Interparticle mesoporous silica as an effective support for enzyme immobilisation

Abstract

Mesoporous silica materials with cylindrical pores (MPSC) and interparticle pore structures (IPMPS) were synthesized by typical sol–gel methods, and their structural properties were characterized. The enzyme immobilised on IPMPS exhibited higher specific reactivity because of an improvement in the substrate affinity of the enzyme immobilised on the pore spaces of the IPMPS support. Fourier transform infrared and circular dichroism spectroscopy indicated that the highly ordered structure of formaldehyde dehydrogenase (FDH) is not altered by binding to IPMPS and MPSC surfaces. Interestingly, after 10 repeated reactions, FDH immobilised on IPMPS exhibited a residual activity higher than that of FDH immobilised on MPSC. The cycle performance of the enzyme immobilised on MPSC decreased because of support aggregation by the released enzyme. Meanwhile, IPMPS has a high-intensity surface electrical charge that is highly dispersible in the presence of enzymes. In addition, enzymes that were inactive because of being buried inside mesopores are quantitatively determined. The dependency of the activity of FDH immobilised on IPMPS and MPSC as a function of substrate (formaldehyde) concentration was also evaluated to determine the potential application of these materials as biosensors. Formaldehyde concentrations of 3.0–500 μM could be detected using FDH immobilised on the IPMPS support.

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1. Introduction

Water pollution is a major cause of damage to the environment. Recently, continuous growth in developing regions such as East Asia has increased both the extent and rate of environmental pollution in these areas. Governments have responded by implementing environmental regulations. For example, in China, regulatory limits for air contaminants are now at levels similar to those expected for developed countries by 2020. Thus, highly sensitive gas sensors must be developed. In the near future, measures for controlling water pollution will also be strengthened, thereby necessitating the development of a high-sensitivity sensor for underwater applications. However, the use of these types of sensors in developing countries is limited because of the high cost of both the sensors and purification catalysts. Hence, the development of low-cost, high-performance sensors and catalysts is urgently needed.

Enzyme biosensors have been used to detect ions and molecules in biological, clinical and environmental monitoring applications, and they have attracted considerable attention because of their simplicity and high sensitivity.¹ However, enzymes are generally unstable at high temperatures and in organic solvents; in addition, they are expensive, thus limiting their practical application. To solve these problems, many researchers have investigated enzyme immobilisation on different types of supports for enhancing enzyme activity and stability under various practical conditions.^2–10 Mesoporous silica (MPSC) materials were first produced in the 1970s and then prepared in the 1990s by researchers in Japan and at Mobil Corporation laboratories.^11,12 These materials have been verified as some of the most effective solid supports for immobilising various enzymes because of their large specific surface areas, large pore volumes and uniform pore sizes.^13–17 Moreover, their high pore volumes also allow for high catalyst adsorption. We previously succeeded in obtaining high enzyme activity and cycling characteristics using MPSC materials.^17–19 Many researchers believe that a large amount of adsorbed catalyst leads to high activity; however, high cost remains an issue with this approach. We proposed that the enzyme is aggregated in the large-volume pores of MPSC materials. The activity of platinum, which is used as an automobile catalyst, is reduced at high temperatures because of aggregation.^20–23 Enzymes, similar to metal catalysts, can possibly exhibit reduced activity upon aggregation in the large pore spaces of MPSC materials. Therefore, we prepared an MPS support with an interparticle pore structure based on a very low volume uniform pore size compared with that of conventional MPSC supports. The aggregation of the enzymes immobilised on this support was spatially suppressed (Scheme 1). Simultaneously, substrate affinity was enhanced in comparison with that of enzymes immobilised on conventional supports. Thus, the enzymes immobilised on an interparticle pore structure MPS (IPMPS) material exhibited a relatively high activity despite the reduced amount of adsorbed enzyme. In addition, further cost reduction was achieved by not using a surfactant in the synthesis. Therefore, the use of IPMPS supports may be a potential approach for solving the problem of the high cost of biocatalysts.

Scheme 1 MPSC-FDH and IPMPS-FDH systems prepared in this study.

