Bifunctional Hybrid Enzyme-Catalytic Metal Organic Framework

Subscriber access provided by Bibliothèque de l'Université Paris-Sud

Biological and Medical Applications of Materials and Interfaces

Bifunctional Hybrid Enzyme-Catalytic Metal Organic Framework Reactor for #-glucosidase Inhibitor Screening Yingying Zhong, Linjin Yu, Qiyi He, Qiuyan Zhu, Chunguo Zhang, Xiping Cui, Junxia Zheng, and Suqing Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b11754 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Bifunctional Hybrid Enzyme-Catalytic Metal Organic Framework Reactor for α-glucosidase Inhibitor Screening Yingying Zhong, Linjin Yu, Qiyi He, Qiuyan Zhu, Chunguo Zhang, Xiping Cui*, Junxia Zheng*, Suqing Zhao* Department of Pharmaceutical Engineering, School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, People’s Republic of China

ABSTRACT: The screening strategy based on α-glucosidase inhibition has been widely employed for the discovery of anti-diabetic drugs, but it still faces some challenges in practical applications, such as poor stability of enzyme, high consumption of test compounds, low sensitivity of screening methods and so on. In this work, a bifunctional hybrid enzymecatalytic metal organic framework reactor (GAA@GOx@Cu-MOF) with a flower-shaped globular structure was innovatively prepared via selfassembling of α-glucosidase (GAA), glucose oxidase (GOx), Cu2+ and 4,4’-bipyridine. It was found that GAA@GOx@Cu-MOF not only enjoyed merits of high stability, selectivity and sensitivity, but also possessed the character of assembly line work, with about 4.58 times enhanced enzyme activity compared with the free enzyme system. Based on the above characteristics, a highly sensitive screening of GAA inhibitors could be achieved with the detection limit of 7.05 nM for acarbose. Furthermore, the proposed method was successfully applied to the screening of oleanolic 1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

acid derivatives as potential anti-diabetic drugs. Therefore, it was expected that this work could provide new insights and inspirations for the screening of clinical anti-diabetic drugs and for further exploration of functional MOF composites. KEYWORDS: hybrid enzyme MOF reactor, α-glucosidase, glucose oxidase, colorimetric sensing, anti-diabetic drug screening INTRODUCTION Diabetes mellitus characterized by high a blood sugar level has become one of the most common chronic diseases with the highest substantial incidence around the world,1-3 and has shown an exponential growth in recent years.4-6 Recently, it was estimated that there were 425 million people with diabetes worldwide, and the number will be projected to 642 million by 2040.7-8 Moreover, diabetes will cause many complications, such as kidney failure, heart disease, microangiopathy, stroke, retinopathy and so on.3,7, 9 After years of research, considerable drugs have been developed to treat diabetes, in which α-glucosidase inhibitor is considered to be one of the most important drugs for the treatment of diabetes. Studies have shown that it could delay the absorption of carbohydrates in the intestine by inhibiting the activity of α-glucosidase, thereby achieving the purpose of controlling the postprandial blood glucose level of the patient.10-11 However, the methods for α-glucosidase (GAA) inhibitors screening remain to be poorly investigated. In vivo 2


Page 2 of 36

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hyperglycemic animal models and in vitro screening (p-nitrophenyl-α-Dglucopyranoside (PNPG) as substrate) are currently two most commonly used approaches, but they suffer from long experimental time, high cost, high consumption of test compounds and low sensitivity.12-13 In order to overcome the above drawbacks, researchers have developed various biosensors based on fluorescent, colorimetric or electrochemical method for the detection of GAA inhibitors. Although these methods have obtained satisfactory short detection time, high sensitivity and less sample consumption, the poor stability of GAA, narrow optimum pH ranges and low tolerance to many metal ions have limited their further applications. Moreover, enzymes themselves may be a source of contamination in the desired product.9,11,13 Therefore, it is highly desirable to develop a screening method with superb stability, suitability, selectivity and sensitivity in practical applications. Immobilized enzyme is effective to enhance stability, suitability, and recyclability of enzyme,14-15 but it generally exhibits lower apparent substrate affinity and catalytic activity than native enzyme.16-17 In order to overcome the above limitations, researchers have shifted their attention to metal-organic frameworks (MOF), which possess large surface area and tunable porous structure and properties in recent years.18-19 It has been demonstrated that MOF hosts not only possessed higher loading efficiency and superior shielding effect than conventional enzyme hosts, but also 3



could improve the catalytic performance of corresponding enzyme.20-24 However, the current studies were mainly focused on improving the actual performance of a single enzyme (such as acetylcholinesterase,25 glucose oxidase,26 OPPA,27-28 Cyt c,29-30 catalase31 and so on) by MOF immobilization, and the examples of multiple enzyme immobilizing simultaneously on MOF were quite rare. To the best of our knowledge, the multi-enzyme systems having been reported were GOx@HRP,24,32 GFP@lipase33 and MP-11@Cyt c,34 while studies on screening αglucosidase inhibitors via simultaneously immobilizing GAA and glucose oxidase (GOX) on MOF have not been reported yet. Moreover, it has been reported that self-assembly of GOx (0.1 mg/mL),17 GOx-HRP (0.25 mg/mL)2,35 or GOx-GAA (0.25 mg/mL)4 with Cu2+ (6 mM) into enzyme@inorganic nanoreactors could improve the affinity of enzyme and substrate,17 detection sensitivity (glucose)2,35 and enzyme catalytic activity.4 Furthermore, Cu-MOF has been confirmed to possess inherent peroxidase-mimic activity, so they are capable of catalyzing the oxidation reaction of the chromogenic substrate 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2.36-38 Therefore, they are expected to replace HRP to reduce cost in bioanalysis applications. In the present study, a new method was innovatively proposed for sensitive screening of α-glucosidase inhibitors based on the dual-functional hybrid enzyme-catalytic MOF reactor mediated colorimetric sensing 4


