A Sensitive and Selective Colorimetric Method Based on the Acetylcholinesterase-like Activity of Zeolitic Imidazolate Framework-8 and Its Applications

In this study, a simple colorimetric method was established to detect copper ion (Cu2+), sulfathiazole (ST), and glucose based on the acetylcholinesterase (AChE)-like activity of zeolitic imidazolate framework-8 (ZIF-8). The AChE-like activity of ZIF-8 can hydrolyze acetylthiocholine chloride (ATCh) to thiocholine (TCh), which will further react with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) to generate 2-nitro-5-thiobenzoic acid (TNB) that has a maximum absorption peak at 405 nm. The effects of different reaction conditions (buffer pH, the volume of ZIF-8, reaction temperature and time, and ATCh concentration) were investigated. Under the optimized conditions, the value of the Michaelis-Menten constant (Km) is measured to be 0.83 mM, which shows a high affinity toward the substrate (ATCh). Meanwhile, the ZIF-8 has good storage stability, which can maintain more than 80.0% of its initial activity after 30 days of storage at room temperature, and the relative standard deviation (RSD) of batch-to-batch (n = 3) is 5.1%. The linear dependences are obtained based on the AChE-like activity of ZIF-8 for the detection of Cu2+, ST, and glucose in the ranges of 0.021–1.34 and 5.38–689.66 µM, 43.10–517.24 µM, and 0.0054–1.40 mM, respectively. The limit of detections (LODs) are calculated to be 20.00 nM, 9.25 µM, and 5.24 µM, respectively. Moreover, the sample spiked recoveries of Cu2+ in lake water, ST in milk, and glucose in strawberry samples were measured, and the results are in the range of 98.4–115.4% with the RSD (n = 3) lower than 3.3%. In addition, the method shows high selectivity in the real sample analysis.


Introduction
Nowadays, enzyme-mimicking catalytic nanomaterial (nanozyme) has been rapidly developed, attracting much attention in many fields. The nanozyme can make up for several shortcomings of biological enzymes, such as poor stability in harsh environments, difficulty in recycling, and high-cost of production. The frequently used nanozymes include metal oxides, metal sulfides, noble metals, and metal-organic frameworks (MOFs) [1][2][3]. MOFs, owning uniform porous structure, high surface area, porosity, and good chemical stability, are composed of organic ligands and metal nodes, which have been intensively used in the analysis of gases, ions, aromatic compounds, and bioactive species based on optical, electrochemical, mechanical or other sensing methods [4]. For example, He, et al. [5] successfully synthesized a novel 3D ruthenium-based metal-organic gels (Ru-MOGs) with fibrillar network structure using a facile one-step strategy at room temperature, which was used in the detection of glucose with a good linearity in the range of 0.02-5 µM, and the limit of detection (LOD) is 9 nM. Sun et al. [6] synthesized an Eu-MOF ([Eu 2 L 2 (DMF)-(H 2 O) 2 ] 2DMF H 2 O) as a turn-off probe for the detection of ferric ion (Fe 3+ ) and copper ion (Cu 2+ ) in N, N-dimethylformamide (DMF), and the LODs are 12.2 µM and 18.3 µM, Therefore, a colorimetric method based on the AChE-like activity of ZIF-8 can be designed to detect Cu 2+ , ST, and glucose. The linear relationship between the Cu 2+ , ST, and glucose concentrations and the inhibition rate of ZIF-8 activity was investigated. Finally, the developed method was applied to detect Cu 2+ , ST, and glucose in lake water, milk, and strawberry samples, respectively.

