Ficin encapsulated in mesoporous metal-organic frameworks with enhanced peroxidase-like activity and colorimetric detection of glucose

Journal Pre-proof Ficin encapsulated in mesoporous metal-organic frameworks with enhanced peroxidase-like activity and colorimetric detection of glucose

Wen Zheng, Jiahui Liu, Danyang Yi, Yadi Pan, Yijuan Long, Huzhi Zheng PII:

S1386-1425(20)30173-6

DOI:

https://doi.org/10.1016/j.saa.2020.118195

Reference:

SAA 118195

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

22 November 2019

Revised date:

21 February 2020

Accepted date:

22 February 2020

Please cite this article as: W. Zheng, J. Liu, D. Yi, et al., Ficin encapsulated in mesoporous metal-organic frameworks with enhanced peroxidase-like activity and colorimetric detection of glucose, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2018), https://doi.org/10.1016/j.saa.2020.118195

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© 2018 Published by Elsevier.

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Ficin encapsulated in mesoporous metal-organic frameworks with enhanced peroxidase-like activity and colorimetric detection of glucose

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Wen Zheng, Jiahui Liu, Danyang Yi, Yadi Pan, Yijuan Long, and Huzhi Zheng*

The Key Laboratory of Luminescent and Real-time Analysis (Southwest University),

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H. Z. Zheng, [email protected]

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Email:

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University, Chongqing 400715, P.R. China

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Ministry of Education, College of Chemistry and Chemical Engineering, Southwest

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Abstract Ficin has been reported to possess peroxidase activity, but its applications in some respects have been limited because of its relatively low activity. Herein, a mesoporous metal-organic framework, PCN-333(Fe), was synthesized, which was selected to encapsulate ficin to form ficin@PCN-333(Fe). Compared with ficin, the

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peroxidase-like activity of ficin@PCN-333(Fe) toward 3,3’,5,5’-tetramethylbenzidine (TMB) oxidation was about 3 times increase in the presence of H2O2, and followed

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classical Michaelis-Menten model. The kinetic parameters showed that stronger

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affinity and higher catalytic constant (Kcat) of ficin@PCN-333(Fe) to both TMB and

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H2O2 compared with ficin, and Kcat of ficin@PCN-333(Fe) was increased by 3.65

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folds and 3.59 folds for TMB and H2O2, respectively. Taking advantages of higher

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catalytic property of ficin@PCN-333(Fe), we developed a colorimetric method with high sensitivity and selectivity to detect glucose, which displayed a good linear

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response toward glucose in the range of 0.5-180 μM with a limit of detection of 97 nM. Furthermore, ficin@PCN-333(Fe) has been proven to successfully detect glucose in human serum, implying its great potentialities and wide applications as peroxidase mimics. Keywords: Ficin; Metal-organic frameworks; Peroxidase-like activity; Glucose detection 1. Introduction Enzymes are a class of biocatalysts, which dominate biological process such as metabolism, nutrition and energy conversion. Nature enzymes, are normally 2

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consisted of protein and RNA, which have been lessened their industrial applications predominantly due to relatively high expense of separation and extraction, stringent reaction condition, and easily denatured [1,2]. In recent years, because artificial enzymes have the advantages of simple synthesis, convenient storage, low cost, and high stability, a lot of great efforts have been made to develop new artificial enzymes

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as substitutes for natural enzymes [3-6]. So far, a variety of nanomaterial-based enzyme mimetics and substances with enhanced enzymatic activity, for example, Pt

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nanoclusters [5], porous Co3O4 nanoplates [7], MnO2 1D nanozyme [8], graphene

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oxide [9,10] and metal-organic framework (MOFs) [11-14], have been reported,

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owing to their large surface-to-volume ratio [15]. Nevertheless, the catalytic activity

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and specificity of nature enzymes are much higher compared with some current

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nanozymes. Thus, the search for enzyme mimetics with improved performance requires a great deal of effort.

