UV-Light-Driven Enhancement of Peroxidase-Like Activity of Mg-Aminoclay-Based Fe 3 O 4 /TiO 2 Hybrids for Colorimetric Detection of Phenolic Compounds

: Light-activated nanozymes possess several advantages, such as light-mediated activity reg-ulation, utilization of molecular oxygen as a green oxidant, and highly enhanced activity; however, the types of light-activated nanozymes are still limited. In this study, we found that Mg aminoclay-based Fe 3 O 4 /TiO 2 hybrids (MgAC-Fe 3 O 4 /TiO 2 ) exhibited peroxidase-like catalytic activity to catalyze the oxidation of the peroxidase substrate 2,2 (cid:48) -azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of H 2 O 2 , which was signiﬁcantly enhanced under ultraviolet (UV)-light irradiation. Compared with MgAC-Fe 3 O 4 and MgAC-TiO 2 , MgAC-Fe 3 O 4 /TiO 2 showed around three-fold enhancement in the absorption intensity corresponding to the oxidized ABTS under UV-light irradiation, presumably due to the synergistic effect between Fe 3 O 4 and TiO 2 , thereby facili-tating photocatalytic electron transfer during the catalytic action. In addition, the MgAC-Fe 3 O 4 /TiO 2 showed vivid stability enhancement in wide range of pH and temperature values compared with natural peroxidase. The UV-light-driven MgAC-Fe 3 O 4 /TiO 2 -based system was successfully applied for the colorimetric detection of phenolic compounds, including pyrocatechol and resorcinol, in a dynamic linear range of 0.15–1.30 mg/mL with a limit of detection as low as 0.1 mg/mL. Further, the system could successfully determine the phenolic compounds in spiked tap water, and thus, it can be used for practical applications. We believe that the UV-light-driven enhancement in the peroxidase-like catalytic performances highlights the potential of MgAC-Fe 3 O 4 /TiO 2 for detecting phenolic compounds as well as other clinically and environmentally important substances. helps the peroxidase-mimicking Fe 3 O 4 -mediated oxidation of ABTS, thereby resulting in an enhanced peroxidase activity of the nanohybrid.


Introduction
Phenolic compounds are considered as one of the most harmful pollutants due to their severe negative impacts on human health and ecosystems, in addition to the difficulty in their biodegradation. They are generally derived from industrial effluents that leach into water reservoirs and result in serious contamination [1]. Therefore, to effectively prevent and treat phenol contamination in water reservoirs, developing a simple, rapid, sensitive, and cost-effective strategy is crucial for the on-site detection of phenolic compounds. To achieve this, various analytical approaches have been proposed, which are based on chromatography [2], electrochemistry [3], fluorometry [4], and colorimetry [5,6]. Among them, colorimetric approach has attracted attention owing to its direct visual response, simple operation, and cost-effectiveness. Typically, colorimetric detection of phenolic compounds is performed through natural peroxidase-mediated oxidative coupling of the target phenols with 4-aminoantipyrine (4-AP) [5]. Although this assay method yields sensitive and accurate detection results, natural peroxidase should be further investigated, as its instability and high-cost significantly limit its practical applications [7].
To overcome the limitations associated with natural enzyme including peroxidase, several types of enzyme mimics have been developed to date. Among them, nanozymes, which are nanomaterials exhibiting enzyme-like activities, have been considered promising for replacing natural enzyme owing to their advantages, such as excellent stability and robustness during long-term operation/storage, low production cost resulting from their facile scale-up synthesis, and easy reutilization resulting from their heterogeneous nature [8,9]. After the first demonstration of intrinsic peroxidase-like activity of magnetic nanoparticles (MNPs), a huge number of nanozymes have been reported and extensively investigated to prove their potential applications in diverse fields [10,11]. Despite the great potentials of nanozymes, their real utilization has been limited majorly by their lower catalytic activity than that of natural enzyme. To resolve the limitation, several strategies to enhance the catalytic activity, such as surface modification with functional ligands, elemental composition engineering, morphology or crystal facet optimization, and development of unique composite, have been reported [12][13][14][15].
