Multifunctional catalytic platform for peroxidase mimicking, enzyme immobilization and biosensing

Author’s Accepted Manuscript Multifunctional Catalytic Platform for Peroxidase Mimicking, Enzyme Immobilization and Biosensing Camila Marchetti Maroneze, Glauco P. dos Santos, Vitoria B. de Moraes, Luiz P. da Costa, Lauro Tatsuo Kubota www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(15)30507-8 http://dx.doi.org/10.1016/j.bios.2015.10.042 BIOS8078

To appear in: Biosensors and Bioelectronic Received date: 24 July 2015 Revised date: 12 October 2015 Accepted date: 14 October 2015 Cite this article as: Camila Marchetti Maroneze, Glauco P. dos Santos, Vitoria B. de Moraes, Luiz P. da Costa and Lauro Tatsuo Kubota, Multifunctional Catalytic Platform for Peroxidase Mimicking, Enzyme Immobilization and Biosensing, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.10.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Multifunctional Catalytic Platform for Peroxidase Mimicking, Enzyme Immobilization and Biosensing Camila Marchetti Maroneze 1,2, Glauco P. dos Santos 1,2, Vitoria B. de Moraes 1,2, Luiz P. da Costa3 and Lauro Tatsuo Kubota1,2 

1

Institute of Chemistry, State University of Campinas (UNICAMP), P.O. Box 6154, 13083-970, Campinas-SP, Brazil. 2

National Institute of Science and Technology of Bioanalytics (INCTBio), Institute of Chemistry - UNICAMP, P.O. Box 6154, 13084-971, Campinas, SP, Brazil. 3

Programa de Pós-graduação em Biotecnologia Industrial – Universidade Tiradentes, Aracaju, SE, Brazil.



Corresponding author: Fax: +55-19-3521-3127

E-mail address: [email protected] (Prof. Lauro T. Kubota)

Abstract A hybrid platform based on ionic liquid-based alkoxysilane functionalized mesoporous silica was applied for the synthesis of supported Pt nanoparticles with peroxidase-like catalytic activity. The positively charged groups (imidazolium) chemically bonded to the surface provide dual-functionality as ion-exchangers to the hybrid material, firstly used for the in situ synthesis of the highly dispersed Pt nanostructures and, secondly, for the immobilization of biological species aiming biosensing purposes. The peroxidase-

1

like catalytic activity of the SiO2/Imi/Pt material was evaluated towards the H2O2mediated oxidation of a chromogenic peroxidase substrate (TMB), allowing the colorimetric detection of H2O2. Finally, to further explore the practical application of this nanomaterial-based artificial system, glucose oxidase (GOx) was immobilized on the catalytic porous platform and a bioassay for the colorimetric determination of glucose was successfully conducted as a model system. The enzyme-like catalytic properties of the SiO2/Imi/Pt as well as its ability to immobilize and keep active biological entities on the porous structure indicate that this hybrid porous platform is potentially useful for the development of biosensing devices.

Keywords: Pt nanoparticles, artificial enzymes, glucose, H2O2, ionic liquids

1. Introduction The design of functional nanomaterials with enzyme-like catalytic activity has been one of the several areas significantly benefited by the successful merging of nanotechnology with biology (Lin et al. 2014c; Wei and Wang 2013). Biomedical diagnostics can be highlighted as a field where these artificial systems have found important practical application, acting in processes that originally make use of natural enzymes, specially horseradish peroxidase (HRP), as labels for the amplified detection of biorecognition events, e.g. the enzyme-like immunosorbent assay (ELISA). The monitoring of H2O2 by using HRP-mediated oxidation reactions of chromogenic substrates is also one the most used strategies in biosensing assays once hydrogen