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MOFs, a kind of prospective organic-inorganic hybrid material, consisted of metal ions/clusters and bridging organic bonding groups, have been received great attentions in catalysis, biosensing and therapy owing to their large specific surface area, adjustable porous morphology, and good biocompatibility [16-21]. These impressive properties make MOFs effective matrixes for immobilizing small molecules, enzymes and proteins [11,22-26]. Immobilization is especially beneficial for enzymes because they are expensive and difficult to reprocess and recycle [25]. Various solid supports for enzymes have been investigated, such as organic microparticles, hydrogels, sol gels, porous or nonporous inorganic carriers and 3

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metal-organic frameworks [27-29]. With its ultra-high porosity and tunable surface function, MOFs are the ideal platform for loading enzymes [30]. PCN-333(Fe), a stable mesoporous MOF, is composed of a trimeric metal clusters and three-terminal linkers, 4,4′,4″-s-triazine-2,4,6-triyl-tribenzoic acid (H3TATB), shows the biggest cage of 5.5 nm [31]. Due to containing single molecule capture for enzymatic

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encapsulation, PCN-333 (Fe) was successfully used to encapsulate HRP, MP-11 and cytochrome c [30,32-35]. In this work, we used PCN-333(Fe) to encapsulate ficin to

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form ficin@PCN-333(Fe), hoping that this method can expand the biological

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application of ficin.

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Ficin, a thiol protease, hold intrinsic peroxidase activity according to reports in

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2017 [36]. However, the applications of ficin in some respects have been limited

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because of its relatively low activity. Considering ficin can be used more widely in analytic detection due to their lower expense and excellent durability against stringent

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temperature and pH, it is highly desirable to escalate the peroxidase-like activity of ficin. Our team has taken some measures to increase the peroxidase activity of ficin [30,37,38], and we should continue to work on this. Considering that PCN-333(Fe) shows the biggest cage of 5.5 nm [31,33], this leads us to explore whether it is possible to increase the peroxidase activity of ficin by encapsulating ficin to PCN-333(Fe). Fortunately, the peroxidase activity of ficin@PCN-333(Fe) which obtained by encapsulating ficin to PCN-333(Fe), toward TMB oxidation by H2O2 was extraordinary increase compared with ficin (Scheme 1A). Glucose is an important source of energy for living things. Under the catalysis of glucose oxidase (GOx), 4

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glucose generates gluconic acid and hydrogen peroxide, and the generated H2O2 can be detected by TMB-H2O2 chromogenic system, which is used to detect the concentration of glucose [39,40]. Taking advantages of higher catalytic property of ficin@PCN-333(Fe), we established a colorimetric method with high selectivity and sensitivity for detecting glucose in the presence of GOx (Scheme 1B). Moreover, the

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detection of glucose in human serum has been successfully illustrated, implying ficin@PCN-333(Fe) has great potentialities for bioassays and biocatalysis in the

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future.

Scheme 1. (A) Schematic of the encapsulation of ficin in PCN-333(Fe) to construct ficin@PCN-333(Fe) with the enhanced peroxidase-like activity. (B) Schematic of detection of glucose by ficin@PCN-333(Fe). 2. Material and methods 2.1 Chemicals and Materials

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4,4′,4″-s-triazine-2,4,6-triyl-tribenzoic acid (H3TATB) was obtained from Bide Pharmatech Ltd (Shanghai, China). Anhydrous ferric chloride and trifluoroacetic acid (TFA) were obtained from Macklin (Shanghai, China). Premium grade ficin was acquired from Sigma-Aldrich (USA). Hydrogen peroxide (H2O2, 30%) was acquired from Chongqing Chuandong Chemical Co., Ltd. (Chongqing, China). N, N-dimethyl formamide (DMF) was purchased from Aladdin Reagents Co., Ltd. (Shanghai, China).

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3,3’,5,5’-Tetramethyl benzidine dihydrochloride (TMB) was procured from Sangon

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Biotech (Shanghai, China). Glucose, maltose, fructose, sucrose and lactose were the

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products of Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All aqueous

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solutions were prepared with ultrapure water (18.2 MΩ) from a Milli-Q ultrapure

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water system (Millipore, USA).

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2.2 Characteizations

Scanning electron microscope (SEM) was acquired using a Supra 55 sapphire

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(Carl Zeiss, Germany). The samples were mixed with potassium bromide with a mass ratio of 1: 100, and then, ground, pressed; at last, fourier transform infrared (FTIR) spectra were carried out by an IRTracer-100 Spectrometer (Shimadzu, Japan). The X-ray diffraction (XRD) patterns were obtained on a Bruker D8 Advance X-ray diffractometer (Bruker, USA) with a Cu Kα anode at 40 kV and 40 mA. After activation at 150 °C for 12 h, N2 adsorption isotherms were obtained on a Quantachrome

Instruments

Quadrasorb

EVO

(Quantachrome,

USA).

