In-Situ Self-Assembly of Zinc / Adenine Hybrid Nanomaterials for Enzyme Immobilization

In this study, a one-step and facile immobilization of enzymes by self-assembly of zinc ions and adenine in aqueous solution with mild conditions was reported. Enzymes, such as glucose oxidase (GOx) and horseradish peroxidase (HRP), could be efficiently encapsulated in Zn/adenine coordination polymers (CPs) with high loading capacity over 90%. When the enzyme was immobilized by CPs, it displayed high catalytic efficiency, high selectivity and enhanced stability due to the protecting effect of the rigid framework. As a result, the relative activity of Zn/adenine nano-CP-immobilized GOx increased by 1.5-fold at pH 3 and 4-fold at 70 to 90 ◦C, compared to free GOx. The immobilized GOx had excellent reusability (more than 90% relative activity after being reused eight times). Furthermore, the use of this system as a glucose biosensor was also demonstrated by co-immobilization of two enzymes, detecting glucose down to 1.84 μM with excellent selectivity. The above work indicated that in-situ self-assembly of Zn/adenine CPs could be a simple and efficient method for biocatalyst immobilization.


Introduction
Enzymes are a typical class of biocatalysts, having been used in a variety of scientific and technical areas.For instance, they have wide applications in the fields of fine and bulk chemicals, foods, pharmaceutical science, cosmetics, textiles and paper industries, due to their high catalytic activity, high selectivity, low toxicity and water solubility [1][2][3][4][5][6].However, the disadvantages of free enzymes, including high cost, poor operational stability and challenges in recovery and reuse, have limited industrial applications of enzymes [7].To solve these issues, immobilization techniques are considered, because binding of free enzymes to supports limits their mobility [8].What is more, some immobilized enzymes could show more robust activity than free enzymes [9][10][11][12].
Conventional immobilization methods are generally divided into four main categories; adsorption, covalent binding, entrapment and cross-linking [13][14][15][16][17][18][19][20][21][22].However, there are several disadvantages in the conventional immobilization methods, such as the lack of effective reusability, difficulties in immobilization, a severe loss of enzymatic activity due to the blocking of the active site of the enzyme, restricted flexibility and mass-transfer limitations between the enzyme and substrate [13].Therefore, some new methods and materials to immobilize enzymes have been developed.
In nature, biomolecules normally have excellent metal-coordination properties [33].Up to now, various types of biomolecules, such as nucleotides [34], proteins [35], peptides [36], amino acids [37] and nucleobases [38] have been used as the ligands to construct CPs.It also has been demonstrated that some CPs are capable of adsorbing and entrapping a broad range of molecules, due to their good biocompatibility and porosity [34,39].Adenine, as an important naturally occurring nitrogen heterocycle present in nucleic acids [40], has multiple possible metal-binding modes [41][42][43].It has been reported that adenine could coordinate with Zn [44], Au [45], Ag [46], Co [47] or Cu [48] to form CPs with diversified morphology and structure.However, most of the researchers just studied the structure of these materials, and very few have studied the encapsulating adaptability of CPs and the activity of guests after they were entrapped.What is more, those CPs that have been reported were formed by harsh reaction conditions, complicated syntheses and with high cost [44][45][46][47][48].
In this work, we reported a convenient, efficient and high-capacity immobilization method for enzymes by the entrapment of glucose oxidase (GOx) and horseradish peroxidase (HRP) within Zn/adenine CPs.We used adenine and zinc in aqueous solution without adding other linkers to form CPs by in-situ self-assembly.Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to characterize the CPs.After the CPs was confirmed with promising encapsulating capacity, we use them to immobilize enzymes.The enzyme activity, pH and thermostability of immobilized enzymes were also investigated.The results revealed that the immobilized enzymes showed high catalytic efficiency, enhanced stability and recyclable usability.Besides, a highly sensitive and selective biosensor for glucose was prepared using the CPs to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system.The Zn/adenine CPs were promising for enzyme immobilization.