Page 4 of 36

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


strategy. Herein, GAA, GOx, Cu2+ and 4,4’-bipyridine were self-assembled to form a spherical hybrid enzyme-MOF reactor with a flower-shaped structure. Maltose was selected as the initial substrate for the reactor, and the absorbance of oxTMB was taken as the signal output. With the operation of the hybrid enzyme reactor, one molecule of maltose was sequentially hydrolyzed and oxidized into two molecules of H 2O2, which was followed by oxidation of TMB to oxTMB with the catalysis of CuMOF. The hybrid enzyme-MOF reactor mediated colorimetric sensing strategy was achieved based on the change in the amount of produced oxTMB. When the inhibitor was introduced into the reactor, the discharge of H2O2 was hindered, resulting in the inhibition of TMB oxidation. Based on those principles, screening of GAA inhibitor could be easily achieved (Scheme 1). Since no toxic elements, extreme harsh conditions and complicated covalent-linkage procedures were involved in the preparation process of GAA@GOx@Cu-MOF, the immobilized GAA and GOx could maintain their original catalytic activity and apparent affinity for the substrate in addition to its enhanced stability, reusability and interference resistance. Furthermore, this sensing method was successfully utilized for GAA inhibitors screening from natural product derivatives.

5



Scheme 1. Schematic illustration of the sensing platform based on the hybrid enzymecatalytic MOF reactor.

EXPERIMENTAL SECTION Materials and Instruments. Glucose oxidase (GOx), αglucosidase (GAA), trypsin, acarbose, amino acids and bicinchoninic acid (BCA) protein quantitation kit were obtained from Sangon Biotech (Shanghai, China). Copper dichloride (CuCl2), 4,4’-bipyridine, maltose, 3,3’,5,5’-tetramethylbenzidine (TMB), hydrogen peroxide, p-nitrophenylα-D-glucopyranoside (PNPG) and other metal salts were purchased from Aladdin Ltd. (Shanghai, China). Ultra-pure water, ethanol, sodium hydroxide, hydrochloric acid and other reagents were offered by Sigma Aldrich (Shanghai, China). The morphology of hybrid enzyme-MOF reactor was characterized by scanning electron microscopy (SEM, SU8220, Hitach, Japan). X-ray 6


Page 6 of 36

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diffractometer (D8 ADVANCE, Bruker, Germany) and Fourier transform infrared (FTIR) spectrometer (Nicolet IS50, Thermofisher, USA) were used to obtain powder Xray diffraction (XRD) patterns and Fourier transform infrared (FTIR) spectra respectively. Specific surface and microporous physical adsorption analyzer (ASAP2020, Mike Instruments, USA) was used to determine the BET specific surface area of GAA@GOx@Cu-MOF and Cu-MOF. Electrophoresis system (DYY-6C, Beijing Liuyi Instrument Factory, China) was used for electrophoresis. The absorption spectrum of oxTMB was measured through a microplate reader (Infinite 200, Tecan, Austria). Preparation of Cu-MOF. Cu-MOF was synthesized according to the reported procedures with minor modifications.37-38 In brief, CuCl2 aqueous (4 mM, 4 mL) and 4,4'-bipyridine ethanol aqueous solution (v/v = 1:1, 16 mM, 4 mL) were simply mixed in 15 mL centrifuge tube and rested at 4 °C for 12 hours. After centrifugation (10 000 rpm, 4 °C, 5 min), the supernatant was removed, and the precipitate was washed three times with 4 mL of ultrapure water. The as-obtained precipitate was finally dispersed in 2 mL ultrapure water. Fabrication of GAA@GOx@Cu-MOF. In the synthesis of the hybrid enzyme-MOF reactor,4,38 4 mL mixture of α-glucosidase (GAA, 2.5 mg/mL, from yeast) and glucose oxidase (GOx, 2.5 mg/mL, from erythromycin) were mixed with 4 mL CuCl2 aqueous solution (4 mM). 7



Page 8 of 36

After a rest at 4 °C for 10 min, 4 mL 4,4’-bipyridyl ethanol aqueous solution was added to trigger the formation of the reactor. Next, the mixture was rested at 4 °C for 12 h followed by centrifugation (10 000 rpm, 4 °C, 5 min) with the supernatant to be removed. Finally, the precipitate was washed three times with 4 mL of ultrapure water and dispersed in 2 mL ultrapure water. Determination of Enzyme Loading Amount. The total enzyme amount loaded in Cu-MOF host was evaluated by classic BCA assay,38 and the GAA amount loaded in Cu-MOF host was determined as follows: (1) 10 μL GAA with different concentrations (125, 62.5, 31.25, 15.63, 7.81, 3.91 μg/mL) and 100 μL 100 mM excessive PNPG were mixed in 96-well plate and incubated at 37 °C for 30 min. Then 100 μL Na 2CO3 (1 M) was added before measuring the absorbance of well at 405 nm. (2) Standard curve was fitted with GAA concentration (mg/mL) as the abscissa and absorbance value as the ordinate. (3) 20 μL GAA@GOx@Cu-MOF was incubated with 100 μL 100 mM PNPG at 37 °C for 30 min and followed by the adding of 100 μL 1 M Na2CO3 to terminate the reaction. After that, the absorbance of well was measured at 405 nm and the GAA amount loaded in Cu-MOF host was calculated according to the standard curve. Cu-MOF and free enzyme system were taken as the control. The loading ratio was calculated as follows：

Loading ratio % 

A1 - A 2  100% A0 8


(1)

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A1, A2 and A0 were the enzyme contents of GAA@GOx@Cu-MOF, Cu-MOF and GAA@GOx respectively. Determination of Enzyme Relative Activity. The detailed information of enzyme relative activity assay was presented in “Supporting Information”. Optimization

of

α-glucosidase

Inhibitor

Screening

Conditions. The detailed information for optimization was shown in “Supporting Information”. Enzymatic Michaelis-Menten Kinetics Investigation. In this work, the enzymatic Michaelis-Menten kinetics assay was performed to explore enzymatic catalytic capabilities of GAA@GOx@Cu-MOF and Cu-MOF according to the previous studies on embedding enzymes in MOF.17,30,31,38 The Michaelis constant Km and the turnover number kcat were calculated via the Lineweaver-Burk plot method (the kcat was the molar extinction coefficient, wherein the kcat value of oxTMB was 3.9×104 M-1 cm-1). The ratio of kcat and Km indicated catalytic efficiency of enzyme, as the higher value of kcat/Km meant higher catalytic efficiency of enzyme. The Km represented the affinity of enzyme for substrate, and a lower Km value indicated a better affinity of enzyme to substrate. In general, the effects of loaded enzyme on the catalytic ability of Cu-MOF were investigated by comparing the Km and kcat/Km values of GAA@GOx@Cu-MOF and CuMOF at different concentrations of TMB or H2O2, and the effect of the Cu9