Thermogravimetric analysis (TGA) was performed on Netzsch STA 449 Jupiter from room temperature to 600 °C at a ramp rate of 10 °C/min in a flowing nitrogen 6

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atmosphere (Netzsch, Germany). 2.3 Preparation of PCN-333(Fe) and ficin@PCN-333(Fe) PCN-333(Fe) was prepared according to the method previously reported [31]. H3TATB (50 mg) and anhydrous FeCl3 (60 mg) were added into 10 ml DMF and sonicated for 5 minutes. Next, 0.5 mL of TFA was added and mixed. The mixture was

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heated in an oven at 150 °C for 12 h until brown precipitate formed. Then the brown precipitate was harvest by centrifugation (10,000 rpm, 8 min) and washed three times

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with fresh DMF and ultrapure water, respectively. Finally, the precipitate was

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dispersed in 5 mL ultrapure water to obtain an aqueous solution of PCN-333(Fe).

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Furthermore, 500 μL of aqueous solution of PCN-333(Fe) was added into 1.0 mL

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of aqueous solution of ficin (1.0 mg mL−1). Next, the mixture was stirred for about 90

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min at room temperature to encapsulate ficin into PCN-333(Fe). The precipitate was harvested by centrifugation (10,000 rpm, 3 min) and washed with ultrapure water for

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five times, and the supernatant was collected to measure the concentration of ficin in ficin@PCN-333(Fe). Scheme 1 shows the encapsulation of ficin in PCN-333 (Fe) to construct ficin @PCN-333 (Fe). The dispersed solid sample was suspended in 1 mL of ultrapure water for further experiments. 2.4 Detection of hydrogen peroxide and glucose H2O2 detection was carried out as follows: 200 μL of 0.20 M PBS (pH=5.5), 200 μL of 0.20 μg mL−1 ficin@PCN-333(Fe) (refering to the concentration of ficin in MOF), 200 μL of 8.0 mM TMB and 200 μL of H2O2 with different concentrations were added into 1200 μL of ultrapure water in turn. Next, the mixture was reacted at 7

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30 °C for 180 min and the standard curve was measured. The detection of Glucose was carried out as follows: first, GOx dissolved in 20 mM PBS (pH=7.0) to prepare 1.0 mg mL−1 GOx solution, then, 200 μL of glucose with different concentrations and 200 μL GOx solution were incubated at 37 °C for 30 min; second, 1000 μL of ultrapure water, 200 μL of 0.20 M PBS (pH=5.5), 200 μL of

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0.20 μg mL-1 ficin@PCN-333(Fe), and 200 μL of 8.0 mM TMB were added into the

min and then the standard curve was measured.

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2.5 The detection of glucose in serum samples

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above mixture solution in sequence; third, the mixtures were reacted at 30 °C for 180

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To assess the glucose content in real biological samples, the serum samples from

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two healthy volunteers were first purified use centrifugal ultrafiltration (Millipore,

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Carrigtwohill, Ireland) at 25 °C and 3,000 rpm for 30 minutes. Then, the supernatant was diluted 5 times. The subsequent operations of glucose detection were the same as

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described above except that the serum sample was used instead of glucose.

3. Results and discussion

3.1 Preparation and characterization of ficin@PCN-333(Fe) PCN-333 (Fe), which is a mesoporous MOF, composed of a trimer metal cluster and a trimer linker, H3TATB [31], exhibits larger cage, making it an extraordinary candidate for ficin encapsulation. In this work, solvothermal reactions of H3TATB with anhydrous ferric chloride afforded crystals of PCN-333(Fe) according to the previous reports [32]. Then, an appropriate amount of PCN-333(Fe) was added into 1.0 mL of ficin aqueous solution (1.0 mg mL−1) to obtain ficin@PCN-333(Fe). The 8

