Spheres-on-sphere silica microspheres as matrix for horseradish peroxidase immobilization and detection of hydrogen peroxide

Abstract

In this work, spheres-on-sphere (SOS) silica microspheres are prepared via a facile one-pot synthesis. After functionalization with carboxyl groups, the carboxylated SOS silica microspheres (SOS-COOH microspheres) can serve as a support for horseradish peroxidase (HRP) covalent immobilization. The obtained enzyme hybrid (SOS-COOH-HRP) is more stable under alkaline conditions than the free counterpart, and exhibits longer-term storage stability and higher resistance toward the denaturing agents such as guanidine hydrochloride (GdmCl) and urea. The Michaelis–Menten constant (K_m) of the immobilized enzyme is decreased slightly while the maximum rate of reaction (V_max) is very close to that of free HRP, resulting in the catalytic efficiency of SOS-COOH-HRP being enhanced significantly. For evaluating its utility, a SOS-COOH-HRP-based colorimetric method has been developed for selectively and sensitively detecting H₂O₂ both in buffer and 10% diluted human serum. Furthermore, the SOS-COOH-HRP displays excellent reusability and reproducibility in cycle analysis. The results demonstrate that the SOS-COOH-HRP has great potential for practical applications in biosensing and industrial fields.

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1 Introduction

Enzymes are highly efficient and selective biocatalysts that have been extensively applied in many research and industrial fields including chemical synthesis, cosmetics, food processing, pharmacology, agrochemicals, and energy production.^1,2 However, the application of native enzyme is often hindered by the lack of long-term stability under storage conditions and difficulties in recovery and reuse of inactivated enzymes. Enzyme immobilization is a widely used strategy to circumvent such problems, which has been proven as an efficient way to enhance the catalytic performance of enzymes, such as activity, stability, specificity, selectivity and reduction of inhibition.³ Enzyme immobilization can be achieved by conjugation of enzymes on solid substrates/supports through a variety of methods/reactions such as physical adsorption,^4,5 covalent attachment,^6,7 and encapsulation/entrapment.^8,9 Electrostatic interaction or physical adsorption immobilization of enzymes on solid substrates/supports is a simple process with the benefits of time saving and reduced complexity of sample preparation. However, the binding is not strong enough to yield stable enzyme conjugates capable of standing the necessary washing steps and incubation conditions in subsequent reactions. Encapsulation/entrapment method is only suitable for the enzyme with small molecular weight substrates and products because it is limited by diffusion property of macromolecule and relatively slow reaction rate. Covalent binding of enzymes with solid substrates/supports is the most commonly used method which offers high stability and is demonstrated to be quite robust: they can provide durable attachment and prevent enzyme leaching from the surface. Recently, the carrier-free immobilization method of cross-linked enzyme aggregates (CLEAs) has also attracted extensive attention due to the simple and robust feature of the strategy.^10,11

The ideal solid substrates/supports for enzyme immobilization should possess characteristics like inertness, favorable physical strength, excellent stability, higher resistance to enzyme aggregation and denaturation, reduced product inhibition and nonspecific adsorption. Kinds of materials including glass,¹² cellulose,¹³ chitosan,¹⁴ mesoporous materials¹⁵ and nanoscaled materials¹⁶ have been employed as matrix for enzyme immobilization. Especially, many natural or synthetic supports with tunable physical and chemical properties have been specifically designed for immobilizing enzymes to satisfy the increasing requirements of different applications.^17,18 For instance, graphene oxide (GO) sheet with an atomically flat surface has been employed to immobilize HRP for removing phenolic compound from aqueous solution.¹⁹ Carboxylethyl- or aminopropyl-functionalized mesoporous silica exhibits high immobilization efficiency and enhanced stability of organophosphorus hydrolase (OPH).¹⁵

Herein, the SOS silica microspheres were synthesized by one-pot strategy using 3-mercaptopropyltrimethoxysilane (MPTMS) as single silicon precursor. The as-prepared SOS silica microspheres are microspheres densely coated with nanospheres, which provide high surface area and easily accessible active sites for surface functionalization. After modification with maleic anhydride, the carboxylated SOS silica microspheres (SOS-COOH microspheres) possess abundant carboxyl groups on the surface which would benefit for the immobilization of enzyme through formation of amide bonds. In addition, the as-prepared SOS-COOH-enzyme composites can be easily separated from reaction mixture for recycling by centrifugation since SOS-COOH microspheres have relative large size (about 5.5 μm in diameter). Because of the wide practical applications, HRP is selected as a model enzyme in the proof of principle experiment. The catalytic kinetics, pH stability, storage stability, and resistance to denaturing agent of the immobilized enzyme have been studied in detail. The results demonstrated that the SOS-COOH microspheres can be used as efficient solid substrates/supports for enzyme immobilization and recycling under controlled conditions.