To the best of our knowledge, the improved enzyme activity of FDH through immobilisation on interparticle pore structures has not been reported yet. Herein, we describe the preparation of two MPS pore structures with pore sizes suitable for FDH (molecular size of 8.6 nm × 8.6 nm × 19.0 nm).¹⁸ The first is MPS material with a cylindrical pore structure, while the second is an MPS support with an interparticle pore structure, which are designated as MPSC and IPMPS, respectively.

First, the effect of the differently structured MPS materials on the enzyme activity of immobilised FDH was investigated. The cycle performance, activity evaluation and characterization of the immobilised enzyme on these two types of pore structure absorbents were reported in a previous communication.²⁴ In this paper, data related to the potential application of the supported enzymes as formaldehyde biosensors, including kinetic studies, further cycle test analysis, determination of the amount of unavailable enzymes buried inside mesopore and advanced structural analysis of the immobilised FDH, are also presented. A new phenomenon discovered from the cycle reaction analysis is reported in this paper. Finally, the dependency of the activity of FDH immobilised on IPMPS and MPSC materials as a function of the substrate (formaldehyde) concentration was evaluated to determine the potential application of these materials as biosensors.

2. Experimental

2.1 Materials

Formaldehyde dehydrogenase (FDH) from Pseudomonas putida was obtained from Toyobo Co. (Tokyo, Japan). The triblock copolymer poly(ethylene glycol)–poly(propylene glycol)–poly(ethylene glycol) (Pluronic P-123, EO₂₀PO₇₀EO₂₀, M_n ca. 5800 g mol⁻¹) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Tetraethoxysilane (TEOS) was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Aqueous ammonia (28%) and normal-decane (n-decane) were purchased from Wako Co. (Tokyo, Japan). All reagents were special grade chemicals. Silica nanoparticles (500 nm) were purchased from Corefront Co. (Tokyo, Japan).

2.2 Methods

2.2.1 Synthesis of MPSC. MPSC material was prepared using Pluronic P-123, TEOS and n-decane as a reagent for controlling morphology. TEOS (2.13 g) was added dropwise to a solution of P-123 (1 g) in 10 M HCl (3.7 mL) and H₂O (31.3 mL). This mixture was stirred at 40 °C for 20 h and then transferred to an autoclave for further reaction at 100 °C for 24 h. The product was filtered, dried at 80 °C for 10 h and then calcined at 550 °C for 4 h in air (heating rate of 1 °C min⁻¹). In some cases, n-decane (7.5 g) was added to the HCl solution containing P-123 before addition of TEOS.

2.2.2 Synthesis of IPMPS. IPMPS materials were prepared as follows: aqueous ammonia (28%, 400 μL) was added to deionized water (2.6 mL). The solution was gently stirred, and TEOS (3 mL) was added to the mixed solution. The resulting mixture was stirred for 1 day at room temperature and then frozen at −30 °C for another day. The frozen sample was then freeze-dried to obtain IPMPS.

2.2.3 Characterization of porous silica materials. The particle morphology of the MPS materials was observed by field-emission scanning electron microscopy (FE-SEM; S-4300, Shimadzu Co., Kyoto, Japan) with an accelerating voltage of 10.0 kV. Transmission electron microscopy (TEM) images were obtained using an instrument (JEOL JEM 2010, JEOL Ltd., Tokyo, Japan) operated at 200 kV. The surface area, pore diameter and pore volume were determined from nitrogen adsorption–desorption measurements using a micrometrics analyser (TriStar 3000; Shimadzu, Kyoto, Japan). Pore diameter distributions were calculated from the desorption branches using the Barrett–Joyner–Halenda (BJH) method. The specific surface area was calculated using the Brunauer–Emmett–Teller (BET) method based on the desorption isotherms. Small-angle X-ray diffraction (SAXRD) spectra were recorded on an XRD (RINT-2550VB3L, Rigaku Co., Tokyo, Japan) with CuKα radiation at 40 kV and 200 mA. The particle diameter and surface potential in the buffer solution were measured by dynamic light scattering (DLS; Otsuka Electronics Co., Tokyo, Japan) and zeta potential (ζ-potential, Otsuka Electronics Co., Tokyo, Japan) analyses, respectively. Ultraviolet-visible (UV-vis) spectra (Beckman Coulter, CA, USA, DU 800 UV-vis spectrophotometer) of the MPS suspensions were also used to verify their redispersion properties.

