignin and ZrO 2 / lignin biomaterials. The best immobilization capacities—24.7 and 27.1 mg g − 1 for Ga 2 O 3 / lignin and ZrO 2 / lignin, respectively—were achieved after a 24 h immobilization process. The Ga 2 O 3 / Lig / GOx and ZrO 2 / Lig / GOx systems were used for the construction of electrochemical biosensor systems, in a dedicated carbon paste electrode (CPE) with the addition of graphite and ferrocene.


Introduction
Hybrid materials have existed on Earth for millions of years. There are many examples of combinations of inorganic and organic components that occur in nature, including shells of mollusks and components of human bones [1][2][3][4][5]. Over time, people took notice that combinations of two or more components could enable the fabrication of a material with new properties that were not exhibited by the separate components. This was just the beginning, as the new properties of hybrid connections are still being explored and their potential has not yet been fully exploited [2][3][4][5][6]. Hybrid materials

Surface Morphology
The techniques that were used to determine the morphological structure of oxides and the obtained hybrid materials were transmission electron microscopy (TEM), scanning electron spectroscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and X-ray diffraction analysis (XRD). The results of transmission electron microscopy are shown in Figure 1. The bare Ga2O3 particles have sizes ranging from 1 to 5 µ m ( Figure 1A). The micrograph of ZrO2 presents spherical nanoparticles from 20 to 50 nm ( Figure 1B). Both images show individual primary particles that tend to agglomerate to form larger clusters. The Ga2O3/lignin and ZrO2/lignin hybrid materials are shown in Figure 1C,D, respectively. The change in the morphology of the material, including the size of the repeated pattern observed in the TEM image, indirectly demonstrates the effectiveness of the combination of APTES-modified metal oxides with activated lignin. Scanning electron microscopy (SEM) was also used to determine the morphology of the components and the obtained hybrid materials (Figure 2). The bare Ga 2 O 3 particles have sizes ranging from 1 to 5 µm ( Figure 1A). The micrograph of ZrO 2 presents spherical nanoparticles from 20 to 50 nm ( Figure 1B). Both images show individual primary particles that tend to agglomerate to form larger clusters. The Ga 2 O 3 /lignin and ZrO 2 /lignin hybrid materials are shown in Figure 1C,D, respectively. The change in the morphology of the material, including the size of the repeated pattern observed in the TEM image, indirectly demonstrates the effectiveness of the combination of APTES-modified metal oxides with activated lignin. Scanning electron microscopy (SEM) was also used to determine the morphology of the components and the obtained hybrid materials (Figure 2).
The SEM micrographs present structures of Ga 2 O 3 , ZrO 2 , Ga 2 O 3 /lignin and ZrO 2 /lignin in a range of about 2-50 µm with the occurrence of irregularly shaped particles. It may be assumed from the shape and size of the particles that the materials will have a relatively small specific surface area and mainly macroscopic pores. The primary microparticles are capable of forming aggregates and agglomerates. The lignin component of the hybrid materials is visible. In the SEM micrographs of the resulting supports, stable irregularly shaped modules of modified gallium oxide or zirconium dioxide and activated lignin have merged together to form clusters of aggregates with no precisely outlined contours.
Energy Dispersive Spectroscopy (EDS) was used for the elements distribution analysis and mapping. The test was carried out for both pristine oxides (ZrO 2   The SEM micrographs present structures of Ga2O3, ZrO2, Ga2O3/lignin and ZrO2/lignin in a range of about 2-50 μm with the occurrence of irregularly shaped particles. It may be assumed from the shape and size of the particles that the materials will have a relatively small specific surface area and mainly macroscopic pores. The primary microparticles are capable of forming aggregates and agglomerates. The lignin component of the hybrid materials is visible. In the SEM micrographs of the resulting supports, stable irregularly shaped modules of modified gallium oxide or zirconium dioxide and activated lignin have merged together to form clusters of aggregates with no precisely outlined contours. Energy Dispersive Spectroscopy (EDS) was used for the elements distribution analysis and mapping. The test was carried out for both pristine oxides (ZrO2, Ga2O3) and for hybrid supports (ZrO2/Lig, Ga2O3/Lig). The obtained results are presented in  The SEM micrographs present structures of Ga2O3, ZrO2, Ga2O3/lignin and ZrO2/lignin in a range of about 2-50 μm with the occurrence of irregularly shaped particles. It may be assumed from the shape and size of the particles that the materials will have a relatively small specific surface area and mainly macroscopic pores. The primary microparticles are capable of forming aggregates and agglomerates. The lignin component of the hybrid materials is visible. In the SEM micrographs of the resulting supports, stable irregularly shaped modules of modified gallium oxide or zirconium dioxide and activated lignin have merged together to form clusters of aggregates with no precisely outlined contours.
Energy Dispersive Spectroscopy (EDS) was used for the elements distribution analysis and mapping. The test was carried out for both pristine oxides (ZrO2, Ga2O3) and for hybrid supports (ZrO2/Lig, Ga2O3/Lig). The obtained results are presented in       The results of EDS also allowed the determination of the percentage content of elements (Ga, Zr, O, C, S, Na, and I). According to the EDS results, a strong peak from C and S are presented in Ga 2 O 3 /Lig and ZrO 2 /Lig materials confirming the existence of incorporated lignin. The values obtained for sulfur and carbon may suggest that in both hybrid materials, it contains similar amounts of lignin. EDS mapping results confirm a uniform of Ga, Zr, S, O, and C elements. The data also reveals the presence of slight amount of sodium and iodine in hybrid Ga 2 O 3 /Lig and ZrO 2 /Lig materials, which may come from the activation of lignin with sodium periodate. The percentage content of elements was collected and summarized in Table 1.    The results of EDS also allowed the determination of the percentage content of elements (Ga, Zr, O, C, S, Na, and I). According to the EDS results, a strong peak from C and S are presented in Ga2O3/Lig and ZrO2/Lig materials confirming the existence of incorporated lignin. The values obtained for sulfur and carbon may suggest that in both hybrid materials, it contains similar amounts of lignin. EDS mapping results confirm a uniform of Ga, Zr, S, O, and C elements. The data also reveals the presence of slight amount of sodium and iodine in hybrid Ga2O3/Lig and ZrO2/Lig materials, which may come from the activation of lignin with sodium periodate. The percentage content of elements was collected and summarized in Table 1. In order to determine the crystal structure, an X-ray diffraction analysis for oxides, hybrid materials, and lignin was performed. The XRD patterns are shown in Figure 7. The XRD patterns of Ga2O3 show characteristic peaks at 2θ values of 31.72°, 35.22°, and 63.89° correspond to the (002), (111), and (512) diffraction peaks, respectively. All obtained XRD patterns are in accordance with those of monoclinic phase of β-Ga2O3 (JCPDS: 41-1103) [39]. Sharp diffraction peaks also indicate the high crystalline quality of the used compounds. The crystalline structures of ZrO2 were also investigated with this analysis. The peaks at 2θ of 28.74°, 31.38°, 50.39°, and 60.47° correspond to the In order to determine the crystal structure, an X-ray diffraction analysis for oxides, hybrid materials, and lignin was performed. The XRD patterns are shown in Figure 7 [40]. The same diffraction reflections from both oxides and hybrid materials are observed. No Bragg peaks have been noticed in the case of lignin because of the amorphous nature of lignin [41]. The study confirmed that the proposed method of synthesis does not affect the crystal structure of the created materials.