2 Experimental section

2.1 Reagents and materials

(3-Mercaptopropyl)trimethoxysilane (MPTMS, 95%), poly(vinyl alcohol) (PVA, M_w 10 000), cetyltrimethylammonium bromide (CTAB, ≥98%), ammonium hydroxide solution (reagent grade, 28–30% NH₃ basis) were purchased from Sigma-Aldrich (USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was obtained from Alfa Aesar (USA). N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) and horseradish peroxidase (HRP, E.C. 1.11.1.7) were purchased from Aladdin (Shanghai, China). p-Phenolsulfonic acid (PSA, 95%) and 4-aminoantipyrine (4-AAP, 98%) were obtained from J&K Scientific Ltd. (Beijing, China). H₂O₂ solution (30%) was purchased from Beijing Chemical Factory (Beijing, China), and a stock solution of H₂O₂ (1 mol L⁻¹) was prepared from the 30% H₂O₂ solution and standardized by titration with potassium permanganate. All other chemical reagents were analytical reagent grade and used as received without further purification. Milli-Q water (18.2 MΩ cm) was used in all experiments.

2.2 Characterization

The morphologies and energy-dispersive X-ray spectroscopy (EDX) analysis of the SOS silica microspheres and SOS-COOH microspheres were examined by a XL30 ESEM FEG scanning electron microscope at an accelerating voltage of 20 kV (FEI, USA). Transmission electron microscopy (TEM) micrographs were obtained on a Hitachi H-600 transmission electron microscope (Hitachi, Japan) with an accelerating voltage of 100 kV. The UV-visible spectra were recorded by Power Wave XS 2 Microplate Spectrophotometer (Biotek, USA). The Fourier transform infrared (FTIR) spectra were recorded on a Bruker Vertex 70 spectrometer (Bruker, Germany). Both the wide-angle X-ray diffraction and small-angle X-ray diffraction patterns of the two microspheres were collected on a D8 ADVANCE X-ray diffractometer (Bruker, Germany) using Cu Kα as radiation, and recorded in the 2θ ranges of 10° to 80° and 0.6° to 5°, respectively. Thermogravimetric analysis (TGA) was carried out on a Perkin-Elmer TGA instrument. The samples were heated with a heating rate of 10°C min⁻¹ from 50 °C to 800 °C under nitrogen atmosphere.

2.3 Synthesis of silica microspheres

SOS and SOS-COOH silica microspheres were synthesized according to our previous work with slight modifications.^20,21 Briefly, 0.25 g PVA and 0.05 g CTAB were dissolved in 5 mL distilled water. Then, 8 mL methanol was added under stirring, followed by adding 2 mL ammonium hydroxide solution with desired concentrations (1.4%, 2.8% or 28%). Reaction mixture was stirred vigorously for 15 min before 0.4 mL MPTMS was added drop-wise over a period of 30 s. The reaction mixture was continuously stirred for 24 h at room temperature. The resulting SOS silica microspheres were collected by centrifugation (5000 rpm, 3 min), and calcined in a furnace at 600 °C for 5 h to remove the organic template. Subsequently, the as-prepared SOS silica microspheres were functionalized with carboxyl group as follows: 0.2 g SOS silica microspheres were added into 30 mL maleic anhydride toluene solution (0.00667 g mL⁻¹) and sonicated for 30 s. After adding 1 mL pyridine, the reaction mixture was incubated at 40 °C and stirred vigorously for 17 h. Finally, the microspheres were washed with toluene (10 mL, 3 times) and methanol (10 mL, 3 times), and dried in a vacuum oven at 80 °C for 12 h. The as-prepared carboxyl functionalized SOS silica microspheres were named SOS-COOH microspheres.

