The TNPs used in this study were synthesised following our previously reported procedure [20] and were characterised as the anatase phase with an average particle size of approximately 20–30 nm. APTES, MPTMS, DCA, TA, glutaraldehyde (GLA), 1-ethyl-3-(3-dimethylamino)propyl carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), H₂O₂, bovine serum albumin (BSA), and catalase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionised water (DW) was purified using a Millipore Milli-Q purification system (Merck Millipore, Molsheim, France).

Amino-TNP or thiol-TNP (TNP-APTES or TNP-MPTMS, respectively) was prepared via silanisation of the TNP surface [22, 23]. Briefly, 50 mg TNPs was added to 20 mL DW containing 0.5 mL APTES or MPTMS (molar ratio of TNP to APTES or MPTMS = 6.25:1), and the reaction mixture was stirred overnight. Carboxylic-TNP (TNP-DCA) was prepared by slowly adding 50 mg TNPs to 10 mL sodium phosphate buffer (0.2 M) containing 10 mg DCA, and the reaction solution was stirred for 1 h at 25 °C. The phenolic-TNP (TNP-TA) intermediates were synthesised by a coordination reaction between the Ti ions on the TNP surface and the phenolic groups of TA [24]. Briefly, 50 mg TNPs were dispersed in 10 mL TA (10 mM) at a molar ratio of 5:1. The mixture was stirred for 1 h at 25 °C. The modified TNP intermediates were separated by centrifugation after washing three times with distilled water and dried at 40 °C under vacuum.

Ultraviolet-visible (UV-Vis) spectra were recorded using a Hitachi U-1900 spectrophotometer (Hitachi High-Tech, Tokyo, Japan). Samples (0.1 mg/mL) were prepared in a 1 cm quartz cuvette and scanned over a wavelength range of 300–600 nm. Fourier-transform infrared (FT-IR) spectra of the pristine and modified TNP intermediates were obtained over the wavenumber range of 400–4000 cm^-1 using a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Catalase was immobilised on the surface of the TNPs using GLA as a crosslinker and covalently attached to the silane-grafted TNP intermediates (Fig. 1). Briefly, 10 mg TNP-APTES was dispersed in sodium phosphate buffer (50 mM, pH 7). Subsequently, 0.5 mL catalase (5 mg/mL) and 50 µL GLA (100 mM) were slowly added over 5 min at 25 °C under stirring. The immobilised catalase was collected by centrifugation (10,000 rpm for 5 min) and washed three times with sodium phosphate buffer. To immobilise catalase on TNP-DCA (10 mg), 3 mg NHS and 1.8 mg EDC were added to 5 mL 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (100 mM, pH 6.5) and stirred for 1 h at room temperature. Subsequently, 0.5 mL catalase (5 mg/mL) in MES buffer (pH 6.5) was added, and the solution was stirred for a further 4 h at 4 °C. The immobilised catalase was collected by centrifugation (10,000 rpm for 5 min) and washed three times with MES buffer. Catalase was immobilised on TNP-TA or TNP-MPTMS by the direct interaction between catalase and the functional groups (phenolic or thiol) on the TNP intermediates. Briefly, 0.5 mL catalase solution (5 mg/mL) in phosphate buffer was slowly added to 10 mL sodium phosphate buffer containing TNP-TA (10 mg) or TNP-MPTMS particles (10 mg). The mixed solution was stirred at 4 °C for 6 h. The immobilised catalase was collected by centrifugation (10,000 rpm for 5 min) and washed three times with sodium phosphate buffer. Finally, the prepared CITNPs were lyophilised and stored at –20 °C.

Fig. 1.

Schematic diagram illustrating the preparation and surface functionalisation of TNPs with four distinct functional groups. (A) amine (using APTES); (B) thiol (using MPTMS); (C) carboxylic (using DCA); and (D) phenolic (using TA). Catalase was subsequently immobilised onto these functionalised TNPs via covalent bonding or surface adsorption, resulting in CITNPs. TNPs, titanium dioxide nanoparticles; APTES, (3-aminopropyl)triethoxysilane; MPTMS, (3-mercaptopropyl)trimethoxysilane; DCA, dihydrocaffeic acid; TA, tannic acid; CITNPs, catalase-immobilised TNPs. The figure was created with ChemDraw Ultra 12.0 (Waltham, MA, USA).

