Nanoscale This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Nanoceria dissolution at acidic pH by breaking off the catalytic loop† Open Access Article. Published on 19 August 2022. Downloaded on 4/17/2023 9:35:46 AM. Cite this: Nanoscale, 2022, 14, 14223 a Dmitry Galyamin, Lena M. Ernst,b Aina Fitó-Parera,a Guillem Mira-Vidal, c c Neus G. Bastús, Neus Sabaté *a,d and Victor Puntes *b,c,d This manuscript proves the reproducibility and robustness of cerium oxide nanoparticles, nanoceria, employed as a chemical reagent with oxidizing capacity (as an electron sink) at acidic pH. Unlike nano- ceria multi-enzyme-mimetic capabilities at neutral or high pH, nanoceria can behave as a stoichiometric reagent at low pH where insoluble Ce4+ ions transform into soluble Ce3+ in the nanocrystal that finally Received 30th June 2022, dissolves. This behaviour can be interpreted as enzyme-like when nanoceria is in excess with respect to Accepted 11th August 2022 the substrate. Under these conditions, the Ce3+/Ce4+ ratio in the NPs can easily be estimated by titration DOI: 10.1039/d2nr03586c with ferrocyanide. This procedure could become a rapid assessment tool for evaluating nanoceria rsc.li/nanoscale capacity in liquid environments. Introduction popularized studies for its application as an anti-inflammatory substance.12 Having the benefits of low cost, excellent stability and toler- The mechanism responsible for the reported behaviour ance to extreme conditions, inorganic nanoparticles (NPs) arises from the ability of Ce atoms to adjust their electronic have revealed promising features that make them reliable and configuration to best fit their immediate electromagnetic robust alternatives to natural enzymes. Since the peroxidase- environment. The main difference between rare earth and like activity of magnetite (Fe3O4) NPs was first described in other metals is having 4f orbitals, whose electrons are shielded 2007,1 there has been exponential growth of studies on by 4d and 5p orbitals. This orbital shield makes 4f electrons enzyme-like NPs, also called nanozymes,2,3 not without contro- weakly bond to the nucleus allowing Ce ions to easily switch versy.4 Among them, nanoceria, cerium oxide (CeO2) NPs, is of between Ce3+ and Ce4+ valence states, creating oxygen particular interest due to its observed multi-enzyme-mimetic vacancies compensating for the positive charge deficiency. capabilities, including antioxidant activity, such as in super- This phenomenon is particularly strong at the nanoscale, oxide dismutase (SOD)5 and catalase,6,7 and pro-oxidant where the very high surface-to-volume ratios withstand a sig- activity such as in peroxidase.8,9 Asati and co-workers recently nificant concentration of oxygen vacancies at the NP surface reported nanoceria activity as an oxidase-like enzyme under accompanied by cerium ions reduced to the Ce3+ state.13 In mildly acidic conditions.10 These nanoceria multi-enzyme-like this redox process, the fluorite (fcc) structure of the crystal is properties have been successfully used for biological detection, maintained, facilitating the oxidation of Ce3+ ions back to Ce4+ substituting the widely used HRP enzyme with a more robust ions and thus enabling the regenerative ability of nanoceria to and reliable alternative.11 Additionally, nanoceria ability to sca- act catalytically.12 The precise amount of oxygen vacancies and venge excess reactive oxygen species (ROS) in living cells has cerium atoms in the Ce3+ state in the NPs increases with a decrease in size,14,15 and can be modified by the synthesis method. This work explores the conditions where this catalytic loop a Instituto de Microelectrónica de Barcelona, IMB-CNM (CSIC). C/dels Til·lers, is broken and Ce3+ ions are not oxidized back to Ce4+. Campus Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain. Systematic experiments evidenced that under the working con- E-mail: neus.sabate@imb-cnm.csic.es b Vall d’Hebron Research Institute (VHIR). Hospital Universitari Vall d’Hebron, ditions (at low pH), nanoceria behave as a pure stoichiometric Passeig de la Vall d’Hebron, 129, 08035 Barcelona, Spain. reagent consumed during the reaction. However, this behav- E-mail: victor.puntes@icn2.cat iour can