Gold-Based Nanoparticles on Amino-Functionalized Mesoporous Silica Supports as Nanozymes for Glucose Oxidation

: The transformation of glucose represents a topic of great interest at di ﬀ erent levels. In the ﬁrst place, glucose is currently conceived as a green feedstock for the sustainable production of chemicals. Secondly, the depletion of glucose at the cellular level is currently envisioned as a promising strategy to treat and alter the erratic metabolism of tumoral cells. The use of natural enzymes o ﬀ ers multiple advantages in terms of speciﬁcity towards the glucose substrate but may lack su ﬃ cient robustness and recyclability beyond the optimal operating conditions of these natural systems. In the present work, we have evaluated the potential use of an inorganic based nanohybrid containing gold nanoparticles supported onto ordered mesoporous supports. We have performed di ﬀ erent assays that corroborate the enzyme-mimicking response of these inorganic surrogates towards the selective conversion of glucose into gluconic acid and hydrogen peroxide. Moreover, we conclude that these enzyme-like mimicking surrogates can operate at di ﬀ erent pH ranges and under mild reaction conditions, can be recycled multiple times and maintain excellent catalytic response in comparison with other gold-based catalysts.


Introduction
Saccharides are abundant, renewable and sustainable chemical compounds that play a key role both at industrial level and in the metabolism of living beings [1,2]. Only in 2017, the United States produced about 27.5 M tons of starch, the most plentiful polysaccharide on Earth, which was further used for industrial and food applications. The hydrolysis of starch over supported metal catalysts produces glucose as main product. For this reason, from a green-feedstock point of view all selective chemical transformations of glucose are industrially attractive to produce high value-added products [1] and have been widely studied [1][2][3][4]. Glucose oxidation yielding gluconic acid possesses a huge industrial importance in the manufacturing of water-soluble cleansing products, food and beverages additives or pharmaceutics and cosmetics. Currently, this industrial production of Analogously, temperature has also shown a positive impact on the rate and selectivity of the glucose oxidation reaction [44,47]. Although the use of a basic medium favors the glucose oxidation reaction and the stability of AuNPs [44], base-free conditions are still desired from the green chemistry, industrial and biomedicine point of view [47,48]. Therefore, it is necessary to improve the AuNPs stability in absence of basic conditions with the selection of suitable stabilizing agents [48] or after immobilization in different functional supports [49].
Several supports have been used for Au NPs deposition and applied in the catalytic Scheme 1. pH dependent equilibrium and Au-mediated catalytic oxidation of glucose.
Analogously, temperature has also shown a positive impact on the rate and selectivity of the glucose oxidation reaction [44,47]. Although the use of a basic medium favors the glucose oxidation reaction and the stability of AuNPs [44], base-free conditions are still desired from the green chemistry, industrial and biomedicine point of view [47,48]. Therefore, it is necessary to improve the AuNPs Catalysts 2020, 10, 333 3 of 15 stability in absence of basic conditions with the selection of suitable stabilizing agents [48] or after immobilization in different functional supports [49].
Several supports have been used for Au NPs deposition and applied in the catalytic oxidation of glucose including carbon-based materials [38,[50][51][52][53], Al 2 O 3 [42,54], CeO 2 [38,39,52], ZrO 2 [55], TiO 2 [28,41], mesoporous SiO 2 [50] or polymers such as poly(methyl methacrylate), polystyrene or polyaniline [43], achieving in most of the cases >80% of glucose conversion and >90% selectivity for gluconic acid. The catalyst preparation method plays a key role in the catalytic activity [40]. The nature of Au-support interactions is also essential to ensure the stability of the catalytic system [36,56]. For instance, supports with high density of -OH groups on their surface promote the anchoring of Au NPs [27,57]. Likewise, -NH 2 surface functional groups can endorse stronger affinity with AuNPs by electrostatic attraction thereby resulting in a more efficient immobilization of both the active metallic nanoparticles and the glucose sustrate [58][59][60].
In this work, we describe the synthesis of an amino-functionalized mesoporous silica nanoplatform decorated with small gold nanoparticles immobilized by electrostatic attraction. This hybrid Au-silica nanohybrid has been evaluated as a potential enzyme surrogate with glucose-oxidase mimicking response. Multiple reaction conditions have been thoroughly explored to identify the best performing scenario and the main reaction products (i.e., gluconic acid and hydrogen peroxide) expected from the selective oxidation of glucose. Furthermore, this work addresses different colorimetric assays to identify these main reaction products and shows competitive catalytic results working under mild, environmentally friendly conditions that are favorable for environmental and biomedical targeted applications. Figure 1a summarizes the different synthesis stages required to fabricate the gold-based nanohybrids. The first step (denoted as (i) in Figure 1a) consisted on the preparation of the gold nanoparticles (Au-NPs). A phosphonium derived salt, tetrakis(hydroxymethyl) phosphonium chloride (THPC), was employed as reducing and stabilizing ligand of the Au NPs. The use of this ionic stabilizing agent has been previously reported by Duff et al. [61] and our group [49,62,63] as a successful alternative to poly-alcohols and other polymeric stabilizers with greater steric hindrance [62]. Cs-corrected Scanning Transmission Electron Microscopy (STEM) images demonstrated the formation of crystalline Au NPs with average diameters below 5 nm (Figure 1c). The second step (denoted as (ii) in Figure 1a) consisted on the synthesis of nanostructured silica hosts containing ordered mesoporous channels as demonstrated by TEM inspection (Figure 1d,e). These silica spheres were functionalized with an amino-grafting agent (see the Materials and Methods section for further details) containing -NH 2 terminal groups (~8% wt. according to thermogravimetric analysis and considered optimal to keep a good colloidal and thermal stability [59,64]) and Si-O bonds able to attach on the surface of the silica spheres [49,65,66].

