Stabilized enzyme immobilization on micron-size PSt–GMA microspheres: different methods to improve the carriers' surface biocompatibility

Abstract

Stabilized immobilization of biomacromolecules on carriers with appropriate orientation and minimum conformational changes is very important in the biochemical and biomedical fields. In this study, PSt–GMA(polystyrene–glycidyl methacrylate) microspheres were utilized as the carriers of immobilizing glucose oxidase (GOD). To improve the carrier's biocompatibility, anionic sulfonate groups and different length of spacer arms were introduced onto the microspheres' surface. The effect of carrier surface properties on the activity and stability of immobilized enzymes was investigated. The results show that the introduction of sulfonate groups and spacer arms could effectively reduce the unspecific adsorption of enzymes and improve their stabilities. Besides, with the increase of the length of spacer arms, both the immobilization amount and stabilities of immobilized GOD could be increased under different experimental conditions.

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1. Introduction

Monodisperse polymer microspheres as absorbents or carriers for biomacromolecules are a kind of versatile material with many important applications in biotechnologies.^1–7 For most of these applications, in particular the modern immunoassays, biomacromolecules need to be immobilized onto tailor-made carriers with specific functionalities, so that the specific binding of receptors and ligands could be “read out” by detecting various signals, including laser, bio- or chem-fluorescence and so forth.^8–13 For example, a glucose biosensor with glucose oxidase (GOD)-functionalized nanomaterial was reported.^13–15 Shi reported GOD-functionalised microspheres by in situ encapsulating GOD loaded functional Fe₃O₄@C nanoparticles with silica.¹⁴ It should be kept in mind that the stabilized immobilization of biomacromolecules on carriers with appropriate orientation and minimum conformational changes is a vital process because it can decisively influence the final quality of detection.^16–19 This is because most proteins are so susceptible to environment changes that they may easily undergo conformational changes and bioactivity losses when attached to solid-phase surfaces. The extent of their denaturation largely depends on a lot of factors, including the structural stability of molecules, the molecule–surface interactions, interfacial crowding, and the immobilization conditions as well.^3,20–23

Till now, various technologies have been developed for protein immobilization, which comprise binding to a carrier, entrapment (encapsulation) and cross-linking.^24–26 Binding to a carrier can be physical (such as van der Waals and hydrophobic interactions), ionic, or covalent. In general, covalent immobilization is superior to physical adsorption, because covalent binding could be effectively avoided the leak of adsorbed proteins during immobilized process through competitive adsorption on exposure to complex biological fluids.²⁷ Among the functional groups on the carrier's surface with for covalent immobilization of proteins, epoxy activated supports have been proposed as very efficient materials for protein immobilization.²⁸ For one thing, the activated supports are very stable during storage and suspension in neutral aqueous media. For another, epoxy-activated supports are easy to form stable covalent linkages with different protein groups (amino, thiol, and phenolic ones) under very mild experimental conditions (e.g. pH 7.0).^29–31 Furthermore, they could be further chemically modified or activated so that various immobilization methods could be created, e.g. the attachment with spacer arms and multipoint immobilization with different groups.³²

It is recently reported that the immobilization of biomacromolecules onto epoxy activated supports follows a two-step mechanism: first a rapid physical adsorption between the proteins and the carriers, and second the covalent reaction between the adsorbed proteins and the epoxy groups. Recently, Roberto Fernández-Lafuente et al. have shown that the modification of epoxy groups with different groups may promote the first-step physical adsorption of proteins.^33–35 Based on these findings, it could be noticed that the control of surface properties has become a quite effective method to regulate proteins' immobilization behaviors.^36,37

In our previous paper, we studied the direct immobilization of glucose oxidase (GOD) onto micron-size PSt–GMA microspheres.³⁸ The results show that only 30% enzymatic activity could be retained as compared with free enzymes. To a large extent, the decreased activity of immobilized GOD might be attributed to their conformational changes, resulting from the strong hydrophobic interactions between the attached molecules and the hydrophobic surface of polystyrene carriers.

To overcome the shortcomings of previous immobilization method, two strategies are put forward. The first one is to introduce additional sulfonate groups onto the carriers' surface. It is hoped that the electrostatic forces between anionic sulfonate groups and the charged proteins could be utilized to (1) regulate the first-step physical adsorption of proteins, (2) control the orientation of adsorbed molecules on the microspheres' surface, and (3) weaken the fierce interactions between proteins and the hydrophobic surface of polystyrene carriers.^39,40 And the second one is to modify epoxy groups with other reagents to generate spacer arms.^23,41 Therefore, in the present work, more attention will be focused on the effects of carriers' physicochemical properties on enzyme's immobilization behaviors as well as the stabilities of immobilized enzymes.

