Luminescent mesoporous nanorods as photocatalytic enzyme-like peroxidase surrogates

Showcasing research from Dr Jose L. Hueso and Professor Jesus Santamaria’s laboratory, Institute of Nanoscience of Aragon, Department of Chemical and Environmental Engineering and CIBER-BBN, University of Zaragoza, Zaragoza, Spain. Luminescent mesoporous nanorods as photocatalytic enzyme-like peroxidase surrogates This work describes the synthesis and use of luminescent mesoporous rods as photo-activated nanozymes. Rod-shaped mesoporous nanoplatforms can be fl ash-thermally activated to promote the formation of carbon dots and Si-based emitting centers with enhanced photocatalytic response as novel metal-free peroxidase-like surrogates upon blue-LED activation. Furthermore, these inorganic photocatalysts are successfully tested as optical sensors for hydrogen peroxide and glucose detection. As featured in: See JoseL. Hueso, Jesus Santamaria et al., Chem. Sci., 2018, 9, 7766. rsc.li/chemical-science Registered charity number: 207890 Luminescent mesoporous nanorods as photocatalytic enzyme-like peroxidase surrogates† M. Carmen Ortega-Liebana, ab Jose L. Hueso, * ab Rodrigo Fernandez-Pacheco, c Silvia Irusta ab and Jesus Santamaria * ab Herein we report on a novel inorganic peroxidase-mimicking nanocatalyst activated under blue LED photoirradiation. A novel flash-pyrolysis method has been developed for the generation of strong blue photoluminescence (PL) centers attributed to silicon and carbon-based sites within a mesoporous SBA15 silica nanorod platform. The type of centers and their PL response can be controlled by varying the flash thermal treatment conditions. By tailoring the operating conditions the system can be driven towards the preferential generation of carbon-based luminescent centers, with or without the simultaneous generation of silicon-based centers. The properties and the nature of these luminescent centers within the mesoporous nanorods have been thoroughly corroborated by a battery of characterization techniques including fluorescence spectroscopy, X-ray photoelectron spectroscopy (XPS) and electron energy loss spectroscopy (EELS) at the local level of the structures combined with scanning transmission electron microscopy (STEM) imaging. In addition, these luminescent mesoporous nanorods have been successfully tested as robust photocatalysts able to display peroxidase-like activity and indirect glucose sensing in a wider range of pH conditions compared to the natural enzyme, especially when carbogenic dots and oxygen-deficient silica centers are simultaneously present in the structure. Introduction Natural enzymes are widely used biocatalysts that combine high substrate specicity with excellent chemo-, regio-, and stereoselectivity under mild reaction conditions. They have lately attracted much interest in medical, biological and industry elds. However, natural enzymes suffer from inherent shortcomings such as high cost of preparation and purication, low stability due to denaturation, lack of sensitivity or catalytic activity due to environmental conditions and limited possibilities for recovery and reutilization. These drawbacks somehow restrain their practical applications. In order to overcome these problems, alternative candidates to mimic the working principles of natural enzymes are being currently explored. These alternative surrogates have been coined as articial enzymes or enzyme-mimicking materials and are considered as promising, highly stable and affordable candidates to natural enzymes in a wide range of applications. 1,2 As a consequence, the last decade has witnessed an increasing research effort devoted to the design and development of efficient articial mimicking enzymes. 3,4 Inorganic nanomaterials provide an appealing alternative due to their attractive properties, including stability, catalytic response and biocompatibility. Among enzymes peroxidases have received special attention due to their key role in multiple metabolic activities and their potential use as biomarkers in multiple diseases. Furthermore, peroxidases include a large family of isoenzymes found in almost all living organisms. These enzymes typically catalyze biological reactions where peroxides are reduced (e.g. hydrogen peroxide, H 2 O 2 ), while a redox substrate acting as an electron donor is simultaneously oxidized. It should be noted that the nature of the electron donor is very much dependent on the structure of the enzyme. Through this catalytic process, peroxidases can scavenge H 2 O 2 , a naturally occurring byproduct of oxygen metabolism in human body, resulting in the formation of water and oxygen. In this way, peroxidases play an important role as highly efficient antioxidants to combat complications engendered by reactive oxygen species (ROS). 5 In addition, peroxidases play a vital role in combination with the glucose oxidase (GOx) to determine glucose concentrations through cascade catalytic reactions. 