Zeta Potential, Mean Hydrodynamic Diameter, PdI, and Elemental Analysis of Microplatforms
The Ga 2 O 3 /lignin and ZrO 2 /lignin supports and their components were exposed to electrokinetic potential measurements to define the dispersion stability of individual supports. The results are shown in Table 1. The potential of the metal oxides changed after modification with APTES from −32.2 to −28.2 mV for Ga 2 O 3 and from −33.5 to −29.4 mV for ZrO 2 , respectively. The results indicate the moderate dispersion stability of Ga 2 O 3 /lignin and ZrO 2 /lignin, for which the electrokinetic potential values are −35.8 and −36.1 mV, respectively. The zeta potential of lignin indicates that the biopolymer is very stable [42]. Moreover, the changes in the zeta potentials of these hybrid platforms provide further confirmation of the binding of lignin to the metal oxide surfaces. Furthermore, there were presented sizes of particles based on non-invasive back scattering (NIBS) measurements and the polydispersity index (PdI) findings of the metal oxides (Ga 2 O 3 , ZrO 2 ), before and after APTES-modification and final hybrid products. The modification of the metal oxides and binding them to the lignin facilitate aggregations and agglomerations of the molecules. The PdI index also increased after both the modification and the hybrid materials synthesis (see Table 2). (111), (200), (220), and (311) diffraction planes, respectively (JCPDS: 79-1771) [40]. The same diffraction reflections from both oxides and hybrid materials are observed. No Bragg peaks have been noticed in the case of lignin because of the amorphous nature of lignin [41]. The study confirmed that the proposed method of synthesis does not affect the crystal structure of the created materials.