2. Materials and methods

2.1 Materials

Styrene (St, Xilong Chemicals Co., China) was retreated with 5% (wt) NaOH solution in order to remove inhibitor hydroquinone completely. 2,2′-Azobisisobutyronitrile (AIBN), poly(N-vinyl pyrrolidone) (PVP, M ∼ 30 000) and ethanol were of analytical grade. Glycidyl methacrylate (GMA, 98%) and sodium sulfonate styrene (NaSS, 93%) were obtained from Jiyuan and Xingzhilian Chemicals Co. (China), respectively. Glucose oxidase from Aspergillus niger (E.C.1.1.3.4, Catalogue number 49178, 22.5 U mg⁻¹) and β-D-(+)-glucose (ACS reagent, EC200-075-1) were purchased from Sigma-Aldrich (Steinheim, Germany). Horseradish peroxidase (E.C.1.11.1.7, ≥150 U mg⁻¹) was obtained from Kaiyon Biological Technology Co. (China). All other chemicals were of analytical grade. Double-distilled water was used throughout this work.

2.2 Preparation and characterization for microspheres

Micron-size monodisperse PSt–GMA and PSt–NaSS–GMA microspheres were prepared by dispersion polymerization, with St as monomer, and GMA or NaSS as comonomers. In brief, 60 mL ethanol, 0.2 g AIBN, 0.4 g PVP, and 14.5 mL St were simultaneously added into a four-neck round-bottom flask (100 mL) fitting with a mechanic stirrer, nitrogen inlet and a reflex condenser. After deaeration with N₂ for 30 min, polymerization reaction was conducted at 70 °C for 5 h under agitation (250 rpm). Then 15 mL ethanol dissolving 0.5 mL GMA (together with 15 mL methanol dissolving 0.4 g NaSS for preparing PSt–NaSS–GMA microspheres) was slowly introduced into the flask within 4 h under an agitation speed of 300 rpm. Thereafter, the copolymerization reaction proceeded at 70 °C until a total reaction time of 12 h ended. After cooling down, the resultant microspheres were purified by repeated centrifugation, decantation and re-dispersion with methanol and water before they were stored at 4 °C for further use.

Microspheres' particle size and size distribution were characterized by a laser diffraction particle size analyzer (Mastersizer 2000, Malvern, Britain), which yielded the volume-average diameter of bulk population, uniformity as a measure for the absolute deviation of median, and average specific surface area for particles. Morphology analysis was performed on a Scanning Electron Microscope (SEM, JSM-6700F, JEOL Ltd, Japan) after the samples were coated with gold under vacuum.

2.3 Introduction of spacer arms

The processes of introducing spacer arms onto the microspheres consisted of two-step reactions (Scheme 1). In the first step, a fixed amount of microspheres were incubated with 60 mL aqueous ammonia (Scheme 1 (A)) or phosphate buffer solutions (PBS, 0.1 M, pH = 8.0) containing 2% diamine (i.e. either ethylenediamine or hexamethylenediamine, Scheme 1 (B)) in a 100 mL single-neck round-bottom flask. Then the mixture was magnetically stirred gently at 50 °C for 12 h, until nearly all the surface epoxy groups were converted to primary amine groups.^42,43 After the reaction, the aminated microspheres were purified by repeated washing with water to remove the residual ammonia or diamine reagents completely. In the second step, the treated microspheres were incubated in a sealed flask with PBS (0.1 M, pH = 8.0) containing 2% (wt) glutaraldehyde at 25 °C for 5 h under mild stirring, and the spacer arms with different length of atoms (n = 8, 11, 15, respectively) were generated through the reaction between amine groups and the aldehyde groups. Finally, the spacer arm attaching microspheres were purified by repeated washing with water.

Scheme 1 Schematic representation of different surface treatment methods for the introduction of spacer arms. (A) Reaction with ammonia (n = 8); (B) reaction with diamine (i = 2 or 6, n = 11 or 15, respectively).

Noted: the length of spacer-arm was taken as the number of atoms between the nitrogen atom of peptide bond and carbon atom of aldehyde. The length of spacer-arm of PSt–NaSS–GMA microspheres was n = 2.

