SCI SINTERING 57 1 2025 08pdf
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  1. Science of Sintering, 57 (2025) 103-113________________________________________________________________________ _____________________________ *) Corresponding author:zorica.mojovic@ihtm.bg.ac.rs https://doi.org/10.2298/SOS231010057RUDK: 661.183.8; 681.586 The Influence of Alumina Type on the Immobilization of Ascorbate Oxidase and Ascorbic Acid Detection Barbara Ramadani1, Sonja Novaković1, Miloš Mojović1, Zorica Mojović2*)1University of Belgrade, Faculty of Physical Chemistry, Studentski trg 12-16, P.O. Box 47, Belgrade, 11158, Republic of Serbia. 2University of Belgrade - Institute of Chemistry, Technology and Metallurgy, Department of Catalysis and Chemical Engineering, Njegoševa 12, Belgrade, Republic of Serbia.Abstract: Two alumina types (trihydrate (T) and anhydrous (G)) were used for the immobilization of ascorbate oxidase (AO) by the adsorption procedure. The samples were characterized by FTIR analysis and EPR spectroscopy to confirm the presence of enzyme in the composite and the activity of immobilized enzyme. The samples of alumina with and without AO were used as modifer of carbon paste electrode and tested by Fe(CN6)3-/4- redox probe using cyclic voltammetry at different scan rates. The response of investigated electrodes toward ascorbic acid was tested in the Britton-Robinson buffer at pH range of 2 to 5. Direct detection of ascorbic acid was tested at G and G+AO electrodes and the calibration curves were constructed by square wave voltammetry under the optimized conditions. The immobilization of AO at anhydrous alumina increased the concentration range and the sensitivity from 0.00617 μM/μA for G to 0.01693 μM/μA for G+AO in the concentration range from 10 μM to 200 μM. Keywords: Alumina; Ascorbate oxidase; Ascorbic acid; Electrochemical sensor. 1. Introduction Alumina, Al2O3, is a well-known catalyst and catalyst support [1, 2], used in various processes. It has also been studied as a biocomposite [3], a reinforcement component of composite-hybrid material [4], or a component of superalloys [5]. In recent years, the application of alumina as an electrode material has been investigated [6-8], despite its low electrical conductivity. The first mention of electrocatalytic properties of alumina came from Zak and Kuwana [9], followed by Compton’s research group's more detailed research [10]. Lima et al. [11] noticed that the type of alumina significantly influences the electrocatalytic performances of alumina-modified electrodes. Alumina is also investigated as a support for enzyme immobilization [12] among other metal oxides. The advantage of metal oxides as support is their stability, both thermal and chemical, and surface chemistry enabling various interactions with enzymes through various surface groups. The adsorption affinity of an enzyme is affected by the electrostatic properties of the metal oxide surface [13]. The alumina surface consists of both positive and negative charges which is shown to be beneficial for enzyme immobilization [14].
  2. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________104Ascorbic acid is one of the compounds with great biomedical importance due to its presence in many biological fluids and widely spread clinical applications [15]. Electrochemical detection of ascorbic acid offers quick and simple quantitative determination [16]. This investigation aimed to establish the suitability of alumina for the development of an ascorbic acid sensor. The alumina was investigated both as an active component and as a support material for biosensors based on ascorbate oxidase. Two types of alumina were used, trihydrate alumina and anhydrous alumina, to investigate the influence of alumina surfacechemistry on its properties for both roles. Ascorbate oxidase was immobilized on the alumina surface by adsorption. The adsorption is the easiest method for enzyme immobilization, and also offers the least disruptive influence on both enzyme structure and support surface chemistry [12]. 2. Materials and Experimental Procedures Two types of industrial alumina (Al2O3) alumina trihydrate (Al2O33H2O, designated as T) and anhydrous alumina (designated as G) were gathered from alumina refinery (Alumina Ltd., Zvornik, B&H). Ascorbate oxidase (from Cucurbita sp., Sigma) was dissolved in 2 ml of deionized water (1.6 mg, 162 units/mg). 