Synthesis and Characterization of Gold-Mesoporous Silica Heteronanostructures
The final step (see (iii) in Figure 1a) involved the gold seeding of the amino-functionalized silica hosts favored by the electrostatic attraction of positively charged -NH 3 + terminal groups and negatively charged Au NPs [49,59,65,66]. The negative charge of these type of Au NPs has been previously reported [67,68] and tentatively attributed to the coexistence of Au-O − surface bonds and chloride stabilizing ions [67][68][69]. TEM and STEM images (Figure 1f,g) confirmed the successful attachment of the Au NPs. Pore size restrictions limited the loading of the bigger Au NPs within the mesochannels. ICP analysis confirmed 1% Au wt. loadings. Following this strategy, Au NPs are endowed with higher catalytic sites availability while preventing them from partially coarsening events typically observed in freestanding Au NPs of this nature [63]. Furthermore, providing a solid and stable silica support can facilitate the recovery of the catalytic Au NPs for their potential reusability and/or regeneration [59].
silica hosts containing ordered mesoporous channels as demonstrated by TEM inspection (Figure 1d,e). These silica spheres were functionalized with an amino-grafting agent (see the Materials and Methods section for further details) containing -NH2 terminal groups (~8% wt. according to thermogravimetric analysis and considered optimal to keep a good colloidal and thermal stability [59,64]) and Si-O bonds able to attach on the surface of the silica spheres [49,65,66].

Colorimetric Assays to Determine the Oxidation of Glucose into Gluconic Acid
Au-MCM-41 hybrids were evaluated as inorganic structured materials mimicking the action of the natural enzyme glucose oxidase (GOx). Our enzyme-like surrogates were tested towards the aerobic oxidation of glucose under mild reaction conditions.
In order to evaluate the successful conversion of glucose into gluconic acid (hereafter GA) and hydrogen peroxide (H 2 O 2 ) (the expected reaction products), a combination of different colorimetric assays were carried out while exploring the influence of different experimental variables. First of all, formation of GA was indirectly validated by UV-Vis spectroscopy. GA can selectively react with hydroxylamine and a trivalent iron salt form a red-colored hydroxamate complex with a broad absorption centered at 505 nm (see Figure 2a,c) [12,70].  The progressive formation of the Fe-complex was monitored at different reaction times under neutral pH conditions. Figure 2b shows the positive influence of increasing the reaction temperature from 25 to 50 • C to increase the selective oxidation of glucose into GA that readily reacts to form the colored complex (see inset in Figure 2a,b). Likewise, the influence of the pH was further evaluated at a fixed reaction temperature of 37 • C. Figure 2d shows the evolution of the absorption band of the hydroxamate complex at different pH values and confirms poorer performance of the Au-MCM-41 catalyst under basic conditions. It is also worth noting that no additional evolution was observed after 60 min of reaction. The formation and quantification of GA as reaction product was additionally determined by a colorimetric titration with sodium hydroxide and phenolphthalein [50,71]. According to the amounts of GA generated (see Figure 2e), the glucose conversion levels reached up to 85% at neutral pH conditions and T = 37 • C after 45 min of reaction. Titration methods confirmed the lower response under more acidic conditions as previously shown by UV-Vis spectroscopy (Figure 2d).