Characterization of ZIF-8 and Feasibility of the Method for Cu 2+ , ST, and Glucose Detection
The morphology and structure of ZIF-8 were characterized by scanning electron microscope (SEM), X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-TR), and Brunner-Emmet-Teller (BET) measurements. As the SEM images shown in Figure 2A,B, the synthesized ZIF-8 nanocrystals are rhombic dodecahedrons with a uniform size of about 1 µm. The XRD spectrum depicted in Figure 2C shows mainly sharp and intense diffraction peaks (2θ = 011°, 002°, 112°, 022°, and 222°), revealing the formation of a crystalline nanostructure, which is consistent with the previous report [17]. In addition, as shown in Figure 2D, the FT-IR spectrum displays the characteristic bands of ZIF-8. The absorption peaks at 3105 cm −1 and 2926 cm −1 are due to the NH and CH stretching vibrations, respectively. The absorption peak at 1574 cm −1 is assigned to the CN stretching vibration. The absorption peaks at 1425 cm −1 and 1308 cm −1 correspond to the bending signals of the imidazole ring, and the band at 401 cm −1 is attributed to the Zn-N stretching vibration [18]. The ZIF-8 exhibits a BET specific surface area of 1455.6 m 2 /g with a total pore volume of 0.4975 cm 3 /g and an average pore diameter of 1.3671 nm ( Figure  2E,F). The above results indicate that ZIF-8 is successfully synthesized.

Characterization of ZIF-8 and Feasibility of the Method for Cu 2+ , ST, and Glucose Detection
The morphology and structure of ZIF-8 were characterized by scanning electron microscope (SEM), X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-TR), and Brunner-Emmet-Teller (BET) measurements. As the SEM images shown in Figure 2A,B, the synthesized ZIF-8 nanocrystals are rhombic dodecahedrons with a uniform size of about 1 µm. The XRD spectrum depicted in Figure 2C shows mainly sharp and intense diffraction peaks (2θ = 011 • , 002 • , 112 • , 022 • , and 222 • ), revealing the formation of a crystalline nanostructure, which is consistent with the previous report [17]. In addition, as shown in Figure 2D, the FT-IR spectrum displays the characteristic bands of ZIF-8. The absorption peaks at 3105 cm −1 and 2926 cm −1 are due to the NH and CH stretching vibrations, respectively. The absorption peak at 1574 cm −1 is assigned to the CN stretching vibration. The absorption peaks at 1425 cm −1 and 1308 cm −1 correspond to the bending signals of the imidazole ring, and the band at 401 cm −1 is attributed to the Zn-N stretching vibration [18]. The ZIF-8 exhibits a BET specific surface area of 1455.6 m 2 /g with a total pore volume of 0.4975 cm 3 /g and an average pore diameter of 1.3671 nm ( Figure 2E,F). The above results indicate that ZIF-8 is successfully synthesized.  As shown in Figure S2, the mixture of 2-methylimidazole, ATCh, and DTNB shows a certain absorption at 405 nm. However, during the synthesis process of ZIF-8, the material will be washed twice with deionized water to remove the unreacted 2-methylimidazole, so its impact on the reaction can be ignored. In addition, the mixture of C4H6O4Zn•2H2O, ATCh, and DTNB shows a negligible effect on the reaction as compared to the mixture of ZIF-8, ATCh, and DTNB. As shown in Figure 3A, the mixture of ZIF-8, As shown in Figure S2, the mixture of 2-methylimidazole, ATCh, and DTNB shows a certain absorption at 405 nm. However, during the synthesis process of ZIF-8, the material will be washed twice with deionized water to remove the unreacted 2-methylimidazole, so its impact on the reaction can be ignored. In addition, the mixture of C 4 H 6 O 4 Zn·2H 2 O, ATCh, and DTNB shows a negligible effect on the reaction as compared to the mixture of ZIF-8, ATCh, and DTNB. As shown in Figure 3A, the mixture of ZIF-8, ATCh, and DTNB shows an obvious absorption at 405 nm. When the Cu 2+ , ST, or GOX and glucose being added to the system, the relative activity of ZIF-8 is obviously weakened. The Cu 2+ slows down the reaction due to its competitive binding with the intermediate TCh [19]. Furthermore, ST weakens the catalytic reaction of ZIF-8 by affecting its enzyme-like activity. In addition, GOX reacts with glucose to generate highly oxidizing H 2 O 2 , which can act with the intermediate TCh to reduce its reaction to DTNB. Therefore, these results indicate that this AChE-like activity is coming from ZIF-8, and the established method based on the AChE-like activity of ZIF-8 used for the Cu 2+ , ST, and glucose detection is practicable.
ATCh, and DTNB shows an obvious absorption at 405 nm. When the Cu 2+ , ST, or GOX and glucose being added to the system, the relative activity of ZIF-8 is obviously weakened. The Cu 2+ slows down the reaction due to its competitive binding with the intermediate TCh [19]. Furthermore, ST weakens the catalytic reaction of ZIF-8 by affecting its enzyme-like activity. In addition, GOX reacts with glucose to generate highly oxidizing H2O2, which can act with the intermediate TCh to reduce its reaction to DTNB. Therefore, these results indicate that this AChE-like activity is coming from ZIF-8, and the established method based on the AChE-like activity of ZIF-8 used for the Cu 2+ , ST, and glucose detection is practicable.