2

peroxide is a product of many oxidoreductases enzymes and acts as an efficient probe for the determination of important analytes like glucose, cholesterol, uric acid and lactate. Recently, various catalytic nanomaterials such as Fe3O4 (Gao et al. 2007; Wei and Wang 2008), janus hematite-SiO2 nanoparticles (Lu et al. 2015), noble metal nanoparticles (Cai et al. 2013; Chen et al. 2011; Ge et al. 2014; He et al. 2011; He et al. 2010; Sun et al. 2015; Wang et al. 2015), cerium oxide nanostructures (Artiglia et al. 2014; Asati et al. 2009; Jiao et al. 2012), MoS2 (Lin et al. 2014a), WS2 (Lin et al. 2014b), VO2 (Nie et al. 2014), V2O5 (André et al. 2011), graphene oxide (Song et al. 2010), and MOFs (Liu et al. 2015) have been used as highly active peroxidase mimetic catalysts in biodetections. Some advantages that boost the utilization of nanomaterials for such purposes are mainly related to the time-consuming and expensive processes that are required for the preparation, purification and storage of natural enzymes in addition to their tendency to denature under environmental changes. Regardless of the advances already achieved concerning to the ability of designing nanomaterials with remarkable enzyme-like activities, it still remains a great challenge to obtain highly stable biomimetic catalyst with remained surface accessibility. Frequently, the catalytic potential of nanostructures still faces some drawbacks like the lowering or loss of the catalytic activity caused by the presence of capping agents, which are usually required in their synthesis in order to stabilize the high surface energies and to avoid particle growth or aggregation (Fedlheim and Foss 2001). The utilization of porous solids with rigid frameworks like silica has been considered a good alternative to overcome these difficulties (Campelo et al. 2009; White et al. 2009). Due to the confinement effects provided by the porous environment, it should be possible to control the size and dispersion degree of supported

3

nanostructures, inhibiting aggregation and producing highly active centers. Besides, the vast possibilities of inserting functional organic groups on the silica surface to obtain hybrid systems (Hoffmann et al. 2006; Soler-Illia and Azzaroni 2011; Van Der Voort et al. 2013) whose properties combine in a single solid the porosity and mechanical stability of the robust inorganic support and the reactive features of the organic entities also make this support material very attractive. A suitable functionality on a silica-based hybrid material may be useful not only to provide a smart basis for building diverse nanostructures but also to allow the immobilization and stabilization of biological species. The efficient combination in a single material of biomolecules and nanomaterials with catalytic properties that are able to provide the transduction of biological phenomena are extremely interesting in the development of novel biosensors. This works describes the synthesis and application of a hybrid material based on mesoporous silica presenting imidazolium cationic groups covalently bonded to the surface. Such groups display a dual-functionality as ion-exchangers in this platform, firstly explored for the in situ synthesis of supported Pt nanoparticles with peroxidaselike activity, evaluated towards the catalytic H2O2-mediated oxidation of 3,3’,5,5’tetramethylbenzidine (TMB), a typical peroxidase substrate (Frey et al. 2000; Josephy et al. 1982). The same cationic groups were further explored in the immobilization of biomolecules aiming to obtain hybrid materials suitable for biosensing purposes. A bioassay involving the immobilization of glucose oxidase (GOx) and the subsequent colorimetric detection of glucose by the H2O2-mediated oxidation of TMB catalyzed by the Pt NPs was conducted as a model system. 2. Experimental 2.1 Synthesis of the imidazolium-based alkoxysilane

4

The synthesis of the imidazolium-based alkoxysilane (Si-Imi) was carried out according to the procedure described elsewhere (Fattori et al. 2012): in a round bottomed flask, a solution containing 6.0 mL of dry toluene, 0.30 mmol of CPTS (3(chloropropyl)-trimethoxysilane, Aldrich, 99%) and 0.30 mmol of 1-methylimidazole (Aldrich, 98%) was stirred for 24 h at 333 K (N2 atmosphere). After that, the reaction mixture was kept under static conditions until the complete separation of the resulting two immiscible liquid phases. The bottom fraction, which corresponds to the Si-Imi product, was then physically separated. 2.2 Functionalization of porous silica with the imidazolium-based alkoxysilane The surface chemical functionalization of the porous silica was carried out by the post-grafting procedure (Figure 1, step (1)), as follows: 1.0 g of porous SiO2 (Aldrich, Davisil Grade 643) was suspended into 100 mL of dry ethanol, followed by addition of 2 mmol of Si-Imi. This mixture was kept under reflux for 24 h. The product was then filtered and washed with ethanol. Finally, the resulting solid was dried at 373 K. The sample obtained will hereafter be designated as SiO2/Imi. 2.3 Synthesis of the supported Pt nanoparticles (Pt NPs) The synthesis of the supported Pt NPs was carried out in two simple steps (Figure 1, steps (2) and (3)). Firstly, the anionic complex PtCl6- is adsorbed onto the functionalized surface by ion-exchange reaction with the positively charged imidazolium groups. The reaction was carried out by immersing 0.2 g of the SiO2/Imi in 20.0 mL of 0.1 x 10-3 mol L-1 aqueous H2PtCl6 solution (H2PtCl6.xH2O, Aldrich). The mixture was kept under stirring for 2h at room temperature. The solid was then centrifuged and washed with water (25 ml) three times. In the second step, the solid with adsorbed PtCl6- ions was submitted to a reduction process by suspending the 5