be interpreted as enzyme-like when the substrate c Instiut Català de Nanociència I Nanotecnologia (ICN2), CSIC and The Barcelona undergoing oxidation is the limiting reagent, not the nano- Institute of Science and Technology (BIST). Campus UAB, 08193 Barcelona, Spain d ceria. Interestingly, under such conditions, unlike enzymes, a Institució Catalana de Recerca I Estudis Avançats (ICREA), 08010 Barcelona, Spain † Electronic supplementary information (ESI) available. See DOI: https://doi.org/ limiting reagent can stop the “enzymatic” analytical reaction at 10.1039/d2nr03586c the desired reading point, facilitating detection. This journal is © The Royal Society of Chemistry 2022 Nanoscale, 2022, 14, 14223–14230 | 14223 View Article Online Paper Nanoscale Results and discussion plays a pure single-phase fluorite (face-centred cubic, fcc) structure (JCPDS No. 34-0394). The diffraction peaks are sub- Nanoceria of ∼3 nm diameter at a concentration of 1.72 mg stantially broadened, reflecting the ultra-small crystal size ml−1 (1.6 × 1016 NPs ml−1) was synthesized using a hydro- (Fig. 1D). In Fig. 1E, the UV-visible spectrum of nanoceria This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. thermal approach with sodium citrate as a complexing and sta- shows a distinct absorption band at 282 nm, which, following bilizing agent. Sodium citrate, a tricarboxylic acid with three Tauc plot, reveals an optical band gap of 3.06 eV. As catalysis is pKa values (3.06, 4.74, and 5.4), provides electrostatic stabiliz- a surface phenomenon, the colloidal stability of nanoceria ation to the NPs at a pH value higher than 3.5, creating an during experiments is critical. Aggregation would lead first to electrostatic stable colloidal solution, as the surface charge vs. a dramatic reduction of the total exposed NP surface and then pH measurements indicate (Fig. S1†). Morphological charac- to the NP precipitation out of the reaction mixture. The col- Open Access Article. Published on 19 August 2022. Downloaded on 4/17/2023 9:35:46 AM. terization by high-angle annular dark-field scanning trans- loidal stability of nanoceria in the working media was mission electron microscopy (HAADF-STEM) reveals the for- measured as a function of time (Fig. 1F). The spectra do not mation of non-aggregated 2.9 ± 0.8 nm quasi-spherical nano- show any significant aggregation or sedimentation, indicating ceria particles of high uniformity and narrow diameter distri- the colloidal and chemical stability of the NPs under experi- bution (Fig. 1A). The hydrodynamic diameter was measured by mental conditions, at pH 4,4, where oxidase-like nanoceria dynamic light scattering (DLS) (Fig. 1B), obtaining a monomo- capacity has been described.10 dal distribution peaking at 4.5 ± 0.2 nm (PDI = 0.26). The zeta In this work, we characterize nanoceria evolution during potential (Fig. 1C) shows that NPs, after purification and resus- the water oxidation process of ferrocyanide ([Fe(CN)6]4−) to fer- pension in 2.2 mM SC, are negatively charged (−47.6 mV at pH ricyanide ([Fe(CN)6]3−) at pH 4.4 as a model of redox colouri- = 9.36 and conductivity = 0.24 mS cm−1), which can be metric reaction.10 Typical species employed as redox mediators ascribed to the citrate molecules reversibly adsorbed onto the in these enzymatic-type reactions are organic dyes such as NP surface. This reversibility is easily proven under dialysis 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2-azinobis(3-ethylben- conditions, where, as the citrate concentration decreases, the zothizoline-6-sulfonic acid) (ABTS), and dopamine (DOPA).10 NPs aggregate (data not shown). The X-ray diffraction patterns These dyes feature high molar extinction coefficients (ε), allow- of the as-synthesized nanoceria confirm that the sample dis- ing high sensitivity on optical measurements at low reagent Fig. 1 Synthesis and characterization of nanoceria. (A) HAADF-STEM images of the as-synthesized NPs. Size distribution (inset). (B) Hydrodynamic profile measured by dynamic light scattering. (C) Zeta potential analysis. (D) X-ray diffraction pattern of the as-synthesized nanoceria and JCPDS No. 34-0394 standards. (E) The UV-visible spectrum of the nanoceria and optical bandgap energy determination using the Tauc equation (inset). (F) Colloidal stability of nanoceria dispersed in the working citrate buffer (100 mM, pH 4.4). 