Colorimetric Assays to Identify the Formation of Hydrogen Peroxide
The other main reaction product expected from an enzyme-like glucose oxidase activity is typically hydrogen peroxide (Figure 3a). In order to assess its presence, two different colorimetric assays were performed [12]. The H 2 O 2 generated during the oxidation of glucose was initially reduced with the aid of a horseradish peroxidase (HRP) enzyme (Figure 3a). In the first colorimetric assay, this reduction step induced the simultaneous oxidation of 3,3,5,5-tetramethylbenzidine (hereafter TMB) into its blueish colored derivative with a maximum absorption at 652 nm (Figure 3a  The other main reaction product expected from an enzyme-like glucose oxidase activity is typically hydrogen peroxide (Figure 3a). In order to assess its presence, two different colorimetric assays were performed [12]. The H2O2 generated during the oxidation of glucose was initially reduced with the aid of a horseradish peroxidase (HRP) enzyme (Figure 3a). In the first colorimetric assay, this reduction step induced the simultaneous oxidation of 3,3,5,5-tetramethylbenzidine (hereafter TMB) into its blueish colored derivative with a maximum absorption at 652 nm (Figure 3a  An analogous colorimetric assay mediated by the HRP enzyme to promote the simultaneous oxidation of a 2,2-azino-bis (3-ethylbenzothiozoline)-6-sulfonic acid salt An analogous colorimetric assay mediated by the HRP enzyme to promote the simultaneous oxidation of a 2,2-azino-bis (3-ethylbenzothiozoline)-6-sulfonic acid salt (ABTS) into its blue colored oxidized derivative was also carried out (see scheme in Figure 4a). A similar behavior regarding the pH influence over time could be observed (Figure 4b). No catalytic activity could be detected at lower pH values of 4 or 5. Furthermore, no additional evolution of the maximum absorption spectrum at 415 nm (Figure 4c) was detected beyond the first 60 min of reaction under neutral or basic conditions (Figure 4b).
Catalysts 2020, 10, x FOR PEER REVIEW 8 of 17 (ABTS) into its blue colored oxidized derivative was also carried out (see scheme in Figure  4a). A similar behavior regarding the pH influence over time could be observed ( Figure  4b). No catalytic activity could be detected at lower pH values of 4 or 5. Furthermore, no additional evolution of the maximum absorption spectrum at 415 nm (Figure 4c) was detected beyond the first 60 min of reaction under neutral or basic conditions (Figure 4b).

Steady-State Kinetics and Recyclability Evaluation
To quantify the glucose-oxidase enzyme mimicking activity of the Au-MCM-41 nanocatalyst, a kinetic study of the glucose conversion into gluconic acid was carried out. The kinetic parameters were determined after performing a series of experiments with increasing concentrations of glucose ( Figure 5a) and adjusting the gluconic acid generated after 30 min of reaction by quantitative colorimetric assays that included pH titration [50,71] and UV-Vis spectroscopy [70,72] (see Experimental section for further details). The data were fitted to a non-linear regression of the Michaelis-Menten equation and the