Page 10 of 36

MOF host on the catalytic performance of loaded enzyme was studied through contrasting the Km and kcat/Km values of GAA@GOx@Cu-MOF and free enzyme system under various concentrations of maltose. The details were shown in “Supporting Information”. Detection of Acarbose Based on GAA@GOx@Cu-MOF. Firstly, 20 μL (216.55 µg/mL) reactor and 10 μL acarbose (dissolved in ultrapure water) with different concentrations (10 000, 5 000, 2 500....19.53 μM) were mixed in 96-well plate and incubated at 37 °C for 10 min, followed by the addition of 80 μL of 40 mM maltose. After incubating at 37 °C for 30 min, 80 μL of 4.15 mM TMB was added and reacted at 37 °C for 20 min. Finally 80 μL of 2 M H2SO4 solution was added to terminate the reaction before the absorbance was measured at 450 nm. In addition, a negative control (without inhibitor) and a blank control (without inhibitor and maltose) were set. The inhibition ratio was calculated as follows: Inhibition % 

ODsample - ODnegative  100% ODnegative

(2)

The absorbance in the formula was the value after the blank control was deducted, and the half maximal inhibitory concentration (IC50) was obtained from the inhibition curve. In addition, the limit of detection (LOD) of the reactor for acarbose was calculated according to the following formula, and the concentrations of acarbose were ranged from 0.05 to1 000 μM in the assay: LOD  3  SD / m 10


(3)

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In the formula, SD was the standard deviation of the blank signal under multiple measurements, m was the slope of the standard curve. Furthermore, the effect of Cu-MOF addition on the linearity (δ) of the reactor was studied via adding Cu-MOF with different concentrations (325, 271, 217, 162, 108 and 54 µg/mL) into the above reaction system. And the linearity of the sensor was calculated as follows: δ = ΔYmax / Y* × 100%

(4)

In the formula, ΔYmax was the maximum deviation between linear fit value and measured value, Y* was the full scale output value of reactor. Application of Screening α-glucosidase Inhibitor from Oleanolic Acid Derivatives. In recent years, considerable interest has been attracted to natural product oleanolic acid and its derivatives because of their hypoglycemic activity. And it was important to screen out the oleanolic acid derivatives with high activity as lead compounds for the development of novel α-glucosidase inhibitors. Therefore, the αglucosidase inhibition activities of oleanolic acid derivatives 4~29 (obtained from our previous work)39 were evaluated via the proposed method in this work, and the experimental process was similar with the detection process of acarbose based on GAA@GOx@Cu-MOF except that the derivatives were dissolved in dimethyl sulfoxide (DMSO) to obtain various concentrations ranging from 0.03 µM to 267 µM. RESULTS AND DISCUSSION 11



Characterization of GAA@GOx@Cu-MOF. Cu-MOF and GAA@GOx@Cu-MOF were characterized by SEM, FTIR, XRD and specific surface and microporous physical adsorption analyzer. Figure 1a, Figure S1 and Figure S2 exhibited that GAA@GOx@Cu-MOF with spherical flower structure had smaller diameter (9.63 µm) and higher specific surface (94.33 m2/g) than the Cu-MOF host (11.77 µm, 18.25 m2/g), which could provide a broader platform for enzymatic reactions. Moreover, GAA@GOx@Cu-MOF had a substantially identical powder XRD pattern with the Cu-MOF host (Figure 1b), which indicated that the integration of GAA and GOx with Cu-MOF had no influence on the main crystal structure of Cu-MOF although the morphology was changed apparently from being square to spherical. Energy-dispersive spectroscopy (EDS) (Figure 1c) showed that GAA@GOx@Cu-MOF contained the elements of C, N, Cl, Cu, O and S, of which the elements O and S were new compared to the elements in the Cu-MOF host (Figure S1). O and S could be attributed to the contribution of the carboxyl and thiol groups of protein, suggesting the presence of GAA, GOx or GAA@GOx in the CuMOF host. That could be confirmed by the characteristic amide band (1 650.79 cm-1) in the FTIR spectrum from GAA@GOx@Cu-MOF (Figure 1d). Moreover, the superimposed FTIR spectra of GAA, GOx and CuMOF were basically consistent with GAA@GOx@Cu-MOF, indicating that GAA and GOx were successfully immobilized on Cu-MOF. 12


Page 12 of 36

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. SEM image (a), XRD patterns (b), EDS spectrum (c), FTIR spectra (d) and EDS mapping images of the elements O and S (e) of GAA@GOx@Cu-MOF.

In order to further investigate how GAA and GOx located in the CuMOF,

SDS-PAGE

analysis

was

performed

firstly (“Supporting

Information”).31 From Figure S3a, it could be found that free enzyme (lane 1) and GAA@GOx@Cu-MOF (lane 2) samples had similar bands, while no obvious band was observed for GAA-GOx-Cu-MOF sample (lane 4). This result suggested that GAA and GOx in GAA @GOx @Cu-MOF were relatively strongly bonded with Cu-MOF and could not be removed by 13



washing, as compared to the enzyme being simply adsorbed on the external surface of Cu-MOF. Moreover, the unchanging morphology of Cu-MOF after being physically mixed with GAA and GOx also indicated that GAA@GOx@Cu-MOF was not formed through the adsorption of GAA and GOx on the external surface of Cu-MOF (Figure S3b). Furthermore, Figure 1e showed that the elements O and S were uniformly dispersed on the cross-section of GAA@GOx@Cu-MOF, which confirmed that GAA and GOx were immobilized in Cu-MOF host rather than being simply adsorbed on the external surface. Optimization for the Ratio of GAA to GOx during the Preparation of Hybrid Enzyme-Catalytic MOF Reactor. In order to obtain a hybrid enzyme-catalytic MOF reactor with high loading rate and strong catalytic activity, the effects of different GAA and GOx ratios on the total enzyme loading rate, GAA loading rate and relative activity of the reactor were investigated. The loading rates were calculated from the standard curve (Figure S4) and the results were shown in Figure 2. Figure 2a exhibited that as the ratio of GAA to GOX decreased (the amount of GAA was constant), the total loading rate of Cu-MOF for enzyme increased firstly and then stabilized, indicating that the loading of enzyme on Cu-MOF reached saturation at the ratio of 1:1, and the total loading rate of enzyme was about 59 % at this moment (Table 1). However, as the ratio decreased, the loading rate of GAA on Cu-MOF stabilized firstly and then 14