2.4 Enzyme immobilization

The typical batch experiments of GOD immobilization were carried out in polypropylene cuvettes immersed in a thermostatic water bath fitting with electromagnetic stirring. In each experiment, 50 mg clean microspheres were incubated in 2 mL PBS (0.1 M, pH = 7.0) containing a predetermined amount of GOD. The sample solution was stirred gently at 25 °C for 8 h. Thereafter, the immobilized GOD was separated from the solution by centrifugation, decantation, and washing with 2 mL fresh buffer solution twice. The separated microspheres loading with immobilized enzymes and the collected supernatants were preserved for further testing.

2.5 Enzymatic assays

The concentration of unbound GOD in the supernatant solutions was determined by a spectrophotometric method.^39,44,45 For each test, a blank experiment was set up (without GOD), allowing correction to be made for counteracting the possible background noise. The amount of immobilized GOD per gram microspheres (MS) was determined by the mass balance of enzymes, and the average of triplicates testing results is presented.

The activity of both free and immobilized GOD was assayed based on a colorimetric method, as was described in our previous work.³⁸ The enzymatic catalysis process consists of two-step reactions as follows:

In order to test the enzyme activity of both free and immobilized GOD, the reaction period of glucose oxidation was strictly controlled at 15 min.³⁸ In brief, a total volume of 4.0 mL phosphate buffer solution (0.1 M, pH = 7.0) containing 0.002 mg free or immobilized GOD and a specific amount of glucose was mixed in a clean polypropylene cuvette, and stirred gently for a suitable time. Then 2.5 mL reactant solution was decanted and mixed with 1 mL 200 mM 4-aminoantipyrine, 1 mL 200 mM phenol, and 0.5 mL horseradish peroxidase (≥20 U mL⁻¹) solution, and stirred quickly for 40 s. The amount of H₂O₂ could be determined by measuring the absorbance of produced quinoneimine dye in solutions at 510 nm.^43,46,47 The activity of GOD was calculated by the amount of H₂O₂ produced divided by reaction time and enzyme amount.

3. Results and discussions

3.1 Characterization for microspheres

PSt–GMA and PSt–NaSS–GMA microspheres were synthesized by dispersion polymerization, and their supposed structures were presented in Fig. 1. The result shows that their average sizes were 5.1 μm and 4.9 μm, and the uniformity reaching 0.265 and 0.257, respectively, suggesting that the microspheres with a narrow size distribution were obtained. Fig. 2 shows the SEM micrographs of these two kinds of microspheres. The smaller size of PSt–NaSS–GMA microspheres was attributed to the usage of additional methanol (with higher polarity than ethanol) as the solvents of NaSS. Previous researches indicated that the increase in the polarity of the dispersion medium in dispersion polymerization usually leads to a reduced particle size of the resultant microspheres.^48,49 However, the specific surface area for both two kinds of microspheres was very similar: 1.24 m² g⁻¹ for PSt–GMA microspheres, and 1.28 m² g⁻¹ for PSt–NaSS–GMA microspheres. Therefore, the approximative specific area allows a quantitative comparison to be made for the immobilization amount of enzymes onto different microspheres.

Fig. 1 Schematic representation of proposed structures of PSt–GMA (a) and PSt–NaSS–GMA (b) microspheres synthesized by dispersion polymerization.

Fig. 2 The SEM micrographs of synthesized PSt–GMA microspheres (a); and PSt–NaSS–GMA microspheres (b).

The morphologies of microspheres with and without spacer arms were shown in Fig. 3. By comparing with blank microspheres (Fig. 3(a)), there were some newly emerged convex structures on the surface of three other samples, and with the increase of spacer arms, both the quantity and area of the convexes would increase, and even formed “island” patches (Fig. 3(c) and (d)). To explain this, one should first keep in mind that the surface of the microspheres via dispersion polymerization is not absolute smooth but “hairy”, as reported by Kawaguchi.⁵⁰ Besides, since the outer layers of microspheres are composed of PGMA grafted onto PSt polymer chains, these chains must provide lots of epoxy-activated sites. Moverover, the epoxy-activated sites were irregular linked onto the surface of PSt–GMA microspheres, and spacer arms were grafted to the epoxy-activated sites, so longer spacer arms were linked molecular brush and might give rise to some fluffy features on the particles' outer layers.