Steady-State Kinetics and Recyclability Evaluation
To quantify the glucose-oxidase enzyme mimicking activity of the Au-MCM-41 nanocatalyst, a kinetic study of the glucose conversion into gluconic acid was carried out. The kinetic parameters were determined after performing a series of experiments with increasing concentrations of glucose ( Figure 5a) and adjusting the gluconic acid generated after 30 min of reaction by quantitative colorimetric assays that included pH titration [50,71] and UV-Vis spectroscopy [70,72] (see Experimental section for further details). The data were fitted to a non-linear regression of the Michaelis-Menten equation and the apparent kinetic parameters were obtained using the Lineweaver-Burk double reciprocal plot as previously suggested by Gao et al. [73] (see inset in Figure 5a).
Catalysts 2020, 10, x FOR PEER REVIEW 9 of 17 apparent kinetic parameters were obtained using the Lineweaver-Burk double reciprocal plot as previously suggested by Gao et al. [73] (see inset in Figure 5a). An apparent Michaelis-Menten constant (defined as Km) of ~55 mM and a maximum velocity (Vmax) of 18 × 10 −6 M/s were determined (inset graph in Figure 5a). This apparent Km constant is higher than other values reported for freestanding gold nanozymes (Km ~7 mM) [71], polymer-coated gold mimics (Km ~0.4 mM) [19], gold-supported mimicking systems (Km ~27 mM) [50] or the glucose oxidase (GOx) natural enzyme (Km ~5 mM) [71]. According to this value, our Au-MCM-41 nanozymes show a slightly weaker affinity towards the glucose substrate [19,50,71]. Nevertheless, the corresponding catalytic activity (Kcat) defined by the coefficient Vmax/[concentration of catalyst] improves the values reported for natural GOx (Kcat ~14.2 s −1 vs. Kcat ~9.7 s −1 , respectively) and it is close to freestanding Au NPs (Kcat ~18.5 s −1 ) reported by Luo et al. [71]. From the catalytic standpoint, the enzyme-like Au-MCM-41 nanohybrids exhibit a very positive response probably due to the good accessibility and homogeneous distribution of the gold active sites in the mesoporous support. The glucose conversion levels achieved (85-92% in less than 1 h of reaction) working under mild reaction conditions (in the absence of co-solvents or basic conditions and without high temperature or working pressure) are extremely competitive with current gold-based systems available in the literature and briefly summarized in Table 1 (vide infra).
It is also plausible that a controlled O2 feeding would have led to an even better performance. Still, it is a very positive outcome if a potential application to modify the microenvironment of tumoral cells (with low O2 concentration levels) is envisioned. As suggested by Cattaneo et al. [46] it is quite plausible that the reaction of glucose is strongly favored by the nucleophilic attack of glucose on Au-O − sites. This reaction would be An apparent Michaelis-Menten constant (defined as K m ) of~55 mM and a maximum velocity (V max ) of 18 × 10 −6 M/s were determined (inset graph in Figure 5a). This apparent K m constant is higher than other values reported for freestanding gold nanozymes (K m~7 mM) [71], polymer-coated gold mimics (K m~0 .4 mM) [19], gold-supported mimicking systems (K m~2 7 mM) [50] or the glucose oxidase (GOx) natural enzyme (K m~5 mM) [71]. According to this value, our Au-MCM-41 nanozymes show a slightly weaker affinity towards the glucose substrate [19,50,71]. Nevertheless, the corresponding catalytic activity (K cat ) defined by the coefficient V max /[concentration of catalyst] improves the values reported for natural GOx (K cat~1 4.2 s −1 vs. K cat~9 .7 s −1 , respectively) and it is close to freestanding Au NPs (K cat~1 8.5 s −1 ) reported by Luo et al. [71]. From the catalytic standpoint, the enzyme-like Au-MCM-41 nanohybrids exhibit a very positive response probably due to the good accessibility and homogeneous distribution of the gold active sites in the mesoporous support. The glucose conversion levels achieved (85-92% in less than 1 h of reaction) working under mild reaction conditions (in the absence of co-solvents or basic conditions and without high temperature or working pressure) are extremely competitive with current gold-based systems available in the literature and briefly summarized in Table 1 (vide infra).
It is also plausible that a controlled O 2 feeding would have led to an even better performance. Still, it is a very positive outcome if a potential application to modify the microenvironment of tumoral cells (with low O 2 concentration levels) is envisioned. As suggested by Cattaneo et al. [46] it is quite plausible that the reaction of glucose is strongly favored by the nucleophilic attack of glucose on Au-O − sites. This reaction would be favored at higher pH and temperatures but those requirements would be still less demanding than those reported in the regular enzymatic pathway, where the hydrolysis of the δ-glucono-lactone is the rate-limiting step for gluconic acid generation [45]. Even though the final product is the same, the Au-based nanozyme may follow a different mechanism. Additional insights with the aid of Nuclear Magnetic Resonance (NMR) techniques and theoretical simulation studies can be noteworthy for the corroboration of these differences in reaction mechanisms between natural enzyme and artificial enzyme systems [45,46,53,[74][75][76]. Furthermore, the silica mesoporous support provides not only a great active surface area for the accommodation of glucose but also a more favorable platform to facilitate the recovery and reutilization of the nanozyme than in freestanding systems. Figure 5b illustrates the capability of these Au-silica hybrids to be reutilized in multiple consecutive reaction cycles with a minimal reduction of the glucose conversion levels, most likely attributable to an incomplete retrieval of all the catalyst from cycle to cycle.

