Rational Design of Mimic Multi-Enzyme Systems in Hierarchically

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Functional Nanostructured Materials (including low-D carbon)

Rational Design of Mimic Multi-Enzyme Systems in Hierarchically Porous Biomimetic Metal-Organic Frameworks Xiao Liu, Wei Qi, Yuefei Wang, Daiwu Lin, Xuejiao Yang, Rongxin Su, and Zhimin He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09388 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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ACS Applied Materials & Interfaces

Rational Design of Mimic Multi-Enzyme Systems in Hierarchically Porous Biomimetic MetalOrganic Frameworks †

Xiao Liu,† Wei Qi,*,†,‡,§ Yuefei Wang,*,†,§ Daiwu Lin,† Xuejiao Yang, Rongxin Su,†,‡,§ and Zhimin He† †State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China ‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, P. R. China §Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, P. R. China

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ABSTRACT: A facile approach was reported to establish mimic multi-enzyme systems with hierarchically porous biomimetic metal–organic frameworks (MOFs) and natural enzymes for tandem catalysis. The hierarchically porous MOF HP-PCN-224(Fe) with peroxidase-like activity and tunable hierarchical porosity was synthesized via a modulator-induced strategy. HP-PCN224(Fe) not only acts as the enzyme immobilization matrix, but also as an effective enzyme mimic, which could cooperate with the immobilized natural enzyme to catalyze the cascade reactions. The mimic multi-enzyme systems were used for the efficiently colorimetric detection of a series of biomolecules, including glucose and uric acid. This work displays the great potential to construct highly functional biocatalysts by integrating the merits of both natural enzymes and MOF mimics, which are promising for applications in biosensing and biomimetic catalysis.

Keywords: metal–organic frameworks; cascade catalysis; multi-enzyme systems; peroxidase mimic; enzyme immobilization

1. INTRODUCTION Enzymes are the most sophisticated biomacromolecule in living organisms. Enzymes can catalyze diverse reactions with excellent efficiency and selectivity.1 In natural systems, multienzyme systems constituted by several kinds of enzymes ensure the process of cascade reactions and help accomplish various physiological processes and superior properties.2,3 Inspired by nature, intense effort has been devoted to developing the mimic multi-enzyme systems, in which


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two or more kinds of natural enzymes are immobilized on the matrix to realize the complex function.4-11 Metal-organic frameworks (MOFs) are constructed through the coordination of metal ions and organic ligands. Owing to their tunable porosity, large surface areas, and excellent chemical/thermal stability, MOFs have drawn extensive research interest in many fields such as gas adsorption, catalysis, optics, electronics and energy storage.12-26 Also, MOFs were important candidates for enzyme immobilization.27-30 A variety of mimic multi-enzyme systems have been constructed in which the MOFs worked as the immobilization matrix containing multiple natural enzymes.31-37 Moreover, with the deeper study and understanding of MOF materials, a variety of MOFs with intrinsic mimic enzyme catalysis have been developed, possessing high catalytic activity and stability.38-42 For example, the integration of metalloporphyrins such as ferriporphyrin into MOFs gave rise to effective peroxidase mimics, such as Fe4SP@HKUST1(Cu),43 MMPF-6,44 and PCN-222.45 These MOF mimics could be utilized for constructing artificial enzyme systems with natural enzymes for tandem catalysis and offer new opportunities to study the cooperation between the natural enzymes and mimics. In recent years, hierarchically porous MOFs (HP-MOFs) have attracted great attention, due to the large mesopores and high surface area which could encapsulate large molecules more efficiently.46-50 In this work, hierarchically porous biomimetic MOF HP-PCN-224(Fe) was successfully synthesized with a modulator/surfactant-induced strategy.50 We introduced dodecanoic acid (DA) as the modulator/surfactant and reduced the amount of ligand to form defect during the synthesis of HP-PCN-224(Fe). The DA was then removed by hydrochloric acid treatment, thus giving rise to relatively large mesoporous. HP-PCN-224(Fe) can immobilize enzymes with high load capacity and excellent stability (Scheme 1). More importantly, HP-


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PCN-224(Fe) not only acts as the enzyme immobilization matrix, but also as an effective peroxidase mimic in the mimic multi-enzyme systems. GOx@HP-PCN-224(Fe) was obtained by immobilizing glucose oxidase (GOx) on HP-PCN-224(Fe), which can effectively catalyze the cascade reaction of glucose and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), used as a glucose detector. By immobilizing uricase on HP-PCN-224(Fe), Uricase@HP-PCN-224(Fe) was also formed, and can effectively catalyze the cascade reaction of uric acid and 4aminophenazone/2,4-dichlorophenol sulfonate (DCPS), used as a uric acid (UA) detector. With this strategy, a strong cooperation of natural enzymes and enzyme mimics is achieved.