6 A variety of nanomaterials a Institute of Nanoscience of Aragon (INA), Department of Chemical Engineering and Environmental Technology, University of Zaragoza, 50018 Zaragoza, Spain. E-mail: [email protected]; jesus.san[email protected] b Nerworking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain c Advanced Microscopy Laboratory (LMA), Institute of Nanoscience of Aragon (INA), University of Zaragoza, 50018 Zaragoza, Spain †Electronic supplementary information (ESI) available: Tables with additional experimental synthesis conditions, additional information regarding the spectroscopic characterization of the catalysts and additional details on the enzymatic assays carried out with the luminescent nanorods. See DOI: 10.1039/c8sc03112f Cite this: Chem. Sci.,2018,9,7766 All publication charges for this article have been paid for by the Royal Society of Chemistry Received 13th July 2018 Accepted 24th August 2018 DOI: 10.1039/c8sc03112f rsc.li/chemical-science 7766 |Chem. Sci.,2018,9, 7766–7778 This journal is © The Royal Society of Chemistry 2018 Chemical Science EDGE ARTICLE Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue including magnetic nanoparticles (NPs), 7–10 metallic NPs, 11–13 carbon nanomaterials, 14,15 silicon 16,17 or their combinations 18,19 have been recently tested as articial inorganic enzymes mimicking the role of their natural peroxidase counterparts in environmental and biomedical elds. Nevertheless, the search for novel, innovative, affordable and stable enzymatic surrogates continues to be the focus of intense research activity. Particularly desirable are novel enzyme-mimicking nanomaterials with photo-enhanced response and capable of remote activation using light, a subject of great interest. 20 Herein we present a novel inorganic nanoplatform based on luminescent mesoporous silica nanorods (hereaer LMS) with enhanced peroxidase-like activity, especially upon blue-light irradiation. This system does not require the presence of any metal or transition metal elements since it is based on the optical response of carbon and silica-based emitting centers. These centers have been generated in situ within the mesoporous network of SBA-15 nanorods. Remarkably, a direct carbonization of the surfactant molecules conned within the ordered mesoporous channels can be achieved aer a few minutes of ash pyrolysis treatment under an inert gas stream. Alternatively, the ash-thermal treatment can be tuned to induce a carbothermic reduction of silica domains when static conditions (no carrier gas supply) are used. A very fast heating to the pyrolysis temperature was necessary to accomplish pyrolysis before the carbon precursors had time leave the silica pores. This fast heating could be achieved by immersion in a uidized bed reactor at 500 C. This method contrasts with previous reports on the literature that generate luminescent mesoporous materials via the ex situ attachment of photoactive species such as quantum dots or organic dyes. These species are typically non-environmentally friendly, require complex functionalization steps and are oen prone to photobleaching. Other impressive reports on mesoporous structures with embedded quantum-conned silicon centers typically require high thermal treatments and/ or subsequent etching with strong acids. 21–25 Furthermore, He et al. reported on the generation of luminescent oxygen de- cient centers by calcination treatment of silica substrates. 26,27 In this work, a different set of optically active centers based on carbon nanodots, Si–C(O) domains and/or oxygen-decient silica centers have been generated within mesoporous silica nanorods by subjecting our silica nanostructures, with their pores still containing the structure-directing agent (SDA) to auidized-bed ash heating process. Species identication has been conrmed by a panoply of techniques: uorescence spectroscopy, XPS, high-resolution (S)TEM imaging and EELS. Moreover, we have shown the ability of this novel platform (made of abundant, environmentally friendly silicon and carbon based materials) to behave as a metal-free enzyme-like photocatalyst, offering a broad optical response in the visible range in combination with excellent thermal and chemical stability. Interestingly, the simultaneous presence of both carbon and silica-based emitting species produced nanostructures with the highest peroxidase-like activity upon LED irradiation. In contrast to previous studies on the peroxidasemimic catalytic activity of freestanding carbon dots 15 and silicon dots, 16 the photo-activated LMSs structures demonstrated high enzyme-like activity over a broad pH range (5.0–7.0) even at neutral pH, which overcomes one of the major shortcomings of currently developed articial enzymes (typically suffering from deactivation when operating at near neutral pH conditions). 28 The high stability of the structures prepared in this work allowed these LMSs platforms to be successfully reused for multiple cycles. It is also worth mentioning that the peroxidase-mimicking role of the LMS photocatalysts was further evaluated in a cascade reaction in combination with other enzyme such as glucose oxidase (GOx). The combination of both systems enabled a rapid and robust quantication of glucose using a colorimetric assay. This new type of metal-free articial enzyme exhibited high sensitive and selective response toward glucose detection only activated under LED blue irradiation. Results and discussion Flash-thermal assisted generation of luminescent mesoporous rod-shaped nanocatalysts The ordered mesoporous silica (MS) nanorods were synthesized using Pluronic P123 as SDA and Tetraethyl orthosilicate (TEOS) as silicon source following a hydrothermal approach reported elsewhere. 