2.4 Immobilizing HRP on SOS-COOH microspheres

HRP was immobilized to the SOS-COOH microspheres by a two-step EDC/NHS coupling procedure. Briefly, the 10 mg mL⁻¹ SOS-COOH microspheres were firstly activated in MES buffer (100 mM, pH 5.70) containing 50 mM EDC and 50 mM sulfo-NHS for 20 min. After the activation, the mixture was centrifuged (5000 rpm, 3 min) and washed by 1 mL coupling buffer (PBS, 100 mM, pH 3.0). Subsequently, the activated SOS-COOH microspheres were reacted with 1.0 mg mL⁻¹ HRP solution in coupling buffer under gentle stirring at 25 °C for 6 h. The microsphere-HRP hybrids were collected by centrifugation (5000 rpm, 3 min) and washed with 1 mL PBS buffer (pH 7.0, 3 times) to remove the unbound enzyme. The hybrids were then re-dispersed in PBS buffer (pH 7.0) and stored at 4 °C. The as-prepared microsphere-HRP hybrids were termed as SOS-COOH-HRP. The immobilization yield of HRP was defined by eqn (1) while the amount of SOS-COOH microsphere kept as constant:

The accurate amount of enzyme in solution was determined by measuring the UV absorbance at 403 nm (extinction coefficient, ε = 1.02 × 10⁵ M⁻¹ cm⁻¹). The actual concentration of SOS microsphere immobilized HRP was defined by eqn (2):

2.5 Assay of enzyme activity

The activity of the immobilized enzyme was determined by monitoring the oxidation of PSA and 4-AAP by SOS-COOH-HRP in the presence of H₂O₂. In a typical experiment, 20 μL of 500 mM PSA, 8 μL of 20 mM 4-AAP and 20 μL of 10 mg mL⁻¹ SOS-COOH-HRP were added into 149.5 μL of 100 mM PBS buffer (pH 7.0) and mixed homogeneously. Then, 2.5 μL of 40 mM H₂O₂ was added into the mixture to initiate the reaction. After incubation at 35 °C for 10 min, the SOS-COOH-HRP was separated by centrifugation (5000 rpm, 3 min). The supernatants were transferred into 96-well plate and read by a Power Wave XS 2 Microplate Spectrophotometer at 490 nm (extinction coefficient, 6450 M⁻¹ cm⁻¹). The kinetic parameters of SOS-COOH-HRP and free HRP, such as K_m, V_max and k_cat, were determined by measuring the initial reaction rate with varying H₂O₂ concentrations as above description.

2.6 H₂O₂ detection assay

Various concentrations of H₂O₂ in PBS buffer (100 mM, pH 7.0), or diluted normal human serum (10% in 100 mM PBS) were reacted with 1.0 mg mL⁻¹ SOS-COOH-HRP, 50 mM PSA and 0.8 mM 4-AAP in a total volume of 200 μL. After incubated at 35 °C for 10 min, the reaction mixture was treated as previous description.