Preparation and Characterization of Zn/Adenine Complexes
We first tested the in-situ self-assembly of zinc ions and adenine.The synthesis of the Zn/adenine composite was performed by mixing zinc chloride solution, adenine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at room temperature.Then, a white solid precipitate quickly appeared.To understand the formation of the coordination complexes, the coordination of Zn 2+ and adenine was studied in different concentrations of HEPES (Figure S1a).The amounts of Zn/adenine composite increased with increasing HEPES concentration (Figure S1b).High HEPES concentration was beneficial to the formation of complexes.The coordination of Zn 2+ and adenine was also studied in different pH and ionic strength.As shown in Figure S1c, the yield of Zn/adenine composites reached the maximum at pH 7.8.Low pH leads to the protonation of adenine [40], which would inhibit the coordination of Zn 2+ ions.Then, the self-assembly reaction was performed at different ionic strengths.It could be found that the amounts of Zn/adenine composite increased with increasing NaCl concentration (Figure S1d).The solubility of adenine in aqueous solution was weakened in high ionic-strength solutions, and it would intensify the reaction of Zn 2+ ions and adenine [34].
To gain a further understanding of the Zn/adenine composite, we characterized CPs by FTIR, XRD, SEM and TEM.As shown in Figure 1a, the shift of the IR band may suggest coordination interactions between Zn 2+ and N 9 (from 1418.4 cm −1 in adenine to 1401.2 cm −1 in the coordination polymer) [45].The 1671.8 cm −1 band of adenine was considered to arise from the NH 2 scissoring vibrational mode [45].The corresponding NH 2 IR band of the coordination polymer was observed at 1643.0 cm −1 .The shift of the NH 2 IR band could be attributed to the C 6 -NH 2 coordination with Zn 2+ .The assignments of FTIR spectra of adenine and Zn/adenine complexes were listed (Table S1).According to the results of XRD (Figure 1b), no sharp diffraction peaks were observed in Zn/adenine complexes, indicating the amorphous nature of Zn/adenine complexes.This may be caused by the asymmetric chemical structure of nucleobases and high coordination flexibility of zinc ions [40].
The SEM photo (Figure 1c) and TEM photo (Figure 1d) revealed that the microstructure of CPs resembles a stack of nanoparticles.From the images, we also can re-confirm the amorphous nature of the nano CPs.Zn 2+ ions and adenine may firstly form nanoparticles, and then these nanoparticles assemble together (Figure S2).Finally, the stoichiometry of Zn/adenine CPs determined by the titration experiment was adenine:Zn 2+ = 1:2 (Figure S3).We considered that adenine bridges four Zn 2+ ions through the N 3 , N 7 , N 9 and NH 2 sites, forming a framework structure (Figure 2).This coordination mode is very similar to the one observed by Hui Wei et al. [45].
Catalysts 2017, 7, 327 3 of 11 SEM photo (Figure 1c) and TEM photo (Figure 1d) revealed that the microstructure of CPs resembles a stack of nanoparticles.From the images, we also can re-confirm the amorphous nature of the nano CPs.Zn 2+ ions and adenine may firstly form nanoparticles, and then these nanoparticles assemble together (Figure S2).Finally, the stoichiometry of Zn/adenine CPs determined by the titration experiment was adenine:Zn 2+ = 1:2 (Figure S3).We considered that adenine bridges four Zn 2+ ions through the N3, N7, N9 and NH2 sites, forming a framework structure (Figure 2).This coordination mode is very similar to the one observed by Hui Wei et al. [45].