In this aspect, light-activated nanozymes have attracted attention due to their capability to catalyze unique photosensitized reactions [16], that can be utilized in diverse applications including chemical synthesis [17], pollutant removal [18], cancer therapy [19], antibacterial applications [20], and biosensing [21]. As light-activated nanozymes, TiO 2 nanoparticles have been representatively investigated since they can efficiently produce hydroxyl or superoxide radicals from the reaction medium under ultraviolet (UV)/visible light irradiation during diverse catalytic reactions, which can be essentially used for oxidizing various organic substances [22]. However, when TiO 2 nanoparticles are used alone, they suffer from low quantum efficiency and narrow light response range [23]. Thus, it is necessary to develop further nanozymes that are capable of efficiently enhancing the catalytic performances under light irradiation.
Herein, we have developed Mg aminoclay-based Fe 3 O 4 /TiO 2 hybrids (MgAC-Fe 3 O 4 / TiO 2 ) as new and efficient light-activated peroxidase-like nanozymes. We previously reported that Fe aminoclay showed intrinsic peroxidase-like activity that can be successfully applied to colorimetrically detect lung cancer cells [24]. As a continuing effort to develop nanozymes, particularly for light-activated ones, MgAC-Fe 3 O 4 /TiO 2 was synthesized via a simple sol-gel reaction. The resulting nanohybrids showed a significant enhancement in peroxidase-like catalytic activity under UV-light irradiation, which is roughly threetimes higher than those of control samples, including MgAC-Fe 3 O 4 and MgAC-TiO 2 . The UV-light-driven MgAC-Fe 3 O 4 /TiO 2 -based system was further applied to colorimetrically determine phenolic compounds, and its analytical features were investigated.

Synthesis of
The synthesized MgAC-TiO 2 , MgAC-Fe 3 O 4 , and MgAC-Fe 3 O 4 /TiO 2 were characterized via transmission electron microscopy (TEM; JEM-2100F, JEOL LTD, Peabody, MA, USA) and scanning electron microscopy (SEM; Hitachi S4700 at the Smart Materials Research Center for IoT at Gachon University for its instrumental support). X-ray diffraction (XRD) analysis (D/MAX-2500, Rigaku Corporation, Tokyo, Japan), equipped with a CuKα radiation generator powered at 40 kV and 30 mA, was used to characterize the crystallinity of the as-prepared samples. The chemical and electronic states of the elements on the surfaces of materials were investigated via X-ray photoelectron spectroscopy (XPS; ES-CALAB250, VG scientific, East Grinstead, UK). The degree of crystallinity (χ c , %) was calculated from the XRD pattern via the following equation [29]: where A c is the area of crystalline peaks and A a is the area of amorphous peaks.

Determination of H 2 O 2 Using MgAC-Fe 3 O 4 /TiO 2 under UV-Light
Peroxidase-like activities of MgAC-Fe 3 O 4 /TiO 2 were examined through the catalytic oxidations of ABTS in the presence of predetermined concentrations of H 2 O 2 . UV-light irradiation at around 312/365 nm was performed using a dual UV transilluminator (Core-Bio, Seoul, Korea). In a typical reaction, MgAC-Fe 3 O 4 /TiO 2 (0.6 mg/mL), ABTS (0.6 mM), and H 2 O 2 at diverse concentrations were added into sodium acetate buffer (0.1 M; pH 4.0), followed by UV-light exposure for 10 min. The control experiments were also carried out following the same assay conditions, but without UV-light exposure. After the reaction, the catalytic materials were separated by centrifugation (10,000 g; 2 min). The absorption intensities corresponding to the oxidized ABTS were measured in a scan mode from 380 to 500 nm or at 417 nm using a microplate reader (Synergy H1, BioTek, Winooski, VT, USA, at the Core-facility for Bionano Materials in Gachon University). The effects of UV irradiation time, reaction buffer pH, and nanohybrid concentration on the peroxidase activity were assessed following the above-mentioned procedures.