3 Results and discussion

3.1 Synthesis and characterization of SOS-COOH microspheres

The SOS silica microspheres were firstly prepared in a facile one-pot synthesis from MPTMS according to our previous report.²⁰ The SOS-COOH microspheres were then prepared by reaction of the SOS silica microspheres with maleic anhydride. The additional –COOH groups were grafted on the SOS microspheres surface through the reactions of hydroxyl groups and maleic anhydride (as shown in the Scheme S1 in ESI†). The morphologies of the as-prepared SOS silica microspheres and SOS-COOH microspheres are characterized by SEM and TEM measurements (as shown in Fig. 1). The SEM micrograph of the microspheres clearly shows that the large microspheres about 5.5 μm in diameter are densely coated by nanospheres around 500 nm in diameter, which can provide a higher surface area. No obvious deformation of morphology was observed after carboxyl functionalization. The TEM micrographs of the SOS silica microspheres and SOS-COOH microspheres (insets of Fig. 1A and B) also indicate that the microspheres have coarse surface. The phenomenon gives additional evidence on that the as-prepared microsphere is assembled by solid microsphere and nanospheres. The EDX analysis indicates that the elements (carbon, oxygen, silicon, and sulfur) are uniformly distributed in the microspheres (as shown in Fig. S1†). The FTIR spectra of SOS silica microspheres and SOS-COOH microspheres are shown in Fig. 1C. The absorption peaks at 470 cm⁻¹ and 803 cm⁻¹ correspond to the Si–O symmetric stretching and bending, respectively. The sharp peak at 1137 cm⁻¹ is attributed to Si–O–Si asymmetry stretching, which is characteristic IR peak of silica. The broad peak at 3435 cm⁻¹ is due to Si–OH stretching vibration, the peak at 2930 cm⁻¹ corresponds to C–H stretching vibration, and the peak at 2570 cm⁻¹ is characteristic vibration of CS-H. All the above peaks clearly show the formation of the SOS silica microspheres. Comparison with FTIR spectrum of SOS silica microspheres, the FTIR spectrum of SOS-COOH microspheres has strong absorption peaks around 1710 cm⁻¹ and 1730 cm⁻¹ (C=O stretching vibration) and weak absorption peak at 3435 cm⁻¹, demonstrating the successful functionalization of SOS silica microspheres with –COOH groups. The XRD patterns of the SOS silica microspheres and SOS-COOH microspheres indicate that the as-prepared microspheres are typical amorphous silica without mesoporous structure (as shown in Fig. 1D).²² The thermal stabilities of the SOS silica microspheres and SOS-COOH microspheres were studied by TGA (as shown in Fig. 1E). The mass loss of SOS-COOH microspheres is greater than that of SOS silica microspheres. The phenomenon is caused by the removal of water molecules from microspheres. The SOS-COOH microspheres can adsorb relatively higher amount of water than that of SOS microspheres since the –COOH groups have high hydrophilicity.

Fig. 1 SEM micrographs of the as-prepared SOS silica microspheres (1.4% ammonium hydroxide solution) (A) and SOS-COOH microspheres (B), the insets of (A) and (B) are the corresponding TEM micrographs. FTIR spectra (C), XRD patterns (D) and TGA curves (E) of the as-prepared microspheres.

3.2 Immobilizing HRP on SOS-COOH

The carboxylic groups on SOS-COOH microspheres provide, in theory, ideal anchoring points for the covalent coupling of proteins using the cross-linker EDC. In this study, HRP is used as model enzyme for addressing the immobilization ability of the SOS-COOH microspheres since immobilizing HRP on solid supports has been extensively studied.^5,6,12,19,23 As shown in Fig. 2, maximum immobilization yield (68.35%) is achieved at pH 3.0 and the immobilization yield is decreased drastically with increasing the solution pH value. After immobilization of HRP, no obvious morphology change of SOS microsphere has been observed by SEM and TEM measurements (Fig. S1F†) since the size of HRP (c.a., 3.7 nm × 4.3 nm × 6.4 nm) is much smaller than that of SOS-COOH microsphere. The EDX spectrum of SOS-COOH-HRP (Fig. S1H†) exhibits a clear peak for nitrogen, demonstrating the successful immobilization of HRP on the microspheres. Importantly, the catalytic activity of SOS-COOH-HRP is higher (1.3 folds) than that of free HRP when they are at the same HRP concentration (as shown in Fig. S2†). The catalytic performance of SOS-COOH-HRP is better than those of immobilized HRP on other silica supports such as mesoporous silica MCF and macroporous silica foam.^24,25 For instance, catalytic activities of immobilized HRP on mesoporous silica MCF or macroporous silica foam are lower than that of free enzymes in solution. Furthermore, because the covalent bonds are generally stronger than the physical interactions (e.g., van der Waals force and encapsulation), the as-prepared SOS-COOH-HRP may exhibit better stability than those of reported HRP–silica composites including SBA-15-HRP and MCM-41-HRP.^24,26 On the other hand, maximum immobilization yield is strongly dependent on the morphology of SOS-COOH microsphere (as shown in Fig. S3†). The SOS-COOH microspheres with high coverage of nanospheres show relative high HRP loading capacity. The experimental result indicates that the SOS-COOH microsphere with densely packed nanospheres can provide higher surface area, which is advantageous for immobilization of biomolecules. In the following study, all SOS-COOH-HRP were prepared by reaction of HRP and SOS-COOH microsphere with high coverage of nanospheres at pH 3.0.

Fig. 2 The effect of pH value on immobilization yield of HRP. The error bars mean standard deviation (n = 3).