14224 | Nanoscale, 2022, 14, 14223–14230 This journal is © The Royal Society of Chemistry 2022 View Article Online Nanoscale Paper concentrations. However, the optical signal saturates approxi- iour and a constant reaction rate, consistent with its widely mately at relatively low concentrations for similar reasons, reported catalytic activity. which restricts kinetic studies (time evolution as a function of If we look at the cerium oxide signal in the UV-vis spectra enzyme vs. dye ratio) to minimal concentration ranges. now, we also see how it does transform during this process. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Because of that, we choose to employ transparent ferrocyanide Fig. 3A shows the recorded spectra after adding 0.47 mM and its redox pair, yellow-coloured ferricyanide (absorbance atomic cerium (in the form of nanoceria) to a solution contain- peak at ∼420 nm), as a redox mediator.16 As ferricyanide pre- ing ferrocyanide in excess (1 mM). The strong absorption sents a much lower ε value than most organic dyes used in the signal has well-defined features in the near-ultraviolet region field (see Table S1 ESI†), it allows for directly measuring a (>400 nm), accounting for the presence of nanoceria.17 As the broader range of nanoceria/ferrocyanide concentration ratios reaction proceeds, the intensity of this band diminishes while Open Access Article. Published on 19 August 2022. Downloaded on 4/17/2023 9:35:46 AM. before spectral saturation, allowing for easy reaction kinetics the characteristic band of ferricyanide arises. These features follow-up. can be attributed to a change in the cerium oxidation state, The performance of nanoceria for the oxidation of a solu- from Ce4+ to Ce3+ (Ce4+ = 320 nm, Ce3+ = 253 and 295 nm)18 tion of 1 mM ferrocyanide was evaluated at five different nano- while ferrocyanide gets oxidized, as nanoceria is consumed. ceria concentrations, ranging from 8.1 × 10−3 to 1.62 × 10−1 We repeated the same experiments with a batch of commercial mg mL−1 (4.7 × 10−2 to 9.4 × 10−1 mM atomic cerium in the nanoceria of similar diameter to test whether the process is form of nanoceria). In these experiments, ferrocyanide is first consistent and independent of nanoceria synthetic conditions. added to a cuvette and then nanoceria to initiate the reaction. Fig. 3B shows the initial and final spectra of both the in-house In our experiments, the pH of the solution was adjusted at pH and commercial nanoceria. Due to the similar concentration = 4.4 (citrate buffer 100 mM) following previous studies that of nanoceria solutions, the initial spectra for both nano- revealed this one as an optimal pH for nanoceria oxidase-like materials (dashed lines) superimpose. However, at the end of behaviour.10 As soon as both species mix, a yellow color the reaction, the decrease of the UV-vis absorption spectra at appears in the transparent solutions evidencing the chemical low wavelengths was more evident when commercial nanoceria reaction progression and the formation of ferricyanide. The was employed. These results suggest that the in-house nano- temporal evolution of the UV-vis absorption spectra is shown ceria present higher oxidation capacity than the commercial in Fig. 2A, where an increase in intensity until saturation after ones (less ceria is consumed), which can be attributed to a 3 hours of reaction can be observed. In Fig. 2B it is shown how lower Ce3+ concentration in the original NPs (see below).19 the final ferricyanide peak intensity depends on the initial As the reaction of cerium to iron is one to one (eqn (1)), one nanoceria concentration, suggesting that nanoceria is acting would expect a stoichiometry of 1: as a limiting reagent, rather than a catalyst, in a first-order reaction, with a rapid increase of conversion at short times CeO2 þ 4Hþ þ ½FeðCNÞ6 4 ! Ce3þ þ 2H2 O þ ½FeðCNÞ6 3 that evolves towards a saturation point and a plateau as it is ð1Þ consumed (Fig. 2C). Under the same experimental conditions, the ferrocyanide oxidation process was compared with laccase However, as the amount of Ce4+ in