The steady-state kinetic analysis was conducted using MgAc-

Determination of Phenolic Compounds Using MgAC-Fe 3 O 4 /TiO 2 under UV-Light
Phenolic compounds were colorimetrically determined by performing the chromogenic reaction for phenolic compounds with 4-AP in the presence of H 2 O 2 . Typically, MgAC-Fe 3 O 4 /TiO 2 (0.6 mg/mL), H 2 O 2 (1 mM), 4-AP (5 mM), and two representative pheno-lic compounds (pyrocatechol and resorcinol) at diverse concentrations were added into sodium acetate buffer (0.1 M, pH 4.0) under UV irradiation. After 10 min, the resulting solutions were centrifuged at 10,000 g for 2 min, and the supernatant was subjected to record the absorption intensity at 500 and 490 nm for pyrocatechol and resorcinol determination, respectively.
The reusability of MgAc-Fe 3 O 4 /TiO 2 was explored through the consecutive runs involving the typical reaction with 1 mg/mL pyrocatechol under UV-light irradiation, centrifugation at 10,000 rpm for 3 min, and supernatant withdrawing with two washes using aqueous buffer (0.1 M sodium acetate, pH 4.0) to remove excess substrates. The hybrid was then reused for up to 9 iterative cycles and the residual detecting activity for pyrocatechol (%) at each round was calculated using the ratio of the residual activity to the initial one.
For determining phenolic compounds in real tap water solution, predetermined amounts of phenolic compounds were added into tap water to obtain spiked water samples. The concentration of pyrocatechol and resorcinol in each spiked sample was measured via the aforementioned procedures. The coefficient of variation [CV (%) = standard deviation (SD)/average × 100] and the recovery rate [recovery (%) = measured value/actual value × 100] were calculated to determine the reproducibility and precision of the assay.

Results and Discussion
For developing efficient light-activated peroxidase-like nanozymes, MgAC-Fe 3 O 4 /TiO 2 was constructed via a simple sol-gel reaction. Fe 3 O 4 composition can catalyze the peroxidaselike action, whereas TiO 2 composition can act as a photocatalyst to facilitate hydroxyl radical generation under UV-light; therefore, we envisioned that the peroxidase activity of the resulting aminoclay-based nanohybrid (MgAC-Fe 3 O 4 /TiO 2 ) will enhance significantly under UV-light irradiation, owing to the expected synergistic effects between Fe 3 O 4 and TiO 2 components (Figure 1a). The resulting UV-light-driven nanohybrid-based system was then used for the colorimetric determination of phenolic compounds, to prove the potential biosensing applicability (Figure 1b). The morphologies of the as-prepared MgAC-Fe3O4/TiO2 were observed and compared with the control samples (MgAC-Fe3O4 and MgAC-TiO2), via TEM and SEM ( Figure  2). The results indicated that MgAC-Fe3O4/TiO2 was successfully synthesized with an average diameter of ~135 ± 45 nm, which is marginally larger than those of control samples. Energy-dispersive X-ray mapping (EDX) analysis from our previous reports indicated  The morphologies of the as-prepared MgAC-Fe 3 O 4 /TiO 2 were observed and compared with the control samples (MgAC-Fe 3 O 4 and MgAC-TiO 2 ), via TEM and SEM ( Figure 2). The results indicated that MgAC-Fe 3 O 4 /TiO 2 was successfully synthesized with an average diameter of~135 ± 45 nm, which is marginally larger than those of control samples. Energy-dispersive X-ray mapping (EDX) analysis from our previous reports indicated that TiO 2 was found to be uniformly distributed on the surface of MgAC-Fe 3 O 4 particles (Supplementary Materials Figure S1) [28].  