To determine the catalase activity, 50 µL catalase (5 mg/mL) or 5 mg CITNPs was added to 500 µL H₂O₂ (5 mM) in sodium phosphate buffer (50 mM, pH 7.0) and incubated for 5 min at 25 °C. The H₂O₂ concentration was measured via UV absorbance at 240 nm [25] using a UV-Vis spectrophotometer (Hitachi U-1900, Tokyo, Japan). The catalase concentration was determined via the Lowry method using a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), with BSA as the standard [26]. The amount of catalase bound to CITNPs was calculated by subtracting the amount of residual catalase in the supernatant after immobilisation from the total amount used initially. The immobilisation efficiency of the CITNPs was calculated using Eqns. 1,2:

(1)$$\begin{array}{c}\hfill \text{Catalase loading}(\%)=(\text{Amount of bound catalase}/\hfill \\ \hfill \text{Amount of used catalase})\times 100\hfill \end{array}$$

(2)$$\begin{array}{c}\hfill \text{Relative activity}(\%)=(\text{Activity of CITNPs}/\hfill \\ \hfill \text{Activity of free catalase})\times 100\hfill \end{array}$$

The effects of temperature and pH on the activity of immobilised catalase were examined at 10–80 °C and pH 3.0–9.0 (pH 3.0–6.0 in 50 mM sodium citrate; pH 6.0–8.0 in 50 mM sodium phosphate; pH 8.0–9.0 in 50 mM Tris-HCl). Each sample was incubated for 5 min under specific conditions and the activity was determined using the enzyme assay described in Section 2.5. The relative percentage activity of catalase was calculated based on the highest observed activity, which was set to 100%.

CITNPs were incubated in sodium phosphate buffer (50 mM, pH 7.0) at 70 °C for 60 min, and the catalase activity was determined using the enzyme assay described in Section 2.5. The reusability of catalase in the CITNPs (10 mg/mL) was evaluated at ambient temperature for 10 min, as described in the catalase activity assay. After each test cycle, the CITNPs were recovered by centrifugation (12,000 rpm for 2 min) and washed three times with phosphate buffer. H₂O₂ solution (5 mM), used as a catalase substrate, was freshly prepared for each assay cycle. The catalase activity at time zero or during the first cycle was assumed to be 100% of the initial catalase activity.

Commercially available dried green tea leaves and coffee powder purchased from a local market (Gwangju, Republic of Korea) were used as model phenolic-containing beverages to prepare beverage extracts [27]. For the tea extract, 2 g dried tea leaves were steeped in 100 mL of DW at 80 °C for 5 min and then filtered through Whatman No. 1 filter paper (GE Healthcare, Chicago, IL, USA). For the coffee extract, 2 g ground coffee powder was brewed with 100 mL boiling DW and filtered. All extracts were cooled to room temperature, diluted to 1:10 (v/v) with 50 mM phosphate buffer (pH 7.0), and further filtered using a Sep-Pak C18 cartridge (Waters Corporation, Milford, MA, USA) followed by a 0.45 µm microfilter (Millipore, Billerica, MA, USA) before analysis. For the H₂O₂ removal assay, CITNPs (or free catalase) were added to the beverage extracts at an equivalent catalase loading of 100 U/mL (enzyme activity determined as described in Section 2.5). The mixtures were gently mixed at 25 °C, and samples were collected at specific time intervals (0, 1, 3, 5, and 10 min) for residual H₂O₂ analyses.

H₂O₂ was indirectly detected via the formation of a ternary complex (Ti-TAR-H₂O₂), as previously described [27]. Briefly, 50 µL of the reaction sample was mixed with 50 µL of 10 mM titanium(III) chloride and 50 µL of 10 mM 4-(2-thiazolylazo)resorcinol (TAR) and incubated at room temperature for 5 min. The resulting mixture containing the ternary complex was injected into a CE system (P/ACE 5500, Beckman Coulter, Kraemer, CA, USA) equipped with a photodiode array detector set at 530 nm ($\lambda$ = 530 nm). The background electrolyte used for the CE analysis consisted of 75 mM boric acid and 35 mM NaOH adjusted to pH 9.1. The peak area corresponding to the Ti-TAR-H₂O₂ complex was quantified using a standard calibration curve to determine the residual H₂O₂ concentration.

The reusability of the CITNPs was evaluated by performing the H₂O₂ removal reaction over 10 consecutive cycles. After each cycle (5 min reaction), the CITNPs were separated by centrifugation (10,000 rpm, 5 min), washed twice with phosphate buffer (pH 7.0), and reused under identical conditions. The relative catalase activity for each cycle was calculated by comparing the H₂O₂ removal rate of that cycle with that of the first cycle.