powder in 20.0 mL of 1.0 x 10-2 mol L-1 aqueous NaBH4 solution at room temperature, keeping the mixture under mechanical stirring for 15 min. The resulting solid (SiO2/Imi/Pt) was then recovered, washed with deionized water and dried at 353 K.

Figure 1. Schematic illustration of the steps involved in the functionalization of the silica surface and in the synthesis of supported Pt NPs.

2.4 Immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx) The immobilization of HRP and GOx on the surface of SiO2/Imi and SiO2/Imi/Pt, respectively, was carried out by immersing 25 mg of the respective powder into 25 mL of 1 mg/mL solutions of HPR (Aldrich) and GOx (Aldrich) in phosphate buffer (pH 7.4). The suspensions were kept under mechanical stirring for 2h at room temperature. After the set time, the solids were centrifuged and washed with phosphate buffer solution (pH 7.4) five times. 2.5 Colorimetric detection of H2O2 by exploiting the peroxidase-like activity of SiO2/Imi/Pt Investigations of the catalytic activity of SiO2/Imi/Pt and its ability of mimicking peroxidase and also the colorimetric detection of hydrogen peroxide were evaluated using the chromogenic substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The reaction was performed by mixing 40 µL of a suspension containing SiO2/Imi/Pt at 25 mg/mL (in deionized water), 100 µL of the TMB substrate solution 0.1 mg/mL (prepared by

6

dissolving 1 mg of TMB in 100 µL of DMSO followed by dilution (1:100) into citric acid-sodium citrate buffer 75 mM at pH 5.0) and 100 µL of a hydrogen peroxide solution (prepared in deionized water) at different concentrations in order to achieve values in the range of 0.1-5 mM. After that, the mixtures were stirred and kept for 5 minutes at room temperature. Then, the solids were centrifuged and 100 µL of the supernatant containing the soluble blue product was mixed with 100 µL of a sulfuric acid solution at 0.5 M, resulting in a yellow color. Finally, the solutions were added to a microplate, which was positioned in the microplate reader (TP-Reader, Thermo Plate), shaken under middle intensity for 30 seconds followed by measurement of the color intensity at 450 nm. The behavior of SiO2/Imi/HRP was also evaluated by applying the same procedure described above but varying the hydrogen peroxide concentration from 0.01 to 0.5 mM. The control experiments were carried out by adding 100 µL of deionized water instead of hydrogen peroxide. All the measurements were carried out in quadruplicate.

2.6 Biosensing of glucose through H2O2-mediated oxidation of TMB catalyzed by Pt NPs The biosensing of glucose was based on a cascade reaction that consists first on the oxidation of glucose by GOx generating H2O2 followed by the H2O2-mediated oxidation of the TMB substrate catalyzed by Pt NPs. This reaction was performed by applying the same conditions, amount and concentration of reagents described in section 2.5 but using the SiO2/Imi/GOx/Pt as the porous platform, varying the concentration of glucose from 0.1 to 2.5 mM instead of hydrogen peroxide and keeping the mixture for 15 minutes at room temperature before centrifugation. The control

7

experiments were carried out by adding 100 µL of deionized water instead of glucose. All the measurements were carried out in quadruplicate.