Fig. 3 SEM micrographs of PSt–NaSS–GMA microspheres after different surface treatment: (a) without treatment; (b) treated with ammonia and glutaraldehyde; (c) treated with ethylenediamine and glutaraldehyde; (d) treated with hexamethylenediamine and glutaraldehyde.

3.2 Effect of sulfonate groups on the stability of immobilized GOD

The stabilities of immobilized GOD on PSt–GMA and PSt–NaSS–GMA microspheres were investigated. In this part of work, a fixed amount of microspheres were incubated in PBS with free GOD for 8 h at 25 °C. Then unbound GOD was washed off by repeated washing with fresh PBS and the residual enzymatic activity of immobilized GOD was assayed (see Fig. 4). The immobilized between the enzyme molecule and the carriers can take place either by adsorption or by formation of covalent bonds between active side of the enzymatic protein and the functional group of the solid carrier. The binding forces of physical adsorption, such as van der Waals binding, hydrophobic interactions or ionic interactions, were too weak to keep the adsorption molecule fixed to the carriers after the repeat washing. However, the binding force via covalent immobilization was too strong, so the enzyme molecule still anchored onto the carrier after the repeat washing. For ease of discussion, it was assumed that the activity of immobilized GOD is proportion to the amount of enzymes. In fact, the activity of immobilized GOD was decreased after repeating washing, and the activity of immobilized GOD was generally higher by adsorption than one by covalent attaching. From the Fig. 4, the activity of immobilized GOD on the PSt–GMA microspheres was higher than one on the PSt–NaSS–GMA microspheres after the first washing. However, the activity of immobilized GOD on the PSt–GMA microspheres was lower than one on the PSt–NaSS–GMA microspheres after the third washing. It was noticed that the decreased ration of the activity of immobilized GOD between the first washing and the third washing was different for the different carriers. This result suggested that the immobilized GOD on the PSt–GMA microspheres leaked more than one on the PSt–NaSS–GMA microspheres, and GOD could bind more easily onto the PSt–GMA by the physical adsorption compared to one onto the PSt–NaSS–GMA microspheres. Therefore it could be noticed that the sulfonate groups of PSt–NaSS–GMA microspheres played important roles. The sulfonate groups may effectively weaken the hydrophobic interactions between enzymes and polystyrene segments, thus effectively reduced the unspecific adsorption and improved the covalent immobilization efficiency. Therefore, in the following work, PSt–NaSS–GMA microspheres were further used.

Fig. 4 The effects of additional sulfonate groups on the stability of enzymes immobilized on microspheres after repeated washing with PBS (compared with PSt–GMA microspheres).

3.3 Effect of spacer arms on the enzyme immobilization

Fig. 5 shows the effect of spacer arms on enzyme immobilization amount of different enzyme bulk concentrations. The increase of spacer arms length resulted in an increase of immobilization amount of GOD, and similar results were reported elsewhere.⁵¹ The difference in GOD's immobilization amount was attributed to the difference of the immobilization efficiency. For no spacer arms or only shorter spacer arms, the mobility of immobilized molecules was poorer. Thus, great steric hindrance influenced binding interactions between enzymes and carriers. But when the length of spacer arms increases, the mobility of immobilized molecules was improved (Scheme 2), and the steric hindrance could be effectively reduced.

Fig. 5 Immobilization amount of glucose oxidase on PSt–NaSS–GMA microspheres with different length of spacer arms (number of atoms n = 8, 11, and 15, respectively, n = 2 indicating no spacer arms were used).

Scheme 2 Physisorption and chemical immobilization of enzyme molecules: (a) direct immobilization or immobilization via very short spacer arms; (b) via a middle length of spacer arms; and (c) via a relatively long spacer arms.

The effect of spacer arms on the activity of immobilized GOD was studied (Fig. 6). It was found that longer spacer arms were superior to shorter ones because of better activity retention. In particular, for the longest spacer arms being used, about 90% activity could be retained as compared with free enzymes, suggesting a very slight denaturation of GOD after immobilization. There are some reasons for the higher activity in the case if immobilization via a longer spacer-arm. Firstly, it has higher mobility of the enzyme molecule on the longer spacer-arm and hence higher collision probability with substrate, so it reduced steric hindrance. Secondly, it avoids the direct contact between enzyme molecules and the rough surface of carrier.

Fig. 6 Activity of both free and immobilized enzymes as a function of glucose concentration at 25 °C and pH 7.0 (the concentration of free or immobilized GOD was fixed at 0.0005 mg mL⁻¹).