Synthesis of Gold Nanoparticles Using THPC as Reducing and Stabilizing Agent (Au-NPs)
Ultrasmall Au-NPs were prepared as described previously elsewhere with slight modifications [62,63]. Successive charging of a 50 mL glass vial, while stirring, with 14 mL of distilled water and then, in order, portions of aqueous solutions of sodium hydroxide (1 M, 165 µL), the reducing agent THPC (65 mM, 333 µL), and the freshly dissolved chloroauric acid salt (3 mg, 1 mL) results in the formation of orange-brown hydrosols of gold. Two minutes was allowed between the addition of THPC and that of chloroauric acid. The reaction mixture was stirred for 1 day at room temperature, protecting the mixture from light to prevent the photothermal decomposition of precursors [79]. Finally, the resulting colloid Au-NPs were stored in a refrigerator at 4 • C until further use.

Synthesis of Amino-Functionalized Mesoporous Silica Supports (MCM-41 Type Material)
Silica materials with hexagonally arranged uniform mesopores were synthesized using the previously reported approach [59,80,81] for base catalysed sol gel condensation of TEOS in the presence of structure directing agent. MCM-41-like mesoporous nanoparticles were synthesized by the following procedure: CTAB (1.00 g, 2.7 mmol) was first dissolved in deionized water (1 L). NaOHaq. (2.00 M, 5 mL) was added to the CTAB solution, and the temperature was adjusted to 80 • C. TEOS (7 mL, 0.05 mmol) was then added drop-wise to the surfactant solution. The mixture was left to stir for 3 h to give a white precipitate. The solid product was centrifuged and washed with deionized water until a neutral pH. Finally, the solid was dried. To prepare the final porous material, the as-synthesized solid were dissolved in acidic EtOH (100 mL, and few drops of HCl 37%) for 12 h to remove the template phase. The precipitate was recovered by centrifugation at 20,000 g and thoroughly washed with distilled H 2 O/EtOH (1:1, 2x) and EtOH (2x). Then the product was dried overnight under reduced pressure. Subsequently, primary amine groups were grafted on the surface of the mesochannels by condensation of 3-aminopropyltriethoxysilane (APTES), to favor electrostatic interactions between the MCM-41 nanoparticles and the Au-NPs [49,59,65,82]. Briefly, 0.50 g of MCM-41 material was dispersed in 10 mL of toluene, 100 µL of TEA and 200 µL of APTES. The reaction was refluxed for 24 h, and the product was centrifuged and washed twice prior to total drying under vacuum for further use.

Synthesis of the Au-MCM-41 Nanohybrids
To prepare the gold-silica nanohybrid, 0.10 g of amino modified MCM-41 nanoparticles was dispersed in 10 mL of the Au-NCs stored suspension. The suspension was adjusted to pH 5 and stirred at 20 • C for 12 h in the dark. Subsequently, the product was collected by centrifugation, washed several times with water, and re-dispersed in water until further use. The synthesis of materials has been performed by the Platform of Production of Biomaterials and Nanoparticles of the NANOBIOSIS ICTS, more specifically by the Nanoparticle Synthesis Unit of the CIBER in BioEngineering, Biomaterials & Nanomedicine (CIBER-BBN).