Page 14 of 36

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


showed a downward trend (Figure 2b), which suggested that GAA could saturate the load on Cu-MOF when the ratio was greater than or equal to 1, and the loading rate of the GAA was about 14% at this time (Table 1). In addition, as displayed in Table 1, the loading efficiency of Cu-MOF for different enzyme was distinct,24 but regardless of the ratio of GAA to GOx, it was clear that Cu-MOF enjoyed higher loading efficiency for GOx than GAA, which meant that GOx was easier to load in Cu-MOF than GAA. Therefore, when the ratio of GAA to GOx was less than 1, the decrease of GAA loading rate under the total saturation loading rate was due to the fact that GOx was more easily loaded on Cu-MOF (Figure 2b). Furthermore, it could be seen from Figure 2c that the reactor had the highest relative activity when the mass ratio of GAA to GOX was 1:1, which could be attributed to the fact that the Cu-MOF host reached the loading saturation state of enzyme at this moment and GAA also achieved the maximum loading rate simultaneously, so that the maltose could be hydrolyzed at the fastest rate to produce glucose provided to GOx hydrolysis. That is, GAA and GOx could best reflect the assembly line performance at this ratio. In summary, the best ratio of GAA to GOx was 1:1.

15



Page 16 of 36

Figure 2. Comparison of total loading rate (a), α-glucosidase loading rate (b) and catalytic activity (c) for different mass ratio of α-glucosidase and GOx.

Table 1. Comparison of total loading rate, α-glucosidase loading rate and catalytic activity for different mass ratio of α-glucosidase and GOx. GAA : GOx (mass ratio) Total loading rate (%) GAA loading rate (%) Relative activity (%)

3:1

2:1

1:1

1:2

1:3

33.81 ±2.79

45.80 ±1.10

59.41 ±2.43

58.01 ±1.23

57.83 ±1.81

13.79 ±0.25

13.62 ±0.28

14.03 ±0.27

8.51 ±0.12

4.28 ±0.08

62.34 ±4.81

82.63 ±4.76

100 ±2.90

92.02 ±3.31

70.84 ±2.70

Enzymatic Catalytic Capabilities of GAA@GOx@Cu-MOF and Cu-MOF. By conducting steady-state kinetic experiments (Table 2), it was found that the Km values of GAA@GOx@Cu-MOF and Cu-MOF were similar no matter when TMB or H2O2 was used as the substrate, implying that the affinity of Cu-MOF for the substrate was not affected by GAA and GOx. Moreover, the ratio of kcat/Km values of the both was close to 1, suggesting that GAA and GOx had almost no effect on the catalytic activity of Cu-MOF. In addition, as shown in Table 2, the Km value of GAA@GOx@Cu-MOF was similar to free enzyme system, which 16


Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


indicated that the Cu-MOF skeleton did not affect the affinity of loaded enzyme to the substrate. Furthermore, it was gratifying that the kcat/Km value of GAA@GOx@Cu-MOF was slightly greater than free enzyme system. The ratio of the two indicated that the catalytic activity of encapsulated enzyme was improved by about 4.58 times compared with the free enzyme system, which might be due to the integration of GAA and GOx in one reactor that endowed them the character of the assembly line work.7

Table 2. Comparison of Km, Vmax and kcat/Km of Cu-MOF, GAA@GOx and GAA@GOx@Cu-MOF under different substrates. Catalyst

Substrate

Km (mM)

Vmax (µM /min)

kcat / Km (mmol-1.s-1)

Cu-MOF

TMB

3.31

3.03

3.91 × 10-2

GAA@GOx@Cu-MOF

TMB

3.58

3.11

3.71 × 10-2

1.05

A1 /A2 Cu-MOF

H2O2

6.84

5.81

3.63 × 10-2

GAA@GOx@Cu-MOF

H2O2

5.83

5.38

3.94 × 10-2

0.92

B1 /B2 GAA@GOx

Maltose

0.87

0.15

3.67 × 102

GAA@GOx@Cu-MOF

Maltose

0.76

1.01

1.68 × 103

4.58

C1 /C2

A1, A2: The kcat / Km of Cu-MOF and GAA@GOx@Cu-MOF under the substrate of TMB; B1, B2: The kcat / Km of Cu-MOF and GAA@GOx@Cu-MOF under the substrate of H2O2; C1, C2: The kcat / Km of GAA@GOx@Cu-MOF and GAA@GOx under the substrate of maltose.