Encapsulation Property of Zn/Adenine Complexes
After confirming the self-assembled properties of Zn 2+ ions and adenine, we next tested the in-situ entrapping ability of the composites using three kinds of guests (water-soluble small dyes, SEM photo (Figure 1c) and TEM photo (Figure 1d) revealed that the microstructure of CPs resembles a stack of nanoparticles.From the images, we also can re-confirm the amorphous nature of the nano CPs.Zn 2+ ions and adenine may firstly form nanoparticles, and then these nanoparticles assemble together (Figure S2).Finally, the stoichiometry of Zn/adenine CPs determined by the titration experiment was adenine:Zn 2+ = 1:2 (Figure S3).We considered that adenine bridges four Zn 2+ ions through the N3, N7, N9 and NH2 sites, forming a framework structure (Figure 2).This coordination mode is very similar to the one observed by Hui Wei et al. [45].

Encapsulation Property of Zn/Adenine Complexes
After confirming the self-assembled properties of Zn 2+ ions and adenine, we next tested the in-situ entrapping ability of the composites using three kinds of guests (water-soluble small dyes,

Encapsulation Property of Zn/Adenine Complexes
After confirming the self-assembled properties of Zn 2+ ions and adenine, we next tested the in-situ entrapping ability of the composites using three kinds of guests (water-soluble small dyes, proteins and nanoparticles).The encapsulation ratios of guests in nano CPs were calculated by measuring the absorption intensity in the supernatant.All of the guests were encapsulated at a high efficiency (Figure 3a).For water-soluble small dyes, both of the Orange G and Amido black 10B could be encapsulated by the CPs, but the degree was different (Figure 3a).In Amido black 10B, the absorption peak almost completely disappeared in the supernatant, while in Orange G the absorption peak was left at ~30% in the supernatant (Figure S4).To study the binding capacity of Zn 2+ /adenine to proteins, fluorescein-labeled bovine serum albumin (BSA, pI = 4.7) was next used.As shown in Figure 3a, more than 79% of BSA was efficiently entrapped.After encapsulating, the fluorescence was almost fully attenuated due to the nano-CP entrapping (Figure S4).This suggested that Zn/adenine complexes could be able to efficiently encapsulate protein molecules.
The above successes in trapping water-soluble small dyes and proteins prompted us to further investigate nanoparticles as guests.Citrate-capped 13 nm Au nanoparticles (NPs) were mixed with adenine and ZnCl 2 .After adding adenine, bluish-violet precipitation formed.The encapsulation ratio of Au NPs in CPs was calculated by measuring the absorption intensity in the supernatant (Figure S4).There was almost no absorption in the supernatant of Au-Zn/adenine complexes, suggesting successful encapsulation.As shown in Figure 3b, the entrapped Au NPs could also be observed by transmission electron microscopy (TEM).The round and dark nanoparticles were the Au NPs, which were entrapped by the Zn/adenine complexes.According to the above results, a diverse range of guest molecules including water-soluble small dyes, proteins and gold NPs could be encapsulated in Zn/adenine nano CPs with high loading capacity.
Catalysts 2017, 7, 327 4 of 11 proteins and nanoparticles).The encapsulation ratios of guests in nano CPs were calculated by measuring the absorption intensity in the supernatant.All of the guests were encapsulated at a high efficiency (Figure 3a).For water-soluble small dyes, both of the Orange G and Amido black 10B could be encapsulated by the CPs, but the degree was different (Figure 3a).In Amido black 10B, the absorption peak almost completely disappeared in the supernatant, while in Orange G the absorption peak was left at ~30% in the supernatant (Figure S4).To study the binding capacity of Zn 2+ /adenine to proteins, fluorescein-labeled bovine serum albumin (BSA, pI = 4.7) was next used.
As shown in Figure 3a, more than 79% of BSA was efficiently entrapped.After encapsulating, the fluorescence was almost fully attenuated due to the nano-CP entrapping (Figure S4).This suggested that Zn/adenine complexes could be able to efficiently encapsulate protein molecules.The above successes in trapping water-soluble small dyes and proteins prompted us to further investigate nanoparticles as guests.Citrate-capped 13 nm Au nanoparticles (NPs) were mixed with adenine and ZnCl2.After adding adenine, bluish-violet precipitation formed.The encapsulation ratio of Au NPs in CPs was calculated by measuring the absorption intensity in the supernatant (Figure S4).There was almost no absorption in the supernatant of Au-Zn/adenine complexes, suggesting successful encapsulation.As shown in Figure 3b, the entrapped Au NPs could also be observed by transmission electron microscopy (TEM).The round and dark nanoparticles were the Au NPs, which were entrapped by the Zn/adenine complexes.According to the above results, a diverse range of guest molecules including water-soluble small dyes, proteins and gold NPs could be encapsulated in Zn/adenine nano CPs with high loading capacity.