3.3 Catalytic activity of SOS-COOH-HRP

A colorimetric assay was employed to evaluate the catalytic activity of the SOS-COOH-HRP using 4-AAP and PSA as substrates.^27,28 PSA can be enzymatically oxidized by hydrogen peroxide to produce a phenyl radical, which then reacts with 4-AAP to form a deeply colored quinone-imine dye, named 4-benzoquinoneimine-2,3-dimethyl-1-phenylpyrazolin-5-one. The absorbance at 490 nm (A₄₉₀) of the colored product is normally used for monitoring catalytic kinetics of enzyme. The Michaelis constants (K_m) and the maximum rate constants (V_max) of SOS-COOH-HRP and free HRP are determined from Lineweaver–Burk analysis (as shown in Fig. S4†). The K_m values of SOS-COOH-HRP and free HRP are 6.732 × 10⁻⁴ M and 9.43 × 10⁻⁴ M, respectively. And the V_max are 1.2424 × 10⁻⁵ M s⁻¹ and 1.4098 × 10⁻⁵ M s⁻¹ for immobilized HRP and free HRP, respectively. Because 1/K_m can approximately represent the affinity between enzyme and substrate, the results demonstrate that the immobilized enzyme has higher affinity to the substrate than free HRP, but it possesses a similar catalysis rate as free HRP. Furthermore, the k_cat/K_m value of SOS-COOH-HRP (9.370 × 10³ M⁻¹ s⁻¹) is higher than that of free HRP (7.591 × 10³ M⁻¹ s⁻¹). The experimental result suggests that catalytic efficiency of HRP is enhanced by immobilizing on the SOS microspheres. Because one microsphere can conjugate with several HRP molecules, the local concentration of immobilized HRP on the SOS-COOH-HRP is much higher than that of free HRP in solution while similar amount of HRP was employed for reaction. This phenomenon leads to that the SOS-COOH-HRP exhibits higher catalytic efficiency than that of free HRP.

3.4 The stability of SOS-COOH-HRP

The outstanding benefit of enzyme immobilization is enhanced stability under both storage and operational conditions. For studying the effect of solution pH on the stabilities of enzymes, the SOS-COOH-HRP and free HRP were pre-incubated in the buffers with various pH values for 1 h, and the enzymatic activities were then tested, respectively. The SOS-COOH-HRP shows the maximum activity at pH 8.0, while the free HRP exhibits maximum activity at pH 7.0 (as shown in Fig. 3A). Furthermore, underbasic reaction condition, the catalytic activity of the SOS-COOH-HRP is much higher than that of free HRP. The phenomenon is consistent with previously reported result.¹⁹ The relative catalytic activity of SOS-COOH-HRP is higher than that of free HRP during the testing period (50 days) when they are stored in PBS buffer (pH 7.0) at 4 °C (as shown in Fig. 3B). For instance, the SOS-COOH-HRP retains 76% of its original activity, while the free HRP only maintains about 45% of its initial activity after 50 days storage. In general, the catalytic activity of enzyme is strongly dependent on the enzyme conformation, i.e. the unfolding of the enzyme molecule can cause enzyme inactivation.²⁹ The unfolding of HRP can be retarded by steric effects when the HRP molecules are rigidly immobilized on the SOS-COOH microsphere, leading to significantly increase the stability of HRP.

Fig. 3 (A) The effect of solution pH value on the enzymatic activities of free HRP and SOS-COOH-HRP, the enzymes have been pre-incubated in the buffers with different pH values for 1 h, respectively; (B) storage stabilities of free HRP and SOS-COOH-HRP at 4 °C; and residual activities of free HRP and SOS-COOH-HRP after incubation with different concentrations of GdmCl (C) and urea (D) for 24 h. The error bars mean standard deviation (n = 3).