29 In order to induce the generation of light-emitting centers within the silica nanorods, these were placed in a thin wall 8 mm diameter quartz tube, which was immersed in a sand uidized-bed reactor at 500 C. The samples were ash-heated to the bed temperature in less than 40 s (Scheme 1 and Table S1†). This ash heating protocol induced luminescent sites within the mesoporous rods while keeping their ordered Scheme 1 Aflash heating of SDA-containing silica nanorods induced the generation of different light-emitting centers within its structure. The conditions could be tailored to obtain only carbon dots in the silica structure (LMS@C) or carbon dots plus silica-based emitting species (LMS@Si@C). A third set of samples containing only luminescent silicon-based species (LMS@Si) obtained by a slow oxidation posttreatment of the LMS@Si@C sample in a conventional furnace. Different reaction atmospheres (flowing N 2 or static atmosphere) were used to tailor the preferential formation of the different luminescent sites during the flash-thermal treatments. This journal is © The Royal Society of Chemistry 2018 Chem. Sci.,2018,9, 7766–7778 | 7767 Edge Article Chemical Science Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online structure (Fig. S1 and Table S2†). Furthermore, the selection of different reaction ambients derived in a different photoluminescence response of the resulting materials (Scheme 1 and Fig. 1). Thus, ushing the samples with nitrogen during pyrolysis led to the preferential generation of carbogenic dots (sample hereaer referred as LMS@C) due to the in situ carbonization of the SDA encapsulated within the mesoporous channels. HRTEM analysis further conrmed the presence of graphitic-like lattice distances (Fig. 1a and S2†). Interestingly, when a static atmosphere with no continuous owing conditions was set, the simultaneous co-generation of a second type of emitting species took place. HR-TEM images and indexing conrmed the coexistence of graphitic dots and additional lattice fringes matching with silicon carbide (see Fig. 1b, S2 and Table S1†). The dynamics (static vs. ow conditions) in the system were even more important than the atmosphere used. Under static conditions (even when air was no removed from the reactor), the P123 surfactant molecules not only generated carbon dots, but also acted as carbonaceous reducing agents (i.e. carbothermic reduction) of the surrounding O 3 Si–O–SiO 3 network that was in close contact (Scheme 1). The partial reduction of the silica units generated defect-luminescent features in the structure stemming from oxygen-decient silica centers 26 and/ or alternatively carbon-doped silica domains 30,31 (sample referred as LMS@Si@C). As a result, a different PL response Fig. 1 Characterization of the luminescent silica nanorods generated under different conditions (top row: flowing N 2 , middle: static conditions, bottom: static conditions plus post-synthesis calcination): (a–c) high resolution TEM images of small crystalline dots identified within the mesostructure of the LMSs networks attributable to graphitic-like carbon dots (black or yellow circles) or alternatively to silicon carbide domains (white circles); (d–f) photoluminescence spectra corresponding to the LMSs at different excitation wavelengths from 365 to 480 nm insets: digital photographs corresponding to the powdered LMSs and their aqueous suspension under irradiation with a UV lamp (l exc ¼365 nm); (g–i) fitted X-ray photoemission contributions in the Si 2p region acknowledging different oxidation states for the LMS@C, LMS@Si@C and LMS@Si samples, respectively. 7768 |Chem. Sci.,2018,9, 7766–7778 This journal is © The Royal Society of Chemistry 2018 Chemical Science Edge Article Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online could be identied (Fig. 1e) in comparison with the rst LMS@C sample (Fig. 1d). In order to clearly identify the emission tentatively attributed to defect-silica and/or carbon-doped silica sites, an additional calcination step was carried out using a slower (5 C min 1 ) temperature ramp in order to remove all carbonaceous species (carbon dots and residues from the pyrolysis of the SDA still present in the pores) from samples LMS@C and LMS@Si@C (Scheme 1). In this way, the contribution of carbon dots could be ruled out. Interestingly, only the LMS@Si@C samples (i.e. those initially prepared under static conditions) were able to maintain the luminescence aer the calcination step (Fig. 1f and Table S1†). Aer the removal of carbon dots, the specic contribution of partially reduced silica emission centers or Si– C–O domains could be clearly discerned, with characteristic emission peaks clearly distinguishable from the carbon dots typical luminescent features. 