Scheme 1. Schematic diagram of the preparation of the mimic multi-enzyme system Enzyme@HP-PCN-224(Fe): (a) the 6-connected D3d symmetric Zr6 and (b) tetratopic TCPP ligand in PCN-224(Fe). (c) Crystal structure of PCN-224(Fe). (d) Schematic of the normal synthesized PCN-224(Fe). (e) The synthesis of hierarchically porous PCN-224(Fe) with a


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modulator/surfactant-induced strategy, (f) the activation of hierarchically porous PCN-224(Fe), and (g) the mimic multi-enzyme systems. 2. EXPERIMENTAL SECTION 2.1 Chemicals Glucose oxidase and uricase were purchased from J&K (Beijing, China). Zirconium chloride (ZrCl4), iron(II) chloride tetrahydrate, 4-aminophenazone, uric acid, fluorescein isothiocyanate (FITC), ABTS, pyrrole, methyl 4-formylbenzoate, 2,4-dichlorophenol sulfonate, dodecanoic acid (DA) and glucose, propionic acid, N, N’-dimethylformamide (DMF) were purchased from Aladdin (Shanghai, China), and used without further purification.2.2 Synthesis of PCN-224(Fe) and HP-PCN-224(Fe) PCN-224(Fe) was prepared according to the reported literature.51 ZrCl4 (300 mg), FeTCPP(TCPP=tetrakis(4-carboxyphenyl)porphyrin) (100 mg), and benzoic acid (4.0 g) were dissolved in 80 mL DMF, and the mixture was heated under 120 °C for 24 h. After cooling down, PCN-224(Fe) crystals were collected by suction-filtration followed by washing with DMF and methanol each for several times. For activation, PCN-224(Fe) crystals were then immersed in a mixture of 80 mL DMF and 1.5 mL concentrated hydrochloric acid. The mixture was stirred at 100 °C for 12 h. PCN-224(Fe) crystals was collected by suction-filtration followed by washing with DMF and methanol each for several 3 times, and dried under vacuum. For the synthesis of HP-PCN-224(Fe), ZrCl4 (300 mg) and Fe-TCPP (50 mg) were dissolved in DMF (80 mL). A certain amount of DA with the molar ratios to ZrCl4 = X (X = 70, 100, 125, 150, 200) was then added. The mixture was heated under 120 °C for 24 h. After cooling down, the precipitate was collected by suction-filtration followed by washing with DMF and methanol each for several times. The activation steps were the same as PCN-224(Fe).


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2.3 Preparation of Enzymes@HP-PCN-224(Fe) HP-PCN-224(Fe) (10 mg) was added into a solution of GOx (2 mL,1.5 mg/mL), and stirred for 3 h under room temperature. Then, the GOx@HP-PCN-224(Fe) was separated by centrifugation, and washed with water. To inspect the immobilization performance of GOx@HP-PCN-224(Fe), HP-PCN-224(Fe) (20 mg) was added into a solution of GOx (4 mL, 1.5 mg/mL) and stirred under room temperature. 100 µL of mixture was taken out and centrifuged, and then measure the GOx concentration of the supernate using coomassie brilliant blue staining.52 FTIC-GOx was synthesized according to our previous work.35 The preparation of FTICGOx@ HP-PCN-224(Fe) was the same as GOx@ HP-PCN-224(Fe). 2.4 Cascade enzymatic activity of Enzymes@HP-PCN-224(Fe) To identify the optimum reaction pH of GOx@HP-PCN-224(Fe), 100 µL GOx@HP-PCN224(Fe) suspension (5 mg/mL), 100 µL glucose solution (6 mM) and 100 µL ABTS solution (12 mM) were added into 2.7 mL buffer at different pH. The change of absorbance value at 420 nm was tested using a UV−vis spectrophotometer.