the NPs is less than the enzyme, from the phenol oxidise group enzymes, at the same total amount of Ce ions, due to oxygen vacancies, we titrated mass concentration (1.62 × 10−4 mg mL−1) (Fig. 2C). Contrary nanoceria with ferrocyanide to determine the actual Ce4+ con- to nanoceria, laccase displays a linear time-dependent behav- centration in the NPs. Fig. 2 (A) UV-vis spectra of 1 mM ferrocyanide solution exposed to 8.1 × 10−2 mg mL−1 (4.7 × 10−1 mM total atomic cerium) nanoceria for 180 min Spectra were recorded every 15 min for 3 hours. (B) Final UV-vis spectra (at 24 h) of the 1 mM ferrocyanide solution exposed at different nanoceria concentrations. (C) and (A) Absorbance values at 420 nm for 1 mM ferrocyanide solutions exposed to different nanoceria concentrations, ranging from 8.10 × 10−3 mg mL−1 to 1.62 × 10−1 mg mL−1 (0.047 to 0.94 mM) and to laccase at a concentration of 1.62 × 10−4 mg mL−1 for 180 minutes. This journal is © The Royal Society of Chemistry 2022 Nanoscale, 2022, 14, 14223–14230 | 14225 View Article Online Paper Nanoscale This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 19 August 2022. Downloaded on 4/17/2023 9:35:46 AM. Fig. 3 (A) Absorbance spectra of 1 mM ferrocyanide solution exposed to nanoceria at a concentration of 8.10 × 10−2 mg mL−1 (0.47 mM) for 60 minutes, (B) comparison of UV-vis spectra between commercial and in-house nanoceria at t = 0 min and after 60 min of reaction. We prepared a set of commercially and in-house syn- the signal at 420 nm while laccase linearly does it. Taking into thesized nanoceria solutions and fixed the nanoceria content account that nanoceria at 0.4 mg mL−1 corresponds to a con- to 0.4 mg mL−1 (2.3 mM atomic cerium). Additional solutions centration of atomic cerium of approximately 2.3 mM, the fer- with 0.5 mg mL−1 laccase enzymes coming from different rocyanide oxidation capacity of these nanoceria solutions batches were also prepared for comparison. The solutions were could be estimated to be around 1.65 mM. This corresponds to mixed with ferrocyanide solutions of varying concentrations, a NP with about 36% of Ce3+ similar to what is obtained from ranging from 0 to 2.4 mM, and allowed to react until the ferro- XPS (Fig. S2†) and it is consistent with the small NP diameter, cyanide was entirely consumed. Fig. 4 shows the values of the the large surface to volume ratio and the corresponding absorption intensity at 420 nm for both enzymatic and nano- accumulation of oxygen vacancies. ceria preparations. As expected, when the ferrocyanide content Under these conditions, Ce4+ atoms in nanoceria act as is low (0 to 1.5 mM), all concentrations tested show a linear be- electron acceptors oxidizing the molecules of interest. In nano- havior between the initial ferrocyanide content and absorption ceria, the formed Ce3+ ions can easily be oxidized again back intensity at saturation, indicating that all the ferrocyanide has to Ce4+, closing the catalytic loop. However, following the been oxidized. At these concentrations, the enzyme perform- Pourbaix diagram,20 cerium atoms in water thermo- ance also superposes that linear dependence. At higher ferro- dynamically prefer to remain in the Ce3+ oxidation state at cyanide concentrations, nanoparticles cannot further increase acidic pH. In this case, the catalytic loop can be broken, and nanoceria behaves as a stoichiometric reagent where Ce4+ transforms into Ce3+ in the NPs that finally dissolves, as pre- viously reported in plant growth media.21 Accordingly, after nanoceria consumption, no NPs were observed by TEM after exhaustive analysis of the sample grid, neither a nanoceria DLS nor UV-vis signal (data not shown). To look closer at nano- ceria evolution during ferrocyanide oxidation, DLS and ICPMS measurements were performed before and after incubation of ferrocyanide and nanoceria at different ratios for 3 hours. 