Immobilization of Single Enzyme
Next, we employed glucose oxidase (GOx) and horseradish peroxidase (HRP) as guest molecules to test the enzyme immobilization property of the Zn/adenine complexes.The loading efficiency of GOx and HRP was 93% and 92%, respectively (Figure 4a).In our system, the immobilized enzymes showed about 20% increase in catalytic activity compared to free enzymes in solution (Figure 4a).High enzyme stability is important in applications [49][50][51].High temperature and extreme pH are the major reasons for enzyme deactivation.The stability of the GOx-Zn/adenine complexes was examined at different pH values (from 3 to 10, Figure 4b) and temperatures (from 30 to 90 °C, Figure 4c) and compared with that of the free GOx in solution.The activity of the GOx-Zn/adenine complexes was more stable compared to that of the free enzymes with respect to pH.Especially at pH 3, the immobilized GOx showed a 1.5-fold increase in relative activity compared to free GOx.When the temperature was higher, the immobilized enzyme exhibited higher activity compared with the free enzyme.As a result, the relative activity of Zn/adenine nano CP-immobilized GOx increased by 4-fold at 70 to 90 °C, compared to free GOx.The confinement of the proteins within the rigid structure of the CPs prevented protein denaturation caused by thermal fluctuations of proteins in solution [52].Thus, the Zn/adenine complexes could protect enzymes from deactivation under heat and acid conditions.We considered that the rigid structure of the nano

Immobilization of Single Enzyme
Next, we employed glucose oxidase (GOx) and horseradish peroxidase (HRP) as guest molecules to test the enzyme immobilization property of the Zn/adenine complexes.The loading efficiency of GOx and HRP was 93% and 92%, respectively (Figure 4a).In our system, the immobilized enzymes showed about 20% increase in catalytic activity compared to free enzymes in solution (Figure 4a).High enzyme stability is important in applications [49][50][51].High temperature and extreme pH are the major reasons for enzyme deactivation.The stability of the GOx-Zn/adenine complexes was examined at different pH values (from 3 to 10, Figure 4b) and temperatures (from 30 to 90 • C, Figure 4c) and compared with that of the free GOx in solution.The activity of the GOx-Zn/adenine complexes was more stable compared to that of the free enzymes with respect to pH.Especially at pH 3, the immobilized GOx showed a 1.5-fold increase in relative activity compared to free GOx.When the temperature was higher, the immobilized enzyme exhibited higher activity compared with the free enzyme.As a result, the relative activity of Zn/adenine nano CP-immobilized GOx increased by 4-fold at 70 to 90 • C, compared to free GOx.The confinement of the proteins within the rigid structure of the CPs prevented protein denaturation caused by thermal fluctuations of proteins in solution [52].Thus, the Zn/adenine complexes could protect enzymes from deactivation under heat and acid conditions.We considered that the rigid structure of the nano CPs would increase the structural stability of the embedded enzymes, thus improving the enzymes' stabilities.We also tested the recycling of the GOx-Zn/adenine complexes.The immobilized enzyme can be easily collected by centrifugation after the reaction, and can be redispersed well by vortex mixing.Results indicated that the GOx-Zn/adenine complexes could reach a relative activity of more than 90% after being reused eight times (Figure 4d).CPs would increase the structural stability of the embedded enzymes, thus improving the enzymes' stabilities.We also tested the recycling of the GOx-Zn/adenine complexes.The immobilized enzyme can be easily collected by centrifugation after the reaction, and can be redispersed well by vortex mixing.Results indicated that the GOx-Zn/adenine complexes could reach a relative activity of more than 90% after being reused eight times (Figure 4d).