2.7 Characterization of the synthesized materials The amount of the functional groups immobilized onto the SiO2 surface was calculated based on the nitrogen content, determined by means of elemental analyses on a Perkin-Elmer 2400 elemental analyzer apparatus. Quantitative determination of platinum in solution was carried out by ICP-OES using a Perkin-Elmer 3000 DV instrument after digestion of the SiO2/Imi/Pt sample with HF (48%) and aqua regia. N2 adsorption-desorption isotherms of SiO2/Imi and SiO2/Imi/Pt materials were measured at 77 K on a Quantachrome Autosorb 1 instrument. The samples were previously outgassed at 353 K for 12 h. The Brunauer-Emmett-Teller (BET) method was employed to calculate the specific surface areas (SBET). Scanning electronic microscopy (SEM) images were acquired in a FEI Quanta FEG 250 scanning electron microscope, operating at 20 kV. The samples were fixed onto double-faced carbon tape and carboncoated in a Bal-Tec MD20 instrument. TEM images were obtained on a Jeol JEM 2100 HTP transmission electron microscope operating at 200 kV. The powders were ultrasonically suspended in deionized water for 30 min and the suspensions were deposited on carbon-coated gold grids.

3. Results and Discussions The amount of imidazolium functional groups chemically bonded on the surface of SiO2/Imi material was determined by means of elemental analyses. Based on the value of N content, the degree of functionalization obtained for SiO2/Imi was 0.5 mmol g-1. The amount of loaded platinum determined by ICP-OES was 0.039 weight %. The

8

porosity of the SiO2/Imi (284 m2/g) and SiO2/Imi/Pt (246 m2/g) materials were evaluated by nitrogen adsorption-desorption, whose isotherms are presented in Figure 2(a). Both are type IV isotherms (according to the IUPAC classification), typical for mesoporous materials (Lowell et al. 2004a, b; Rouquerol et al. 1999a, b). The observed hysteresis loops are H2 type, often associated with porous materials consisting of disordered porous structure and broad pore size distribution. No obstruction or blockage of the pores was observed for the material presenting the supported Pt, suggesting that the metallic structures may be available for further catalytic reactions. The SEM image presented in Figure 2(b) shows that the SiO2/Imi/Pt sample is composed of agglomerates of irregular and undefined shape and with a broad particle size distribution. The transmission electron microscopy (TEM) shown in Figure 2(c) certifies that the Pt structures are present as nanoparticles (particle size around 2.5 nm) well dispersed and sparsely found into the porous structure, which may be strongly related to the low concentration of the noble metal (0.039 wt %). No evidence of Pt NPs on the outer surface of the silica particles was observed.

9

Figure 2. N2 adsorption-desorption isotherms of SiO2/Imi and SiO2/Imi/Pt in (a), a SEM image of the SiO2/Imi/Pt sample in (b) and a TEM image in (c).

The catalytic properties of the SiO2/Imi/Pt porous platform and the ability of Pt NPs of mimicking the HRP activity were evaluated toward the H2O2-mediated oxidation of TMB (peroxidase substrate). For sake of comparison, the enzymatic catalytic activity was also evaluated by immobilizing HRP on the Pt-free SiO2/Imi material. Upon addition of SiO2/Imi/HRP or SiO2/Imi/Pt material to solutions containing TMB and H2O2, the catalytic oxidation of the substrate yielded a typical blue color reaction for both samples (Josephy et al. 1982), which turned yellow after addition of sulfuric acid and the pH lowering. The absorbances at 450 nm for solutions with increasing concentrations of H2O2 are shown in Figure 3. The catalytic activities of SiO2/Imi/HRP and SiO2/Imi/Pt towards TMB oxidation were H2O2-concentration-dependent, making possible the development of a colorimetric method for hydrogen peroxide detection. The SiO2/Imi/Pt nanostructured-based artificial system exhibited a linear behavior within an H2O2 concentration range ten times higher than the enzyme-based SiO2/Imi/HRP, and a limit of detection (LOD) of 7.5 x 10-5 M. The calculated Michaelis-Menten apparent Km values toward H2O2 for SiO2/Imi/HRP and SiO2/Imi/Pt are 0.48 mM and 5.85 mM respectively, indicating that in the studied conditions the HRP has higher affinity for the substrate than the Pt nanostructures, and also that higher concentrations of H2O2 are required for artificial system to achieve maximal activity.