30–34 This sample containing only silicon-related emitting species was labeled as LMS@Si for subsequent use and evaluation (Scheme 1). To corroborate that the generation of luminescent centers was due to the use of the ash pyrolysis method, slow conventional calcination treatments were carried out for comparison under identical reaction atmospheres and temperatures (Table S1†). None of the control experiments under conventional heating conditions rendered any luminescent nanostructures (Table S1†). HR-TEM images also identied the presence of encased crystalline nanodomains (Fig. 1a and b). Previous researchers have also identied luminescent Si centers by using a variety of calcination procedures on Si-containing materials. 34,35 Our previous research had already shown that it is possible to use a nanoporous structure to load organic precursors that, upon rapid pyrolysis, yield carbon dots conned within the pore channels. 36 In this work, instead of loading an external Ccontaining species, we have used as precursor the SDA already present in the pores, since it is used during the synthesis of the silica nanostructure. The SDA has the advantage of being already conned in the pores and to have a much stronger interaction with the silica walls (unlike adsorbed organic molecules, it cannot be simply desorbed: removing it from the structure requires calcination at temperatures in excess of 500 C).Asaresult,theP123micellesencapsulated within the mesoporous channels of LMSs are carbonized in situ, yielding conned carbon dots and generating oxygen-decient silica centers, while preserving the silica mesostructure. On the other hand, the chemical structure of the SDA may also play a role in the process, since organic groups are known to preferentially induce dehydrogenation events in surrounding/neighboring Si–O networks. This induces the formation of multiple oxygen vacancies, surface defects and the appearance of oxygen decient centers as previously postulated in previous works. 26,30,31,33,34 Spectroscopic analysis of the silicon and carbon-based emitting centers present in the rod-shaped luminescent mesoporous nanocatalysts The optical properties of the different LMSs were evaluated by uorescence spectroscopy. The as-prepared rods exhibited no luminescent properties (Table S1†). However, aer the ashthermal treatment, remarkable PL properties of LMSs were observed. As displayed in Fig. 1d and e, as the excitation wavelength increased from 365 to 480 nm, the emission peak gradually red shied from 434 to 516 nm, which suggests that the photo-luminescence of the LMS@C and LMS@Si@C are excitation wavelength dependent. Previous reports have attributed this optical behavior and blue emission to the presence of carbogenic dots subjected to quantum connement effects. 37,38 In order to corroborate this hypothesis, different control experiments were carried out. Both the LMS@C and LMS@Si@C and the non-heated nanorods were digested with NaOH to etch the mesoporous structure (Fig. S3†). Aer a puri- cation process (centrifugation and dialysis), a yellow clear suspension was obtained. This suspension also exhibited PL response, that is, a strong luminescence remained aer etching the silica supports (Fig. S3†). Strong photoluminescence was also observed when the aqueous suspension was irradiated under a UV lamp. Finally, TEM analysis conrmed the presence of the nanoparticles still present aer the basic etching treatment (Fig. S3†) in the ash-thermally activated LMSs samples. These results and the absence of optical response in the nontreated nanorods (Fig. S3†) further conrmed the in situ generation of carbon nanodots within the mesochannels, as a consequence of the fast pyrolysis of the co-directing surfactant molecules present within the mesochannels. Previous theoretical and experimental reports have suggested a variety of potential mechanisms behind the PL response of carbon dots such as the presence of surface state defects, 39 molecule-like state, 40 sp 2 -carbon networks 41 and quantum size effects, 42 etc. Mechanistically, the photoluminescence from@C may be attributed to the presence of surface energy traps that become emissive upon stabilization as a result of the surface passivation. 43 On the other hand, the emission spectra of the LMS@Si (Fig. 1f) exhibited a different structure tentatively associated to the formation of oxygen-decient silica centers. Here, the formation process is attributed to the partial reduction of surface species favored by the strong interaction of the P123 surfactant molecules with the surrounding silica units that upon pyrolysis in addition to generating carbon dots acted as carbonaceous reducing agents of the O 3 Si–O–SiO 3 network. An analogous control experiment digesting the LMS@Si sample with NaOH did not render any PL response, thereby conrming the origin of the emission on silica-related centers which are no longer available aer the chemical etching treatment (see Fig. S4†). In summary, the blue photoluminescence observed can be ascribed to oxygen-decient silica centers with Si–Si bonds. The identication of the actual centers responsible for luminescence remains under discussion. Previous reports on blue photoluminescence of treated silica have been attributed to the surface passivation of small silicon nanocrystalline domains; 32,44 or the presence of Si–Si–O 3 species on the silicon NPs networks. 