In order to further verify its stability, SOS-COOH-HRP was treated by two most common chemical denaturants, guanidine hydrochloride (GdmCl) and urea, respectively. After treated with low concentration (≤1 M) of GdmCl, the SOS-COOH-HRP exhibits similar inactivation behavior as free HRP. However, the SOS-COOH-HRP shows higher stability than that of free HRP in high concentration of GdmCl (as shown in Fig. 3C). The Coulomb interaction between GdmCl and the protein is normally thought to be involved in the denaturation process.³⁰ The free amine residues of HRP are decreased after covalent immobilization reaction, resulting in Coulomb interaction of GdmCl with SOS-COOH-HRP is weaker than that of GdmCl with free HRP. The phenomenon makes that the immobilized HRP is uneasily denatured by GdmCl. In the urea case, both the SOS-COOH-HRP and free HRP suffer from a gradual decrease of catalytic activity. However, the retained catalytic activity of the SOS-COOH-HRP is always higher than that of free HRP (as shown in Fig. 3D). In the presence of 1 M urea, the relative catalytic activity of SOS-COOH-HRP is moderately enhanced since the low concentration of urea is beneficial to maintain the active conformation of HRP through improving the stability of partly unfolded polypeptide chains in proteins.³¹ In 5 M urea, the SOS-COOH-HRP retains 49% catalytic activity, while the catalytic activity of the free HRP drops to 9%. Urea is found to interact directly with protein by pulling away water surrounding the protein.³² The SOS-COOH microsphere has abundant hydrophilic groups (e.g., sulfhydryl groups and carboxyl groups) on the surface which can attract water molecules to serve as part of the hydration shell of immobilized enzyme. As a consequence, the hydration shell can improve the stability of immobilized HRP under urea treatment.

3.5 Determination of H₂O₂

In order to test its utility, a SOS-COOH-HRP-based colorimetric assay has been developed for detecting H₂O₂. H₂O₂ is one of important reactive oxygen species, which accumulates in the mitochondria, and is closely related with a variety of diseases, such as cancer, Alzheimer's, Parkinson's diseases.^33–36 Various methods have been developed for the analysis of H₂O₂ including spectrophotometry,³⁷ fluorometry,³⁸ chemiluminescence³⁹ and electrochemistry.⁴⁰ In particular, nanomaterial-based enzymeless electrochemical biosensors exhibit significant superiorities on H₂O₂ detection including low-cost and long-term stability.⁴⁰ However, other electroactive species coexisting in the biological sample could also be oxidized under same experimental conditions, resulting in severely interfering with H₂O₂ detection. The enzyme-based colorimetric assay may overcome part of the limitations of the electrochemical read out format, and provides reasonable selectivity of H₂O₂ detection.³⁷ Several experiment parameters including reaction temperature and time, pH value of buffer and the concentrations of SOS-COOH-HRP, 4-AAP and PSA are investigated systematically to establish optimized conditions for H₂O₂ detection by monitoring the difference of A₄₉₀. As shown in Fig. 4A and B, the maximum value of A₄₉₀ is obtained at 35 °C and pH 7.0. The A₄₉₀ is increased with increasing the reaction time and the concentrations of SOS-COOH-HRP, PSA and 4-AAP, and tends to reach a plateau above 10 min of reaction time, 1.0 mg mL⁻¹ of SOS-COOH-HRP, 50 mM of PSA and 0.8 mM of 4-AAP, respectively (as shown in Fig. 4C to F). Under the optimized conditions, the A₄₉₀ is increased by increasing the concentration of H₂O₂ (as shown in Fig. 5). The A₄₉₀ shows a linear correlation to the concentration of H₂O₂ within the range from 1 μM to 1 mM (as shown in inset of Fig. 5), and the regression equation is A₄₉₀ = 0.03497 + 0.00156C with a correlation coefficient of 0.99746. The detection limit (3 times standard deviation) is experimentally determined to be 0.7 μM, which satisfies the requirement for early clinical diagnosis (e.g., threshold cell damage H₂O₂ concentration is 50 μM).⁴¹ The detection limit is lower than or equal to those of the previously reported methods (e.g., spectrophotometry- and electrochemistry-based assays), and the proposed method is also superior in the relatively wider linear range.^42–45 In particular, good stability and the excellent recycling/reusability of SOS-COOH-HRP can greatly decrease the cost of H₂O₂ detection.

Fig. 4 Effect of reaction temperature (A), solution pH value (B), reaction time (C), SOS-COOH-HRP concentration (D), PSA concentration (E) and 4-AAP concentration (F) on the absorbance at 490 nm (A₄₉₀), respectively. The error bars mean standard deviation (n = 3).