2.2.4 Immobilisation of FDH onto MPS materials. The facile preparation of immobilised FDH was performed as follows. One hundred microlitres of an FDH (0.5 mg)/50 mM phosphate buffer solution (pH 7.0) was added to 700 μL of the same buffer solution. The porous silica material (1.5 mg) was then added to the FDH solution, and the mixture was stirred overnight at 4 °C. The precipitate was separated by centrifugation (12 000 rpm) at 4 °C for 10 min. FDH immobilised on the support was then washed with cold deionized water and stored at 4 °C.

2.2.5 Assay of FDH activity. A sample of immobilised FDH (1.5 mg) was added to a mixture containing a 7.68 mM formaldehyde solution (100 μL) as the substrate, a nicotinamide adenine dinucleotide (NAD) solution (100 μL, 4 mg mL⁻¹) and a 50 mM phosphate buffer solution (pH 7.0, 800 μL). This mixture was stirred at room temperature for 8 min. The immobilised enzyme was then separated by centrifugation (12 000 rpm) at 4 °C for 2 min. The amount of NADH generated by the enzymatic reaction shown in eqn (1) was determined using a UV-vis spectrophotometer at a fixed wavelength of 340 nm (BioSpec-1600, Shimadzu, Japan).

HCHO + NAD⁺ + H₂O → HCOOH + NADH + H⁺ (I)

The activity of any non-immobilised FDH (native FDH) was measured as a positive control. The enzyme activity (unit) of FDH was determined by eqn (II):

enzyme (unit) = (absorbance value) × (molecular weight of NADH)⁻¹ × (amount of enzyme adsorption)⁻¹ × (reaction time)⁻¹ (II)

2.2.6 Analysis of the structural order of immobilised FDH. The secondary structures of native and immobilised FDH were analysed by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectra of native FDH and immobilised FDH were recorded at room temperature on a spectrometer system (Perkin-Elmer Spectrum GX FT-IR) using a mercury–cadmium–tellurium detector (resolution, 4 cm⁻¹; number of scans, 64; MA, USA).

The secondary structure of native and immobilised FDH was determined by circular dichroism (CD) (J-820K, JASCO Co., Tokyo, Japan) in the wavelength range of 190–260 nm using a quartz cell (optical path length, 0.1 cm) and an integration number of 16. CD spectra were measured with concentrations of native FDH and FDH immobilised on MPSC and IPMPS prepared at 6.0 × 10⁻⁷ and 4.1 × 10⁻⁶ and 4.1 × 10⁻⁶ M, respectively.

3. Results and discussion

3.1 Properties of MPS materials

The structures of IPMPS and MPSC samples were characterized on the basis of their BET surface areas, BJH pore size distributions, TEM and FE-SEM images, ζ-potentials and UV-vis spectra. The structural properties of IPMPS and MPSC materials as determined by BET and ζ-potential measurements and SAXRD analyses are summarized in Table S1.†

The pore size distribution curves and nitrogen adsorption–desorption isotherms obtained using the BJH and BET methods for the IPMPS (circles) and MPSC (squares) samples are plotted in Fig. S1.† The MPSC material exhibited type IV (H1) curves, indicating the presence of a cylindrical mesoporous structure on its surface.²⁵ In contrast, IPMPS did not exhibit a type IV (H1) structure. However, both of the synthesized MPSC and IPMPS materials exhibited very high surface areas of 723.7 and 291.8 m² g⁻¹ and pore volumes of 1.8 and 0.9 cm³ g⁻¹, respectively. The average pore diameters of the MPSC and IPMPS samples were controlled at approximately 13 and 14 nm, respectively. The suitability of this MPS pore size for the molecular size of FDH has been demonstrated previously,¹⁸ and both samples had an optimum pore size for FDH immobilisation. Fig. S1† shows that the IPMPS material had a significantly reduced pore volume because of its non-cylindrical pore structure. FE-SEM images of the MPSC and IPMPS materials are shown in Fig. 1(a) and (b). The MPSC sample was composed of large particles (∼500 nm), whereas the IPMPS material was composed of aggregates of differently sized particles (100–400 nm). This result indicates that a heterotypic sol–gel reaction occurs because of the presence of an excessive amount of ammonia in the case of the IPMPS material. The TEM images of the MPSC and IPMPS materials are shown in Fig. 1(A) and (B), respectively. As can be seen in these images, the MPS material was formed of uniform and small domains of cylindrical small pores (Fig. 1(A)), while the mesopores of IPMPS were not regularly arranged (Fig. 1(B)). Thus, the porous structure of the IPMPS sample was determined from the TEM images. Subsequently, the pore structures were investigated by SAXRD analysis, and the results are shown in Table S2.† The MPSC material exhibited diffraction peaks (100, 110 and 200) corresponding to an ordered two-dimensional hexagonal mesostructure (P6mm).²⁶ By contrast, the mesoporous structure of the IPMPS material was not well ordered (Fig. S2†). In conclusion, the shape of the pore structure of the IPMPS material is regular but with varying pore spaces. Therefore, the IPMPS sample appears to have a ‘disordered pore structure’ based on the SAXRD analysis.