33,45 More recent reports with similar temperature treatments showed that the PL response is highly dependent on the heating environment. Uchino et al. demonstrated that the This journal is © The Royal Society of Chemistry 2018 Chem. Sci.,2018,9, 7766–7778 | 7769 Edge Article Chemical Science Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online dehydroxylation reaction under air resulted in the formation of a defect pair consisting of dioxasilirane (]Si(O 2 )) and silylene (]Si:). 46 Additional authors also correlated the appearance of PL properties to the appearance of oxygen defects and vacancies under air heating conditions. 33,35 Alternatively, different authors have associated the origin of the different emission peaks to the presence of Si–C–O domains. 30,31,47–49 To further explore the ash-thermal treatment effects, the different luminescent nanorods were also analyzed by X-ray photoelectron spectroscopy (XPS). High-resolution scanning of the Si 2p core level spectra is shown in Fig. 1g–i. The Si 2p region corresponding to the LMS@C sample was tted into two main components (Fig. 1g), the one at higher binding energy (102.8 eV) associated to Si–O bonds and the peak at 102.0 eV attributed to Si–CH x bonds. 47 This latter component would conrm the presence of carbon-enriched silica species on the material surface. Previous reports 50 have also formed carbonaceous deposits inside mesoporous silica by calcination at a high temperature (900 C for 3 h) under nitrogen protection. However in this case no luminescent carbon dots were observed, instead, signicant carbonaceous deposits helped to maintain the silica structure in place. In a different work carbon dots were generated by calcination of carbon-containing precursors inside narrower silica pores, but a complex procedure was used involving extraction of the SDA under saturated solution of HCl, followed by functionalization of the silica previous to calcination (at 400 C for 2 h). No luminescent Si centers were observed. 51 The LMS@Si@C sample also showed two main contributions in the Si 2p spectrum at 101.9 and 101.1 eV, respectively (see Fig. 1h). Besides the Si–CH x bonds, the contribution at lower binding energy would indicate the presence of silicon atoms forming Si– Si bonds 34 and conrming Si reduction as a result of partial dehydroxylation events in the silica network (Scheme 1). 31,33 This is in agreement with our previous assumption that the P123 surfactant molecules act as reducing agent generating oxygendecient silica centers. Aer slow conventional calcination treatments, the Si 2p contribution of LMS@Si evolved into three components at 101.1, 101.9 and 103.3 eV indicating that some reoxidation occurred, as shown in Fig. 1i (see also Table S3†). The analysis of C 1s and O 1s signals conrmed the presence of Si– CH x ,C–OandC–OH surface groups (Table S3†). 47,52,53 In order to analyze the chemical structure of the different materials at the nanoscale level, electron energy loss spectra were acquired in a probe-corrected microscope in scanning transmission (STEM) mode. The sample was scanned with a very thin probe (around 0.1 nm in diameter) so that High Angle Annular Dark Field (HAADF) images and EELS spectra were collected simultaneously for each point. The analysis of the lowloss region of the EEL spectrum is probably the most direct way to identify different silicon-based species. 54–56 Different material phases were identied by measuring the plasmon energy of the obtained materials and comparing those to external ref. 57 (see Fig. 2). In the case of LMS@C, the presence of a plasmon peak at 22.2 eV matched well with the presence of silicon carbide (Si–C) and carbon structures (Fig. 2a). These assignations agree with the XPS analysis previous described (vide supra). For the other structures, the overlapping of several peaks in a very narrow region increased the difficulty to clearly distinguish one single material. Therefore, a combination of several phases is the most probable scenario. Thus, for the LMS@Si@C sample, there is a broadening of the plasmon signals. This can be explained as a sum of the plasmon peak of SiO 2 at 23.5 eV and a broad shoulder most probably attributabletothepresenceofSiinareducedstateat16.8eV. 58 This is clearly visible throughout the analysis of multiple points of a single nanorod, either in the outer or inner areas (Fig. 2b). Regarding the LMS@Si structure, no presence of carbon or SiC contribution was observed. Instead, a combination of more reducedSisignalwasmainlydetectedintheinnerareas whereas the outer fraction of the nanorod was more compatiblewiththepresenceofmoreoxidizedphaseswiththeplasmon peak of SiO 2 (Fig. 2c). This latter outcome reinforces the hypothesis of a dehydroxylation pathway to render SiO 2 and Si–Si domains. 