Zeta Potential, Mean Hydrodynamic Diameter, PdI, and Elemental Analysis of Microplatforms
The Ga2O3/lignin and ZrO2/lignin supports and their components were exposed to electrokinetic potential measurements to define the dispersion stability of individual supports. The results are shown in Table 1. The potential of the metal oxides changed after modification with APTES from −32.2 to −28.2 mV for Ga2O3 and from −33.5 to −29.4 mV for ZrO2, respectively. The results indicate the moderate dispersion stability of Ga2O3/lignin and ZrO2/lignin, for which the electrokinetic potential values are −35.8 and −36.1 mV, respectively. The zeta potential of lignin indicates that the biopolymer is very stable [42]. Moreover, the changes in the zeta potentials of these hybrid platforms provide further confirmation of the binding of lignin to the metal oxide surfaces. Furthermore, there were presented sizes of particles based on non-invasive back scattering (NIBS) measurements and the polydispersity index (PdI) findings of the metal oxides (Ga2O3, ZrO2), before and after APTESmodification and final hybrid products. The modification of the metal oxides and binding them to the lignin facilitate aggregations and agglomerations of the molecules. The PdI index also increased after both the modification and the hybrid materials synthesis (see Table 2). Table 2. Zeta potential, mean particle size, and polydispersity character of studied samples. The next test to confirm the chemical composition of synthesized samples was elemental analysis. This analysis allowed the determination of the nitrogen (N), carbon (C), hydrogen (H), and sulfur (S) contents. The presence of nitrogen content in the modified metal oxides after APTES-  Table 2. Zeta potential, mean particle size, and polydispersity character of studied samples. The next test to confirm the chemical composition of synthesized samples was elemental analysis. This analysis allowed the determination of the nitrogen (N), carbon (C), hydrogen (H), and sulfur (S) contents. The presence of nitrogen content in the modified metal oxides after APTES-modification were observed. The obtained results are similar for both hybrid materials (Ga 2 O 3 /lignin; ZrO 2 /lignin). The obtained results were presented in Table 3.

Fourier Transform Infrared Spectroscopy (FT-IR) Analysis
FT-IR spectra are shown in Figure 8A,B. The band of stretching vibrations of −OH groups lies in the wavenumber range 3600-3400 cm −1 [5]. In the range 3000-2800 cm −1 , peaks originating from stretching vibrations of −CH groups (CH 3 and CH 2 ) are noticeable [5]. The signals in the range 1600-1475 cm −1 are characteristic for stretching vibrations of C Ar = C Ar groups originating from the aromatic rings of lignin [5].

Fourier Transform Infrared Spectroscopy (FT-IR) Analysis
FT-IR spectra are shown in Figure 8A,B. The band of stretching vibrations of −OH groups lies in the wavenumber range 3600-3400 cm −1 [5]. In the range 3000-2800 cm −1 , peaks originating from stretching vibrations of −CH groups (CH3 and CH2) are noticeable [5]. The signals in the range 1600-1475 cm −1 are characteristic for stretching vibrations of CAr = CAr groups originating from the aromatic rings of lignin [5].  Figure 8A shows the FT-IR spectrum of Ga2O3 after modification. As a result of the modification, −NH2 groups are introduced [43]. The spectrum confirms the effectiveness of the modification due to the presence of a band in the wavenumber range 3400-3200 cm −1 , derived from stretching vibrations of −NH bonds [5,43]. The spectrum also includes bands corresponding to the vibrations of functional groups originating from Ga2O3. The vibrations of Ga-O bonds produce a signal in the wavenumber range 650-700 cm −1 [43]. Figure 8A also shows the FT-IR spectrum of the Ga2O3/lignin hybrid. In the range 3200-3600 cm −1 , increase in the intensity of bands originating from stretching vibrations of −OH and −NH, at wavenumber range 3400-3200 cm −1 , groups are visible, indicating effective obtaining of hybrid [5].
In the spectrum of zirconium dioxide after modification, which is shown in Figure 8B, attention should be paid to the signals in the wavenumber range 3600-3400 cm −1 generated by stretching vibrations of hydroxyl groups, and the band in the region around 500 cm −1 , characteristic for Zr-O  Figure 8A shows the FT-IR spectrum of Ga 2 O 3 after modification. As a result of the modification, −NH 2 groups are introduced [43]. The spectrum confirms the effectiveness of the modification due to the presence of a band in the wavenumber range 3400-3200 cm −1 , derived from stretching vibrations of −NH bonds [5,43]. The spectrum also includes bands corresponding to the vibrations of functional groups originating from Ga 2 O 3 . The vibrations of Ga-O bonds produce a signal in the wavenumber range 650-700 cm −1 [43]. Figure 8A also shows the FT-IR spectrum of the Ga 2 O 3 /lignin hybrid. In the range 3200-3600 cm −1 , increase in the intensity of bands originating from stretching vibrations of −OH and −NH, at wavenumber range 3400-3200 cm −1 , groups are visible, indicating effective obtaining of hybrid [5].
In the spectrum of zirconium dioxide after modification, which is shown in Figure 8B, attention should be paid to the signals in the wavenumber range 3600-3400 cm −1 generated by stretching vibrations of hydroxyl groups, and the band in the region around 500 cm −1 , characteristic for Zr-O groups [43]. Hydroxyl and amine groups bands increased in intensity following modification with APTES. After binding of the structure of lignin to the metal oxide, bands corresponding to vibrations of C-H bonds are presented at about 2800 cm −1 , and signals in the range 1500-1425 cm −1 indicate the C Ar -C Ar bonds originating from the aromatic structures occurring in the structure of lignin [43].