2.35 mM atomic cerium in the form of nanoceria was exposed to increased concentrations of ferrocyanide (0.77, 1.16, 1.54 and 2.3 mM). DLS and the corresponding NP concentration analyses show the progressive dissolution of the NPs maintain- ing the NP number rather constant until disintegration at high ferrocyanide concentrations (Fig. 5A). For ICPMS analysis, the samples were purified by centrifu- gation–filtration before analysis to separate NPs from cerium ions. Fig. 5C shows how the presence of Ce3+ ions in solution Fig. 4 Absorbance values at 420 nm after 60 minutes for a set of solu- increases as the amount of ferrocyanide increases. The discre- tions with a fixed concentration of 0.4 mg mL−1 (equivalent to 2.3 mM pancy between the expected and elemental measured values is atomic cerium) for commercially and in-house synthesized nanoceria and different initial concentrations of ferrocyanide, ranging from 0 to attributed to some retention in the purification filters and the 2.4 mM. Spectra between commercial and in-house nanoceria at t = spontaneous oxidation of ferrocyanide during the experi- 0 min and after 60 min of reaction. mental time window. 14226 | Nanoscale, 2022, 14, 14223–14230 This journal is © The Royal Society of Chemistry 2022 View Article Online Nanoscale Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 19 August 2022. Downloaded on 4/17/2023 9:35:46 AM. Fig. 5 In-house synthesized cerium NPs at a concentration of 0.4 mg ml−1 (equivalent to 2.3 mM atomic cerium) reacting for 3 hours with different concentrations of ferrocyanide, ranging from 0 to 2.4 mM. (A) Hydrodynamic diameter by intensity profiles measured by DLS plotted against K4[Fe (CN)6] concentrations. (B) CeO2 NP concentrations measured by DLS. (C) Dissolved cerium ions measured by ICPMS, plotted against K4[Fe(CN)6] concentrations. This translates into nanoceria being able to uptake a finite Finally, when comparing inorganic nanoparticles and bio- number of electrons while maintaining its fluorite crystal logical enzymes, reproducibility and robustness have largely structure and NP integrity before disintegration. Note that Ce3+ been described as features that make NPs desirable candidates ions are soluble in water. Therefore, if Ce3+ ions and the to substitute enzymes. We evaluated these two aspects with corresponding oxygen vacancies are not recycled back to Ce4+ two different batches of laccase enzymes and the two different and oxygen absorbed, the increasing concentration of these ceria NPs (in-house and commercial NPs) tested under the oxygen vacancies in the NPs will lead to cation repulsion and same conditions (Fig. 6A). It can be seen that while enzymes irreversible disintegration of the NPs. Despite that, it is well show very different oxidation rates per mass, the nanoceria known that the dissolution of cerium oxide in aqueous solu- data superimposes. This difference in response between tions is hindered by the great stability of the crystalline enzymes is a widespread phenomenon as the complexity of cal- network, the same that endures the cyclic formation of oxygen culating the activity per mass of protein itself, which is highly vacancies and cerium reduction.22 However, nanoceria around variable. Another interesting feature of nanoceria is that, 2–3 nm is very close to the calculated thermodynamically unlike enzymes, it does not require any specific storage con- stable crystal size23 facilitating NP dissolution. This process ditions or handling. A batch of in-house synthesized nanoceria accelerates as pH decreases. We did control experiments where solutions was stored at room temperature and under ambient nanoceria was incubated at pH 4.4 and pH 1.7 (Fig. S3†). In light exposure for one month. Ferrocyanide oxidation measure- the absence of ferrocyanide, a good electron source, the NP ments were performed on days 1, 15, and 30 for the five tested dissolution takes about 60 hours, while at pH 1.7, it takes less concentrations. The results are shown in Fig. 6B. The nano- than 3 hours. ceria oxidative capability remains unaltered ( p < 0,05), Fig. 6 (A) Oxidation reaction rate for different concentrations of two different batches of laccase, commercial and in-house nanoceria in the pres- ence of 1 mM ferrocyanide. (B) Absorbance measurements for the in-house batch of nanoceria for over a month (n = 3). The results show remark- able reproducibility and statistically non-significant differences ( p < 0.05). This journal is © The Royal Society of Chemistry 2022 Nanoscale, 2022, 14, 14223–14230 | 14227 View Article Online Paper Nanoscale