33,46 In conclusion, all the spectroscopic techniques further suggest that the origin of the induced photoluminescent properties in the rod-shaped mesoporous supports can be tentatively ascribed to the combined generation of carbogenic centers, silica domains in a reduced oxidation state (i.e. generation of oxygen vacancies) and/or the appearance of Si–C(O) centers. Indeed, only ash pyrolysis provides the effect sought, while conventional treatments at the same temperature did not show generation of luminescent centers. The very high speed of heating achieved by uidized-bed aided ash heating is the key to understand this differential behavior. Under ash heating conditions, the material reaches the desired temperature almost instantly. This means that the organic molecules trapped inside the silica pores are quickly pyrolyzed and the decomposition products reach a high temperature before they have time to diffuse out of the pores, a process retarded also by the rod-like shape of the mesoporous support. It should be noted that these organic molecules present a close interaction with the surrounding silica units, since during synthesis they as structure-directing agents around which the silica pores are formed. During that brief heating period at 500 C the structure directing agents are rapidly pyrolyzed, forming carbon-based nanodomains and giving carbon-based pyrolysis products. At high temperatures both species can act as reducers, affecting the neighboring Si–O centers present in the amorphous silica network of the rods. In contrast, under conventional heating conditions, we start from room temperature, with an increase of 10 C min 1 until reaching 500 C. This means that there is ample time for the gaseous species to diffuse out of the pores before they reach temperatures where they could act as in situ reducers. As a consequence, this treatment in spite of its longer duration (in total 2.8 h), did not induce reduction in the nanostructure (see Table S1†). Luminescent mesoporous rods as enzyme-like peroxidase-like photocatalysts Inspired by previous works on the use of freestanding carbon and silicon nanoparticles as peroxidase-mimicking articial 7770 |Chem. Sci.,2018,9, 7766–7778 This journal is © The Royal Society of Chemistry 2018 Chemical Science Edge Article Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online enzymes, 4,15–17,59,60 our luminescent nanorods were tested as a novel metal-free inorganic peroxidase surrogate with high surface area and visible light response. In order to assess the peroxidase-like activity of the LMSs, the 3,30,5,50-tetramethylbenzidine (TMB)–H 2 O 2 assay was used as model reaction. The catalytic oxidation of the peroxidase substrate TMB was monitored in the presence of H 2 O 2 under light excitation. The maximum absorbance of the blue generated TMB oxidation product occurs at 652 nm and is typically ascribed to the chargetransfer complexes of diamine and diimine derived from the one-electron oxidation of TMB (see Fig. 3c and d). In previous studies, it was suggested that the wavelength of the LED used to trigger the oxidation of TMB should match the optimal excitation wavelength of the sensitizer. 60 We initially selected the blue-LED at 405 nm for irradiation to maximize the performance of our photocatalysts based on the maximum absorption/emission spectra (see Fig. 2 and experimental setup in Fig. S9†). Fig. 3a (see also Fig. S5†), compares the enzyme-like activity of LMS@C, LMS@Si and LMS@Si@C. The LMSs showed no evidences of catalytic activity towards TMB oxidation in the absence of light aer 30 min of reaction (data not shown). However, a slight catalytic activity for TMB oxidation under aerobic condition could be detected for expanded periods of time as expected from the charge transfer role of carbon and silicon nanodomains. 15,16 On the other hand, LMS@C, LMS@Si and LMS@Si@C showed increasing activity. In particular, the LMS@Si@C sample exhibited the highest peroxidasemimicking response (Fig. 3a and S5†), with an apparent synergistic effect that suggests the benecial role of combining both type of emitting centers. Furthermore, no signicant oxidation of TMB occurred in the control experiments (see Fig. S6†and inset in Fig. 3a). It is especially remarkable that no response was detected in the absence of H 2 O 2 , thereby con- rming the selective peroxidase-like behaviour of our catalysts (see Fig. S7†). This further conrms that the carbogenic centers encased within our mesoporous supports do not resemble other graphene-like materials with selective photosensitizing capabilities to oxidize TMB in the absence of H 2 O 2 . 