17



Properties of GAA@GOx@Cu-MOF. In order to verify the properties of the synthesized hybrid enzyme-catalytic MOF reactor, the bioactivities of GAA@GOx@Cu-MOF under different conditions were investigated and compared with the free GAA and GOx systems. As shown in Figure 3a and Figure S7, GAA@GOx@Cu-MOF could maintain nearly 100 % of its original activity whether being stored at room temperature for 7 days or at 4 oC for 30 days, while free GAA and GOx lost more than 80 % of the original activity, indicating that encapsulation of GAA and GOx in Cu-MOF could significantly improve their storage stability. By treating GAA@GOx for 60 minutes at different temperatures, it was found that the bioactivity of GAA@GOx decreased with the increase of temperature, and more than 80 % of the original activity lost when the temperature reached 70 °C. However, almost no change in the activity of GAA@GOx@Cu-MOF was observed under the same conditions, and it could maintain nearly 90 % of the original bioactivity even at 80 °C (Figure 3b), which revealed that the improved thermal stability of GAA and GOx could be achieved via being loaded in Cu-MOF. As seen from Figure 3c, both GAA@GOx@Cu-MOF and GAA@GOx achieved the highest activity at pH 7, but became inactivated in peracid and overbased environments. However, the activity loss of GAA@GOx@Cu-MOF was less than GAA@GOx. In particular at pH 4, 18


Page 18 of 36

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the activity of GAA@GOx@Cu-MOF was much higher than that of GAA@GOx, which was probably because the catalytic activity and structural integrity of Cu-MOF were highly dependent on pH and reached its highest activity and most intact structure at pH 4.38 SEM image in Figure S6a confirmed that GAA@GOx@Cu-MOF had an intact structure at this moment, which could provide effective protection for enzyme, thereby to avoid the inactivation of enzyme in peracid environments. However, the Cu-MOF catalytic activity was completely lost at pH 2 or 1238 and GAA@GOx@Cu-MOF was also completely collapsed upon the time (Figure S8b, c), resulting in the enzyme originally encapsulated in the skeleton being released and inactivated in peracid and overbased environments. On the whole, Cu-MOF could increase chemical stability of loaded enzyme in a certain pH range. After 13 hours of treatment with trypsin at 37 °C, more than 65 % of the initial bioactivity was still observed from GAA@GOx@Cu-MOF. However, only 25 % of the bioactivity was retained in GAA@GOx under the same conditions (Figure 3d), which indicated that the Cu-MOF host could provide effective shielding for GAA and GOx against biodegradation.

19



Figure 3. Comparison of different types of stability under different conditions of GAA@GOx and GAA@GOx@Cu-MOF: storage stability at room temperature (a), thermal stability (b), chemical stability (c), and biological stability (d).

The reusability of reactor was also confirmed. As shown in Figure 4a and Figure S9, GAA@GOx@Cu-MOF before and after recycling had substantially identical powder XRD patterns and SEM images, which indicated that the framework structure of the reactor had hardly changed after 5 cycles of use and confirmed that the reactor possessed considerable stability. Moreover, after three cycles of use, GAA@GOx@Cu-MOF could still maintain nearly 80 % of its original activity, which suggested that the reactor could be recycled at least three times, while the free enzyme system was difficult to separate and reuse after the enzymatic reaction (Figure 4b 20


Page 20 of 36

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and Table S1). Furthermore, it could also be seen from Figure 4b that the relative activity and mass of the reactor exhibited consistent change trend as the number of cycles increased, which revealed that the reduced relative activity of reactor in the process of repeated use may be attributed to the loss of GAA@GOx@Cu-MOF during this process. On the one hand, the loss of the reactor was probably due to the fact that the reactor located in the upper layer of the precipitate was easy to be removed together with the supernatant when the supernatant was poured or aspirated after each centrifugation. On the other hand, it was perhaps owing to the too short centrifugation time that the reactor remained in the supernatant.

Figure 4. XRD patterns before and after recycling (a) and the reusability (b) of GAA@GOx@Cu-MOF.

To further demonstrate the reliability and application potential of the reactor

for

α-glucosidase

inhibitors

screening,

anti-interference

performance was explored by adding 20 μL of 20 μM different metal ions (Ba2+, Mg2+, Al3+, Zn2+, Fe2+, Fe3+, Ca2+, Na+, Cd2+, Cu2+ and K+) or 200 21



Page 22 of 36

μM of various amino acids (Phe, Met, Try, Tyr, Ger, Cys, Gla and Gly) to the sensing system. It could be seen from Figure 5 that relative activity of each test group was close to 100 % in the presence of interference factors, which proved that GAA@GOx@Cu-MOF had strong anti-interference performance and good selectivity.

Figure 5. Anti-interference performance of GAA@GOx@Cu-MOF: anti-interference ability to metal ions (a), anti-interference ability to amino acid (b).

Optimization

of

α-glucosidase

Inhibitor

Screening

Conditions. For purpose of realizing the best sensing performance of the hybrid enzyme-catalytic MOF reactor, the detection conditions including pH of the reaction system, concentrations of GAA@GOx@Cu-MOF and enzymatic reaction time were optimized. According to the reported studies on α-glucosidase and glucose oxidase activity assay,9,41 the reaction system with different pH (5, 6, 6.4, 6.7, 7.0, 7.3, 8 and 9) was evaluated, and the results showed that pH = 7.0 gave the best catalytic activity of the reactor (Figure 6a). Therefore, pH = 7.0 was considered to be the optimal pH for 22


Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the α-glucosidase inhibitor screening assays. Figure 6b exhibited that as the concentration of GAA@GOx@Cu-MOF increased, the catalytic efficiency of the reactor was enhanced gradually until up to 216.55 µg/mL, after which the increase of concentration would no longer affect the catalytic activity, indicating that the concentration of 216.55 μg/mL of GAA@GOx@Cu-MOF was sufficient to catalyze the decomposition of substrate maltose in the reaction system. Moreover, as shown in Figure 6c, the catalytic activity of the reactor increased gradually with the incubation time prolonged, and after 30 min the catalytic efficiency of the reactor ceased to change with enzymatic reaction time, indicating that 30 min was the optimal reaction time.

Figure 6. Optimization of α-glucosidase inhibitor screening conditions including pH of the reaction system (a), concentrations of GAA@GOx@Cu-MOF (b) and enzymatic reaction time (c).

Application

to

α-glucosidase

Inhibitor

Investigation.