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enzyme immobilization efficiency of PCN-333(Fe) is about 43%, which is calculated by using the standard BCA protein assay kit to compare the fixed ficin content in PCN-333(Fe) to the initial ficin content (Fig. S1). To illustrate the successful preparation of ficin@PCN-333(Fe), a series of characterizations were carried out. SEM images of PCN-333(Fe) (Fig. 1A) and ficin@PCN-333(Fe) (Fig. 1B) show that they are all regular octahedral structures with edge length of 3.25 ± 0.31 μm and 4.03

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± 0.31 μm, respectively (50 particles are counted at random for both materials), and

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the introduction of ficin make the surface of PCN-333(Fe) smoother compared with

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free MOF. The elemental mappings of PCN-333(Fe) (Fig. S2) and fcin@PCN-333(Fe)

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(Fig. S3) demonstrate that both contain C, N, O, F, and Fe elements, and

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fcin@PCN-333(Fe) also has S and P elements compared with PCN-333(Fe). This

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proves that ficin is successfully encapsulated and evenly distributed in PCN-333(Fe). Additionally, the crystalline structures of PCN-333(Fe) and ficin@PCN-333(Fe)

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were examined by XRD as shown in Fig. 1C. There is no difference between PCN-333 (Fe) and ficin@PCN-333 (Fe) based on the positions and corresponding intensities of all diffraction peaks, indicating the crystalline structure of PCN-333(Fe) has no change after encapsulating ficin. The FTIR spectra of the ficin, PCN-333(Fe) and ficin@PCN-333(Fe) were shown in Fig. 1D. The typical characteristic peaks at 1413 cm−1, 1653 cm−1 and 2960 cm−1 are corresponding to the stretching vibrations of N−H, C=O and O−H in the ficin [30]. The typical characteristic peaks at 1608 cm−1, 1520 cm−1 and 1014 cm−1 are corresponding to the stretching vibrations of C=N, C=C (in the benzene ring) and C−F in PCN-333(Fe). However, after encapsulating the ficin 9

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into PCN-333(Fe), no new absorption peak is found in the ficin@PCN-333(Fe) compared with pure PCN-333(Fe), indicating the coordinating environment of H3TATB in ficin@PCN-333(Fe) is consistent with that in PCN-333(Fe). It may be due to the fact that ficin is immobilized in PCN-333(Fe) via physical adsorption process instead of covalent bonding. PCN-333(Fe) and ficin@PCN-333(Fe) were further characterized by nitrogen adsorption isotherms study at 77 K (Fig. S4), leading to

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BET surface areas of 2410.5 m2 g−1 and 1434.3 m2 g−1, respectively. This loss of

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surface area of about 1000 m2 g−1 is consistent with the presence of ficin within the

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pores of PCN-333(Fe). In addition, the pore size distribution calculated by the density

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function theory (DFT) indicates that the mesopore volume is significantly reduced

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from 1.72 cm3 g−1 for PCN-333(Fe) to 0.95 cm3 g−1 for ficin@PCN-333(Fe), showing

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ficin is encapsulated inside PCN-333(Fe). Thermal gravimetric analysis (TGA) also revealed the loading of ficin in PCN-333(Fe) (Fig. S5), and proved that PCN-333(Fe)

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had remarkable thermal stability [41]. These results demonstrate the successful preparation of ficin@PCN-333(Fe).

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Journal Pre-proof Fig. 1. SEM images (Scale bar, 1μm) of PCN-333(Fe) (A) and ficin@PCN-333(Fe) (B). (C) XRD patterns of ficin@PCN-333(Fe) and PCN-333(Fe). (D) FTIR spectra of the ficin, PCN-333(Fe) and ficin@PCN-333(Fe). 3.2 Ficin@PCN-333(Fe) with enhanced peroxidase activity The peroxidase activity of ficin@PCN-333(Fe) and ficin was examined by performing typical reactions using TMB as a substrate in the presence of H2O2 to

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produce oxTMB which had maximal absorption peak at 370 nm and 652 nm [42]. As

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displayed in Fig. 2A, in the absence of peroxidase mimics, TMB-H2O2 system

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produces almost no absorption band at 370 nm and 652 nm (curve 1 in Fig. 2A).