showing robustness under low demanding storage conditions. Experimental For larger storage times, we recommend doing it in the dark to avoid citrate sodium degradation. Synthesis of nanoceria Several works have described the mode of action of nano- Nanoceria was synthesized by a hydrothermal method based This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. ceria, sometimes reaching contradictory conclusions about its on the basic precipitation of CeNO3 in the presence of SC. In catalytic nature. Variability in pH has been proved to be one of detail, a TMAOH solution (50 mL, 50 mM) was added to a the most relevant parameters directly influencing nanoceria 50 mL solution containing CeNO3 (20 mM) and sodium citrate activity.10,24 For instance, nanoceria presents excellent anti- (40 mM). The final concentrations were 25 mM TMAOH, oxidant capacity in water at neutral or high pH, while oxidative 10 mM CeNO3, and 20 mM SC. The reaction mixture was left capacities of nanoceria have been observed preferably under under stirring overnight at room temperature. Later, the Open Access Article. Published on 19 August 2022. Downloaded on 4/17/2023 9:35:46 AM. acidic conditions, at pH 4 to 6.10,24 Yang and co-workers mixture was transferred to a three-necked round bottom flask attempted to elucidate the pH-dependent oxidizing mechanism (250 mL) and left under refluxing at 100 °C for 4 hours. The and the role of oxygen in the reaction,25 while presenting mixed resulting mixture was a stable, well-dispersed solution of articles reporting both catalytic-like and reagent-like activity. 3.5 nm CeO2 NPs at a concentration of 1.72 mg mL−1 CeO2. Citrate 100 mM buffer preparation Conclusions Citrate buffer at pH = 4.4 was prepared by mixing 0.2 M citric acid solution (Panreac), 0.2 M sodium citrate solution (Probus) The association of the redox activity of nanoceria as a reagent and deionized water in a ratio of 28 : 23 : 49. or a catalyst is not straightforward. The fact that the catalytic activity of nanoceria has been reported under very distinct con- Scanning transmission electron microscopy (STEM) ditions of pH, ionic strength, NP size and/or concentration CeO2 NPs were visualized using STEM (Tecnai F20 S/TEM). hinders the direct comparison between the reported data. 10 µL of the as-synthesized solutions were drop-cast onto a Indeed, it depends on the particular testing conditions. Here, carbon-coated 200 mesh copper grid and left to dry for at least when comparing nanoceria and laccase, it is clearly shown 24 h in the air at room temperature. The average diameter and that under some conditions, nanoceria is a chemical reagent diameter distribution of the samples were measured using with oxidizing capacity. Accordingly, the reaction transforms the Image Analysis software by counting at least 2000 particles. insoluble Ce4+ ions into soluble Ce3+, consuming nanoceria. When NPs are in excess with respect to the substrate, enzymes UV-visible spectra and NPs appear to behave similarly, which is illusory. It is also UV-vis spectra were acquired with a Cary 60 spectrophotometer worth noting that we proved the robustness of nanoceria after (Agilent) using 1.5 mL plastic cuvettes ranging from 250 nm to many measurements over a month without much controlling 800 nm. the storage conditions. Therefore, it can also be stated that nanoceria has outstanding reproducibility and stability, thanks Dynamic light scattering to the robustness of inorganic nanomaterials in front of protein A Malvern ZetaSizer Ultra (Malvern Instruments, UK) operating structures like enzymes. Finally, we would like to point out that at a light source wavelength of 532 nm and a fixed scattering the Ce3+/4+ ratio in a nanoceria NP has been easily estimated by angle of 173° was used to measure the hydrodynamic size of titration with ferrocyanide, a procedure that could become a CeO2 NPs. Measurements were conducted in 1 cm path cell rapid assessment tool for the evaluation of nanoceria in liquid and at 25 °C. environments. Normally, the determination of the Ce3+/Ce4+ ratio is achieved by X-ray photoelectron spectroscopy (XPS)26 Inductively coupled plasma mass spectrometry (ICPMS) and eventually electron magnetic resonance (EPR);27 