Co-Immobilization of GOx and HRP
After establishing the feasibility of using the Zn/adenine complexes for single-enzyme immobilization, co-immobilization of multiple enzymes was further performed.Co-immobilization of multiple enzymes could enhance the overall reaction efficiency and specificity, and omit the isolation of reaction intermediates [10].GOx specifically converts glucose to gluconic acid and produces H2O2 as a byproduct, which is a co-substrate for HRP to oxidize 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) [52].Therefore, based on the above studies, GOx and HRP were chosen for a cascade reaction.The total immobilization ratio was over 90%, measured by the Bradford assay.The catalytic activities of the co-localized GOx and HRP in the Zn/adenine complexes were evaluated by reacting with glucose using ABTS as a chromogenic substrate, and they were compared with the same concentration of free GOx and HRP.However, the relative activity of single enzymes on their own was just half of the activity of the co-immobilized enzymes.The selectivity for glucose was confirmed by monitoring the absorbance at 414 nm in the presence of various competing compounds (Figure 5a).Different concentrations of glucose were used to measure the sensitivity of the sensor.Figure 5b illustrates a good linearity between the absorbance and the concentration of glucose in the range of 0-100 μM (R 2 = 0.995).The concentration of limit of detection CLOD can be expressed as a function of S (slope of the curve) and SB (a standard deviation): CLOD = 3SB/S [35].In the experiment, SB was

Co-Immobilization of GOx and HRP
After establishing the feasibility of using the Zn/adenine complexes for single-enzyme immobilization, co-immobilization of multiple enzymes was further performed.Co-immobilization of multiple enzymes could enhance the overall reaction efficiency and specificity, and omit the isolation of reaction intermediates [10].GOx specifically converts glucose to gluconic acid and produces H 2 O 2 as a byproduct, which is a co-substrate for HRP to oxidize 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) [52].Therefore, based on the above studies, GOx and HRP were chosen for a cascade reaction.The total immobilization ratio was over 90%, measured by the Bradford assay.The catalytic activities of the co-localized GOx and HRP in the Zn/adenine complexes were evaluated by reacting with glucose using ABTS as a chromogenic substrate, and they were compared with the same concentration of free GOx and HRP.However, the relative activity of single enzymes on their own was just half of the activity of the co-immobilized enzymes.The selectivity for glucose was confirmed by monitoring the absorbance at 414 nm in the presence of various competing compounds (Figure 5a).Different concentrations of glucose were used to measure the sensitivity of the sensor.Figure 5b illustrates a good linearity between the absorbance and the concentration of glucose in the range of 0-100 µM (R 2 = 0.995).The concentration of limit of detection C LOD can be expressed as a function of S (slope of the curve) and S B (a standard deviation): C LOD = 3S B /S [35].In the experiment, S B was determined to be calculated by three sets of blank signals, and S was 0.0049.Calculated according to the formula, the limit of detection (LOD) was determined to be 1.84 µM, which is lower than some of the previously reported colorimetric glucose sensors [35,53].
Catalysts 2017, 7, 327 6 of 11 the formula, the limit of detection (LOD) was determined to be 1.84 μM, which is lower than some of the previously reported colorimetric glucose sensors [35,53].