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When PCN-333(Fe) is added to the TMB-H2O2 system, negligible absorption peaks

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are found at 370 nm and 652 nm (curve 2 in Fig. 2A), indicating that PCN-333(Fe) has ignorable peroxidase-like activity at low concentrations. When ficin or

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ficin@PCN-333(Fe) is added to the system, the absorbance values at 370 and 652 nm

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increase significantly, and the absorbance at 652 nm in the presence of ficin is about 3 folds lower than that in the presence of ficin@PCN-333(Fe) (curve 3, 5 in Fig. 2A), proving that ficin@PCN-333(Fe) possesses enhanced peroxidase activity. In addition, in order to prove that ficin@PCN-333(Fe) obtained by ficin encapsulated in PCN-333 has enhanced peroxidase activity, we add both ficin and free PCN-333(Fe) to TMB-H2O2 system, and the results show that the absorbance at 652 nm is not significantly different compared with that of ficin alone (curve 4 in Fig. 2A). In Fig. 2B, we also recorded the absorbance over time at 652 nm, which varied with different catalysts catalytic systems. In the same conditions, the TMB-H2O2 system catalyzed by ficin@PCN-333(Fe) shows an evidently higher reaction velocity than that by ficin. 11

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Thus, it can be inferred that the immobilized ficin in PCN-333(Fe) can increase the

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peroxidase-like activity of ficin.

Fig. 2. (A) The UV-Vis absorption spectra of TMB-H2O2 chromogenic system

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catalyzed by different catalysts. (B) Time-dependent absorbance at 652 nm varied

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with different catalysts catalytic reaction systems: (1) TMB + H2O2, (2) TMB + H2O2 + PCN-333(Fe), (3) TMB + H2O2 + ficin, (4) TMB + H2O2 + ficin + PCN-333(Fe), (5) +

H2O2

+

ficin@PCN-333(Fe).

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TMB

The

concentration

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ficin

or

ficin@PCN-333(Fe) was 20 ng mL−1. Experimental conditions: 0.20 M PBS, pH=5.5;

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0.80 mM H2O2 and TMB; incubated at 30 °C; incubated for 180 min.

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3.3 Optimization of the catalytic reaction conditions The peroxidase-like activity of ficin@PCN-333(Fe) and ficin also depended on many experimental conditions, such as the pH, incubation temperature, concentration of H2O2, and incubation time (Fig. 3). Compared with neutral and weakly alkaline pH, both ficin@PCN-333(Fe) and ficin exhibit higher catalytic activity at a weakly acidic pH, and the maximal catalytic activity is observed at pH 5.5 (Fig. 3A). Ficin@PCN-333(Fe) and ficin yield a maximal absorbance at 30 °C when varying the temperature from 5 °C to 60 °C, so 30 °C is chosen as the optimal temperature (Fig. 3B). The influence of concentration of H2O2 on catalytic activity was studied, and the 12

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most suitable concentration of H2O2 was 0.80 mM and the activity of ficin@PCN-333(Fe) and ficin was inhibited at higher H2O2 concentration (Fig. 3C). We also studied the effect of incubation time on the peroxidase-like activity of ficin@PCN-333(Fe) and ficin (Fig. 3D), as the reaction time increased, the absorbance at 652 nm gradually increased and reached the plateau after 180 min

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incubation, so 180 min was selected as the optimal reaction time. Thence, the optimal pH, temperature, H2O2 concentration, and incubation time were as follows: 5.5, 30 °C,

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0.80 mM, 180 min. Moreover, we also investigated the stability of catalytic activity of

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ficin@PCN-333(Fe) after storage at 4 °C and room temperature (Fig. S6), and it was

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found that the peroxidase activity of ficin@PCN-333(Fe) remained at 70% and 50%

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after 15 days, respectively.

Fig. 3. The peroxidase-like activity of ficin@PCN-333(Fe) and ficin was pH (A), temperature (B), H2O2 concentration (C) and time (D) dependent. The concentration of ficin or ficin@PCN-333(Fe) was 20 ng mL−1. ΔA =A - A0, where A and A0 are the absorbance at 652 nm in the presence and absence of peroxidase mimic, respectively. 13

Journal Pre-proof Error bars represent the standard deviations of three independent experiments. 3.4 Steady-state kinetics of catalytic reaction Based on the enzyme kinetics theory and methods, the steady-state kinetics of ficin@PCN-333(Fe) were investigated for the TMB-H2O2 chromogenic system to further comprehend the immobilized ficin in the PCN-333(Fe) could exhibit much

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higher catalytic activity than free ficin. As shown in Fig. 4, the typical Michaelis-Menten curves could be observed by varying the concentration of H2O2

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(Fig. 4A) or the concentration of TMB (Fig. 4B), and the double reciprocal

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Lineweaver-Burk plots were constructed for either H2O2 or TMB as the substrate (Fig.