The kinetics parameters of free and immobilized GOD were determined by using the classic Michaelis–Menten equations:

Plotting 1/V versus 1/[S] yields an appropriate linear plot, which is known as Lineweaver–Burk plot (see Fig. 7). Two important kinetics parameters Michaelis constant (K_m) and maximum reaction rate (V_max) were calculated from the slope and intercept of the plot of 1/V vs. 1/[S], respectively; and the results were listed in Table 1. Here, it is necessary to mention that the value 1/K_m reflects the affinity of enzymes to substrates: the larger value of 1/K_m, the stronger affinity of enzymes to substrates. In the case of free GOD, the value of K_m was 21.537 mM for free enzymes. Although the process of immobilization could generally increase the value of K_m, longer spacer arms might counteract this tendency. Similar results were reported in other literature^38,52 Understandably, enzymes immobilized onto the spacer arms have higher affinity to substrates than enzymes directly immobilized, which results from the increased frequency and probability of molecular collision and the less extent of inactivation. By comparison between free enzymes and enzymes immobilized on the longest spacer arms (n = 15), their K_m was already very approximative. On the other hand, the value of V_max was almost unchanged for all systems, which implies that the barriers of the diffusion of substrate and product could be negligible in all cases.

Fig. 7 Lineweaver–Burk plots of the enzymatic activity of free and GOD immobilized directly onto microspheres or onto different spacer arms. n indicates the length of spacer arms (i.e. atom number).

Table 1 Kinetics parameters of free and immobilized GOD at 25 °C and pH 7.0

GOD	Km (mM)	Vmax (M min^−1 mg^−1 GOD)
Free	21.537	25.575
No spacer arms (n = 2)	57.385	25.316
On spacer arms (n = 8)	50.726	25.641
On spacer arms (n = 11)	46.747	25.773
On spacer arms (n = 15)	24.094	24.752

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Compared to free enzyme, an important advantage of immobilized enzymes is the improvement of the thermal stability.^53,54 As known, there are two steps for the immobilized process: the physical adsorption and covalent attachment. Due to the complexity of the immobilized process, it is usually difficult to make a distinction quantitative between the physical adsorption and covalent attachment. However, it is known that the physical adsorption is too weak to retain the interaction of the enzyme and carrier, so the stability of physical adsorption enzyme is lower compared to that of covalent immobilized enzyme.

For technical applications, it is usually required that immobilized enzymes should tolerate relatively tough environmental conditions and keep enzymes' activity at a wider pH or temperature range.^55,56 Therefore, the thermal stability of immobilized enzymes at 50 °C was first analyzed. As reflected from Fig. 8, while free enzymes suffered from great activity loss after incubating at 50 °C for 150 min, the activity changes were not so obvious for immobilized enzymes, in particular, only 20% activity was lost for enzymes immobilized onto long spacer arms (n = 15). The good thermal stability of immobilized enzymes could be attributed to the fixation of enzyme molecules to hairy chains or spacer arms of microspheres, which prevent autodigestion and the maintenance of the weak intramolecular forces in the GOD molecules, and/or thermal inactivation of glucose oxidase. Due to the immobilization reaction and the limited movement of enzyme molecule, the stability of immobilized enzyme is improved after immobilized process. From the Scheme 2, it could obtain that the longer spacer-arm could entraps more GOD molecules and limits more their movement than the short spacer-arm did. It can be understand that the less movement of GOD immobilized on the spacer-arm modified microspheres, the higher thermal stability. Moreover, the thermal stability of immobilized GOD illustrated the interaction strength between the enzyme and carriers. Compared to shorter spacer-arm, the interaction force between the enzyme and carrier with longer spacer-arm was stronger. In a word, the good thermal stability of immobilized enzymes could be attributed to the fixation of enzyme molecules to hairy chains or spacer arms of microspheres, which prevent autodigestion and the maintenance of the weak intramolecular forces in the GOD molecules, and/or thermal inactivation of glucose oxidase.

Fig. 8 Thermal stability of glucose oxidase immobilized onto microspheres or different spacer arms. Both free and immobilized enzymes were incubated at 50 °C in PBS (0.1 M, pH 7.0). At different time intervals aliquots were taken to measure the enzymatic activity with glucose as substrates.