Figure S4a,c) [30]. Furthermore, in both MgAC-Fe 3 O 4 /TiO 2 and MgAC-TiO 2 , the peaks corresponding to Ti 2p 3/2 and Ti 2p 1/2 at 458.2 and 464.07 eV, respectively, were observed, confirming the presence of Ti 4+ state for TiO 2 (Figure S4b,d) [31]. All these characterizations demonstrate that the nanohybrid was synthesized by successfully combining MgAC, Fe 3 O 4 , and TiO 2 .  We performed typical ABTS oxidation reaction in the presence of H2O2, with or with out UV-light irradiation, to examine the peroxidase activity of MgAC-Fe3O4/TiO2 and compared it with those of MgAC-Fe3O4 and MgAC-TiO2. The experimental results clearly suggest that MgAC-Fe3O4/TiO2 exhibited a significant enhancement in the peroxidase ac tivity under UV-light irradiation (Figure 3). Compared with MgAC-Fe3O4 and MgAC TiO2, the enhancement in the nanohybrid activity by UV-light irradiation was ~3-fold higher, presumably due to the expected synergistic effect of MgAC-Fe3O4/TiO2. TiO within the nanohybrid would catalyze hydroxyl radical generation by UV-light irradia tion, which helps the peroxidase-mimicking Fe3O4-mediated oxidation of ABTS, thereby resulting in an enhanced peroxidase activity of the nanohybrid. out UV-light irradiation, to examine the peroxidase activity of MgAC-Fe3O4/TiO2 an compared it with those of MgAC-Fe3O4 and MgAC-TiO2. The experimental results clearl suggest that MgAC-Fe3O4/TiO2 exhibited a significant enhancement in the peroxidase ac tivity under UV-light irradiation (Figure 3). Compared with MgAC-Fe3O4 and MgAC TiO2, the enhancement in the nanohybrid activity by UV-light irradiation was ~3-fol higher, presumably due to the expected synergistic effect of MgAC-Fe3O4/TiO2. TiO within the nanohybrid would catalyze hydroxyl radical generation by UV-light irradia tion, which helps the peroxidase-mimicking Fe3O4-mediated oxidation of ABTS, thereb resulting in an enhanced peroxidase activity of the nanohybrid. To better understand the peroxidase-like activity, the steady-state kinetic assays o MgAc-Fe3O4/TiO2 were performed in the presence and absence of UV-light conditions us ing ABTS and H2O2 as substrate. MgAc-Fe3O4/TiO2 showed the typical Michalis-Mente kinetics toward H2O2 and ABTS in both cases ( Figure S5). Its Michaelis-Menten constan (Km) and the maximal reaction velocity (Vmax) values were determined based on the dou ble-reciprocal Lineweaver-Burk plots (Table S1). Under the UV-light irradiation, the K values for both ABTS and H2O2 were lower than those without UV-light condition, prov ing the significant enhancement of the nanohybrid's affinity toward both substrates. Fur thermore, the Vmax values of MgAc-Fe3O4/TiO2 toward ABTS and H2O2 in the presence o  (Table S1). Under the UV-light irradiation, the K m values for both ABTS and H 2 O 2 were lower than those without UV-light condition, proving the significant enhancement of the nanohybrid's affinity toward both substrates. Furthermore, the V max values of MgAc-Fe 3 O 4 /TiO 2 toward ABTS and H 2 O 2 in the presence of UV-light irradiation were~7.4 and~3.4-fold higher than those observed in the absence of UV-light, respectively. These results provide the convincing evidence that UV-light acts as a superb inducer to impressively intensify the catalytic activity of MgAc-Fe 3 O 4 /TiO 2 .