10

Moreover, comparing the Km values for the immobilized HRP and soluble HRP (Gao et al. 2007; Lu et al. 2015), the similarity of the values suggests that hydrogen peroxide diffusion is not affected in a significant way by the material, and also indicates that the immobilization procedure is not damaging the enzyme. The inhibition of the peroxidase-like catalytic activity of SiO2/Imi/Pt at higher H2O2 concentrations followed the same behavior of other nanomaterials-based peroxidase mimetics already reported in the literature, as is also observed for the enzyme catalyzed reaction (Gao et al. 2007; Gao et al. 2014; Lin et al. 2014b; Su et al. 2015; Wang et al. 2015; Zhang et al. 2014). According to the previous studies involving Pt and other metallic nanostructures, the electron transfer from TMB to OH• radicals which are generated on the surface of Pt due to the hemolytic cleavage of O-O bond of adsorbed H2O2 (Mckee 1969) are the responsible for the catalytic activity and the mimetic comportment.

11

Figure 3. Absorvances at 450 nm for solutions with increasing concentrations of H 2O2 in the presence of TMB chromogenic substrate and different catalysts. (a) and (b) refer to the natural enzyme HRP immobilized on the SiO2/Imi sample and (c) and (d) to the artificial system based on the SiO2/Imi/Pt nanomaterial.

A bioassay for the determination of glucose was carried out as a proof of concept to demonstrate the potentiality of the developed platform for biosensing applications. The immobilization of enzymes, in the present case glucose oxidase (GOx), was performed by ion-exchange reactions with the imidazolium groups attached to the surface. By adjusting the pH to values above the isoelectric point of the enzyme to be immobilized, the predominance of negative charges on the surface of the proteic structure allows efficient electrostatic interactions between the enzyme and the functional positively charged groups. The biological species are kept strongly adsorbed on the surface, as evidenced by the bioassay results which were obtained after five washing/centrifuging cycles of SiO2/Imi/Pt/GOx material with phosphate buffer solution (pH 7.4). Figure 4(a) shows a schematic illustration of the cascade reactions that take place into the porous environment upon addition of a solution containing glucose and TMB. In the first step (reaction 1), glucose is oxidized by GOx in the presence of dissolved O2, yielding an equimolar amount of H2O2 as one of the products of the enzymatic reaction. Subsequently, TMB is oxidized by the OH• radicals

12

originated from the catalytic decomposition of H2O2 on the surface of Pt NPs, producing the blue color reaction as discussed previously.

Glucose

N

N

N

N

Cl-

Cl-

TMBox Si OO O

Si OO O

SiO2

GOx

Pt NP

SiO2/Imi/GOx/Pt

H2O2

(1) Oxidation of glucose by glucose oxidase (GOx), yielding H2O2 (2) H2O2-mediated oxidation of TMB catalyzed by Pt NPs

Figure 4. (a) Schematic illustration of the cascade reactions involved in the colorimetric detection of glucose ; (b) and (c) show the absorvances at 450 nm for solutions with increasing concentrations of glucose after the H2O2-mediated oxidation of TMB by using the hybrid SiO2/Imi/Pt/GOx catalytic biosensing platform.

The absorbances for solutions with increasing concentrations of glucose are shown in Figures 4(b) and 4(c). The SiO2/Imi/Pt/GOx hybrid material exhibited good performance towards the colorimetric detection of glucose, showing a LOD for glucose of 16.3 x 10-5 M and a wide linear range of response, as can be observed in the Fig 4(c). The results clearly show that both catalytic sites, those on the biological specie (GOx) as well as those on surface of the confined Pt NPs are not obstructed or inactivated due 13

to enzyme immobilization, allowing the interest species (glucose, H2O2 and TMB molecules) to diffuse inside the porous structure and the cascade reactions to proceed normally. Possible conformational changes on the proteic structure that may happen owing to the immobilization process and that cause inactivation of the enzymatic activity were not observed in this study, indicating the efficiency of the imidazolium cationic groups to keep active biological units attached to the functionalized surface. The catalytic platform containing the Pt NPs has shown no significant alterations on the catalytic activity towards the colorimetric sensing of H2O2 after six months of its preparation.