100 mg of each alumina type (T and G) was dispersed in 1 ml of AO solution and placed in a rotator set for a duration of 2 h with a rotation speed of about 50 rpm. After the immobilization step the dispersion was filtered and repeatedly rinsed with deionized water to wash trace of enzyme that was not attached to the alumina. After drying in air and at room temperature the obtained samples were designated with T+AO and G+AO. FTIR–ATR spectra were collected on a Thermo Scientific FT-IR iS5 spectrometer equipped with an ATR (attenuated total reflectance) detector using a diamond cell in the wavelength range of 4000–600 cm−1, at a spectral resolution of 4 cm−1 over 16 scans. A background of the clean diamond cell was performed for each analysis undertaken. The diamond cell requires only minute amounts of sample material. Also, spectra were recorded by contacting the ATR crystal directly onto the polished surfaces of the mounted cross sections. The electron paramagnetic resonance (EPR) spectra were recorded at room temperature employing Bruker Elexsys II E540 spectrometer operating at X-band (9.5 GHz) using the following settings: modulation amplitude 2 G; modulation frequency 100 kHz; microwave power 10 mW. All spectra were recorded and analyzed using the Xepr software (Bruker BioSpin Germany). 2 mg of each sample was dispersed in 50 μl of 2.5 mM solution of ascorbic acid in phosphate buffer pH 7. After an incubation time of 3 minutes, thesuspension was centrifugated and 30 μl of supernatant was exploited. The electrochemical properties of samples were performed in the form of a modified carbon paste electrode. The sample and carbon black (Vulcan-XC 72R) in the 1:1 weight ratio were mixed with paraffin oil. A Teflon tube (diameter 2 mm) filled with paste and equipped with contact was used as a working electrode. Three electrode cell was composed of as described working electrode, reference electrode (Ag/AgCl in 3 M KCl), and auxiliary electrode (Pt rod). The measurements were conducted using an Autolab electrochemical workstation (Autolab PGSTAT302N, Metrohm-Autolab BV, Netherlands). The response of electrodes toward ascorbic acid (≥99 %, Sigma-Aldrich) in pH range 2-5 was tested in 0.1 M Britton-Robinson buffer. The buffers were freshly prepared and measured using a “Jenway” 3320 pH meter.
  3. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________1053. Results and Discussion 3.1. FTIR analysis Fig. 1. FTIR spectra of alumina samples with and without immobilized ascorbate oxidase. Fig. 1 shows FTIR spectra of alumina samples with and without AO. The characteristic bands of trihydrate (T) alumina are visible in two regions: from 4000 cm-1 to 3300 cm-1 and from 1000 cm-1 to 500 cm-1. The bands in the first region are associated with the hydrogen bond between hydroxyl groups and with the adsorbed water [17, 18]. The bands in the second group are associated with stretching and bending vibrations of Al-O and AlO-H [19]. These bands are preserved in the T sample modified with the enzyme. The FTIR spectrum of the G sample was rather poor in the characteristic bands in comparison to T alumina. The characteristic band corresponding to Al-O-Al banding vibration in the AlO6 unit is present at 670 cm-1 [19]. The bands in the region 1500-1700 cm-1cаracteristic of the amide I and the amide II are visible in the spectra of both alumina-AO composites. The characteristic band of AO corresponding to the N−H band [20] appeared at around 1520 cm−1 at spectra of both alumina samples modified with AO. The second characteristic band at 1687 cm−1corresponding to the stretching vibration of C=O in the peptide linkage [21] was also present in the spectra of alumina-AO composites. The FTIR spectra of both samples containing AO contained additional bands at 3743 cm-1 and 3856 cm-1. These bands originate from the oscillation of the amide fine resonance structure [22]. The band at 2366 cm−1 appeared in the spectra of all four samples and is associated with the presence of ambient CO2 [23]. 3.2. EPR analysis The EPR spectroscopy was used for the detection and quantification of ascorbate radical formed under the influence of alumina, with and without immobilized ascorbate oxidase. All obtained EPR spectra showed characteristic ascorbyl radical doublet with g = 2.005 (Fig. 2), as same as reported previously [24].
  4. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________106The signal of ascorbate radical generated in the presence of T alumina was significantly higher than in the presence of G alumina indicating the ability of T alumina to oxidize the ascorbic acid to ascorbate radical. The alumina samples with immobilized enzyme showed increased signal in comparison to their paternal alumina indicating that the activity of ascorbate oxidase was preserved after immobilization on the alumina. Fig. 2. EPR spectra obtained in the solution of ascorbic acid after an incubation period of 3 minutes with investigated samples. 3.3. Electrochemical measurements The electrochemical characterization of starting alumina samples and alumina bio-composites was performed by cyclic voltammetry in a 1 mM solution of Fe(CN6)3-/4- redox probe in 0.2 M KCl. A smaller peak-to-peak separation value obtained for the G electrode (Fig. 3) showed that the process of charge transfer was more facile than on the T electrode. Fig. 3. Cyclic voltammograms recorded on investigated electrodes in 1mM Fe(CN)63-/4- in 0.2 M KCl at the scan rate 100 mVs-1.