Preparation of Zn and Adenine Coordinated Complexes
In a typical experiment, the Zn/adenine complexes were prepared by mixing 100 μL ZnCl2 (45 mM), 100 μL adenine (15 mM) and 500 μL HEPES buffer (100 mM, pH 7.4).The volume of the system was 1 mL, and water was added to make up.After 2 h at room temperature, the samples were centrifuged at 10,000 rpm for 5 min and washed with Milli-Q water to remove remaining chemicals.

Study of the Zn/Adenine Complexes at Different Concentrations and pH of Buffer, as Well as Different Ionic Strengths
Different concentrations of HEPES buffer (pH 7.4, 0.1 M) and different pH of HEPES buffer were prepared.The Zn/adenine complexes were prepared by 500 μL buffer mixed with 100 μL adenine solution (15 mM) and 100 μL ZnCl2 (45 mM).To quantify the weight of precipitations, all the samples were centrifuged, dried under 60 °C and weighed.The content of adenine remained in the supernatant was measured using UV-vis spectroscopy at 260 nm by the standard curve (Figure S5).The influence of ionic strength was also studied.Appropriate NaCl solution (2 mM) was added into the reaction system to enhance ionic strength.The weight of precipitation and residue ratio of adenine were measured by the aforementioned method.In the experiments, the concentration of HEPES was 10, 20, 30, 40 or 50 mM.The different pH values were 6.8, 7.2, 7.4, 7.8 or 8.2.The

Preparation of Zn and Adenine Coordinated Complexes
In a typical experiment, the Zn/adenine complexes were prepared by mixing 100 µL ZnCl 2 (45 mM), 100 µL adenine (15 mM) and 500 µL HEPES buffer (100 mM, pH 7.4).The volume of the system was 1 mL, and water was added to make up.After 2 h at room temperature, the samples were centrifuged at 10,000 rpm for 5 min and washed with Milli-Q water to remove remaining chemicals.

Study of the Zn/Adenine Complexes at Different Concentrations and pH of Buffer, as Well as Different Ionic Strengths
Different concentrations of HEPES buffer (pH 7.4, 0.1 M) and different pH of HEPES buffer were prepared.The Zn/adenine complexes were prepared by 500 µL buffer mixed with 100 µL adenine solution (15 mM) and 100 µL ZnCl 2 (45 mM).To quantify the weight of precipitations, all the samples were centrifuged, dried under 60 • C and weighed.The content of adenine remained in the supernatant was measured using UV-vis spectroscopy at 260 nm by the standard curve (Figure S5).The influence of ionic strength was also studied.Appropriate NaCl solution (2 mM) was added into the reaction system to enhance ionic strength.The weight of precipitation and residue ratio of adenine were measured by the aforementioned method.In the experiments, the concentration of HEPES was 10, 20, 30, 40 or 50 mM.The different pH values were 6.8, 7.2, 7.4, 7.8 or 8.2.The concentration of NaCl was 100, 200, 300, 400 or 500 mM.

Stoichiometry and Structural Characterization of Zn/Adenine Complexes
The reaction ratio of adenine obtained by mixing aqueous ZnCl 2 (5 mL in water) and aqueous adenine (5 mL in 0.1 M HEPES buffer at pH 7.4) was plotted as a function of mixed ratio.Conditions (in reaction mixtures): [adenine] = 1.5 mM, [ZnCl 2 ] = 0, 0.75, 1.5, 2.25, 3.0, 4.5, 6.0, 7.5 and 9.0 mM.It was mixed for about two hours, and centrifuged to separate the supernatant and precipitate.The amount of adenine remained in the supernatant was measured using UV-vis spectroscopy at 260 nm.
Fourier transform infrared spectroscopy (FTIR) spectra of adenine and Zn/adenine complexes were obtained on a FTIR spectrometer (8700/Continuum XL Imaging Microscope, Nicolet, Waltham, MA, USA) with measuring wavelength ranging from 4000 to 550 cm −1 .
Scanning electron microscopy (SEM) images of samples were taken on a S-4700 scanning electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 10.0 kV.Samples for SEM measurements were prepared by pipetting a drop of the solution of the coordination complexes onto a cover glass and drying on a filter paper.
Transmission electron microscopy (TEM) was performed on a Hitachi H-800 transmission electron microscope (Hitachi, Tokyo, Japan).The sample was prepared by pipetting a drop of the solution of the Zn/adenine complexes onto a 230 mesh carbon copper grid and drying on a filter paper.The Au-Zn/adenine complexes were prepared by the same method.