Optimization of the Detection Conditions
In order to obtain a good sensitivity of the developed method for Cu 2+ , ST, and glucose detection, several experimental parameters were optimized, including buffer pH, the volume of ZIF-8, reaction temperature, reaction time, and the concentration of ATCh. As shown in Figure 3B, the catalytic activity of ZIF-8 changes with the buffer pH (from 7 to 9), and reaches the highest at pH 8, which was selected for the subsequent experiments (The maximum point in each curve was set as 100%).
Then, the effect of the volume of ZIF-8 was investigated. The catalytic activity increases with the increase in the volume of ZIF-8 from 30 to 100 µL ( Figure 3C) but increases slowly from 50 µL and remains stable after 80 µL. Therefore, 80 µL was selected for the subsequent experiments. Furthermore, as shown in Figure 3D, the effect of reaction temperature on the catalytic activity of ZIF-8 was investigated. As the increase in reaction temperature from 30 to 60 • C, the catalytic activity keeps steady above 50 • C, which was selected as the optimum reaction temperature. In addition, the catalytic activity increases with the increase in reaction time (5,8,10, and 13 min), and increases slowly after 10 min ( Figure 3E). Therefore, further experiments were performed for 10 min. Finally, the effect of the concentration of ATCh was investigated. The catalytic activity increases with the increase in ATCh concentration (1.40, 3.45, 5.17, and 6.90 mM), and increases slowly after 5.17 mM ( Figure 3F). Therefore, 5.17 mM of substrate (ATCh) is enough for the reaction. In summary, the optimized reaction conditions for the detection of Cu 2+ , ST, and glucose based on the AChE-like activity of ZIF-8 are as follows. The buffer pH, volume of ZIF-8, reaction temperature, reaction time, and the concentration of ATCh are 8, 80 µL, 50 • C, 10 min, and 5.17 mM, respectively.

Kinetics Study and Stability of ZIF-8
The steady-state kinetic study for the AChE-like activity of ZIF-8 was performed. As shown in Figure 4A, a typical Lineweaver-Burk plot curve was obtained based on the different ATCh concentrations (0.17, 0.34, 0.69, 1.03, and 1.38 mM). Through Equation (1), the obtained linear regression equation of the Lineweaver-Burk plot is y = 0.4256x + 0.5099 and R 2 = 0.9927, where x and y are the reciprocal of ATCh (substrate) concentration and reaction velocity (the absorbance of product), respectively. A smaller K m value indicates a stronger affinity between the enzyme and the substrate. The K m value of the AChE-like activity of ZIF-8 in this study (0.83 mM) is similar to that of natural AChE (0.23 mM) [20], which indicates that the ZIF-8 has a good affinity toward ATCh and exhibits excellent AChE-like catalytic activity. In addition, a batch of ZIF-8 was synthesized and kept at room temperature for 30 days under dry conditions. The relative AChE-like activity of ZIF-8 maintains more than 80.0% of its initial activity after 30 days of storage, and the relative standard deviation (RSD) of batch-to-batch (n = 3) is 5.1%, indicating that the synthesized ZIF-8 is stable ( Figure 4B).