4. Conclusions The proposed hybrid platform based on supported Pt nanoparticles highly dispersed on imidazolium functionalized mesoporous silica has shown peroxidase enzyme-like catalytic properties, here evaluated towards the H2O2-mediated oxidation of 3,3’,5,5’tetramethylbenzidine (TMB). This attribute was explored for the colorimetric detection of H2O2, whose results indicate a linear response range for the artificial system (SiO2/Imi/Pt) which is ten times wider than the natural one that makes use of HRP enzyme immobilized on the Pt-free material (SiO2/Imi/HRP). A bioassay involving the colorimetric determination of glucose was also successfully performed by immobilizing glucose oxidase enzyme (GOx) on the catalytic platform SiO2/Imi/Pt. Upon addition of glucose and TMB, a cascade reaction initiates with the oxidation of glucose, followed by the formation of H2O2 and the subsequent H2O2-mediated catalytic oxidation of the TMB chromogenic substrate on the surface of the metallic Pt nanostructures. The catalytic sites remain accessible even after the immobilization of the biological species, as evidenced by the tested bioassay. The enzyme-like catalytic 14

properties of the SiO2/Imi/Pt as well as its ability to immobilize and keep active biological entities on the porous structure indicate that this hybrid porous platform is potentially useful for the development of biosensing devices.

Acknowledgments The authors thank the financial support from São Paulo Research Foundation (FAPESP), National Council for Scientific and Technological Development (CNPq), Coordination for the Improvement of Higher Level Education Personnel (CAPES) and National Institute of Science and Technology in Bioanalytics (INCTBio). The authors are indebted to their respective founding agencies, CMM (CNPq), VBM (CNPq) and GPS (FAPESP). We are also grateful to LNNano/CNPEM (Campinas-SP) for the TEM measurements.

References André, R., Natálio, F., Humanes, M., Leppin, J., Heinze, K., Wever, R., Schröder, H.C., Müller, W.E.G., Tremel, W., 2011. Adv. Funct. Mater. 21(3), 501-509. Artiglia, L., Agnoli, S., Paganini, M.C., Cattelan, M., Granozzi, G., 2014. Acs Appl. Mater. Interf. 6(22), 20130-20136. Asati, A., Santra, S., Kaittanis, C., Nath, S., Perez, J.M., 2009. Angew. Chem. Int. Edit. 48(13), 2308-2312. Cai, K., Lv, Z., Chen, K., Huang, L., Wang, J., Shao, F., Wang, Y., Han, H., 2013. Chem. Commun. 49(54), 6024-6026. Campelo, J.M., Luna, D., Luque, R., Marinas, J.M., Romero, A.A., 2009. Chemsuschem 2(1), 18-45. Chen, H., Li, Y., Zhang, F., Zhang, G., Fan, X., 2011. J. Mater. Chem. 21(44), 17658-17661. Fattori, N., Maroneze, C.M., Da Costa, L.P., Strauss, M., Sigoli, F.A., Mazali, I.O., Gushikem, Y., 2012. Langmuir 28(27), 10281-10288. Fedlheim, D.L., Foss, C.A., 2001. Metal Nanoparticles: Synthesis, Characterization, and Applications. Taylor & Francis. Frey, A., Meckelein, B., Externest, D., Schmidt, M.A., 2000. J. Immunol. Methods 233(1-2), 47-56. Gao, L., Zhuang, J., Nie, L., Zhang, J., Zhang, Y., Gu, N., Wang, T., Feng, J., Yang, D., Perrett, S., Yan, X., 2007. Nat. Nanotechnol. 2(9), 577-583. Gao, Z., Hou, L., Xu, M., Tang, D., 2014. Sci. Rep. 4, 3966. Ge, S., Liu, F., Liu, W., Yan, M., Song, X., Yu, J., 2014. Chem. Commun. 50(4), 475-477. He, W., Liu, Y., Yuan, J., Yin, J.J., Wu, X., Hu, X., Zhang, K., Liu, J., Chen, C., Ji, Y., Guo, Y., 2011. Biomaterials 32(4), 1139-1147.