The effects of duration of UV-light irradiation, pH of reaction buffer, and concentration of the employed MgAC-Fe 3 O 4 /TiO 2 on the peroxidase activity were examined. As the duration of UV-light irradiation increased, the catalytic activity correspondingly increased. Considering the favorable detection speed and that the 10 min irradiation yielded sufficiently high ABTS oxidation (over 70%) compared to 20 min irradiation, UV-light exposure for 10 min was adopted for further studies (Figure 4a). MgAC-Fe 3 O 4 /TiO 2 exhibited peroxidase activity at acidic pH conditions, and sodium acetate buffer at pH = 4 was adopted as the standard reaction buffer for our assay, since extreme acidic conditions could result in Fe ion leaching (Figure 4b) [8]. According to the peroxidase activity vs. the amount of MgAC-Fe 3 O 4 /TiO 2 , 0.6 mg/mL of MgAC-Fe 3 O 4 /TiO 2 was the optimal amount and used for further studies (Figure 4c). Under the selected assay conditions, H 2 O 2 was successfully determined from 2 to 25 µM with the limit of detection (LOD) as low as 1 µM (Figure 4d), which is among the best values reported so far for the colorimetric detection of H 2 O 2 [32].
In contrast with natural peroxidase, MgAC-Fe 3 O 4 /TiO 2 are expected to be stable. To demonstrate this, the nanohybrid and HRP were incubated in a wide pH (3)(4)(5)(6)(7)(8)(9)(10)(11) and temperature (20-90 • C) range for 3 h, and the remaining activities were evaluated ( Figure 5). According to the results, MgAC-Fe 3 O 4 /TiO 2 efficiently maintained its activity under extreme pH and temperature conditions, while HRP faced a significant loss in its activity at both acidic and basic pH values or high temperatures (over 60 • C). Therefore, the excellent stability of MgAC-Fe 3 O 4 /TiO 2 is considered beneficial for the practical applications. adopted as the standard reaction buffer for our assay, since extreme acidic conditions could result in Fe ion leaching (Figure 4b) [8]. According to the peroxidase activity vs. the amount of MgAC-Fe3O4/TiO2, 0.6 mg/mL of MgAC-Fe3O4/TiO2 was the optimal amount and used for further studies (Figure 4c). Under the selected assay conditions, H2O2 was successfully determined from 2 to 25 µM with the limit of detection (LOD) as low as 1 µM (Figure 4d), which is among the best values reported so far for the colorimetric detection of H2O2 [32]. In contrast with natural peroxidase, MgAC-Fe3O4/TiO2 are expected to be stable. To demonstrate this, the nanohybrid and HRP were incubated in a wide pH (3)(4)(5)(6)(7)(8)(9)(10)(11) and temperature (20-90 °C) range for 3 h, and the remaining activities were evaluated ( Figure 5). According to the results, MgAC-Fe3O4/TiO2 efficiently maintained its activity under extreme pH and temperature conditions, while HRP faced a significant loss in its activity at both acidic and basic pH values or high temperatures (over 60 °C). Therefore, the excellent stability of MgAC-Fe3O4/TiO2 is considered beneficial for the practical applications. Finally, we used our MgAC-Fe3O4/TiO2 to colorimetrically determine the selected phenolic compounds of pyrocatechol and resorcinol. Under the UV-light irradiation, our nanohybrid efficiently catalyzed the decomposition of H2O2 to produce hydroxyl radicals, which induced the oxidation of target phenolic compounds to generate quinoid radicals. The radicals further reacted with 4-AP to produce colored products, which can be observed by the naked eye and quantified by spectrophotometry [6]. The colors of the final products depended on the kind of phenol species, and thus, pyrocatechol and resorcinol induced slightly different colors and were quantitatively determined at different absorption intensities of 500 and 490 nm, respectively. We first performed experiments to verify the effects of UV-light on the detection of phenolic compounds. The results clearly showed that the absorption intensities toward phenolic compounds were significantly enhanced when irradiated with UV-light, indicating that UV-light-mediated activation of MgAC-Fe3O4/TiO2 would be crucial to sensitively determine phenolic compounds (Figure 6a,b). From the analysis of dose-response curves, both pyrocatechol and resorcinol were successfully determined with the same linear range of 