Colorimetric Detection of Cu 2+ , ST, and Glucose
In Figure 4C, based on the AChE-like activity of ZIF-8, with the increase in concentration of Cu 2+ , the absorbance at 405 nm is decreased because the inhibition rate of ZIF-8 activity is increased. Good linear dependence is obtained in the ranges of 0.021-1.34 µM (calibration curve: y = 16.3204x + 28.0815, R 2 = 0.9884), and 5.38-689.66 µM (y = 0.05109x + 55.4258, R 2 = 0.9904). The LOD is calculated based on 3σ/s, where σ is the standard deviation of the blank signal (n = 11) and s is the slope of the regression line. The LOD is calculated to be 20.00 nM. As shown in Figure 4D, good linear dependence is obtained based on the AChElike activity of ZIF-8 for the ST detection in the range of 43.10-517.24 µM (calibration curve: y = 0.06929x + 18.2985, R 2 = 0.9953), and the LOD is calculated to be 9.25 µM. In addition, a linear equation for the detection of glucose is obtained in the range of 0.0054-1.40 mM (calibration curve: y = 19.4178x + 30.4700, R 2 = 0.9987). The LOD is calculated to be 5.24 µM ( Figure 4E). As compared with previous studies (Tables 1-3), most of the reported methods require a long time for the synthesis of materials, or a high temperature, and many organic reagents, which is not cost-effective and environmentally friendly. The developed method in this study for the detection of Cu 2+ , ST, and glucose shows some advantages, such as simplicity of the material synthesis process, high efficiency, low LOD, and wide linear range.     For the purpose of assessing the selectivity of the sensing system for Cu 2+ , ST, and glucose detection, some potential interfering substances, including amino acids, small biomolecules, and common ions, were systematically investigated in their effects on the detection. As shown in Figure 4F, the potential substances in the lake water samples (detection of Cu 2+ ) such as Co 2+ , Ni 2+ , K + , Mn 2+ , and Na + , the potential substances in the milk sample (detection of ST) such as oxytetracycline, VB1, doxycycline, BSA, L-glutamic acid, and L-tyrosine, the potential substances in fruit sample (detection of glucose) such as VB1, VB3, VB5, VB6, sucrose, citric acid, and Na + , were investigated. The results indicate that only Cu 2+ , ST, and glucose have obvious effects on the AChE-like activity of ZIF-8, indicating that the sensor has a high specificity for the detection of Cu 2+ , ST, and glucose.

Detection of Cu 2+ , ST, and Glucose in Real Samples
To evaluate the practical application of this sensing platform, the real samples were analyzed on the basis of the standard addition method. As shown in Table 4, the established method was used for the detection of Cu 2+ , ST, and glucose in the lake water, milk, and strawberry samples. A standard addition method was adopted by adding Cu 2+ into the lake water samples to reach the final concentrations of 0.084, 21.55, and 172.41 µM, and the sample spiked recoveries are within the range of 81.4-112.8% with the RSD (n = 3) lower than 8.1%. Furthermore, the ST was added into the milk sample with the final concentrations of 43.10, 86.21, and 172.41 µM, and analyzed by the developed method, the sample spiked recoveries are in the range of 98.4-115.4% with the RSD lower than 3.3%. Finally, standard addition of glucose to the strawberry sample with the final concentration of 0.086, 0.17, and 0.34 mM was performed, the sample spiked recoveries are in the range of 93.0-112.8% with the RSD lower than 1.8%. These results indicate that the developed method has good analytical performance and can be applied in the real sample analysis. In addition, this Cu 2+ , ST, and glucose sensor shows reasonable repeatability and reliability for their low RSD% value.