15

He, W., Wu, X., Liu, J., Hu, X., Zhang, K., Hou, S., Zhou, W., Xie, S., 2010. Chem. Mater. 22(9), 2988-2994. Hoffmann, F., Cornelius, M., Morell, J., Froba, M., 2006. Angew. Chem. Int. Edit. 45(20), 3216-3251. Jiao, X., Song, H., Zhao, H., Bai, W., Zhang, L., Lv, Y., 2012. Anal. Methods 4(10), 32613267. Josephy, P.D., Eling, T., Mason, R.P., 1982. J. Biol. Chem. 257(7), 3669-3675. Lin, T., Zhong, L., Guo, L., Fu, F., Chen, G., 2014a. Nanoscale 6(20), 11856-11862. Lin, T., Zhong, L., Song, Z., Guo, L., Wu, H., Guo, Q., Chen, Y., Fu, F., Chen, G., 2014b. Biosens. Bioelectron. 62, 302-307. Lin, Y., Ren, J., Qu, X., 2014c. Acc. Chem. Res. 47(4), 1097-1105. Liu, Y.L., Fu, W.L., Li, C.M., Huang, C.Z., Li, Y.F., 2015. Anal. Chim. Acta 861(0), 55-61. Lowell, S., Shields, J., Thomas, M., Thommes, M., 2004a. Adsorption Isotherms. Characterization of Porous Solids and Powders: Surface Area, Pore Size and Density, pp. 1114. Springer Netherlands. Lowell, S., Shields, J., Thomas, M., Thommes, M., 2004b. Mesopore Analysis. Characterization of Porous Solids and Powders: Surface Area, Pore Size and Density, pp. 101-128. Springer Netherlands. Lu, C., Liu, X., Li, Y., Yu, F., Tang, L., Hu, Y., Ying, Y., 2015. Acs Appl. Mater. Interf. 7(28), 15395-15402. Mckee, D.W., 1969. J. Catal. 14(4), 355-&. Nie, G., Zhang, L., Lei, J., Yang, L., Zhang, Z., Lu, X., Wang, C., 2014. J. Mater. Chem. A 2(9), 2910-2914. Rouquerol, F., Rouquerol, J., Sing, K., 1999a. Chapter 4 - Interpretation of Physisorption Isotherms at the Gas-Solid Interface. In: Rouquerol, F., Rouquerol, J., Sing, K. (Eds.), Adsorption by Powders and Porous Solids, pp. 93-115. Academic Press, London. Rouquerol, F., Rouquerol, J., Sing, K., 1999b. Chapter 7 - Assessment of Mesoporosity. In: Rouquerol, F., Rouquerol, J., Sing, K. (Eds.), Adsorption by Powders and Porous Solids, pp. 191-217. Academic Press, London. Soler-Illia, G.J.A.A., Azzaroni, O., 2011. Chem. Soc. Rev. 40(2), 1107-1150. Song, Y., Qu, K., Zhao, C., Ren, J., Qu, X., 2010. Adv. Mater. 22(19), 2206-2210. Su, L., Qin, W., Zhang, H., Rahman, Z.U., Ren, C., Ma, S., Chen, X., 2015. Biosens. Bioelectron. 63, 384-391. Sun, Y., He, K., Zhang, Z., Zhou, A., Duan, H., 2015. Biosens. Bioelectron. 68(0), 358-364. Van Der Voort, P., Esquivel, D., De Canck, E., Goethals, F., Van Driessche, I., RomeroSalguero, F.J., 2013. Chem. Soc. Rev. 42(9), 3913-3955. Wang, Z., Yang, X., Yang, J., Jiang, Y., He, N., 2015. Anal. Chim. Acta 862(0), 53-63. Wei, H., Wang, E., 2008. Anal. Chem. 80(6), 2250-2254. Wei, H., Wang, E., 2013. Chem. Soc. Rev. 42(14), 6060-6093. White, R.J., Luque, R., Budarin, V.L., Clark, J.H., MacQuarrie, D.J., 2009. Chem. Soc. Rev. 38(2), 481-494. Zhang, L.N., Deng, H.H., Lin, F.L., Xu, X.W., Weng, S.H., Liu, A.L., Lin, X.H., Xia, X.H., Chen, W., 2014. Anal. Chem. 86(5), 2711-2718.

Highlights

Supported Pt nanoparticles were successfully synthesized on a porous platform based on silica chemically functionalized with cationic imidazolium groups.

16

The resulting hybrid material has shown peroxidase-like catalytic activity that allowed its application in the colorimetric sensing of H2O2 as a mimetic system. A bioassay for the colorimetric determination of glucose was successfully conducted as a model system through the immobilization of glucose oxidase on the platform.

17