As previously reported [20], X-ray diffraction analyses and transmission electron microscopy observations confirmed that the TNPs employed in this study exhibited a well-defined anatase crystal structure and possessed a relatively uniform particle size distribution with diameters of approximately 20–30 nm. To prepare the TNP intermediates, the TNP surface was modified with four types of functional groups (amine, thiol, carboxylic acid, and phenolic groups), abbreviated as APTES, MPTMS, DCA, and TA, respectively. As shown in Fig. 2, the functional groups on the TNP intermediates were confirmed using UV-Vis and FT-IR spectroscopy as well as the sedimentation behaviour. A rapid increase in UV absorbance was observed for TNP-TA and TNP-DCA at 300–400 nm, indicating the presence of phenolic groups on the modified surfaces, whereas both TNP-APTES and TNP-MPTMS exhibited weak UV absorbance (Fig. 2A). As shown in Fig. 2B, the FT-IR spectra of all modified TNP intermediates exhibited a characteristic Ti-O-Ti stretching band at 483–683 cm^-1. TNP-APTES and TNP-MPTMS exhibited Ti-O-Si asymmetric stretching bands with a small peak at 985 cm^-1 and weak peaks at 2853 and 2925 cm^-1 corresponding to the C-H asymmetric and symmetric stretching vibrations of the coupling agents [28]. TNP-TA exhibited the characteristic bands of TA at 1000–1700 cm^-1 owing to the chemical interactions between TA and the TiO₂ surface. Moreover, the sedimentation behaviours of TNP-APTES and TNP-MPTMS differed from those of TNP-TA and TNP-DCA. That is, the TNP-APTES or TNP-MPTMS suspensions in water aggregated and precipitated after 1 h, whereas those of TNP-DCA and TNP-TA did not (Fig. 2C).

Fig. 2.

Characterisation of the TNP intermediates functionalised with various surface groups. (A) UV-Vis spectra: TNP functionalised with APTES (amine), MPTMS (thiol), DCA (carboxylic acid), and TA (phenolic) exhibited distinct absorption profiles. TNP-TA and TNP-DCA exhibited strong absorbance at 300–400 nm, attributable to phenolic group interactions. (B) FT-IR spectra: all surface-modified TNPs exhibited the characteristic Ti-O-Ti stretching band (483–683 cm^-1). TNP-APTES and TNP-MPTMS also exhibited Ti-O-Si asymmetric stretching (~985 cm^-1) and C–H stretching vibrations (~2853 and 2925 cm^-1), confirming successful organosilane coupling. TNP-TA exhibited distinct bands at 1000–1700 cm^-1, corresponding to phenolic TA structures. (C) Photographic images of the TNP suspension stability: TNP-APTES and TNP-MPTMS aggregated and precipitated within 1 h, whereas TNP-DCA and TNP-TA remained well-dispersed, indicating improved colloidal stability. UV-Vis, ultraviolet-visible; FT-IR, Fourier-transform infrared.

Catalase was immobilised on the TNP intermediates, each possessing a specific functional group (amine, thiol, carboxylic, or phenolic) on its surface. Table 1 compares the catalase loading capacity and relative activity of the catalases after the immobilisation reaction with the TNP intermediates (TNP-APTES, TNP-MPTMS, TNP-DCA, and TNP-TA). The catalase loading on TNP-APTES, TNP-MPTMS, TNP-DCA, and TNP-TA followed the order B $>$ A $>$ C $>$ D (87.3%, 85.5%, 78.5% and 75.5%, respectively). Moreover, the relative activity of the catalases followed the order A $>$ B $>$ C $>$ D (77.2%, 73.9%, 70.6%, and 60.2%, respectively).

The effect of temperature and pH on the activity of the catalase immobilised on the TNPs was investigated by determining the relative catalase activity over a broad range of temperatures (10–80 °C) and pH values (2.0–9.0). As shown in Fig. 3, both free and immobilised catalase exhibit maximum enzyme activity at approximately 40 °C. However, the CITNPs are thermally more stable than free catalase, retaining higher catalase activity over a broader temperature range, particularly at higher temperatures (50–80 °C) (Fig. 3A). Additionally, CITNP catalase, as compared with free catalase, exhibits high pH tolerance, maintaining substantial activity across a wide pH range (pH 2.0–9.0) (Fig. 3B). CITNPs demonstrated optimal catalytic activity at 40 °C and pH 6.0–7.0. Although enhanced stability after immobilisation is typically expected, the extent of activity retention varies across different surface functional groups, indicating that the immobilisation chemistry influences enzyme performance independently of simple loading effects.