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4C, D). The apparent kinetics parameters, Michaelis-Menten constant (Km) and

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maximum initial velocity (Vmax), were obtained from Lineweaver-Burk plots. Km is

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equal to the substrate concentration at which conversion rate is half of Vmax. Km also shows the affinity of the enzyme for the substrate: a higher Km value means a lower

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affinity. The apparent kinetic parameters of ficin@PCN-333(Fe) and ficin [30] are compared (Table S1). The Km value of ficin@PCN-333(Fe) towards TMB and H2O2 are 0.08 mM and 0.25 mM, respectively, both are lower than that of ficin. It certificates a stronger affinity of ficin@PCN-333(Fe) to both TMB and H2O2 than that of ficin. The enhanced substrate affinity may be due to the large surface area of the PCN-333(Fe), which promotes contact of the substrate molecules with the active site of ficin, allowing more substrate to concentrate around the ficin@PCN-333(Fe). Thus, ficin embedded in PCN-333(Fe) could effectively increase its peroxidase activity. Additionally, Kcat of ficin@PCN-333(Fe) was higher than ficin, which increased by 14

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3.65 folds and 3.59 folds for TMB and H2O2, respectively. Therefore, the decreased Km and the enhanced Kcat clearly suggest that after encapsulating ficin in

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PCN-333(Fe), the peroxidase-like activity of ficin have been improved significantly.

Fig. 4. Steady-state kinetic assay and catalytic mechanism of ficin@PCN-333(Fe).

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The velocity (v) of the reaction was surveyed by using 20 ng mL−1 ficin@PCN-333(Fe) under optimal conditions. The error bars express that the

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standard deviations derived from three repeated measurements. (A) The concentration of TMB was 0.10 mM and the concentration of H2O2 as a substrate was varied. (B) The concentration of H2O2 was 0.40 mM and the TMB concentration as a substrate was

varied.

(C)

Double

reciprocal

plots

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peroxidase-like

activity

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ficin@PCN-333(Fe) with the concentration of TMB fixed and H2O2 varied, (D) the concentration of H2O2 fixed and TMB varied. 3.5 Detection of H2O2 and glucose Since ficin@PCN-333(Fe) can catalyze H2O2 to oxidize TMB to produce oxTMB with a blue color change, and the catalytic activity is dependent on the concentration of H2O2. So, the concentration of H2O2 was measured using 15

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ficin@PCN-333(Fe). As shown in Fig. S7, as the concentration of H2O2 gradually increases, the absorbance of oxTMB at 652 nm gradually increases. The linear equation is A = 0.0044 c + 0.0598 (R2 = 0.9855), with the linear range of 0.5 to 220 μM, c represents the concentration of H2O2 and A represents the absorbance at 652 nm.

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Glucose can be oxidized to produce gluconic acid and H2O2 in the presence of GOx and O2. The generated H2O2 can be further utilized by peroxidase to oxidize

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TMB. So, current sensitive H2O2 response system make it possible to further build a

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terrace for detecting glucose, which is a crucial biological analyte. As the glucose

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concentration increases, the absorbance at 652 nm rises gradually, and the color of the

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system gradually turns blue (Fig. 5A, B). A good linear relationship (R2=0.9909) is

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acquired over the concentration range from 0.5 to 180 μM (Fig. 5B), and the limit of detection (LOD) is calculated to be 96 nM (3σ/k). Compared with previously reported

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analytical methods, the determination of glucose based on ficin@PCN-333(Fe) displays higher sensitivity (Table S2). In addition, the selectivity and specificity of the proposed method were examined using some glucose analogues (fructose, lactose, sucrose, galactose, and maltose) as potential interfering substances. Compared with blank, other interfering species were found to not cause any obvious change of absorbance at 652 nm even though each of their concentration was 4 times higher than glucose (Fig. 5C). Furthermore, the effect of coexistence substances on the detection of glucose were also researched (Fig. 5D). Fructose, lactose, sucrose, galactose, and maltose have negligible interference on the 16

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detection of glucose, even if these interfering substances are added to the system at the same time. These results indicate that we proposed method of glucose detection has significant sensitivity and selectivity and offers potential applications for

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biomedical testing.