Acarbose as a clinical anti-diabetic drug targeting at GAA was employed as a model for the examination of practical application potential of the 23



sensing system developed in this work. As shown in Figure 7a, the IC50 value of acarbose was calculated as 539.65 ± 21.97 μM, which was consistent with the literature reports (Table 3), indicating that the hybrid enzyme reactor possessed high reliability for screening α-glucosidase inhibitors. Moreover, it could be seen from Figure 7b that the inhibition rate was enhanced with the increase of acarbose concentration, and showed a good linear relationship in a wide dynamic range (12.5~800 μM). The fitted linear equation was y = 30.00 x - 29.06 (R2 = 0.997, δ = 0.4218%). The LOD was estimated to be 7.05 nM at S/N = 3, which was lower than what was found in the literature.6 The result suggested that the GAA@GOx@Cu-MOF hybrid enzyme reactor exhibited highly sensitivity for screening GAA inhibitor. Furthermore, the result in Figure 7c showed that as the concentration of added Cu-MOF increased, the linearity of the reactor was almost the same as that without Cu-MOF, which indicated that Cu-MOF addition had no effect on the linearity of the sensor.

Figure 7. The inhibition curve of acarbose (a), the calibration curve of acarbose (b) and the effect of Cu-MOF addition on the linearity of the sensor (c).

24


Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. The comparison of detection limits and/or IC50 of acarbose with different detection methods. Sensing system

Method

Substrate

LOD (nM)

IC50 (µM)

Reference

GAA@GOx@Cu2+AgNPs

SPR

maltose

5

-

4

GAA- PFP

fluorescent

PNPG

-

600

5

GAA- N-doped CDot

fluorescent

4-nitrophenyl-a-Dglucopyranoside (NGP)

10

586.80

6

GAA-(N-doped CDot)-CoOOH

fluorescent

L-ascorbicacid-2-O-α-Dglucopyranosyl(AAG)

1

557

9

GAA-GOx-CysAuNPs

colorimetric

maltose

-

587

13

GAA

colorimetric

PNPG

-

579.15

40

GAA@GOx@CuMOF

colorimetric

maltose

7.05

539.65

This work

Screening of Potential Anti-diabetic Drugs from Oleanolic Acid Derivatives. In view of the excellent sensitivity and practical reliability of the reactor, it has been successfully used in the screening of oleanolic acid derivatives as potential α-glucosidase inhibitors, and the results were compared with conventional methods (i.e. GAA activity was detected by measuring the absorbance of p-nitrophenol (PNP) produced by GAA hydrolysis of PNPG). As shown in Table S2, it could be found that compound 10 with the IC50 value of 0.29 ± 0.03 μM exhibited the most inhibition activity, which was consistent with the result in our previous work.39 Moreover, as it could be seen from Figure 8, the IC50 values of 25



these oleanolic acid analogues obtained from the proposed method and commonly used method39 showed a good correlation (R2 = 0.9971), which further proved the feasibility and practicability of this strategy. Furthermore, according to the above results, the proposed strategy was expected to be applied in the screening of α-glucosidase inhibitors from natural products and the work would be performed in our later research with the steps as follows: firstly, the active ligands in the crude extract of natural products were interacted with GAA@GOx@Cu-MOF at 37 °C for certain time, being followed by centrifugation and washing to remove the unbound impurities. Next, the ligands combined on GAA@GOx@CuMOF were released and the supernatant was finally separated through HPLC to obtain the single active ligand as a potential α-glucosidase inhibitor.

Figure 8. The comparison of the inhibiting results of the proposed method and the common method.

26


Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSIONS In this study, a bifunctional GAA@GOx@Cu-MOF hybrid enzymecatalytic MOF reactor for α-glucosidase inhibitors screening by using GAA, GOx and Cu-MOF as models was successfully prepared. The reactor not only possessed similar sensing performance to GAA-GOx-HRP, but also displayed an excellent anti-interference performance and high sensitivity toward GAA inhibitors screening, and the detection limit of acarbose was 7.05 nM. More importantly, Cu-MOF could effectively enhance the catalytic activity, storage stability, thermal stability, chemical stability, biostability and recyclability of GAA and GOx, which could greatly broaden the application of enzyme so that they were no longer confined to mild environments. Furthermore, the proposed method, with excellent accuracy, was successfully applied to the screening of oleanolic acid derivatives as potential anti-diabetic drugs and expected to be applied in the screening of α-glucosidase inhibitors from natural products. In summary, it was expected that the prepared hybrid enzyme-catalytic MOF reactor had great potential in the screening of anti-diabetic drugs. Meanwhile, this work also provided some insights and inspirations for further research on multifunctional MOF composites. ASSOCIATED CONTENT Supporting Information. The supporting information was available free of charge and the contents of the file was briefly described as follows: 27



The

detailed

information

included:

Page 28 of 36

SDS-PAGE

analysis,

determination of enzyme relative activity, enzymatic Michaelis-Menten kinetics assay and optimization of α-glucosidase inhibitor screening conditions; SEM image, EDS image, DLS data, BET surface area and enzymatic catalytic capability of Cu-MOF; optical density standard curve of BSA and α-glucosidase; SEM image, DLS data, electropherogram, BET surface area, reusability, storage stabilities and enzymatic catalytic capability of GAA@GOx@Cu-MOF; the results of potential anti-diabetic drug screening based on GAA@GOx@Cu-MOF. (PDF) AUTHOR INFORMATION *Corresponding Author Dr. Xiping Cui, Prof. Junxia Zheng and Prof. Suqing Zhao E-mail addresses: [email protected], [email protected], [email protected] E-mail addresses for other authors [email protected]

(Yingying

Zhong),

[email protected]

(Linjin Yu), [email protected] (Qiyi He), [email protected] (Qiuyan Zhu), [email protected] (Chunguo Zhang) Author Contributions The manuscript was written with contributions of all authors who have given approval to the final version of the manuscript. In this work, Yingying Zhong and Linjin Yu conducted the experimental work and 28


Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


drafted the manuscript. Suqing Zhao, Junxia Zheng and Xiping Cui were the directors and designers of the work. Qiyi He, Qiu-Yan Zhu and ChunGuo Zhang participated in the synthesis assay work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors appreciate the financial support from the Guangzhou Science and Technology Foundation (2016201604030025, 201902010430100011), Guangdong Science and Technology Foundation (2017A050501034).