Instruments
For the characterization of material, the SEM images were obtained through a fieldemission SEM (JSM-7600F, JEOL Ltd., Tokyo, Japan); The XRD pattern was obtained using a X' pert Powder diffractometer (Malvern Panalytical Ltd., Malvern, The Netherlands) with secondary beam graphite monochromated Cu Kα radiation; The FT-TR pattern was obtained through a Nicolet iS50 (Thermo Scientific Inc., Waltham, MA, USA); And the BET studies were performed on a Quadrasorb 2 MP (Kantar, New York, NY, USA) specific surface and aperture analyzer. The ultrapure water used for all experiments was purified by a water purification system (ATSelem 1820A, Antesheng Environmental Protection Equipment, Chongqing, China). The UV-Vis analysis was performed on a UV-5500 PC spectrophotometer (Shanghai Metash Instruments Co., Ltd., Shanghai, China). A tabletop low-speed centrifuge L420 was purchased from Hunan Xiang Yi Laboratory Instrument Development Co., Ltd. (Changsha, China).

Synthesis of ZIF-8
The ZIF-8 was synthesized according to the previously reported method [41]. Firstly, the mixture solution of 4 mL of C 4 H 6 O 4 Zn·2H 2 O (0.40 M) and 40 mL of 2-methylimidazole (0.80 M) was placed in an oven and reacted at 30 • C for 2 h. The obtained ZIF-8 was purified by centrifugation for 5 min at 3800 rpm (2259× g) and rinsed with ultra-pure water twice. Finally, the prepared ZIF-8 was re-suspended in 8 mL of deionized water and stored in a 4 • C refrigerator.

ZIF-8 Activity Assays
ZIF-8 can catalyze ATCh to TCh, which will further react with DTNB to generate TNB that has a maximum absorption peak at 405 nm. In a 0.5 mL centrifuge tube, 80 µL of ZIF-8 (dispersed in deionized water), 100 µL of PBS (prepared in 10.00 mM of phosphate buffer, pH 8), 200 µL of 5.17 mM ATCh (prepared in 10.00 mM phosphate buffer, pH 8), and 200 µL of 1.38 mM DTNB (prepared in ethanol) were mixed together, and then incubated at 50 • C with shaken at 160 rpm for 10 min. After being centrifuged by a handheld mini centrifuge for 2 min, the absorbance of the supernatant at 405 nm was recorded. Each sample was measured three times.

Enzyme Kinetics
The K m is an important parameter of an enzyme kinetic reaction and reflects the affinity between the enzyme and substrate, which can be calculated by the Lineweaver-Burk Equation (1) [42].
where V and V max are the initial and maximum rate of the enzyme reaction, respectively,   ) were mixed together into a 0.5 mL centrifuge tube, and then incubated at 50 • C with shaken at 160 rpm for 10 min, respectively. The absorbance at 405 nm of the mixture supernatant was recorded. Each sample was measured three times.

Real Sample Analysis
To validate its feasibility and practicability, the method based on the AChE-like activity of ZIF-8 was used to detect the Cu 2+ , ST, and glucose in lake water, milk, and strawberry samples, respectively.  10.00 mM phosphate buffer, pH 8), and 200 µL of 1.38 mM DTNB (prepared in ethanol) were mixed well, and then incubated at 50 • C with shaken at 160 rpm for 10 min, respectively. The absorbance at 405 nm of the mixture supernatant was recorded. The spiked recoveries in lake water, milk, and strawberry samples of Cu 2+ , ST, and glucose were calculated by the linear relationship between the inhibition rates and their concentrations, respectively.

Conclusions
In summary, the AChE-like activity of ZIF-8 was successfully applied in the sensitive detection of Cu 2+ , ST, and glucose. ZIF-8 has high AChE activity and shows good affinity to the substrate, which also has good storage stability. As compared with the other methods, this method has the advantages of a low LOD and wide detection range, which has the potential as a convenient method for the rapid and sensitive detection of Cu 2+ , ST, and glucose. Moreover, the ZIF-8 is of high stability, and low cost, which can be synthesized through a simple method under mild conditions. In addition, ZIF-8 has a series of enzymelike activities, which can be further applied in the analysis of different analytes. In short, this study provides a feasible detection method for Cu 2+ , ST, and glucose, which may be applied in monitoring their levels in some health problems, such as Alzheimer's disease, Menke's disease, Wilson's disease, resistance to antibiotics, allergic reactions, and toxic effects, and Diabetes.

Data Availability Statement:
The data presented in this study are contained within the article.