Fig. 1 FE-SEM (a and b) and TEM (A and B) images of the synthesized mesoporous silica materials. (A and a) MPSC and (B and b) IPMPS.

The catalytic activity of an enzyme immobilised on a carrier is influenced by the particle size and dispersion of the absorbent.^27,28 Thus, the particle diameter and dispersion performance of the MPS materials with two different pore structures were measured by DLS and UV-vis spectroscopy, respectively. The particle diameters of the IPMPS and MPSC materials were approximately 480.6 and 288.5 nm, respectively (Fig. S3†). These results correspond to the sizes of the aggregates observed in the SEM images. The UV-vis spectra of suspensions of the two porous silica materials were then obtained to verify their degree of dispersion. According to Beer's law, the absorbance at a characteristic peak exhibits a linear relationship with concentration when a homogeneous solution of a substance is formed.^29,30 A good linear relationship (R = 0.95) between the absorbance at 357 nm and the concentration of the two MPS material was observed (see Fig. S4†), confirming that each is well dispersed in a 50 mM phosphate buffer solution (pH = 7.0). Results for dispersion performance confirmed that the two porous silica materials are similar.

When an enzyme is physically adsorbed on a support, adsorption is mainly dependent on electrostatic interactions. Therefore, to study the difference in the electrostatic charges of the two MPS materials, the surface potentials of the IPMPS and MPSC samples were obtained by measuring their ζ-potentials. The IPMPS and MPSC materials exhibited ζ-potential values of −30.9 and −13.8 mV, respectively (Table S1†).

The FTIR spectra showed the presence of ammonia used in the IPMPS synthesis. The FTIR spectra of MPSC and IPMPS are shown in Fig. S5.† The bands at 980 and 1620 cm⁻¹ are attributable to the binding of Si–O and the bending vibration of NH₃, respectively. The peak of NH₃ was not observed in the FTIR spectra of IPMPS because it evaporated during freeze-drying. Two peaks in the spectrum of IPMPS-FDH and MPSC-FDH near the amide I (1656 cm⁻¹) and II (1528 cm⁻¹) bands, shown in Fig. 2(a), indicated that FDH is immobilised on IPMPS-FDH.

Fig. 2 (a) FTIR spectra of the native and immobilised FDH on various MPS materials. Native FDH is denoted by the solid line. FDH on MPSC and IPMPS is denoted by dashed and dotted lines, respectively. (b) Circular dichroism spectra of the native and immobilised FDH on various absorbents. Native FDH is denoted by the solid line. FDH on MPSC and IPMPS is denoted by the dashed and dotted lines, respectively.

3.2 Structural changes in immobilised FDH

We then analysed the structure of FDH to study the influence of the electrostatic interactions between the enzyme and silica materials. Changes in the secondary structure of FDH immobilised onto the two MPS materials were analysed by FTIR and CD.

According to literature,^31–35 the contribution of the β-sheet, unordered and α-helix content can be identified on the basis of the amide I peaks at approximately 1670, 1660 and 1650 and 1640 cm⁻¹, respectively, whereas peaks in the 1680 cm⁻¹ to 1690 cm⁻¹ region reflect the contribution of the antiparallel β-sheet. Fig. 2 shows the amide I region of FTIR spectra measured over the wavelength range of 1700–1580 cm⁻¹ for native and immobilised FDH samples, respectively. The α-helix and β-sheet conformation of FDH are shown in Scheme S1.† The bands at 1650, 1690 and 1630 cm⁻¹ suggest the presence of the α-helix, antiparallel β-sheet and β-sheet, respectively.^36,37 The band indicating the presence of a random coil was observed near 1540 cm⁻¹. The maximum peak for native FDH (solid), FDH immobilised on IPMPS (dotted line) and FDH immobilised on MPSC (dashed line) was observed at 1650 cm⁻¹, suggesting a high content of the α-helix. In addition, the full-width at half-maximum of the peaks of the samples was similar. These results suggest that the enzyme does not undergo any structural changes when immobilised on the two pore structures.