To identify the optimum reaction pH for

Uricase@HP-PCN-224(Fe), 100 µL Uricase@HP-PCN-224(Fe) suspension (5 mg/mL), 100 µL UA solution (6 mM), 100 µL 4-aminophenazone (12 mM) and 100 µL DCPS (48 mM) were added into 2.6 mL buffer at different pH. The change of absorbance value at 505 nm was tested with a UV−vis spectrophotometer. The thermal stability of GOx@HP-PCN-224(Fe) and Uricase@HP-PCN-224(Fe) was determined by putting GOx@HP-PCN-224(Fe) solution (5 mg/mL) and Uricase@HP-PCN224(Fe) solution (5 mg/mL) in a 60 °C oven, followed by detecting their activity using the above methods hourly.


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2.5 Spectrometric measurements of glucose and uric acid For the measurements of glucose, different concentrations of glucose solutions (1.5 mL) each containing 5 mg GOx@HP-PCN-224(Fe) and 1 mM ABTS were incubated at 37 °C for 60 min. GOx@HP-PCN-224(Fe) was removed by centrifugation, and the absorption spectra of supernate was measured. For the measurements of UA, different concentrations of UA solutions (1.5 mL) each containing 5 mg Uricase@HP-PCN-224(Fe), 1 mM 4-aminophenazone and 4 mM DCPS were incubated at 37 °C for 60 min. Uricase@HP-PCN-224(Fe) was removed by centrifugation, and the absorption spectra of supernate was measured. 2.6 Characterization Scanning electron microscopy (SEM) images were taken on a Hitachi S-4800 field-emission microscope. Powder X-ray diffraction (XRD) patterns were recorded using a Bruker D8 advance X-ray powder diffractometer. UV-vis spectra were obtained using a Purkinje General TU-1810 UV-vis spectrophotometer. Fourier transform infrared (FT-IR) data were recorded on a American Nicolet AVATAR 360 FT-IR spectrometer. N2 isotherms at 77K were measured by a Quantachrome QUADRASORB SI automated surface area & pore size analyzer. The pore size and pore volume was calculated based on the density functional theory (DFT) method. Wide angle X-ray diffraction (WAXD) data with a synchrotron X-ray source were measured at beamline 1W2A of the Beijing Synchrotron Radiation Facility (BSRF). For data collection, the sampling distance from Mar165-CCD to the beam was set at 179 mm, and the wavelength of rays was λ = 0.154 nm.

The confocal fluorescence images were taken by an Olympus

FV1000confocal laser scanning microscopy (CLSM).


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3. RESULTS AND DISCUSSION The hierarchically porous MOF HP-PCN-224(Fe) was successfully synthesized by introducing excess DA to an insufficient amount of ligand (molar ratio of DA/ZrCl4 is 125). SEM images revealed the formation of spherical particles that were 3-5 µm in diameter (Figure 1a). Highmagnification SEM images demonstrated the presence of large amounts of irregular mesoporous at both surface and inside of the spherical particles (Figure 1b, c). The porosity of the HP-PCN224(Fe) was evaluated by nitrogen adsorption studies at 77 K. The pore size distributions of HPPCN-224(Fe) calculated by density functional theory (DFT) are shown in Figure 1d, only micropores were observed in PCN-224(Fe), which is consistent with the crystal structure, featuring 3-D channels 1.9 nm in width.51 However, a large amount of mesoporous ranging from 2 nm to 35 nm in size were formed in HP-PCN-224(Fe). It could be ascribed to the DA-induced defect formation which gave rise to large mesoporous in the progress of synthesis.

Figure 1. SEM images of (a) HP-PCN-224(Fe) spherical particles and (b) their porous surface. (c) SEM images a broken HP-PCN-224(Fe) particle. The inset image showed the porous structure inside the particles. (d) DFT pore size distributions of as-synthesized PCN-224(Fe) and HP-PCN-224(Fe).