Thermal Stability
Thermogravimetric analysis (TG) was used to determine the thermal stability of the prepared hybrids ( Figure 9).
Measurements were performed in the temperature range up to 1000 • C. The TG curves of Ga 2 O 3 before and after modification show insignificant mass loss [44] associated with the loss of water that was connected to the oxide surface with physical bonds. The analysis shows the high thermal stability of Ga 2 O 3 in a wide range of temperatures. From the curve for the Ga 2 O 3 /lignin hybrid system, it can be concluded that the thermal decomposition of the sample consists of three main steps. In the temperature range from 25 • C to about 250 • C, there is a slight decrease (around 10%) in the mass of the sample, related to the evaporation of water on matrix surface. There is then a rapid decrease in mass by about 30% in a temperature range of approximately 300-600 • C [44]. This is related to the thermal decomposition of the lignin present in the Ga 2 O 3 /lignin hybrid system. At temperatures above 600 • C, the sample passes along a further loss of mass of about 15%, caused by the thermal degradation of lignin fragments that occur in combination with carbon. Nevertheless, it can be concluded that the Ga 2 O 3 /lignin hybrid system has relatively high thermal stability [4,5,44]. groups [43]. Hydroxyl and amine groups bands increased in intensity following modification with APTES. After binding of the structure of lignin to the metal oxide, bands corresponding to vibrations of C-H bonds are presented at about 2800 cm −1 , and signals in the range 1500-1425 cm −1 indicate the CAr-CAr bonds originating from the aromatic structures occurring in the structure of lignin [43].

Thermal Stability
Thermogravimetric analysis (TG) was used to determine the thermal stability of the prepared hybrids ( Figure 9). Measurements were performed in the temperature range up to 1000 °C. The TG curves of Ga2O3 before and after modification show insignificant mass loss [44] associated with the loss of water that was connected to the oxide surface with physical bonds. The analysis shows the high thermal stability of Ga2O3 in a wide range of temperatures. From the curve for the Ga2O3/lignin hybrid system, it can be concluded that the thermal decomposition of the sample consists of three main steps. In the temperature range from 25 °C to about 250 °C, there is a slight decrease (around 10%) in the mass of the sample, related to the evaporation of water on matrix surface. There is then a rapid decrease in mass by about 30% in a temperature range of approximately 300-600 °C [44]. This is related to the thermal decomposition of the lignin present in the Ga2O3/lignin hybrid system. At temperatures above 600 °C, the sample passes along a further loss of mass of about 15%, caused by the thermal degradation of lignin fragments that occur in combination with carbon. Nevertheless, it can be concluded that the Ga2O3/lignin hybrid system has relatively high thermal stability [4,5,44].
On the graph in Figure 9B, the ZrO2/lignin hybrid system is observed to undergo a significant loss of mass of heating compared with unmodified and modified zirconium dioxide. The first mass loss, connected with the elimination of water physically absorbed on the surface of lignin, occurred in the temperature range 25-200 °C and amounted to about 8%. The second larger loss occurred in the range 280-320 °C. This was a loss of about 25%, associated with the loss of crystalline water trapped in the structure of the ZrO2/lignin hybrid material [4,5]. Thermal treatment in the third stage above 350 °C (up to 1000 °C) causes a partial loss of lignin fragments associated with carbon decomposition (a mass loss of about 30%), which results from fragmentation of the molecule due to unclear and uncontrolled reactions [3,4].

Efficiency of Glucose Oxidase (GOx) Immobilization
To measure the efficiency of enzyme immobilization, the Bradford method was used. The assay is a spectrophotometric measurement at wavelength λ = 595 nm, using a dye called Coomassie Brillant Blue G-250, which makes a specific combination with amino acid residues containing a positive charge, including mainly arginine, as well as histidine, lysine, proline, tryptophan, and On the graph in Figure 9B, the ZrO 2 /lignin hybrid system is observed to undergo a significant loss of mass of heating compared with unmodified and modified zirconium dioxide. The first mass loss, connected with the elimination of water physically absorbed on the surface of lignin, occurred in the temperature range 25-200 • C and amounted to about 8%. The second larger loss occurred in the range 280-320 • C. This was a loss of about 25%, associated with the loss of crystalline water trapped in the structure of the ZrO 2 /lignin hybrid material [4,5]. Thermal treatment in the third stage above 350 • C (up to 1000 • C) causes a partial loss of lignin fragments associated with carbon decomposition (a mass loss of about 30%), which results from fragmentation of the molecule due to unclear and uncontrolled reactions [3,4].