Fig. 3.

Effect of temperature and pH on the catalase activity of the CITNPs. (A) Temperature profile: the catalase activity of the CITNPs derived from each TNP intermediate was measured at 10–80 °C. The activities are expressed relative to the maximum observed at 40 °C. Compared to free catalase, the CITNPs exhibited broader thermal tolerance, retaining higher relative activity at elevated temperatures. (B) pH profile: the catalase activity was evaluated at pH 2.0–9.0 using appropriate buffer systems. The activities are expressed relative to the maximum observed at pH 6. The CITNPs retained higher activity than free catalase under both acidic and alkaline conditions, indicating enhanced pH stability after immobilisation. Data is presented as mean $\pm$ SD (n = 3).

The thermal stability of catalase was examined by incubating CITNPs in 50 mM phosphate buffer at 70 °C for 60 min. Fig. 4A shows that the catalase activity of CITNPs prepared using various TNP intermediates decreases with increasing incubation time. Notably, the CITNPs exhibit a slower catalase activity reduction rate than free catalase, retaining 9%–28.9% of the initial catalase activity, depending on the preparation method, after 60 min of incubation at 70 °C. Additionally, CITNP catalase prepared with TNP-APTES demonstrates high reusability, as compared with free catalase, retaining 41.8%–76.3% of the initial catalase activity even after 10 reuse cycles (Fig. 4B), depending on the TNP intermediate. Notably, differences in operational stability were observed despite comparable immobilisation efficiencies, suggesting that enzyme–support interactions play a critical role in long-term performance.

Fig. 4.

Thermal stability and reusability of CITNPs. (A) Thermal stability: CITNPs were incubated at 70 °C in 50 mM phosphate buffer (pH 7.0) for up to 60 min. The residual activity was measured and expressed relative to the untreated controls. The CITNPs retained significantly higher residual activity than free catalase, ranging from 9%–28.9% after 60 min. (B) Reusability: the catalytic activity of the free catalase and CITNPs was assessed over 10 consecutive reaction cycles. The activity was measured after each cycle and expressed as a percentage of the initial activity (cycle 1). The CITNPs retained 41.8%–76.3% activity after 10 cycles, while the activity of free catalase declined by $>$90% within three cycles. Data represent mean $\pm$ SD (n = 3).

Since the CITNPs prepared using TNP-APTES (method A) exhibited superior catalase activity than the other TNP intermediates, it was selected for further evaluation in removing H₂O₂ from tea or coffee extracts through the use of a CE system (indirectly detected the Ti-TAR-H₂O₂ complex). As shown in Fig. 5A,B, the H₂O₂ peak rapidly decreases with an increasing CITNP treatment time with the extract of tea or coffee, degrading $>$90% of H₂O₂ in 10 min. To further confirm CITNP reusability, H₂O₂ degradation by the CITNPs was examined over 10 consecutive cycles in tea or coffee extracts. Notably, CITNP retained a $>$70% catalytic activity after 10 cycles (Fig. 5C).

Fig. 5.

Catalytic activity and reusability of CITNPs in the removal of H₂O₂ from tea and coffee extracts. (A) CE analysis of the H₂O₂ concentration in tea extract before and after treatment with CITNPs. The peak corresponding to the Ti-TAR-H₂O₂ complex (indicated by an arrow at a migration time of 7.4 min) decreased progressively following treatment with CITNPs (10 µg/mL) at 0, 1 and 10 min. (B) Time-course of H₂O₂ removal during 10 min of incubation at 40 °C. Residual H₂O₂ concentrations are expressed as a percentage of the initial concentration in both tea and coffee extracts. (C) Reusability of CITNPs for H₂O₂ removal. The enzyme activity was measured after each cycle and expressed as a percentage of the initial activity (cycle 1). The CITNPs retained $>$76% of their activity after 10 cycles. Data were collected using CITNPs prepared from TNP-APTES (method A). Data is presented as mean $\pm$ SD (n = 3).

Tea and coffee extracts were selected as representative complex aqueous matrices rather than simple buffer systems. These beverages contain endogenously generated H₂O₂ resulting from polyphenol auto-oxidation during heating and storage, as previously reported by our group using CE. Therefore, their use enables the validation of catalytic performance and analytical robustness under realistic matrix conditions. Quantitative kinetic parameters, such as K_m and V_max, were not evaluated in this study because accurate kinetic analyses of immobilised enzymes require accounting for substrate diffusion and mass-transfer limitations. Future work will involve detailed kinetic modelling to further elucidate the catalytic behaviour of the immobilised system.