29



REFERENCES (1) Association, A.D. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 2012, 35, 64-71. (2) Perry, R. J.; Samuel, V. T.; Petersen, K. F.; Shulman, G. I. The Role of Hepatic Lipids in Hepatic Insulin Resistance and Type 2 Diabetes. Nature 2014, 510, 84-91. (3) Veiseh, O.; Langer, R. Diabetes: a Smart Insulin Patch, Nature 2015, 524, 39-40. (4) Kong, W.; Wu, D.; Hu, N.; Li, N.; Dai, C.; Chen, X.; Suo, Y.; Li, G.; Wu, Y. Robust Hybrid Enzyme Nanoreactor Mediated Plasmonic Sensing Strategy for Ultrasensitive Screening of Anti-Diabetic Drug. Biosens. Bioelectron. 2018, 99, 653-659. (5) Cao, A.; Tang, Y.; Liu, Y. Novel Fluorescent Biosensor for Alpha-Glucosidase Inhibitor Screening Based on Cationic Conjugated Polymers. ACS Appl. Mater. Interfaces 2012, 4, 3773-3778. (6) Li, J.; He, G.; Wang, B.; Shi, L.; Gao, T.; Li, G. Fabrication of Reusable Electrochemical Biosensor and Its Application for the Assay of Alpha-Glucosidase Activity. Anal. Chim. Acta 2018, 1026, 140-146. (7) Pulungan, A. B.; Afifa, I. T.; Annisa, D. Type 2 Diabetes Mellitus in Children and Adolescent: an Indonesian Perspective. Ann. Pediatr. Endocrinol. Metab. 2018, 23, 119-125. (8) Veiseh, O.; Langer, R. Diabetes: a Smart Insulin Patch. Nature 2015, 524, 39–40. (9) Li, G.; Kong, W.; Zhao, M.; Lu, S.; Gong, P.; Chen, G.; Xia, L.; Wang, H.; You, J.; Wu, Y. A Fluorescence Resonance Energy Transfer (FRET) Based "Turn-On" Nanofluorescence Sensor Using a Nitrogen-Doped Carbon Dot-Hexagonal Cobalt 30


Page 30 of 36

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Oxyhydroxide Nanosheet Architecture and Application to Alpha-Glucosidase Inhibitor Screening. Biosens. Bioelectron. 2016, 79, 728-35. (10) Zhang, J.; Liu, Y.; Lv, J.; Li, G. A Colorimetric Method for Alpha-Glucosidase Activity Assay and Its Inhibitor Screening Based on Aggregation of Gold Nanoparticles Induced by Specific Recognition between Phenylenediboronic Acid and 4Aminophenyl-α-d-Glucopyranoside. Nano Res. 2014, 8, 920-930. (11) Zhang, J.; Liu, Y.; Wang, X.; Chen, Y.; Li, G. Electrochemical Assay of AlphaGlucosidase Activity and the Inhibitor Screening in Cell Medium. Biosens. Bioelectron. 2015, 74, 666-672. (12) Sun, J.; Yang, F.; Zhao, D.; Yang, X. Highly Sensitive Realtime Assay of Inorganic Pyrophosphatase Activity Based on the Fluorescent Gold Nanoclusters. Anal. Chem. 2014, 86, 7883-7889. (13) Chen, H.; Zhang, J.; Wu, H.; Koh, K.; Yin, Y. Sensitive Colorimetric Assays for Alpha-Glucosidase Activity and Inhibitor Screening Based on Unmodified Gold Nanoparticles. Anal. Chim. Acta 2015, 875, 92-98. (14) Sheldon, R. A.; van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What and How. Chem. Soc. Rev. 2013, 42, 6223-6235. (15) Garcia-Galan, C.; Berenguer-Murcia, Á.; Fernandez-Lafuente, R.; Rodrigues, R. C. Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance. Adv. Synth. Catal. 2011, 353, 2885-2904. (26) Jiang, J.; Yu, Y.; Wang, L.; Li, J.; Ling, J.; Li, Y.; Duan, G. Enzyme Immobilized on Polyamidoamine-Coated Magnetic Microspheres for Alpha-Glucosidase Tnhibitors 31



Screening from Radix Paeoniae Rubra Extracts Accompanied with Molecular Modeling. Talanta 2019, 195, 127-136. (17) Li, Z.; Ding, Y.; Li, S.; Jiang, Y.; Liu, Z.; Ge, J. Highly Active, Stable and SelfAntimicrobial Enzyme Catalysts Prepared by Biomimetic Mineralization of Copper Hydroxysulfate. Nanoscale 2016, 8, 17440-17445. (18) Furukawa, H.; Cordova, K. E.; O'Keeffe, M.; Yaghi, O. M. The Chemistry and Applications of Metal-Organic Frameworks. Science 2013, 341, 1-12. (19) Zhou, H. C.; Long, J. R.; Yaghi, O. M. Introduction to Metal-Organic Frameworks. Chem. Rev. 2012, 112, 673-674. (20) Hou, C.; Wang, Y.; Ding, Q.; Jiang, L.; Li, M.; Zhu, W.; Pan, D.; Zhu, H.; Liu, M. Facile Synthesis of Enzyme-Embedded Magnetic Metal-Organic Frameworks as a Reusable Mimic Multi-Enzyme System: Mimetic Peroxidase Properties and Colorimetric Sensor. Nanoscale 2015, 7, 18770-18779. (21) Gao, J.; Wang, C.; Tan, H. Lanthanide/Nucleotide Coordination Polymers: an Excellent Host Platform for Encapsulating Enzymes and Fluorescent Nanoparticles to Enhance Ratiometric Sensing. J. Mater. Chem. B 2017, 5, 7692-7700. (22) Lyu, F.; Zhang, Y.; Zare, R. N.; Ge, J.; Liu, Z. One-Pot Synthesis of ProteinEmbedded Metal-Organic Frameworks with Enhanced Biological Activities. Nano Lett. 2014, 14, 5761-5765. (23) Zhang, C.; Wang, X.; Hou, M.; Li, X.; Wu, X.; Ge, J. Immobilization on MetalOrganic Framework Engenders High Sensitivity for Enzymatic Electrochemical Detection. ACS Appl. Mater. Interfaces 2017, 9, 13831-13836. 32