Efficiency of Glucose Oxidase (GOx) Immobilization
To measure the efficiency of enzyme immobilization, the Bradford method was used. The assay is a spectrophotometric measurement at wavelength λ = 595 nm, using a dye called Coomassie Brillant Blue G-250, which makes a specific combination with amino acid residues containing a positive charge, including mainly arginine, as well as histidine, lysine, proline, tryptophan, and tyrosine. The used G-250 dye links with amino acids that change the colour from brown to blue, which it can be observed at UV-Vis spectrophotometer. The Bradford test is a comparatively universal and effective assay. The adsorption immobilization of GOx on Ga 2 O 3 /lignin or ZrO 2 /lignin hybrid materials was conducted at pH 5.0 and involved the formation of hydrogen bonds between amine groups in the enzyme and the carbonyl groups present in lignin. Studies of the quantity of immobilized GOx was performed using supernatants from presented hybrid materials after immobilization and unbound enzyme. The results are shown in Figure 10.
It may be observed from Figure 10 that the quantity of enzyme immobilized on the material grows as the process time increases up to 24 h. Extending the duration of the process, such as 72 h or 96 h, does not increase the quantity of enzyme adsorbed on the support. This may be due to the saturation of the surface active sites capable of immobilizing the enzyme. The maximum quantities of immobilized glucose oxidase on the Ga 2 O 3 , ZrO 2 , Ga 2 O 3 /lignin, and ZrO 2 /lignin hybrid materials were obtained after 24 h of the process, and reached 9.7, 11.6, 24.7, and 27.1 mg g −1 , respectively.
The obtained results were compared with our previously created systems and literature data and are summarized in Table 4.
tyrosine. The used G-250 dye links with amino acids that change the colour from brown to blue, which it can be observed at UV-Vis spectrophotometer. The Bradford test is a comparatively universal and effective assay. The adsorption immobilization of GOx on Ga2O3/lignin or ZrO2/lignin hybrid materials was conducted at pH 5.0 and involved the formation of hydrogen bonds between amine groups in the enzyme and the carbonyl groups present in lignin. Studies of the quantity of immobilized GOx was performed using supernatants from presented hybrid materials after immobilization and unbound enzyme. The results are shown in Figure 10. It may be observed from Figure 10 that the quantity of enzyme immobilized on the material grows as the process time increases up to 24 h. Extending the duration of the process, such as 72 h or 96 h, does not increase the quantity of enzyme adsorbed on the support. This may be due to the saturation of the surface active sites capable of immobilizing the enzyme. The maximum quantities of immobilized glucose oxidase on the Ga2O3, ZrO2, Ga2O3/lignin, and ZrO2/lignin hybrid materials were obtained after 24 h of the process, and reached 9.7, 11.6, 24.7, and 27.1 mg g −1 , respectively.
The obtained results were compared with our previously created systems and literature data and are summarized in Table 4. To confirm the immobilization of GOx on the surfaces of the hybrids and to examine changes in the surface topography, AFM measurements were carried out ( Figure 11).  To confirm the immobilization of GOx on the surfaces of the hybrids and to examine changes in the surface topography, AFM measurements were carried out ( Figure 11). Variable changes take place in the samples before and after the immobilization process. The parameter Z on the AFM images increases for the systems containing enzyme. In addition, the surface structure of the matrix becomes rougher, which may suggest that the surface is covered with objects of a larger size in relation to the native roughness of the lignin. Variable changes take place in the samples before and after the immobilization process. The parameter Z on the AFM images increases for the systems containing enzyme. In addition, the surface structure of the matrix becomes rougher, which may suggest that the surface is covered with objects of a larger size in relation to the native roughness of the lignin. Figure 12A shows cyclic voltammograms of CPE/Ga 2 O 3 /lignin-GOx in PBS in the absence of glucose and in the presence of 1, 2, 5, and 10 mM glucose. Ferrocene mediator was incorporated into the electrode to provide electrochemical communication between GOx active sites and the electrode surface. It can be seen that the oxidation was accomplished based on a clear increase in the anodic current at a potential close to the formal potential for ferrocene. This behavior indicates that the CPE/Ga 2 O 3 /lignin-GOx electrode is able to catalyze the oxidation of glucose using Fc+/Fc as an effective mediator. As shown by the plot in Figure 11B, the anodic current increased with increasing glucose concentration. All electrochemical tests were repeated three times and were characterized by high repeatability. Similar results were obtained for the CPE/ZrO2/lignin-GOx modified electrode system ( Figure  13). It is observed that the higher the glucose concentration, the higher the anodic current recorded at the CPE/ZrO2/lignin-GOx. Moreover, the shape of the I vs. c curve in Figure 13B suggests that the enzymatic reaction follows Michaelis-Menten kinetics [49]. Similar results were obtained for the CPE/ZrO 2 /lignin-GOx modified electrode system ( Figure 13). It is observed that the higher the glucose concentration, the higher the anodic current recorded at the CPE/ZrO 2 /lignin-GOx. Moreover, the shape of the I vs. c curve in Figure 13B suggests that the enzymatic reaction follows Michaelis-Menten kinetics [49]. current dependence on glucose concentration (B). Similar results were obtained for the CPE/ZrO2/lignin-GOx modified electrode system ( Figure  13). It is observed that the higher the glucose concentration, the higher the anodic current recorded at the CPE/ZrO2/lignin-GOx. Moreover, the shape of the I vs. c curve in Figure 13B suggests that the enzymatic reaction follows Michaelis-Menten kinetics [49].