  5. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________107The oxidation peak potential for both bio-composites shifted toward a less positive value and their peak-to-peak separation values decreased when compared to their corresponding alumina samples. These results indicated that adding ascorbate oxidase to the alumina enhanced the kinetics of the charge transfer process. The influence of scan rate on the process of charge transfer on these electrodes was tested in the scan range from 20 mVs-1 to 300 mVs-1 (Fig. 4a-d). Fig. 4. Cyclic voltammograms recorded on investigated electrodes in 1mM Fe(CN)63-/4- in 0.2 M KCl at different scan rates in the range 20 – 300 mVs-1 at a) T; b) G; c) T+AO and d) G+AO electrode.; e) Plot of the peak current dependence on the square root of scan rate. The plot of peak current vs. square of scan rate (Fig. 4е) showed linear dependence and the plot log Ip vs. log v (not shown) produced linear regression with slopes for all electrodes in the range from 0.45 to 0.65 showing that the electrode process was controlled by diffusion. The peak-to-peak separation value was significantly higher than 59 mV expected for a reversible one-electron process. Therefore, the electroactive surface area was calculated by the Randles-Ševćik equation for an irreversible process [25]:= 2.99105(′)1/21/21/2 (1) where: n is the total number of electrons transferred per molecule in the electrochemical process and n’ is the number of electrons transferred per molecule in the rate-determining step, while the other variables have their usual meaning. The estimated electroactive surface area was 0.020 cm2 for T, 0.025 cm2 for G, 0.030 cm2 for T+AO and 0.031 cm2 for G+AO. The activity of alumina-based electrodes toward oxidation of ascorbic acid was tested in the Britton-Robinson buffer in a pH range from 2 to 5 (Fig. 5).
  6. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________108Fig. 5. Cyclic voltammograms recorded in 1 mM AA in 0.1 M Britton-Robinson buffer at a) T; b) G; c) T+AO and d) G+AO electrode. e) The plot of dependence of peak potential on pH value. f) The plot of dependence of peak current on pH value. The modification of both alumina resulted in a changed sensitivity toward ascorbic acid. The dependence of peak potential and the peak current on pH are presented in Fig.5e and 5f. The peak potential dependence on the pH is defined by the equation [26]: = − (2) where R is the gas constant, T is the temperature in K, F is the Faraday constant, and m and n are the number of protons and electrons involved in the oxidation, respectively. The value of Ep vs. pH slope amounted to 51 mV pH-1 and 56 mV pH-1 for T and T+AO, respectively, showing that oxidation of ascorbic acid at these electrodes proceeded
  7. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________109through a mechanism involving an equal number of protons and electrons. The value obtained for the G electrode was 125 mV pH-1 expected for the 2H+1e- mechanism. The value obtained for the G+AO electrode was 78 mV pH-1 expected for the 3H+2e- mechanism. In the previous research [27], it was shown that specific surface groups of G alumina can affect the oxidation mechanism of proton-coupled electron transfer reactions. The surface of investigated alumina samples, trihydrate and anhydrous, contains several types of OH groups. The main types are double-coordinated basal groups (hydroxyl groups bound to two Al atoms, ≡Al2OH) and singly coordinated edge sites at edges (hydroxyl groups bound to one Al atom, ≡AlOH) [28]. The pKa values for the first type are in the range of 2-4, while the second type has higher pKa values, usually 8-10 [29]. The number of thesegroups is decreased in the anhydrous alumina upon calcination and the number of Al-O-Al bridges as well as the number of Lewis acid sites is increased [30]. Fig. 6. a) Superimposed SWV curves of ascorbic acid oxidation with different concentrations (10, 20, 40, 100, 200, 400, 1000, 2000 and 4000 μM); a) for G electrode in the Britton-Robinson buffer pH 5 and b) G+AO electrode in the Britton-Robinson buffer pH 5 (the inset figures shows SWV curves for low concentrations); the linear relationship between the peak current and the concentration of ascorbic acid for c) G electrode and d) G+AO electrode. Ascorbic acid has two pKa values: 4.04 and 11.7 [31]. Monoanion formed after the first deprotonation step is a substrate for ascorbate oxidase [32]. The electrostatic repulsion and attraction between ascorbic acid species and surface groups at the alumina samples formed at different pHs influence the response of the investigated electrodes. Trihydrate alumina is richer than anhydrous alumina in AlOH2+ groups that can serve as binding sites for ascorbate monoanion. Therefore, slightly higher oxidation currents are