Page 32 of 36

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24) Wu, X. L.; Ge, J.; Yang, C.; Hou, M.; Liu, Z. Facile Synthesis of Multiple EnzymeContaining Metal-Organic Frameworks in a Biomolecule-Friendly Environment. Chem. Commun. 2015, 51, 13408-13411. (25) Dong, S.; Peng, L.; Wei, W.; Huang, T. Three MOF-Templated Carbon Nanocomposites for Potential Platforms of Enzyme Immobilization with Improved Electrochemical Performance. ACS Appl. Mater. Interfaces 2018, 10, 14665-14672. (26) Wu, X.; Yang, C.; Ge, J.; Liu, Z. Polydopamine Tethered Enzyme/Metal-Organic Framework Composites with High Stability and Reusability. Nanoscale 2015, 7, 18883-18886. (27) Li, P.; Moon, S. Y.; Guelta, M. A.; Harvey, S. P.; Hupp, J. T.; Farha, O. K. Encapsulation of a Nerve Agent Detoxifying Enzyme by a Mesoporous Zirconium Metal-Organic Framework Engenders Thermal and Long-Term Stability. J. Am. Chem. Soc. 2016, 138, 8052-8055. (28) Li, P.; Moon, S. Y.; Guelta, M. A.; Lin, L.; Gomez-Gualdron, D. A.; Snurr, R. Q.; Harvey, S. P.; Hupp, J. T.; Farha, O. K. Nanosizing a Metal-Organic Framework Enzyme Carrier for Accelerating Nerve Agent Hydrolysis. ACS Nano 2016, 10, 91749182. (29) Chen, Y.; Lykourinou, V.; Vetromile, C.; Hoang, T.; Ming, L. J.; Larsen, R. W.; Ma, S. How Can Proteins Enter the Interior of a MOF? Investigation of Cytochrome c Translocation into a MOF Consisting of Mesoporous Cages with Microporous Windows. J. Am. Chem. Soc. 2012, 134, 13188-13191. (30) Lyu, F. J.; Zhang, Y. F.; Zare, R. N.; Ge, J.; Liu, Z. One-Pot Synthesis of Protein33



Embedded Metal−Organic Frameworks with Enhanced Biological Activities. Nano Lett. 2014, 14, 5761-5765. (31) Shieh, F. K.; Wang, S. C.; Yen, C. I.; Wu, C. C.; Dutta, S.; Chou, L. Y.; Morabito, J. V.; Hu, P.; Hsu, M. H.; Wu, K. C. W.; Tsung, C. K. Imparting Functionality to Biocatalysts via Embedding Enzymes into Nanoporous Materials by a de Novo Approach: Size-Selective Sheltering of Catalase in Metal−Organic Framework Microcrystals. J. Am. Chem. Soc. 2015, 137, 4276-4279. (32) Lian, X. Z.; Chen, Y. P.; Liu, T. F.; Zhou, H. C. Coupling Two Enzymes into a Tandem Nanoreactor Utilizing a Hierarchically Structured MOF. Chem. Sci. 2016, 7, 6969-6973. (33) Jung, S.; Kim, Y.; Kim, S. J.; Kwon, T. H.; Huh, S.; Park, S. Bio-Functionalization of Metal-Organic Frameworks by Covalent Protein Conjugation. Chem. Commun. 2011, 47, 2904-2906. (34) Feng, D.; Liu, T. F.; Su, J.; Bosch, M.; Wei, Z. W.; Wan, W.; Yuan, D. Q.; Chen, Y, P.; Wang, X.; Wang, K. C.; Lian, X. Z.; Gu, Z. Y.; Park, J.; Zou, X. D.; Zhou, H. C. Stable Metal-Organic Frameworks Containing Single-Molecule Traps for Enzyme Encapsulation. Nat. Commun. 2015, 6, 5979. (35) Sun, J. Y.; Ge, J. C.; Liu, W. M.; Lan, M. H.; Zhang, H. Y.; Wang, P. F.; Wang, Y. M.; Niu, Z. W. Multi-Enzyme Co-Embedded Organic-Inorganic Hybrid Nanoflowers: Synthesis and Application as a Colorimetric Sensor. Nanoscale. 2014, 6, 255-262. (36) Lykourinou, V.; Chen, Y.; Wang, X. S.; Meng, L.; Hoang, T.; Ming, L. J.; Musselman, R. L.; Ma, S. Immobilization of MP-11 into a Mesoporous Metal-Organic 34


Page 34 of 36

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Framework, MP-11@mesoMOF: a New Platform for Enzymatic Catalysis. J. Am. Chem. Soc. 2011, 133, 10382-10385. (37) Wu, J.; Wang, X.; Wang, Q.; Lou, Z.; Li, S.; Zhu, Y.; Qin, L.; Wei, H. Nanomaterials with Enzyme-Like Characteristics (Nanozymes): Next-Generation Artificial Enzymes (II). Chem. Soc. Rev. 2019, 48, 1004-1076. (38) Wang, C.; Gao, J.; Tan, H. Integrated Antibody with Catalytic Metal-Organic Framework for Colorimetric Immunoassay. ACS Appl. Mater. Interfaces. 2018, 10, 25113-25120. (39) Zhong, Y. Y.; Chen, H. S.; Wu, P. P.; Zhang, B. J.; Yang, Y.; Zhu, Q. Y.; Zhang, C. G.; Zhao, S. Q. Synthesis and Biological Evaluation of Novel Oleanolic Acid Analogues as Potential Alpha-Glucosidase Inhibitors. Eur. J. Med. Chem. 2019, 164, 706-716. (40) Wu, P. P.; Zhang, K.; Lu, Y. J.; He, P.; Zhao, S. Q. In Vitro and In Vivo Evaluation of the Antidiabetic Activity of Ursolic Acid Derivatives. Eur. J. Med. Chem. 2014, 80, 502-508. (41) Jiang, X.; Sun, C. J.; Guo, Y.; Nie, G. J.; Xu, L. Peroxidase-Like Activity of Apoferritin Paired Gold Clusters for Glucose Detection. Biosens. Bioelectron. 2015, 64, 165-170.

35



Graphical abstract (For Table of Contents Only)

36


Page 36 of 36

Bifunctional Hybrid Enzyme-Catalytic Metal Organic Framework

Recommend Documents