59 Interestingly, the irradiation of the catalysts (in the presence of TMB– H 2 O 2 ) with other LED wavelengths (Fig. 3b and also Fig. S8†) rendered a similar catalytic trend as the observed for the former experiments carried out at 405 nm. In contrast, green (532 nm) or red-emitting (740 nm) LEDs rendered a very limited or negligible response. This seems to conrm that the minimum Fig. 2 STEM images (left) and localized EELS low-loss spectra (right) at specific positions of the different luminescent nanorods: (a) LMS@C; (b) LMS@Si@C; (c) LMS@Si samples. EEL spectra are compared with references of C, Si, SiC and SiO 2 , respectively. This journal is © The Royal Society of Chemistry 2018 Chem. Sci.,2018,9, 7766–7778 | 7771 Edge Article Chemical Science Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online threshold energy for photoactivation of our catalysts is given by wavelengths at 460 nm. Taking into account the reaction mechanism reported in previous works 15,61 we attribute the peroxidase-mimicking contribution of the carbogenic dots embedded within the mesochannels to their charge-transfer dual capacity to simultaneously accept electrons from the lonely pair available in the amine group of TMB and donate electrons from their conduction band to H 2 O 2 that is reduced to water (Fig. 3c). The presence of light to induce electron–hole separation further accelerates the complete cycle TMB–H 2 O 2 oxidation–reduction. In contrast, additional active sites that have rendered a major light-driven peroxidase activity (see Fig. 3a) must be associated to the preferential presence of O 3 Si–SiO 3 ,Si–O, Si–OH dangling bonds including multiple surface and reactive defects (induced by the rapid pyrolysis conditions). According to previous studies 62–64 H 2 O 2 preferentially adsorbs onto Si/SiO x sites and reacts to form Si–O electrophilic intermediates that withdraw electron from TMB and promote its oxidation 16,62 (see Fig. 3d). It seems also quite plausible that H 2 O 2 alternatively or simultaneously reacts aer a hemolytic cleavage of strained threemembered Si–O rings 63 that ends up with the production of cOH radicals. This cOH radical generation was conrmed by using disodium terephthalate (NaTA) as a selective probe that especically reacts with cOH radical groups to form 2-hydroxyl disodium terephthalate, a uorescent compound emitting at 425 nm (Fig. S10a†and schematic reaction in Fig. S10b†). These cOH could further react with additional H 2 O 2 molecules thereby accelerating the whole peroxidase-mimicking response. It is also well established that the enzymatic activity can be strongly dependent on the reaction conditions. Therefore, we studied the inuence on the observed activity of LMS@Si@C samples of the concentration of nanomaterials, pH and temperature. The pH was varied from 2 to 9 and the temperature from 20 to 65 C. As shown in Fig. 4a, the catalytic activity increased in the pH range of 5–6, indicating that the oxidation reaction of TMB occurred easily under weakly acidic conditions. Likewise, the optimal performance was estimated at pH 5 for concentrations of 4 mgmL 1 of nanohybrid under irradiation at 405 nm (see Fig. S11†). Thus, unlike natural enzymes such as HRP that operate in a narrow interval of pH, 60 our hybrid materials could perform and remain active in a wide range of pH (Fig. 4a) with an optimal performance around pH 5. In addition, to compare with other studies reported where there is no photo-enzymatic reaction, we evaluated the activity of the hybrid as a function of temperature in the range of 22– 65 C without irradiation. Fig. S12†shows the temperature dependent response curves. The optimal temperature was approximately 45–50 C (as determined from the conversion aer 45 min reaction time). It can be seen that the activity of the nanohybrid was much higher when working at room temperature under LED irradiation compared to thermal activation: 5 minutes of irradiation at room temperature produced a conversion well above that obtained at any working temperature without illumination (see Fig. 4b). It is also worth mentioning that the photo-catalyst also exhibited a high stability aer several LED irradiation cycles (see Fig. S13†). The very small decrease observed per cycle is attributed to the loss of catalyst in the handling between samples. In order to quantify the peroxidase-like activity of the LMS@Si@C a kinetic study of the oxidation of TMB by reduction of H 2 O 2 was carried out. The kinetic parameters were obtained by performing a series of experiments changing the concentrations of one substrate and keeping constant the concentration of the other. To acquire the kinetics parameters, the data were tted to the Michaelis–Menten equation and typical double-reciprocal Lineweaver–Burk plots were displayed (Fig. 5). The parallel trends observed at different substrate concentrations were identied as a clear signal of a so-called Fig. 3 Evaluation of the peroxidase-like activity of the different LMSs. (a) Time-dependent absorbance changes TMB at 652 nm for the different luminescent mesoporous nanorods inset: digital image of wells containing reaction mixtures after 10 min or reaction with no light (1) or under irradiation with blue LED (2, A, B and C): (1) TMB + H 2 O 2 ; (2) TMB + H 2 O 2 ; (A) LMS@C; (B) LMS@Si and (C) LMS@Si@C; (b) Evaluation of the influence of irradiating the LMS@Si@C catalyst with different LED wavelengths. Experimental conditions: [catalyst] ¼4mg mL 1 ; [TMB] ¼0.16 mM; [H 2 O 2 ]¼10 mM; pH ¼7.4 (0.2 M NaAc buffer); total volume ¼2 mL; irradiation time ¼5 min (inset: digital image of wells containing reaction mixtures after 5 min under irradiation with UV LED, blue LED-405, blue LED-460, green LED-532, red LED-740 and white LED, respectively). The error bars represent the standard deviation of three measurements; (c) proposed mechanism in the TMB–H 2 O 2 involving the carbon dots embedded within the mesoporous template; (d) proposed peroxidase-like photoactivation mechanism involving Si–O active sites involving the generation of Si–O intermediates with affinity to withdraw electrons from TMB substrates. 