  8. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________110obtained on the T electrode than on the G electrode at lower pH values. Additionally, G alumina is rich in Al-O-Al bridges which repell monoanion with its negative charge. In the pH range 2-4 AlOH2+ groups deprotonate and the currents obtained on the T electrode decrease at pH >4. On the other hand, increased Lewis acid sites in the G electrode led to increased current values at higher pH values when LH- concentration increases. The immobilization of AO on T alumina led to decreased currents across all investigated pH values. This is probably mainly the result of the adsorption of the enzyme on the alumina active sites. On the other hand, G+AO showed higher currents at lower pH and a markedly decreased current at pH 4, at a value where the concentration of ascorbate anion increases because of the first deprotonation step. This drop in the current is the confirmation of the enzyme activity. Ascorbate oxidase catalyzes the reaction of ascorbic acid with oxygen to dehydroascorbate. The consumption of oxygen can be used for the construction of biosensors of ascorbic acid. However, in this paper, we have investigated the influence of adsorbed AO on the ability of alumina to be used for direct detection of ascorbic acid. Further investigation involved testing the sensing performance of G and G+AO electrodes by square wave voltammetry. The optimized parameters for the G electrode were pH = 5, deposition potential -0.5 V, deposition time 10 s, and amplitude 0.08 V. The optimized parameters for the G+AO electrode were pH = 2, deposition potential -0.3 V, deposition time 10 s, and amplitude 0.15V. SWV recorded at these electrodes for AAconcentration from 10 M to 4000 M, under their corresponding optimized parameters are given in Fig. 6a and 6b. A slight shift in the peak potential to higher values was evident at SWV recorded at the G electrode. At SWV recorded at the G+AO electrode, this shift was not present. This result together with a smaller peak width indicated better G+AO electrode performance. The respective calibration plots are presented in Fig. 6c and 6d. The current for the calibration curve was determined at the potential of 0.5 V for the G electrode and 0.75 V for the G+AO electrode. The G electrode showed a linear relationship in the concentration range of 10–2000 M, while the G+AO electrode showed a linear relationship in the concentration range of 10–4000 M, albeit with two slopes. The sensitivity of the G+AO electrode (0.01693 μM/μA in the concentration range from 10 M to 200 M and 0.01021 μM/μA for the concentration range 200 M to 4000 M) was significantly improved in comparison to the sensitivity of the G electrode (0.00617 μM/μA). The limit of detection (LOD) of 9 μM for the G electrode and 7 μM and the G+AO electrode was estimated from the triple value of standard deviation. The results showed that the presence of AO on the surface of G alumina led to the increased sensitivity of the electrode toward ascorbic acid. The result is the consequence of the presence of AO which occupied binding sites changing the pattern of surface charges. 4. Conclusion Two alumina types (trihydrate and anhydrous) were used for the immobilization ofascorbate oxidase (AO). FTIR analysis showed that the ascorbate oxidase was successfully immobilized at the surface of alumina. The EPR analysis confirmed that the immobilized enzyme retained its activity. Electrochemical characterization showed that the presence of AO at the surface of both alumina enhances charge transfer of Fe(CN6)3-/4- redox probe. The electrochemical oxidation of ascorbic acid at different pH in the range 2-5 depended on the alumina type and the presence of AO. The anhydrous alumina was shown to be a better choice for immobilization of the enzyme. The immobilization of AO at anhydrous alumina led to the shift of the highest electrode activity to the lower pH values and increased