Determination of Activity and Steady-State Kinetics of the Au-MCM-41 Nanohybrids
The glucose oxidation reactions were carried out with temperature control in a thermoplate, equipped with a mechanical stirrer operating at 750 rpm, using a total volume of 100 mL and different glucose concentrations from 10 up to 180 mM. The concentration of the Au-MCM-41 nanocatalyst was 0.025 mg/mL. Concentration of dissolved O 2 was typically in the range of 6-8 mg/L (determined with an Orion Optical Dissolved O 2 sensor, Thermo Scientific). The pH of the reaction mixture was held constant at 7.4. The kinetics of the catalytic reaction could be well adjusted to a typical Michaelis-Menten curve [70,72]. The Michaelis-Menten constant was calculated using the Lineweaver-Burk plot: where V 0 is the initial velocity, V max is the maximal reaction velocity, and [S] is the concentration of the substrate. The rate of the catalytic reaction was defined as the amount of generated product, gluconic acid, in a fixed time interval of 30 min. The concentration of gluconic acid was determined via titration with NaOH (0.1 M) using phenolphthalein at pH 8.2 as colorimetric pH indicator. Likewise, this reaction product, (i.e., gluconic acid), was also verified by reaction with hydroxylamine and a trivalent Fe III salt, leading to a red complex hydroxamate-Fe 3+ (with a maximum absorbance at 505 nm) [70]. Namely, 125 µL of solution 1 (5 mmol/L EDTA and 0.15 mmol/L triethylamine in water) and 12.5 µL of solution 2 (3 mol/L NH 2 OH in water) were added to 200 µL of the supernatant centrifuged reaction solution, and the mixture was allowed to react for 25 min. Finally, 62.5 µL of solution 3 (1 mol/L HCl, 0.1 mol/L FeCl 3 , and 0.25 mol/L CCl 3 COOH in water) was added to the reaction medium, and reaction was incubated for another 5 min before spectral acquisition. Absorbance values from the Fe-complex at different reaction times and total gluconic acid concentrations were correlated using the Lambert-Beer law and a molar absorption coefficient of 61.2 [50,[70][71][72]. In order to prevent the influence of the nanoparticles, the suspensions were previously centrifuged at 6000 rpm in 2 mL Eppendorfs.
Hydrogen peroxide (H 2 O 2 ) was indirectly tested with the aid of a horseradish peroxidase (HRP) enzyme that selectively favored the reduction of the H 2 O 2 present in solution and the simultaneous oxidation of ABTS or TMB with specific absorbances at 415 and 652 nm, respectively [12,70,72]. In a standard procedure, 300 µL of the filtered catalytic reaction solution was added to 50 µL of 8 mM ABTS and 50 µL of 0.4 µg/mL HRP. The oxidation produced a green color with a major absorbance peak at 415 nm (final concentrations after dilutions for UV-Vis measurements were 1 mM ABTS and 0.05 µg/mL HRP). Also, 350 µL of the filtered catalytic reaction solution was added to 8 µL of 40 mM TMB and 50 µL of 0.4 µg/mL HRP (final concentrations after dilutions for UV-Vis measurements were 0.8 mM TMB and 0.05 µg/mL HRP). The oxidation produced a blue color with a maximum absorbance centered at 652 nm. Acetate/Acetic acid buffer solutions (0.2 M, 0.2 mL) were prepared combining 5.4 g sodium acetate trihydrate +1.2 mL glacial acetic acid in 198.8 mL water. Recycling experiments carried out after one hour reaction runs included 2 centrifuge cycles and redispersion in water prior to the addition of fresh glucose solutions. This process was repeated prior to each reuse.

Catalyst Characterization
The textural properties of the MCM-41 materials were determined from nitrogen adsorption isotherms measured by an ASAP 2020 instrument (Micromeritics, Norcross, GA, USA). The samples were out-gassed at 26.7 Pa and 623 K for 6 h before the measurement. The amount of amine groups grafted onto silica surface was determined by thermogravimetric analysis (SDTA-851 equipment, Mettler Toledo, Barcelona, Spain). Samples of ca. 3 mg were heated at 5 • C/min up to 800 • C) under synthetic air flow. The morphologies, size distribution and crystalline structure of these materials were determined by Transmission Electron Microscopy (TEM) in a T20 (Tecnai, Thermo Fisher, Hillboro, OR, USA) and Titan Cube (FEI, Thermo Fisher, Hillboro, OR, USA) system operated at 200-300 kV, respectively, and coupled with a CCD camera (Gatan, CA, USA). The samples for TEM analysis were prepared by suspension of the corresponding sample in water, dropping the suspension onto a 200-mesh carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA, USA) and letting it dry under ambient air with the aid of anti-capillary tweezers. The UV-vis absorption spectra of the products of reaction were systematically recorded using a V-67 UV/Visible spectrophotometer (JASCO, Madrid, Spain) using a quartz cell of 1 cm light path.

Conclusions
Au NPs supported onto mesoporous silica supports via electrostatic attraction represents a feasible and straightforward strategy to fabricate glucose-oxidase enzyme-like inorganic platforms able to deliver a successful performance under mild reaction conditions (neutral pH and temperature).