Modification of Metal Oxide Materials
To obtain modified oxides (gallium oxide and zirconium (IV) oxide) a solution consisting of (3-aminopropyl) triethoxysilane (APTES):water:methanol in the ratio 1:4:16 was prepared. The modification was carried out using 6 g of one of the oxides and 6.3 mL of the modifying solution (0.3 mL of APTES, 1.2 mL of H 2 O, 4.8 mL of MeOH). The modifying solution was added to the oxide with constant stirring. The final paste product was dried at 105 • C for 24 h. The last stage of the modification consisted in grinding and sieving the obtained modified oxide through a sieve with a mesh size of 64 µm. The mechanism of modification of selected oxides with the APTES is presented in Figure 14.
modification was carried out using 6 g of one of the oxides and 6.3 mL of the modifying solution (0.3 mL of APTES, 1.2 mL of H2O, 4.8 mL of MeOH). The modifying solution was added to the oxide with constant stirring. The final paste product was dried at 105 °C for 24 h. The last stage of the modification consisted in grinding and sieving the obtained modified oxide through a sieve with a mesh size of 64 μm. The mechanism of modification of selected oxides with the APTES is presented in Figure 14.

Activation of Lignin and Synthesis of Ga2O3/Lignin and ZrO2/Lignin Supports
In a 500 mL glass reactor equipped with a peristaltic pump, 4 g of lignin, 200 mL of dioxane, and 22.5 mL of distilled water were mixed. Next, 6 g of sodium iodate dissolved in 120 mL of water was added at a rate of 12 mL min −1 . To ensure that the lignin oxidized only under the influence of the oxidant, the reactor was sealed with silver foil. This caused the oxidation process to occur only under the impact of sodium periodate (NaIO4), excluding oxidation under the influence of light. The reaction of chemical activation of lignin lasted 80 min. The mechanism of this synthesis is presented in Figure 15.

Activation of Lignin and Synthesis of Ga 2 O 3 /Lignin and ZrO 2 /Lignin Supports
In a 500 mL glass reactor equipped with a peristaltic pump, 4 g of lignin, 200 mL of dioxane, and 22.5 mL of distilled water were mixed. Next, 6 g of sodium iodate dissolved in 120 mL of water was added at a rate of 12 mL min −1 . To ensure that the lignin oxidized only under the influence of the oxidant, the reactor was sealed with silver foil. This caused the oxidation process to occur only under the impact of sodium periodate (NaIO 4 ), excluding oxidation under the influence of light. The reaction of chemical activation of lignin lasted 80 min. The mechanism of this synthesis is presented in Figure 15. modification was carried out using 6 g of one of the oxides and 6.3 mL of the modifying solution (0.3 mL of APTES, 1.2 mL of H2O, 4.8 mL of MeOH). The modifying solution was added to the oxide with constant stirring. The final paste product was dried at 105 °C for 24 h. The last stage of the modification consisted in grinding and sieving the obtained modified oxide through a sieve with a mesh size of 64 μm. The mechanism of modification of selected oxides with the APTES is presented in Figure 14.

Activation of Lignin and Synthesis of Ga2O3/Lignin and ZrO2/Lignin Supports
In a 500 mL glass reactor equipped with a peristaltic pump, 4 g of lignin, 200 mL of dioxane, and 22.5 mL of distilled water were mixed. Next, 6 g of sodium iodate dissolved in 120 mL of water was added at a rate of 12 mL min −1 . To ensure that the lignin oxidized only under the influence of the oxidant, the reactor was sealed with silver foil. This caused the oxidation process to occur only under the impact of sodium periodate (NaIO4), excluding oxidation under the influence of light. The reaction of chemical activation of lignin lasted 80 min. The mechanism of this synthesis is presented in Figure 15.  In the next step, the 4 g of modified oxide (Ga 2 O 3 or ZrO 2 ) was added to the reaction mixture. Media synthesis reactions were carried out for 1 h with continuous stirring. The next step was evaporation of the solvents in an evaporator. The obtained dry powder was washed with distilled water and left in an oven at 105 • C for 24 h. This step is schematically shown in Figure 16.