Immobilization of Single Enzymes
The aqueous solution of GOx (1 mg/mL) and the aqueous solution of HRP (1 mg/mL) was prepared and stored at 4 • C. Immobilization of the enzymes within the Zn/adenine complexes was performed by firstly mixing 100 µL of 15 mM adenine aqueous solution, 500 µL of HEPES buffer (0.1 M, pH 7.4), and 100 µL of enzymes.Then, 100 µL of ZnCl 2 (45 mM) in water was quickly added and mixed.After 2 h, the immobilized enzymes were collected by centrifugation at 10,000 rpm for 5 min.The amounts of protein incorporated into the Zn/adenine complexes were measured by the Coomassie brilliant blue method.
For the GOx activity assay, 200 µL of glucose (20 mM) solution and 200 µL of ABTS (0.5 mM) were mixed with 20 µL of free GOx (100 µg/mL) or 22 µL of the suspension of the immobilized GOx (containing the same amount of protein compared with free GOx).Then, 40 µL of HRP (100 µg/mL) were added.The mixed samples were incubated at room temperature for 5 min.The reaction was monitored with a UV/vis spectrometer at 414 nm.For the HRP activity assay, 200 µL ABTS (0.5 mM) and 200 µL H 2 O 2 (0.9 mM) were added into 0.5 µg free enzyme and equivalent immobilized enzyme, respectively.The mixed samples were incubated at room temperature for 5 min.The absorbance was recorded at 414 nm.

Enzyme Stability Test
For stability test at different pH values, the free GOx and the suspension of GOx-Zn/adenine complexes were added into 1 mL of various pH solutions for 4 h.Then, the enzymatic activity was measured by recording the absorbance at 414 nm.To test stability at different temperatures, free and immobilized enzymes were incubated at 30-90 • C for 30 min.To test the recycling of the GOx-Zn/adenine complexes, the reaction was performed for 5 min, and the immobilized enzyme was separated by centrifugation.The supernatant was measured by recording the absorbance at 414 nm.Then, new substrate and other solution were added to start the new cycle of the reaction for 5 min.The above steps were repeated several times to observe the change of the activity.In all the experiments, the error bars were calculated based on the standard deviation from three independent measurements.

Co-Immobilization of GOx and HRP
Co-immobilization of enzymes within the Zn/adenine complexes was performed by mixing 100 µL 15 mM adenine aqueous solution with 500 µL HEPES (100 mM, pH 7.4).Then, 50 µL GOx and HRP (1 mg/mL each) were added by vortex mixing.Finally, 100 µL ZnCl 2 (45 mM) was quickly added and mixed.After 2 h, the immobilized enzymes were collected by centrifugation at 10,000 rpm for 5 min.In co-immobilization, the total immobilized protein ratio (i.e., percentage of immobilized protein) was measured by the Coomassie brilliant blue method.
3.9.Glucose Detection with GOx-HRP-Zn/Adenine Complexes Different concentrations of glucose (750 µL) and 1 mM ABTS (750 µL) were added into 500 µL of the suspension of GOx-HRP-Zn/adenine complexes.The samples were then incubated at room temperature for 10 min.The reaction solution was centrifuged at 10,000 rpm for 3 min, and the absorbance of the supernatants at 414 nm was measured by using a UV-1100 spectrophotometer.The selectivity was determined by the absorbance of the supernatants using 100 µM glucose as the substrate, compared with 100 µM xylose, fructose, mannose, or galactose, or 1 mg/mL BSA.