7772 |Chem. Sci.,2018,9, 7766–7778 This journal is © The Royal Society of Chemistry 2018 Chemical Science Edge Article Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online ping–pong mechanism, 15,16 where a transitory catalyst complex is formed during the catalytic reaction. This indicates that, the LMS@Si@C, like horseradish peroxidase (HRP), binds and reacts with the rst substrate (either TMB or H 2 O 2 ), then releases the rst product before reacting with the second substrate. As observed from the kinetic parameters, the active sites of LMS@Si@C created reagent intermediates for TMB oxidation. The combination of @Si@C species facilitated the formation of such intermediates, leading to enhanced catalytic activity. This ping–pong mechanism is also observed in analogous HRPbased systems. Important enzyme catalytic parameters such as the Michaelis–Menten constant (K m ) and the maximum initial velocity (V max ) were obtained. K m is recognized as an indicator of the affinity of an enzyme to its substrate. A strong affinity of the nanoenzyme-substrate is reected by a smaller K m and vice versa. 9 The apparent K m and V max values of LMS@Si@C and other enzyme-like nanomaterials have been summarized for comparison in Table S4.†The low K m values for the LMS@Si@C catalyst indicate high affinity towards both TMB and H 2 O 2 .Itis also worth mentioning that K m values relative to both TMB and H 2 O 2 were remarkably lower than in previously reported arti- cial enzymes with analogous compositions and/or photocatalytic response (Table S4†). This may be due to the fact that photoactive centers are abundant and homogeneously distributed on the high surface area of the mesoporous nanorods. 19 Hence, the lower magnitude of K m values for H 2 O 2 also suggests that a lower concentration of H 2 O 2 was required to successfully complete the TMB oxidation in comparison with many other enzyme-like peroxidase systems. 19,65 The apparent K m and the maximum reaction rate value of photo-activated LMS@Si@C with TMB as substrate was 0.0525 mM and 1.488 nM s 1 , respectively. Compared with that of HRP (K m ¼0.155 mM), 15 the K m value of the photo-activated LMS@Si@C with TMB as substrate was much lower, indicating that LMS@Si@C possess a higher affinity towards the TMB substrate than the natural counterpart (HRP). In addition, the LMS@Si@C with TMB as the substrate had much smaller K m value than other reported nanomaterials with peroxidase-like activities, such as freestanding Si-dots (K m ¼1.502 mM), 16 graphene oxide (GO)- AuNCs nanocomposites (K m ¼0.16 mM) 66 or Fe 3 O 4 @carbon (K m ¼0.072 mM). 66 Furthermore, it is worth mentioning that our photocatalyst exhibit negligible enzymatic activity in the absence of H 2 O 2 (Fig. S7†) or light external stimuli (Fig. S6–S8†). While DNA-complexes have reported an analogous response, 60 Fig. 4 Evaluation of the overall peroxidase-mimicking performance of the LMS@Si@C sample against different reaction parameters: (a) influence of pH (experiments at 19–20 C under irradiation with a blue LED at 405 nm for 5 min); and (b) comparison of the evolution of relative activity with time. Conversion under LED irradiation compared to thermally-driven reaction under optimal conditions (45 C). The experiments were carried out using 4 mgmL 1 of catalyst in 2 mL of 0.2 M NaAc buffer with 0.16 mM TMB as substrate. The H 2 O 2 concentration was 10 mM at pH 7.4 unless otherwise stated. The relative activity in each graph is referred to the maximum reaction rate observed for the experiments represented. The error bars represent the standard deviation of three measurements. Fig. 5 Steady-state kinetic assays using double-reciprocal Lineweaver–Burk plots and determination of the main kinetic constants during the blue-LED induced oxidation of TMB with H 2 O 2 using LMS@Si@C as enzyme-like peroxidase photocatalyst. The velocity (v) of the reaction was measured using 4 mgmL 1 LMS@Si@C in 2 mL of 0.2 M NaAc buffer at pH 6 and photosensitization. The error bars represent the standard error derived from three repeated measurements. Experimental conditions in (a) [TMB] ¼0.8 mM and [H 2 O 2 ] was systematically varied; (b) [H 2 O 2 ]¼50 mM and [TMB] was systematically varied; (c and d) double reciprocal plots of activity of NPs with the concentration of one substrate (H 2 O 2 or TMB) fixed and the other varied. This journal is © The Royal Society of Chemistry 2018 Chem. Sci.,2018,9, 7766–7778 | 7773 Edge Article Chemical Science Open Access Article. Published on 24 August 2018. Downloaded on 9/20/2024 8:50:39 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online