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We investigated the influence of the amount of DA on mesoporous formation by adjusting the feed ratio of DA/ZrCl4(X). As shown in Figure 2 and Figure S1, the normal synthesized PCN224(Fe) crystal presents a cubic shape with a dense surface. As the amount of modulator was increased, the morphology of HP-PCN-224(Fe) became dodecahedral and then more and more spherical. The possible synthetic mechanism could be divided into two regimes depending on the amount of DA added. When with low amount of DA, the HP-PCN-224(Fe) were prepared through a modulator-induced defect-formation strategy (Scheme S2b). The carboxylic acid modulators firstly coordinated to the Zr ion to form modulator-capped Zr-oxo clusters. Then the Zr-oxo clusters assembled to the 3D crystal via reversible ligand exchange with the TCPP ligands. The presence of residual modulators due to the insufficient amount of TCPP ligands lead to the formation defects, so the HP-PCN-224(Fe) had the morphological structure of polyhedron (Figure 2b). However, when with higher amount of DA, the synthetic mechanism turned to a surfactant-induced defect-formation strategy (Scheme S2c). The Zr-oxo clusters assembled to the small nanocrystals possibly due to the high concentration of DA. Then the large spheroidal particles were formed by the stacking of small nanocrystals, which also formed the stacking mesopores. In this process, the DA might act as a surfactant. The N2 sorption isotherms and pore diameter distributions of the PCN-224(Fe) and HP-PCN-224(Fe) are shown in Figure S2. Large amounts of mesoporous were formed in HP-PCN-224(Fe), which are sufficient for encapsulating natural enzymes. By increasing the amount of the modulator added during synthesis, the mesoporosity of HP-PCN-224(Fe) increased until the ratio of modulator/Zr reached 150, after which point the mesoporosity of HP-PCN-224(Fe)-150 began to decrease. The XRD patterns of the as-synthesized PCN-224(Fe) and HP-PCN-224(Fe) were in good agreement with the simulated pattern of PCN-224 (Figure S3), confirming the crystalline structures of the as-


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synthesized materials. The crystalline structures of the as-synthesized PCN-224(Fe) and HPPCN-224(Fe) were also confirmed by wide angle X-ray diffraction (WAXD) using a synchrotron X-ray source (Figure S4).

Figure 2. Illustrations and SEM images showing the morphology changes of PCN-224(Fe) and HP-PCN-224(Fe)-X with increasing amounts of modulator (X refers to the ratio of modulator and Zr): (a) PCN-224(Fe); (b) HP-PCN-224(Fe)-70; (C) HP-PCN-224(Fe)-100; (d) HP-PCN224(Fe)-125; (e) HP-PCN-224(Fe)-150. The scale bars are 500 nm.


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The porous HP-PCN-224(Fe) is appealing for enzymatic immobilization, and we chose GOx as a test case. HP-PCN-224(Fe) with different pore size was used for the immobilization of GOx. As shown in Figure 4a, only 25.3 mg GOx were immobilized on per gram of PCN-224(Fe), as the small micropores of PCN-224(Fe) were not able to encapsulate GOx molecules. Conversely, HP-PCN-224(Fe) showed excellent enzyme immobilization ability. By increasing the amount of modulator added during synthesis, the enzyme immobilization capacity of HP-PCN-224(Fe) also increased, up to a maximum immobilization capacity of 192.6 mg/g for HP-PCN-224(Fe)-125. When modulator/Zr reached 150, the enzyme immobilization capacity of HP-PCN-224(Fe)-150 began to decrease, which was consistent with the decreased mesoporous porosity (Figure 2e and Figure S2a). Hence, HP-PCN-224(Fe)-125 was most suitable for the enzyme immobilization matrix, and subsequently abbreviated as HP-PCN-224(Fe). The kinetics of the adsorption of GOx into HP-PCN-224(Fe) was studied by monitoring the residual GOx concentration of the supernate. As shown in Figure S5, the load amount of GOx increased with time, and reached a maximum immobilization capacity of GOx (192.6 mg/g) after approximately 150 min. To confirm that the GOx was immobilized on HP-PCN-224(Fe), the FITC-labeled GOx was used. The confocal laser scanning microscopy (CLSM) images indicated the successful immobilization of FITC-GOx on HP-PCN-224(Fe) (Figure 3a and Figure S6). The monodispersed green fluorescent spherical particles and clean background demonstrated the stable immobilization of enzyme on HP-PCN-224(Fe). To further confirm that GOx was immobilized in HP-PCN224(Fe), the FT-IR test was carried out. Compared to HP-PCN-224(Fe), the FT-IR spectra of GOx@HP-PCN-224(Fe) showed bands at 1650–1550 cm−1 in the amide I and amide II regions, which could be attributed to the vibrational stretching of C=O and –NH–. The bands observed at 2975–2845 cm−1 were consistent with the C–H stretching of –CH2– and –CH3 in GOx (Figure