CD spectral measurements were conducted to analyse the changes in the secondary structure of the enzyme. Fig. 2(b) shows the CD spectra measured in the wavelength range of 190–260 nm for native and immobilised FDH suspended in a 50 mM (pH 7.0) phosphate buffer solution. Broad negative peaks were observed from 208 to 222 nm in the spectrum of the native FDH (solid line), suggesting a combination of two different structures: α-helix with inverted peaks at 222 and 208 nm and β-sheets with an inverted peak at 217 nm. Similar to the FTIR analysis results, FDH that immobilised on two pore structure supports did not undergo a structural change.

3.3 FDH adsorption values and activity of the immobilised enzyme on the two MPS materials

To estimate the influence of the pore structure on enzyme activity, FDH was immobilised via physical adsorption on the MPSC and IPMPS samples. Fig. S6(A)† shows the dependence of the amount of adsorbed FDH on the two adsorbents at different enzyme concentrations. Any FDH adsorbed on the outside of the pores was washed away with deionized water prior to the measurement. No FDH was detected in the supernatant when the supports containing immobilised enzyme were washed two times (Fig. S7†); therefore, the immobilised samples were washed once for all experiments. The amount of the immobilised enzyme was calculated using the amount of enzyme detected in the supernatant and in the washing solution. Both adsorbents with adsorbed enzyme exhibited L-type isotherms (Fig. S6(A) and S8†), which were easily rectified using Langmuir coordinates.⁴³ The amount of adsorbed enzyme on the MPSC material was higher than that on the IPMPS sample.

This result indicates that the carrier with a larger pore volume adsorbs a greater amount of enzyme. In fact, the pore volume of the MPSC material was approximately two times that of the IPMPS material. On the other hand, the enzyme immobilised on the IPMPS sample exhibited a higher specific activity (Fig. S6(B)†) than that immobilised on the MPSC material. The higher reactivity of the IPMPS sample is believed to be due to the improved dispersion of the immobilised enzyme on IPMPS material. Thus, FDH, when surrounded by the interparticle pore structure, has improved substrate affinity. Therefore, although both the amount of adsorbed enzyme on the IPMPS sample and pore volume of the IPMPS material are much lower than those of the MPSC sample, the activity is higher. As a result, the dispersibility and substrate affinity of the immobilised enzyme on the IPMPS material are enhanced.

3.4 Kinetic studies of immobilised FDH

The kinetic properties of FDH immobilised on each MPS material, including the Michaelis constant K_m, the maximum rate V_max and the turnover number (K_cat), were determined for the reactions of the enzyme with formaldehyde (Table 1; Fig. S9†).^39–42 The K_m value for FDH immobilised on the IPMPS sample was much smaller than that for FDH immobilised on the MPSC material. These results indicate that interparticle pore spaces have a greater effect on the substrate affinity of the enzyme than cylindrical pore spaces. Consequently, FDH immobilised on the IPMPS material exhibited high activity at a low enzyme adsorption level. Similar to the results of the K_m value, the K_cat value for the IPMPS material with immobilised FDH was higher than that for MPSC. This result suggests that the enzymatic reaction effectively proceeds when FDH was immobilised on the IPMPS materials with better contact between the enzyme and formaldehyde.

Table 1 Apparent K_m, K_cat, and K_cat/K_m values for MPS materials with two pore structures shown in Fig. S9,† calculated using the Michaelis–Menten equation

Sample name	Km [mM]	Kcat [min^−1]	Kcat/Km
Native	0.249	12.1	48.6
IPMPS	0.285	6.18	21.7
MPSC	0.509	3.62	7.11

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3.5 Cycle test

As the substrate affinity of the enzyme increased as a result of immobilisation on the IPMPS material, the enzyme could be released into the reaction solution because of the increase of contact with the substrate. To determine if enzyme release occurred, therefore, the immobilised enzyme on the two MPS materials was subjected to repeated reactions, and the results are illustrated in Fig. 3. After each reaction cycle, the residual activity was recorded. Native FDH and both immobilised FDH samples retained ∼90% and ∼60%, respectively, of their initial activity, even after 10 repeated reactions.