0.15-1.30 mg/mL and LOD as low as 0.1 mg/mL, proving the high sensitivity for the determination of phenolic compounds (Figure 6c,d) [5]. In addition, the MgAc-Fe3O4/TiO2 were efficiently reused by simple centrifugation, by preserving over 90% of the original detecting activity for pyrocatechol even after nine consecutive cycles, proving the reusability of MgAc-Fe3O4/TiO2 ( Figure S6). Finally, we used our MgAC-Fe 3 O 4 /TiO 2 to colorimetrically determine the selected phenolic compounds of pyrocatechol and resorcinol. Under the UV-light irradiation, our nanohybrid efficiently catalyzed the decomposition of H 2 O 2 to produce hydroxyl radicals, which induced the oxidation of target phenolic compounds to generate quinoid radicals. The radicals further reacted with 4-AP to produce colored products, which can be observed by the naked eye and quantified by spectrophotometry [6]. The colors of the final products depended on the kind of phenol species, and thus, pyrocatechol and resorcinol induced slightly different colors and were quantitatively determined at different absorption intensities of 500 and 490 nm, respectively. We first performed experiments to verify the effects of UVlight on the detection of phenolic compounds. The results clearly showed that the absorption intensities toward phenolic compounds were significantly enhanced when irradiated with UVlight, indicating that UV-light-mediated activation of MgAC-Fe 3 O 4 /TiO 2 would be crucial to sensitively determine phenolic compounds (Figure 6a,b). From the analysis of doseresponse curves, both pyrocatechol and resorcinol were successfully determined with the same linear range of 0.15-1.30 mg/mL and LOD as low as 0.1 mg/mL, proving the high sensitivity for the determination of phenolic compounds (Figure 6c,d) [5]. In addition, the MgAc-Fe 3 O 4 /TiO 2 were efficiently reused by simple centrifugation, by preserving over 90% of the original detecting activity for pyrocatechol even after nine consecutive cycles, proving the reusability of MgAc-Fe 3 O 4 /TiO 2 ( Figure S6).
Fe3O4/TiO2 would be crucial to sensitively determine phenolic compounds (Figure 6a,b). From the analysis of dose-response curves, both pyrocatechol and resorcinol were successfully determined with the same linear range of 0.15-1.30 mg/mL and LOD as low as 0.1 mg/mL, proving the high sensitivity for the determination of phenolic compounds (Figure 6c,d) [5]. In addition, the MgAc-Fe3O4/TiO2 were efficiently reused by simple centrifugation, by preserving over 90% of the original detecting activity for pyrocatechol even after nine consecutive cycles, proving the reusability of MgAc-Fe3O4/TiO2 ( Figure S6). The practical utility of UV-light-driven MgAC-Fe 3 O 4 /TiO 2 -based system was demonstrated by determining several different levels of pyrocatechol and resorcinol in spiked tap water solutions. The system yielded acceptable CVs below 7% and recoveries of 97-109% (Table 1), validating the excellent reproducibility and reliability of this assay method. The CVs and recovery values of our system were comparable to those of previous nanomaterial-based strategies for phenolic compound detection (Table S2). These results demonstrated that the current method can be employed for the on-site practical determination of phenolic compounds.

Conclusions
Herein, we developed a novel UV-light-activated peroxidase-like nanozyme, MgAC-Fe 3 O 4 /TiO 2 , which was synthesized easily via sol-gel reaction. The activity of the developed nanozyme enhanced significantly in the presence of UV-light, owing to the synergistic effect between Fe 3 O 4 and TiO 2 . With the enhanced activity, selected phenolic compounds of pyrocatechol and resorcinol were successfully determined with high sensitivity down to 0.1 mg/mL with excellent stability and reusability. The practical utility of UV-light-driven MgAC-Fe 3 O 4 /TiO 2 -based system was successfully demonstrated by precisely determining the concentrations of phenolic compounds in spiked tap water solution. The constructed nanohybrids could serve as new and efficient light-activated nanozymes, and we believe that they have great potential to be utilized in practical biosensing applications for detecting clinically and environmentally important substances.