Immobilization of Glucose Oxidase on Ga 2 O 3 /Lignin and ZrO 2 /Lignin Supports
Before the adsorption immobilization process, an enzyme solution was prepared in a 10 mg mL −1 PBS phosphate saline buffer. Next, 50 mg of the support and 20 mL of the previously prepared solution of glucose oxidase were combined. The reaction system was stirred using a magnetic stirrer for 1, 2, 3, 24, 72, or 96 h. Immobilization was carried out at ambient temperature. After the immobilization process was completed, the solution was poured out and centrifuged for 15 min at 15,000 rpm. The resulting filtrate was then separated and the precipitate was dried at ambient temperature for 24 h. Furthermore, the Bradford method was a technique that could not be used to determine the efficiency of immobilization for kraft lignin as a component. As presented in previous works, the kraft lignin is well-soluble in water, which made it impossible to use the Bradford method [50]. Figure 17 shows a schematic process of glucose oxidase immobilization on the Ga 2 O 3 /lignin and ZrO 2 /lignin matrix surfaces.
In the next step, the 4 g of modified oxide (Ga2O3 or ZrO2) was added to the reaction mixture. Media synthesis reactions were carried out for 1 h with continuous stirring. The next step was evaporation of the solvents in an evaporator. The obtained dry powder was washed with distilled water and left in an oven at 105 °C for 24 h. This step is schematically shown in Figure 16.

Immobilization of Glucose Oxidase on Ga2O3/Lignin and ZrO2/Lignin Supports
Before the adsorption immobilization process, an enzyme solution was prepared in a 10 mg mL −1 PBS phosphate saline buffer. Next, 50 mg of the support and 20 mL of the previously prepared solution of glucose oxidase were combined. The reaction system was stirred using a magnetic stirrer for 1, 2, 3, 24, 72, or 96 h. Immobilization was carried out at ambient temperature. After the immobilization process was completed, the solution was poured out and centrifuged for 15 min at 15,000 rpm. The resulting filtrate was then separated and the precipitate was dried at ambient temperature for 24 h. Furthermore, the Bradford method was a technique that could not be used to determine the efficiency of immobilization for kraft lignin as a component. As presented in previous works, the kraft lignin is well-soluble in water, which made it impossible to use the Bradford method [50]. Figure 17 shows a schematic process of glucose oxidase immobilization on the Ga2O3/lignin and ZrO2/lignin matrix surfaces

Immobilization of Glucose Oxidase on Ga2O3/Lignin and ZrO2/Lignin Supports
Before the adsorption immobilization process, an enzyme solution was prepared in a 10 mg mL −1 PBS phosphate saline buffer. Next, 50 mg of the support and 20 mL of the previously prepared solution of glucose oxidase were combined. The reaction system was stirred using a magnetic stirrer for 1, 2, 3, 24, 72, or 96 h. Immobilization was carried out at ambient temperature. After the immobilization process was completed, the solution was poured out and centrifuged for 15 min at 15,000 rpm. The resulting filtrate was then separated and the precipitate was dried at ambient temperature for 24 h. Furthermore, the Bradford method was a technique that could not be used to determine the efficiency of immobilization for kraft lignin as a component. As presented in previous works, the kraft lignin is well-soluble in water, which made it impossible to use the Bradford method [50]. Figure 17 shows a schematic process of glucose oxidase immobilization on the Ga2O3/lignin and ZrO2/lignin matrix surfaces  The mechanism by which enzyme binds to lignin has been presented previously in the literature [50,51]. These studies have shown that the activity of the immobilized enzyme on the matrix surface slightly decreases, which is determined by the formation of hydrogen bonds and ionic interactions between the enzyme and matrix [51,52].

Construction of an Enzymatic Biosensor
To construct an enzyme biosensor, a paste composed of 15 mg of Ga 2 O 3 /lignin/GOx or ZrO 2 /lignin/GOx, 15 mg of graphite and 2 mg of ferrocene mediator was hand-mixed in a mortar, with one drop of paraffin oil used as a binder. Freshly made paste was used to construct the carbon paste electrode, as shown in Figure 18.

Construction of an Enzymatic Biosensor
To construct an enzyme biosensor, a paste composed of 15 mg of Ga2O3/lignin/GOx or ZrO2/lignin/GOx, 15 mg of graphite and 2 mg of ferrocene mediator was hand-mixed in a mortar, with one drop of paraffin oil used as a binder. Freshly made paste was used to construct the carbon paste electrode, as shown in Figure 18.