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S7). XRD pattern of GOx@HP-PCN-224(Fe) was in good agreement with the HP-PCN-224(Fe) and simulated pattern of PCN-224, indicating that the crystal structure of HP-PCN-224(Fe) after enzyme immobilization was completely unaffected by this process (Figure 1f). Moreover, like other Zr-based MOFs materials, the HP-PCN-224(Fe) has excellent water and pH stability, which is crucial for enzyme immobilization. As shown in Figure S8a, HP-PCN-224(Fe)-125 was treated with aqueous solutions of different pH value for 12 h. The XRD patterns before and after treatment indicated no structural degradation of HP-PCN-224(Fe) after treatment, suggesting the outstanding water and pH stability of HP-PCN-224(Fe) (Figure S8b).

Figure 3. (a) CLSM image showing the green fluorescence of the FITC-GOx@HP-PCN224(Fe). (b) XRD patterns of as-synthesized PCN-224(Fe), HP-PCN-224(Fe) and the MOF– enzyme composite GOx@HP-PCN-224(Fe). Compared with other enzyme immobilization system, the biomimetic HP-PCN-224(Fe) are appealing not only for its high enzyme load capability, excellent stability and tunable pore sizes, but also for its intrinsic peroxidase-like activity presented by the metalloporphyrins. ABTS was used as the reactant to test the peroxidase-like activity of HP-PCN-224(Fe). As illustrated in Scheme S1 and Figure S9a, HP-PCN-224(Fe) could catalyze the oxidation of ABTS with H2O2 to generate the oxidized ABTS radical (ABTS+), which showed an intense characteristic absorbance at 420 nm after reacting for 10 min (Figure S9b, blue curve). However,


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inappreciable absorbance was observed in the absence of HP-PCN-224(Fe), indicating that no oxidation reaction between ABTS and H2O2 (Figure S9b, black curve). This indicated that the as-prepared HP-PCN-224(Fe) possessed intrinsic peroxidase-like activity. The peroxidase-like activity of HP-PCN-224(Fe) allowed for us to design a catalytic cascade reaction with the GOx@HP-PCN-224(Fe). As shown in Figure 5a, glucose could react with O2 under the catalysis of GOx, generating gluconic acid and H2O2. ABTS could be oxidized to ABTS+ by the generated H2O2 with catalysis of HP-PCN-224(Fe). The corresponding equations are shown in Scheme S1a. The effect of pH on the catalytic cascade reaction was studied (Figure 4b). The optimum pH for GOx@HP-PCN-224(Fe) was 5.5–6.5, consistent with an oxidase-peroxidase coupled enzyme system (GOx&HRP). However, in more acidic or alkaline conditions, GOx@HP-PCN-224(Fe) exhibits higher residual activity than that of free GOx, and shows a certain pH protection of HP-PCN-224(Fe) for GOx. This protective effect is also present for uricase when immobilized on HP-PCN-224(Fe) (Figure S10). The thermal stability of GOx@HP-PCN-224(Fe) was also studied as shown in Figure 4c. In comparing the activity retention between GOx@HP-PCN-224(Fe) and free GOx incubated at 60 °C for 15 h, the free GOx had an extremely low residual activity of 16%, while GOx@HP-PCN-224(Fe) remained at approximately 70% activity. Similarly, after incubation at 60 °C for 15 h, Uricase@HP-PCN224(Fe) had much higher residual activity than free uricase (Figure S11). The reusability of the immobilized enzyme is also a crucial factor for such a system. To study the reusability, GOx@HP-PCN-224(Fe) was regained through centrifugation after reaction. The reclaimed GOx@HP-PCN-224(Fe) was reused in a fresh reaction solution. As shown in Figure 4d, GOx@HP-PCN-224(Fe) retained about 90 % residual activity after five cycles, showing excellent reusability. The leaching of the immobilized enzymes in these composite materials


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after catalysis was studied (Figure S12). Only about 5 % of the immobilized enzymes leached from the composite materials after each catalysis.