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Fig. 5. (A) The absorption spectra corresponding to various concentrations of glucose (from bottom to top: 0.5, 1, 5, 10, 20, 40, 60, 80, 100, 120, 150, 160, 180 μM). (B)

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The linear regression to plots of the absorbance at 652 nm with the glucose. (C) The selectivity analysis for glucose determination by detecting the absorbance change at 652 nm. (D) Effects of coexisting substances on the determination of glucose. The concentration of glucose was 100 μM, and the other interferential substances were 400 μM. Experiment conditions: 20 ng mL−1 of ficin@PCN-333(Fe), 0.80 mM TMB, 30 °C, pH 5.5, 180 min. 3.6 Detection of glucose in human serum Based on the use of ficin@PCN-333(Fe) to detect glucose with excellent linearity and selectivity, we explored the practical application of our sensing system in human serum supplied by two healthy volunteers. The original serum samples are finally diluted 100 folds to reduce the possible effects of complex components and to 17

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ensure that the glucose concentration is suitable for the range of linear we have established. And the results obtained are shown in Fig. 6, the results obtained by our method were no significant differences with the results provided by a local hospital (P = 0.90). Therefore, we can use the proposed method to detect glucose in human serum, which provides a new method for the detection of blood glucose in the clinic. These

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results showed that our proposed method had excellent accuracy and reliability for

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further practical application.

Fig. 6. The average glucose concentration in blood serum obtained from this method

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is compared with results from Southwest University hospital. The error bars represent the standard deviation of three measurements. 4. Conclusion

In conclusion, PCN-333(Fe) is chosen to encapsulate ficin to form ficin@PCN-333(Fe), which possesses a higher peroxidase-like activity compared with ficin. Due to the large surface area of ficin@PCN-333(Fe), more contact of the substrate molecules with the active site of the ficin is promoted, which increases the catalytic activity of ficin@PCN-333(Fe) by about 3 times. In addition, the catalytic mechanism of ficin@PCN-333(Fe) follows the classical Michaelis-Menten model, and the kinetic parameters indicate that ficin@PCN-333(Fe) has better affinity and 18

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higher catalytic constant for both TMB and H2O2. Based on the excellent catalytic property of ficin@PCN-333(Fe), we developed a colorimetric method with high selectivity and sensitivity for the determination of glucose with a wide linear range and low detection limit. Moreover, the glucose content in human serum has been detected successfully, suggesting ficin@PCN-333(Fe) has broad prospects as

Conflicts of interest

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Acknowledgements

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There are no conflicts to declare.

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peroxidase mimics in various fields.

This research was financially supported by the National Natural Science

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Foundation of China (Nos. 21405124, 21175110) and the Fundamental Research Funds for the Central Universities (Nos. XDJK2013A022, XDJK2014C173, XDJK2017D052).

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References

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Credit Author Statement

Wen Zheng: Conceptualization, Data curation, Writing-original draft. Jiahui Liu: Resources, Investigation, Writing-original draft. Danyang Yi: Software, Methodology, Validation. Yadi Pan: Supervision, Writing-review & editing.

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Yijuan Long: Investigation, Formal analysis.

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Huzhi Zheng: Funding acquisition, Project administration.

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Graphical abstract

Scheme 1. (A) Schematic of the encapsulation of ficin in PCN-333(Fe) to construct

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ficin@PCN-333(Fe) with the enhanced peroxidase-like activity. (B) Schematic of

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detection of glucose by ficin@PCN-333(Fe).

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Highlights  PCN-333(Fe) was selected to encapsulate ficin to form ficin@PCN-333(Fe).  Ficin@PCN-333(Fe) possessed higher peroxidase-like activity than ficin.  An excellent selective and sensitive colorimetric sensor for glucose was provided.

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 We detected the concentration of glucose in human serum successfully.

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