  9. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________111sensitivity. The effect is the consequence of the changed pattern of surface charges by the presence of the adsorbed enzyme. Acknowledgments This work was financially supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant No. 451-03-47/2023-01/200026 and Grant no.451-03-47/2023-01/200146).ORCID numbers: Barbara Ramadani, https://orcid.org/0009-0004-7305-8534Miloš Mojović, https://orcid.org/0000-0002-1868-9913Zorica Mojović, https://orcid.org/0000-0003-4804-07765. References1.H. Pines, W. O. Haag, J. Am. Chem. Soc., 82 (1960) 2471. https://doi.org/10.1021/ja01495a0212.M. Trueba, S. P. Trasatti, Eur. J. Inorg. Chem., 17 (2005) 3393. https://doi.org/10.1002/ejic.2005003483.R. P. Alvarez-Carrizal, E. Refugio-García, J. A. Rodríguez-García, J. G. MirandaHernández, E. Rocha-Rangel, Sci. Sinter., 54 (2022) 415. https://doi.org/10.2298/SOS2204415A4.Z. Özkan, H. Gökmeşe, U. Gökmen, Sci. Sinter., 54 (2022) 177.https://doi.org/10.2298/SOS2202177O5.C. V. Prică, N. A. Sechel, B. V. Neamțu, T. F. Marinca, Fl. Popa, H. F. Chicinaș, I. Chicinaș, Sci. Sinter., 54 (2022) 335. https://doi.org/10.2298/SOS2203335P6.J. Zhang, X. Mao, B. Pan, J. Xu, X. Ding, N. Han, L. Wang, Y. Wang, Y. Li, Nano Res., 16 (2023) 4685. https://doi.org/10.1007/s12274-022-5128-27.S. Pandiyarajan, S. S. M. Manickaraj, A-H. Liao, A. R. P. Selvam, K-Y. Lee, H-C. Chuang, J. Alloys Compd., 936 (2023) 168213. https://doi.org/10.1016/j.jallcom.2022.1682138.Moutcine, C. Laghlimi, O. Ifguis, M. A. Smaini, S. E. El Qouatli, M. Hammi, A. Chtaini, Diam. Relat. Mater., 104 (2020) 107747. https://doi.org/10.1016/j.diamond.2020.1077479.J. Zak, T. Kuwana, J. Am. Chem. Soc., 104 (1982) 5514. https://doi.org/10.1021/ja00384a05610.Q. Lin, Q. Li, C. Batchelor-McAuley, R.G. Compton, J. Phys. Chem. C, 119 (2015) 1489. https://doi.org/10.1021/jp511414b11.A.P. Lima, R.C. Souza, M.N.T. Silva, R.F. Gonçalves, E. Nossol, E.M. Richter, R.C. Lima, R.A.A. Munoz, Sensor Actuat. B-Chem. 262 (2018) 646. https://doi.org/10.1016/j.snb.2018.02.02812.Y. Satyawali, S. Van Roy, A. Roevens, V. Meynen, S. Mullens, P. Jochems, W. Doyen, L. Cauwenberghs, W. Dejonghe, RSC Adv., 3 (2013) 24054. https://doi.org/10.1039/C3RA45107K13.S. Fukuzaki, H. Urano, K. Nagata, J. Ferment. Bioeng., 81 (1996) 163.
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  11. B. Ramadani et al.,/Science of Sintering, 57(2025)103-113 ___________________________________________________________________________113Сажетак: Два типа алумина (трихидрате (Т) и анхидрована (G)) су коришћене за имобилизацију аскорбат оксидазе (АО) методом адсорпције. Узорци су окарактерисани ФТИР анализом и ЕПР спектроскопијом како би се потврдило присуство ензима у композитима и активност имобилисаног ензима. Узорци алумине са и без АО су употребљени као модификатори угљеничне паста електроде и тестиран је њихов одговор на Fe(CN6)3-/4-на редокс пробу методом цикличне волтаметрије при различитим брзинама поларизације. Одговор испитиваних електрода на аскорбинску киселину је тестиран у Бритон-Робинсон пуферу у pH опсегу 2-5. Тестирана је активност електрода са и без аскорбат оксидазе за директну детекцију аскорбинске киселине и калибрационе криве су конструсиане помоћу волтамерије са правоугаоно задатим напоном спровођеном под оптималним условима снимања. Имобилизација АО на анхидрованој алумини је довела до побољшања опсега концентрација и осетљивости са 0.00617 μM/μA за G електроду на 0.01693 μM/μA за G+AO електроду у опсегу концентрација од 10 μМ до 200 μМ..Кључне речи: Алумина; Аскорбат-оксидаза; Аскорбинска киселина; Електрохемијски сензор.© 2025 Authors. Published by association for ETRAN Society. This article is an open access article distributed under the terms and conditions of the Creative Commons — Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/).
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