Fig. 3 Residual activity of FDH immobilised on MPSC and IPMPS as a function of catalyst recycle number.

The structure of the enzyme after 10 reaction cycles was estimated by FTIR and CD spectra. Results show no change (Fig. S10†); however, FDH was slightly inactivated after 10 cycle reaction (Fig. 3). These results indicate that FDH immobilized on the IPMPS material can be used repeatedly, as can the enzyme immobilized on the traditional cylindrical MPS material. Interestingly, after three repeated reactions, FDH immobilised on the IPMPS sample exhibited a residual activity higher than that of FDH immobilised on the MPSC material.

The reason for the decreased cycle test activity is generally enzyme release (Table S2†). Hence, the enzyme released in the washing solution was quantified after enzyme immobilised on such supports was repeatedly washed with buffer (Table S2†). The enzyme release amount of the enzyme immobilised on MPSC and IPMPS was approximately 6.78% and 18.9%, respectively, of the total adsorption amount. From the above results, reduced activity in the reaction cycle cannot be explained only by the enzyme release amount.

Deka et al. reported that support aggregation arises in cases when the enzyme acts as a binder.⁴³ Closer analyses of the DLS results (Fig. 4) indicated that not only mean particle size is increased but also larger agglomerates are formed at the expense of smaller particles. In other words, the systematic shift in the average particle size indicated preferential agglomeration of the larger particles.

Fig. 4 Dynamic light scattering (DLS)-based particle size distribution of various pore structure silica materials after enzyme immobilisation on silica materials after 10 cycle reactions. (a) IPMPS and (b) MPSC.

In addition, agglomerates having an order of magnitude higher than the original ones were formed. The ζ-potential of MPSC and IPMPS in buffer showed a value of −13.8 and −30.9 mV (Table 2). On the other hand, the values for MPSC and IPMPS in the presence of 0.5 mg mL⁻¹ protein were −16.5 and −30.2 mV, respectively. Particles with ζ-potential values more positive than +30.0 mV or more negative than −30.0 mV are normally considered stable.⁴³ Protein that was released from MPSC was used for support aggregation, which was also evident from the DLS results measured even at higher protein concentrations by repeated washing. Based on the above results, we suggest that the activity of the cycle test mainly decreases because of the decrease of the substrate diffusion rate due to support aggregation and enzyme release.

Table 2 Zeta potential of FDH, three silica materials and FDH–silica composite

Sample name	ζ-Potential [mV]
FDH	−17.4
IPMPS	−30.9
FDH immobilised on IPMPS	−30.2
MPSC	−13.8
FDH immobilised on MPSC	−16.5
Silica particle (diameter 500 nm)	−68.6
FDH immobilized on silica particle	−56.8

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3.6 Determination of activity and inactive enzymes on MPS materials

Kinetic studies and enzyme activity analysis indicated that it is difficult for the enzyme buried inside an MPSC mesopore to make contact with the substrate. Thus, the determination of buried enzymes in the pores was conducted. The method was conducted in four steps. (1) The calibration curve was plotted with absorbance on the y-axis and the amount of enzyme on the x-axis. This experiment used silica nanoparticles with sizes similar to MPSC and IPMPS supports. The enzyme activity of native enzyme and enzymes immobilised on 500 nm silica nanoparticles is shown in Fig. 5. Enzymes immobilised on silica nano particle are all active enzyme, because it was adsorbed on silica surface (Scheme 2).

Fig. 5 Absorbance calibration curve of native enzymes and enzymes immobilised on silica nanoparticles (500 nm).

Scheme 2 Model of MPSC-FDH and silica nano particle–FDH.