however, The levels of elemental cerium were analyzed using ICPMS these techniques are complex and costly. (7900 ICPMS, Agilent) in Servei d’Anàlisi Química (UAB, This is relevant in the field of biotechnology and biological Barcelona). In brief, samples were centrifuged with 3 kDa cen- enzymes and their outstanding specificity and high catalytic trifugal filter units (Amicon-Ultra-15, Merck, Germany) in activity.28–30 Since they present intrinsic limitations due to order to separate ionic dissolved cerium from the NP fraction. their proteic nature31 making their catalytic performance sus- Thereafter, microwave assisted digestion (Metrohm, Milestone ceptible to temperature, ionic concentration, and the electro- Ethos Easy, Milestone Systems, Denmark) using HNO3 at static environment where they operate.32 Additionally, they are 200 °C was performed. costly and require careful handling due to intrinsic perform- ance variability, all in all pushing for the search for technologi- Kinetic studies for laccase enzyme and nanoparticles with cal alternatives.33 In this context, nanoparticle oxides may ferricyanide as a substrate overcome many of the limitations of natural enzymes and con- A 96-well plate (flat bottom, 400 µL per well, Sigma-Aldrich) tribute to developments in drug development, diagnosis and was used to perform an optical inspection of the redox reac- therapy, food processing and preservation, and bioremediation tion. The columns were filled with consecutive half-dilutions applications. of the enzymes and nanoparticles starting from a concen- 14228 | Nanoscale, 2022, 14, 14223–14230 This journal is © The Royal Society of Chemistry 2022 View Article Online Nanoscale Paper tration of 0.1 mg mL−1 for the least active laccase (L1, laccase MCIN/AEI (CONCORD, PCI2019-103436) cofunded by the from Trametes versicolor purchased from Sigma-Aldrich EC European Union and Generalitat de Catalunya 1.10.3.2, reference 38429), 0.01 mg mL−1 for the most active (2017-SGR-1431). ICN2 is supported by the Severo Ochoa laccase (L2, laccase from Trametes versicolor purchased from program from Spanish MINECO (SEV-2017-0706) and is This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Sigma-Aldrich EC 1.10.3.2, reference 51639) and 0.4 mg mL−1 funded by the CERCA Programme/Generalitat de Catalunya. for both the commercial (PlasmaChem, cerium oxide nano- Dmitry Galyamin thanks the doctoral program particles, 5 wt% aqueous suspension, average diameter size of “Electroquímica. Ciència i Tecnologia” of the Universitat 4 nm) and synthesized NPs (average diameter size of 3.5 nm). Autònoma de Barcelona (UAB). Citrate buffer (adjusted to pH 4.4) was used as the medium and solvent for the solution preparations. Potassium ferricya- Open Access Article. Published on 19 August 2022. Downloaded on 4/17/2023 9:35:46 AM. nide (K3[Fe(CN)6], Sigma-Aldrich) was added to the wells at a Notes and references final concentration of 1 mM to start the chemical reaction. Kinetic measurements were performed with an ELISA plate 1 L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, reader (Varioskan Flash from Thermo Scientific) at a wave- T. Wang, J. Feng, D. Yang, S. Perrett and X. Yan, Nat. length of 420 nm every 30 seconds for over an hour. Note that Nanotechnol., 2007, 2, 577–583. an initial incubation time of 2 min was needed in order to fill 2 J. Wu, X. Wang, Q. Wang, Z. Lou, S. Li, Y. Zhu, L. Qin and all the wells of the plate with the ferricyanide solution (this H. Wei, Chem. Soc. Rev., 2019, 48, 1004–1076. initial time is included in the graphs). 3 H. Wei, L. Gao, K. Fan, J. Liu, J. He, X. Qu, S. Dong, E. Wang and X. Yan, Nano Today, 2021, 40, X-ray diffraction analysis of nanoceria 101269. X-ray diffraction (XRD) data were collected on a PANalytical 4 S. Scott, H. Zhao, A. Dey and T. B. Gunnoe, ACS Catal., X’Pert diffractometer using a Cu Kα radiation source (λ = 2020, 10, 14315–14317. 1.541 Å). In a typical experiment, the 2θ diffraction (Bragg) 5 C. Korsvik, S. Patil, S. Seal and W. T. Self, Chem. Commun., angles were measured by scanning the goniometer from 10° to 2007, 10, 1056–1058. 95°. The samples were prepared by centrifugation to precipi- 6 J. 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