The effect of pH values on the enzymatic activity changes was further investigated (see Fig. 9). No shift of enzymatic optimum pH value was observed for both free and immobilized GOD. Apart from this, it was found that all employed spacer arms could noticeably enhance the stability of glucose oxidase at a wider range of pH value, as compared with the case of free enzymes. This implies that the spacer arms could protect immobilized enzymes from inactivation in solutions apart from the optimum pH value.

Fig. 9 Enzymatic activity changes as a function of pH values (25 °C). The concentration of free or immobilized GOD was 0.5 mM and the concentration of glucose was 43.75 mM. Other conditions were kept constant.

Some researchers reported that the optimal reaction pH of immobilized enzyme has been changed compared to free enzyme.⁵⁷ Generally, the optimal reaction pH of immobilized enzyme onto cationic carrier moves toward the lower pH, in contrast, it moves toward the higher pH if enzyme was immobilized onto anionic carriers. No shift of the enzymatic optimum pH value can be illustrated that the microenvironment of these carriers did not change. Compared to free enzyme, the immobilized enzyme enhances the pH profile because of the limited movement and multipoint linked. For longer spacer-arm, it can entrap the GOD molecule and provide more interaction forces, so the conformational change is lower when the pH changes. This can explain that the GOD immobilized on longer spacer-arm modified microspheres possesses the broader pH profile.

The operational stability for immobilized GOD was also investigated. A fixed amount of immobilized enzymes were treated by repeated washing with PBS (0.1 M, pH 7.0), and the residual activity was measured, which was calculated by dividing the actual activity by the initial value. Plotting residual activity versus washing times yields four curves for different enzyme systems. As was shown in Fig. 10, with the increase of the length of spacer arms, the stability of immobilized enzymes was obviously improved. For example, in the case of no spacer arms, less than 60% activity was retained after eight cycles of washing. However, when the longest spacer arms were used, the residual activity decreased slowly after each cycle, and 85% of that was retained finally. This proved again that, the longer spacer arms being used, the better stability achieved for immobilized enzymes.

Fig. 10 The relationships between residual activity of immobilized enzymes and washing times with phosphate buffer solution (0.1 M, pH 7.0).

The different operational stability of immobilized enzyme could be attributed to at least two factors. Firstly, physical-adsorbed GOD leaked more easily than covalent bond did. Compared to the microspheres with longer spacer-arms, the enzyme amount by physical-adsorbed was more than one by covalent immobilized for the microspheres with shorter spacer-arms. So, after undergoing repeated washing, the microspheres with shorter spacer-arm have less of immobilized GOD, so less enzymatic activity. However, the lost amount of GOD on the longer spacer-arm is less than that on the shorter spacer-arm, so the GOD on the longer spacer-arm possesses higher enzymatic activity. Secondly, the conformation of immobilized enzyme molecule may generally change after repeated washing, however the longer spacer-arm could protect better the conformation of enzyme molecule. In a word, the longer spacer arms and covalent binding formed between the GOD molecules and the functional groups on the microspheres' surface were believed to be two positive factors in preserving enzymes and protecting GOD's three-dimensional structures from severe changes or damage. Therefore, immobilized GOD onto PSt–GMA microspheres with longer spacer-arm still preserved sufficient native enzymatic activity.

4. Conclusion

In the present work, micron-size PSt microspheres with different groups or different length of spacer arms were used as carriers for a systematic study of GOD immobilization. It was found that the introduction of different groups on the PSt microspheres could influence the immobilized amount and the stabilities of bound enzymes. Further investigations have also shown that immobilization of GOD onto spacer arms is a better method than direct immobilization, because the spacer arms could effectively increase the immobilization amount and avoid the inactivation of GOD. Furthermore, longer spacer arms are preferable to short ones in conjugating GOD. This was proved by several stabilities improvement of immobilized GOD in varying environmental conditions (temperature, pH value, repeated washing, etc) as compared to free enzyme. Therefore, the combination of the above two methods has successfully proved in effectively improving the biocompatibility of resultant microspheres. In the future, a kind of functional microspheres with spacer arms, particularly, the GOD-functionalized microspheres are going to apply in biomedical or biochemical fields, such as glucose biosensing, biocatalysis.

Acknowledgements

We thank Science Foundation of Hengyang Normal University (13B29), Hunan Provincial Natural Science Foundation of China (2016JJ6014), Aid programs for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province and the Key Discipline of Hunan Province for financial support of this work.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra18126k

‡ These authors equally contributed to the work.

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