Physicochemical Analysis
To determine the functional groups present in the structure of the hybrid materials, they were subjected to Fourier transform infrared spectroscopy (FT-IR). FT-IR spectra were collected using a Vertex 70 spectrometer (Bruker, Billerica, MA, USA). Materials for analysis were tested in the form of tablets, previously prepared by mixing 2 mg of the test substance and 250 mg of anhydrous KBr at a pressure of 10 MPa. The FT-IR analyses were performed at a resolution of 0.5 cm −1 in the wavenumber range of 4000-500 cm −1 . TEM analysis records the electrons passing through the sample. A Jeol analyzer (JEM-1400) was used for the analysis, with 120 kV maximum acceleration and 2 nm resolution. Atomic Force Microscopy was performed using an Agilent 5500 atomic force microscope in intermittent contact mode in ambient conditions. The test material from the solution was applied to the surface of mica, which was earlier cleaned by mechanical removal of stripping. The test material was applied to the substrate by spin coating. An Allin One cantilever (BudgetSensors, Sofia, Bulgaria) with a resonance frequency of approximately 150 kHz was used for scanning. The surface morphology of the samples were determined using scanning electron microscope with energy dispersive spectroscopy (SEM-EDS). The tests were performed on a Jeol 7001TTLS (Jeol SAS, Croissy, France) with 30 kV maximum acceleration and 1.5 nm resolution. To assess the stability of materials in liquid solvent, zeta potential (ZP) analysis was performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) with a range of 0.6-6000 nm. Thermogravimetric analysis (TGA) was carried out using a Jupiter STA 449 F3 instrument (Netzsch, Selb, Germany). The XRD analysis was made using a D8 Advance X-ray diffractometer (Bruker, Billerica, MA, USA). The elemental analysis was made using a Vario EL Cube apparatus (Elementar Analysensysteme GmbH, Langenselbold,

Physicochemical Analysis
To determine the functional groups present in the structure of the hybrid materials, they were subjected to Fourier transform infrared spectroscopy (FT-IR). FT-IR spectra were collected using a Vertex 70 spectrometer (Bruker, Billerica, MA, USA). Materials for analysis were tested in the form of tablets, previously prepared by mixing 2 mg of the test substance and 250 mg of anhydrous KBr at a pressure of 10 MPa. The FT-IR analyses were performed at a resolution of 0.5 cm −1 in the wavenumber range of 4000-500 cm −1 . TEM analysis records the electrons passing through the sample. A Jeol analyzer (JEM-1400) was used for the analysis, with 120 kV maximum acceleration and 2 nm resolution. Atomic Force Microscopy was performed using an Agilent 5500 atomic force microscope in intermittent contact mode in ambient conditions. The test material from the solution was applied to the surface of mica, which was earlier cleaned by mechanical removal of stripping. The test material was applied to the substrate by spin coating. An Allin One cantilever (BudgetSensors, Sofia, Bulgaria) with a resonance frequency of approximately 150 kHz was used for scanning. The surface morphology of the samples were determined using scanning electron microscope with energy dispersive spectroscopy (SEM-EDS). The tests were performed on a Jeol 7001TTLS (Jeol SAS, Croissy, France) with 30 kV maximum acceleration and 1.5 nm resolution. To assess the stability of materials in liquid solvent, zeta potential (ZP) analysis was performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) with a range of 0.6-6000 nm. Thermogravimetric analysis (TGA) was carried out using a Jupiter STA 449 F3 instrument (Netzsch, Selb, Germany). The XRD analysis was made using a D8 Advance X-ray diffractometer (Bruker, Billerica, MA, USA). The elemental analysis was made using a Vario EL Cube apparatus (Elementar Analysensysteme GmbH, Langenselbold, Germany). A 10 mg of sample was used for analysis. During the process, a sample was combusted in an oxygen atmosphere, and after that, transferred onto the reduction column. The resulting gases were separated in an adsorption column, and then recorded using a detector. The results are given as averages of three measurements, each accurate to 0.0001%.

Electrochemical Study
Electrochemical tests were carried out in a three-electrode system using an Autolab (PGSTAT-30) electrochemical analyzer (Eco Chemie, Utrecht, Netherlands). A carbon paste electrode (BASi, West Lafayette, IN, USA) was used for the working electrodes (CPE/Ga 2 O 3 /lignin/GOx/Fc and CPE/ZrO 2 /lignin/GOx/Fc). Silver chlorine electrode Ag/AgCl (3 M KCl) was the reference electrode and platinum wire the counter electrode. All electrochemical tests were conducted in ambient temperature in at phosphate buffer pH 7.4.

Conclusions
In this paper, we have presented attractive alternative microplatforms for enzyme immobilization and biosensing. The support obtained exhibited good affinity for the immobilization of glucose oxidase (GOx). A higher amount of the GOx (27.1 mg g −1 ) was immobilized on ZrO 2 /lignin carrier than on Ga 2 O 3 /lignin (24.7 mg g −1 ). The Ga 2 O 3 /lignin-GOx and ZrO 2 /lignin-GOx systems, with ferrocene and carbon paste (BASi), exhibited satisfied electrochemical properties in the catalytic oxidation of glucose. The microplatforms are suitable materials for the preparation of efficient and cheap supports for biocatalysts and for biosensor application.
A review of the literature indicates the higher interest of research on metal oxides and the development of microcarriers for industrial and environmental applications indeed. An interesting application may be the use of an enzyme biosensor for glucose detection based on the aforementioned micromaterials, as produced in this study.