Enzyme activity is reduced by simple immobilisation on silica. Therefore, (1) the amount of the buried enzyme was determined using a calibration curve of the enzymes immobilised on the silica spheres. (2) FDH immobilised on MPSC materials was measured on the basis of the enzymatic activity of FDH. (3) The amount of active enzyme (X) was determined from the calibration curve of enzyme immobilised on silica nanoparticles. (4) The amount of the buried inactive enzyme (Y) was calculated by subtracting the amount of active enzymes from the total adsorption amount (Table 3). Enzymes immobilised on MPSC were highly inactive compared with those on IPMPS. Conversely, enzymes immobilised on IPMPS were almost active. We believe that the high efficiency originates from the interparticle mesopore structure.

Table 3 Amount of enzyme attached to absorbents and active (X) and the amount attached to absorbents but inactive (Y)

	Absorbance^a^,^b	Adsorbed amount of enzyme^b [mg: protein/1.5 mg: MPSs]	X^c [mg: protein]	Y^d [mg: protein]
a Enzyme activity of FDH immobilised on MPS materials.b Immobilised method shown in Section 4.2.4.c X value was calculated using the calibration curve of FDH immobilised on silica nanoparticles of Fig. 5.d Y value was calculated by subtracting the X value from the enzyme-adsorbed amount.
IPMPS	1.27	0.16	0.15	0.01
MPSC	1.61	0.32	0.20	0.12

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3.7 Application as biosensor

Given the small amount of enzyme adsorbed on the IPMPS sample, the two catalysts showed similar activities, even at low concentrations (Fig. 6). This result indicates that FDH immobilised on the IPMPS and MPSC materials can detect trace amounts of formaldehyde ranging from 3 to 500 μM. Commercially available formaldehyde sensors detect formaldehyde concentrations within the 3–150 mM range.^44,45 Therefore, the IPMPS system prepared herein is more sensitive than any other conventional formaldehyde detector and has great potential as a formaldehyde biosensor.

Fig. 6 Activity profiles of immobilised FDH on IPMPS and MPSC depending on various substrate concentrations.

4. Conclusions

FDH was immobilised by physical adsorption onto carefully controlled, size-optimized pores of two absorbents. The performance of these catalysts was evaluated on the basis of their enzyme activity, cycling characteristics, kinetic behaviour and enzyme structure analyses to determine the optimal structure for the carrier. FDH immobilised on the IPMPS support exhibited a higher activity than that on the MPSC material because of an increase in the substrate affinity resulting from the interparticle pore space. In addition, FDH immobilised on the IPMPS support exhibited high activity at a low enzyme adsorption level, thus indicating potential to enable the realization of low-cost biosensors. Furthermore, biocatalysts consisting of FDH immobilised on both the MPSC and IPMPS materials remained stable through repeated reaction cycles.

Finally, FDH immobilised on the IPMPS support detected formaldehyde at concentrations as low as 3.0 μM, exhibiting a higher sensitivity than any other conventional formaldehyde sensors. Therefore, this FDH/IPMPS system is a promising candidate for application as formaldehyde sensors. In addition, the potential of new IPMPS immobilisation carriers was also demonstrated.

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Footnote

† Electronic supplementary information (ESI) available: Nitrogen adsorption–desorption isotherms and BJH analysis (Fig. S1). Small-angle XRD patterns (Fig. S2). Average diameters of porous silica materials (Fig. S3). Correlation of the absorbance at 356 nm with concentration (Fig. S4). FTIR spectra of various MPS materials (Fig. S5). (A) Isotherms of FDH adsorption on different immobilisation concentrations: MPS (squares) and IPMPS (circles). (B) Relative activities of FDH immobilised on each mesoporous material: MPS (squares) and IPMPS (circles) (Fig. S6). Amount of enzyme in the supernatant after immobilisation and washing with an aqueous solution (Fig. S7). Langmuir and Freundlich plots of enzyme adsorption onto porous silica materials (Fig. S8). Kinetic parameters of immobilised FDH on each MPS material (Fig. S9). (a) FTIR spectra of the native and immobilised FDH on various MPS materials and spectra after 10 cycle reactions. (b) CD spectra of the native and immobilised FDH on various MPS materials and spectra after 10 cycle reactions (Fig. S10). Structural properties of MPS materials (Table S1). Enzyme release amount by the washing of immobilised FDH on each MPS material (Table S2). α-Helix (red) and β-sheet (blue) in FDH. The structural data (Scheme S1). See DOI: 10.1039/c3ra46122j

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