Fig. 5 UV-visible spectra of colorimetric sensing for H₂O₂ under varying concentrations in PBS (pH 7.0). The inset is the calibration curve of A₄₉₀ versus concentration of H₂O₂. The error bars mean standard deviation (n = 3).

3.6 Interference study

To further evaluate the selectivity of SOS-COOH-HRP-based colorimetric method for H₂O₂ determination, we investigate the A₄₉₀ responses to 500 μM H₂O₂ in the presence of possible interfering substances including inorganic ions, sugars and amino acids. As shown in Table S1,† the substances have little effect on the H₂O₂ determination. The experimental result indicates that the proposed SOS-COOH-HRP-based colorimetric method has relative high selectivity for H₂O₂ determination and great potential for detecting H₂O₂ in practical analysis.

3.7 Detecting H₂O₂ in real sample

The ability of the SOS-COOH-HRP has also been tested in H₂O₂ spiked 10% human serum. As shown in Fig. 6, the A₄₉₀ is linearly increased with increasing the concentration of H₂O₂ from 1 μM to 1000 μM, and the linear regression equation is A₄₉₀ = 0.06054 + 0.00145C with a correlation coefficient of 0.99961. H₂O₂ in 10% human serum can be detected as low as 2 μM. In addition, the result shows that the recovery rates are 94.45–108.35%, and the relative standard deviations (RSD) are less than 6.41% (as shown in Table S2†). The experimental result suggests that the SOS-COOH-HRP-based colorimetric method is reliable and reproducible, and can be employed for detecting H₂O₂ in real sample.

Fig. 6 UV-visible spectra of colorimetric sensing for H₂O₂ under varying concentrations in 10% human serum. The inset is the calibration curve of A₄₉₀ versus concentration of H₂O₂. The error bars mean standard deviation (n = 3).

3.8 Reusability of the immobilized enzyme

The immobilized enzyme can be easily separated from the reaction system and reused, which would greatly reduce the cost of practical use. Fig. 7 illustrates the residual catalytic activity of SOS-COOH-HRP after multiple recycling reactions. The catalytic activity of the immobilized HRP is slowly decreased with increasing the number of reacting cycles. However, the SOS-COOH-HRP still keeps 73% of initial activity after the 10th reacting cycle, which is much better than other reported solid supports including graphene (GO) (25% of its initial activity after 7 cycles),¹⁹ periodic mesoporous organosilicas (40% residual activity of the immobilized HRP on MI100 in the 6th recycle),⁴⁶ p-HRP@CaCO₃ (51% of its original activity after 7 uses).⁴⁷ The results demonstrate that the SOS-COOH-HRP has good reusability and stability. The decreasing of catalytic activity may due to lost trace amount of HRP from the SOS-COOH microsphere and/or the steric hindrance of accumulating reaction products in the reacting cycles.⁴⁸

Fig. 7 Reusability of SOS-COOH-HRP in PBS (100 mM, pH 7.0). The error bars mean standard deviation (n = 3).

4 Conclusions

In conclusion, carboxyl functionalized SOS (SOS-COOH) microspheres have been synthesized and employed as solid matrix for enzyme immobilization. Compare to free HRP, the SOS-COOH-HRP hybrid shows improved stability towards high pH values, denaturing agent and relative long storage time. The experimental result demonstrates that SOS-COOH microspheres can be used as ideal carriers for immobilization of enzymes. The covalent immobilization of enzymes on SOS-COOH microspheres preserves the native structure of enzyme and the increase of local enzyme concentration in reaction solution, resulting in much greater enhancement of enzymatic activity and stability in contrast to unbound enzyme. Furthermore, SOS-COOH-HRP can be easily recycled by centrifugation. This study provides a practical strategy for cost-effective applications of enzyme in biocatalysis and biosensing.

Acknowledgements

The authors would like to thank the National Natural Science Foundation of China (Grant no. 21075118) and Jilin Provincial Science and Technology Department (Grant no. 20100701) for financial supports.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: The schematic representation of the synthesis SOS-COOH-HRP, EDX analysis of SOS-COOH microspheres and SOS-COOH-HRP, additional spectra data, SEM images, double reciprocal plots, and tolerance of coexisting substance, recovery rate of real sample analysis. See DOI: 10.1039/c5ra03755g

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