Figure 4. (a) Immobilization capacity of HP-PCN-224(Fe)-X (X = 70, 100, 125, 150) for GOx. (b) Relative activities of GOx@HP-PCN-224(Fe) and free GOx under different pH values. (c) Thermal stability of immobilized GOx and free GOx. The thermal inactivation was carried out at 60 °C for a period of 15 h. (d) Reusability of the GOx@HP-PCN-224(Fe). As a proof-of-concept application, GOx@HP-PCN-224(Fe) was used as a colorimetric glucose detector.33, 53 As shown in Figure 5b, c, as the concentration of glucose increased, the absorbance of solution at 420 nm gradually increased, and the solution color changed from light to dark green (Figure 5d). As a result, the absorption value at 420 nm displayed a good linear relationship to the glucose concentration from 5–300 µM, giving a detection limit of 0.87 µM (Figure 5c). To estimate the feasibility of GOx@HP-PCN-224(Fe) as a glucose detector, the specificity is one of the most crucial requirements. The specificity of GOx@HP-PCN-224(Fe) was studied through introducing various interferential analytes. Chlorinated salts (NaCl and


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CaCl2), saccharides (fructose, maltose and mannose) and biomacromolecule (BSA) were introduced into the reaction system, and negligible absorbance of the solutions was observed (Figure 5e). This demonstrates the nice specificity of GOx@HP-PCN-224(Fe) as a colorimetric glucose detector, which was akin to the fantastic specificity of natural enzymes. To demonstrate the universality of this strategy, a colorimetric UA detector was further established by immobilizing uricase on HP-PCN-224(Fe). We used 4-aminophenazone and DCPS as the substrate to study the activity of Uricase@HP-PCN-224(Fe) and detect the UA concentration.10 As shown in Scheme S1b, UA could react with O2 under the catalysis of uricase, generating allantoin and H2O2. 4-aminophenazone and DCPS could then be oxidized to a kind of red quinoneimine dye by the generated H2O2 with the catalysis of HP-PCN-224(Fe), and show an intense characteristic absorbance at 505 nm. As shown in Figure S13a-c, as the concentration of UA increased, the absorbance of solution at 505 nm gradually increased, and the absorption value displayed a good linear relationship to the UA concentration from 5–100 µM, giving a detection limit of 1.8 µM. To evaluate the specificity of Uricase@HP-PCN-224(Fe), several interferential analytes such as NaCl, glutamic acid, ascorbic acid, urea, glucose and maltose were introduced. As shown in Figure S13d, inappreciable absorbance of the solutions was observed towards the interferential analytes, and demonstrating the the feasibility of Uricase@HP-PCN224(Fe) as a colorimetric UA detector.


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Figure 5. (a) Schematic diagram showing the tandem catalysis of GOx@HP-PCN-224(Fe). (b) UV-Vis absorption data of solutions with the increase of glucose concentration from 5–300 µM. (c) Plot of A420 versus glucose concentration. (d) Colorimetric comparison of ABTS solutions after reaction with the catalysis of GOx@HP-PCN-224(Fe) and different concentrations of glucose. GOx@HP-PCN-224(Fe) was removed by centrifugation. (e) Absorbance value of the solutions after reaction with 1 mM NaCl, 1 mM CaCl2, 300 µM fructose, 300 µM maltose, 300 µM mannose, 1 µgL−1 BSA and 300 µM glucose. 4. CONCLUTION In summary, we report a facile approach to establish mimic multi-enzyme systems with biomimetic HP-MOFs and natural enzymes for tandem catalysis. By utilizing the peroxidase-like activity of HP-PCN-224(Fe) and the catalytic activity for glucose oxidation of GOx and uricase,


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we fabricated two colorimetric sensors for the detection of glucose and UA based on the mimic multi-enzyme

systems

GOx@HP-PCN-224(Fe)

and

Uricase@HP-PCN-224(Fe).

The

immobilized enzymes exhibited excellent pH and thermal stability compared with free enzyme. The results not only provided a simple, highly efficient method for enzyme immobilization but also established a bridge between natural enzymes and mimics. This allowed us to integrate the merits of both components for the construction of highly functional biocatalysts that have great potential applications in biosensing, biomimetic catalysis, biomedical and biofuel cells.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed material synthesis and characterization data (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This work was supported by the Natural Science Foundation of China (Nos. 21476165, 21606166, 51773149, 21621004), the Beiyang Young Scholar of Tianjin University (2012), and the State Key Laboratory of Chemical Engineering (Nos. SKL-ChE-08B01). The authors thank Prof. Zhonghua Wu and Dr. Guang Mo of BSRF for assistance with the X-ray scattering measurement. REFERENCES [1]

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Rational Design of Mimic Multi-Enzyme Systems in Hierarchically

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