Conclusions
In summary, we presented a one-step, facile and general method for immobilization of enzymes by a typical metal-organic nano coordination polymer.The zinc/adenine hybrid nanomaterials were formed by self-assembly of zinc ions and adenine in aqueous solution with mild conditions.The Zn/adenine CPs showed a good adaptive encapsulating ability.A diverse range of guests, including water-soluble small dyes, proteins and nanoparticles, could be encapsulated in the nano CPs.All these guests were loaded at a high capacity.Indeed, the loading efficiency of enzymes was over 90%.The GOx-Zn/adenine complexes displayed high catalytic efficiency, high selectivity and enhanced stability due to the protecting effect of the rigid framework.As a result, the relative activity of Zn/adenine nano-CP-immobilized GOx increased by 1.5-fold at pH 3 and 4-fold at 70 to 90 • C, compared to free GOx.Moreover, the immobilized GOx could reach a relative activity of more than 90% after being reused eight times.The use of this system as a glucose biosensor was also demonstrated by co-immobilization of two enzymes, detecting glucose down to 1.84 µM with excellent selectivity.The high sensitivity, stability and recyclable usability of the immobilized enzymes against free enzymes make this method promising for biocatalyst immobilization.

Supplementary Materials:
The following are available online at www.mdpi.com/2073-4344/7/11/327/s1, Figure S1: (a) a photograph of Zn 2+ reacting with adenine in different concentrations of pH 7.4 HEPES buffer.The CP precipitant weight and the adenine percentage remained in the supernatant after Zn 2+ reacted with adenine

Figure 3 .
Figure 3. (a) The encapsulation ratios of different guests in the nano CPs; (b) the TEM image of the Au nanoparticles entrapped by Zn/adenine complexes.

Figure 3 .
Figure 3. (a) The encapsulation ratios of different guests in the nano CPs; (b) the TEM image of the Au nanoparticles entrapped by Zn/adenine complexes.

Figure 4 .
Figure 4. (a) Ratio of encapsulated glucose oxidase (GOx) and horseradish peroxidase (HRP) by the Zn/adenine complexes, and the relative activity of the two immobilized enzymes compared to the free enzymes; stability of the GOx-Zn/adenine complexes compared with the equivalent free enzymes (b) at different pH values at 25 °C; (c) at different temperatures; (d) relative activity of GOx-Zn/adenine complexes after reusing for 8 cycles.

Figure 4 .
Figure 4. (a) Ratio of encapsulated glucose oxidase (GOx) and horseradish peroxidase (HRP) by the Zn/adenine complexes, and the relative activity of the two immobilized enzymes compared to the free enzymes; stability of the GOx-Zn/adenine complexes compared with the equivalent free enzymes (b) at different pH values at 25 • C; (c) at different temperatures; (d) relative activity of GOx-Zn/adenine complexes after reusing for 8 cycles.

Figure 5 .
Figure 5. (a) The selectivity of the GOx-HRP-Zn/adenine complexes for 100 μM glucose in comparison to 100 μM fructose, mannose, xylose, maltose and 1 mg/mL bovine serum albumin (BSA) (inset: photographs of the samples.);(b) detection of glucose in solutions with glucose concentrations of 0-100 μM (absorbance at 414 nm was measured after incubation in solution for 10 min at room temperature) (inset: photographs of the samples).

Figure 5 .
Figure 5. (a) The selectivity of the GOx-HRP-Zn/adenine complexes for 100 µM glucose in comparison to 100 µM fructose, mannose, xylose, maltose and 1 mg/mL bovine serum albumin (BSA) (inset: photographs of the samples.);(b) detection of glucose in solutions with glucose concentrations of 0-100 µM (absorbance at 414 nm was measured after incubation in solution for 10 min at room temperature) (inset: photographs of the samples).