Method for rapidly screening acetylcholin esterase inhibitor based on Fe-N-C monatomic nano-enzyme acidic hydrogel

Abstract

The invention discloses a method for rapidly screening an acetylcholin esterase inhibitor based on Fe-N-C monatomic nano-enzyme acidic hydrogel, and belongs to the field of drug screening and biological detection. The method comprises the following steps: firstly, preparing Fe-N-C monatomic nano-enzyme acidic hydrogel, and preassembling the Fe-N-C monatomic nano-enzyme acidic hydrogel on a centrifugal tube cover; then constructing a screening reaction system in the centrifugal tube body, sequentially adding acetylcholin esterase and a substance to be screened for incubation and combination, and then adding acetylthiocholine for reaction; then adding 3, 3 ', 5, 5'-tetramethyl benzidine, covering a tube cover preloaded with hydrogel, inverting a centrifugal tube, diffusing a reaction system into the hydrogel to perform a chromogenic reaction, judging whether the to-be-screened substance has acetylcholin esterase inhibitory activity or not by acquiring color information of the hydrogel, and indicating that the to-be-screened substance has the inhibitory activity when the hydrogel is blue. According to the method, the efficient catalytic performance of the Fe-N-C monatomic nano-enzyme and the stable carrier characteristic of the acidic hydrogel are combined, portable, rapid and high-sensitivity screening is achieved through the centrifugal tube inversion integrated design, and the method is suitable for efficient screening of acetylcholin esterase inhibitors in natural products.

Description

Technical Field

[0001] This invention belongs to the field of drug screening and biological detection, specifically relating to a method for rapid screening of acetylcholinesterase inhibitors based on Fe-NC single-atom nanozyme acidic hydrogel. Background Technology

[0002] Acetylcholinesterase (AChE) is crucial for maintaining neurotransmitter homeostasis by hydrolyzing acetylcholine, and its activity is closely related to neurodegenerative diseases such as Alzheimer's disease. Therefore, acetylcholinesterase inhibitors (AChEIs) have become a key strategy for treating this disease.

[0003] Currently, screening methods for AChEIs include colorimetric methods, fluorescence methods, chemiluminescence methods, and electrochemical methods. For example, Chinese patent CN202210449997.3, "A cellulose-based sensor material for detecting and screening acetylcholinesterase inhibitors, its preparation method, and its application," discloses a colorimetric sensor material based on a three-dimensional network cellulose matrix. It immobilizes acetylcholinesterase through chemical bonds and physically adsorbs a chromogenic agent, utilizing the color intensity changes of the substrate hydrolysis reaction to efficiently detect the inhibitory intensity of acetylcholinesterase inhibitors. Chinese patent CN202310278712.9, "A fluorescent probe for screening acetylcholinesterase inhibitors, its preparation method, and its application," discloses a fluorescent probe for acetylcholinesterase inhibitors. This probe possesses good water solubility, stability, high sensitivity, and near-infrared emission spectroscopy. It also exhibits good cell permeability, few side effects, and low synthesis cost, making it suitable for screening effective substances in traditional Chinese medicine. Chinese patent CN201310421008.0, entitled "A High-Throughput Screening Method for Acetylcholinesterase Inhibitors," discloses a high-throughput fluorescence screening method based on an enzyme cascade reaction. This method assesses acetylcholinesterase activity and inhibitory efficacy by detecting fluorescence intensity. The steps include model establishment and optimization, and positive validation, offering advantages such as simplicity, speed, high sensitivity, and stable and reliable results. However, the aforementioned method typically relies on large, specialized instruments and requires professional operation, and its accuracy and sensitivity are limited. Therefore, there is an urgent need to develop a simple, stable, sensitive, portable, and rapid detection method to efficiently screen novel AChEIs from natural products. This invention, for the first time, combines an acidic hydrogel detection matrix of Fe-N-CSAzyme to construct a cascade signal amplification strategy, improving the stability and sensitivity of the detection system in complex environments. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapidly screening acetylcholinesterase inhibitors based on Fe-NC single-atom nanozyme acidic hydrogel.

[0005] A method for rapid screening of acetylcholinesterase inhibitors based on Fe-NC single-atom nanozyme acidic hydrogel includes the following steps: (1) Preparation of Fe-NC single-atom nanozyme acidic hydrogel: Dissolve agarose in HAc-NaAc buffer, heat to 80-120℃ to form a transparent and clear agarose solution, add Fe-NC single-atom nanozyme and mix thoroughly, take the mixed solution and place it in the cap of a centrifuge tube to cool naturally into a gel, and obtain Fe-NC single-atom nanozyme acidic hydrogel.

[0006] The pH of the HAc-NaAc buffer solution is 3.0-5.0; the mass ratio of agarose to Fe-NC single-atom nanozyme is 8:1-12:1.

[0007] The preparation method of Fe-NC single-atom nanozymes includes the following steps: Zn(NO3)2·6H2O, Fe(NO3)3·9H2O and 2-methylimidazole were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous mixed solution. The mixed solution was reacted at 135-145 ℃ for 20-25 hours. After the reaction was completed, the solution was naturally cooled to room temperature, washed and dried to obtain Fe / Zn-ZIF nanomaterials. The molar ratio of Fe(NO3)3·9H2O, Zn(NO3)2·6H2O and 2-methylimidazole was 1:(2-4):(3-5). Fe / Zn-ZIF was heated to 1000-1200℃ under N2 protection at a heating rate of 10℃ / min and calcined at a constant temperature for 1-2 hours. After calcination, it was naturally cooled to room temperature, and then acid-washed, filtered, washed and dried to obtain Fe-NC single-atom nanozyme (Fe-NC SAzyme).

[0008] (2) Preparation of the screening reaction system: Add acetylcholinesterase (AChE) solution and the substance to be screened into the centrifuge tube, and incubate at 35-40℃ to allow the substance to be screened to fully bind with acetylcholinesterase (AChE). Then add acetylthiocholine (ATCh) and react at 35-40℃ for 25-35 minutes to allow acetylcholinesterase (AChE) to act on the substrate acetylthiocholine (ATCh). The concentration of acetylcholinesterase (AChE) in the reaction system is 1-50 mU / mL, the concentration of the compound to be screened in the reaction system is 0.01-1 mM, the concentration of acetylthiocholine in the reaction system is 0.01-1 mM, and the substance to be screened is an alkaloid compound.

[0009] (3) Colorimetric reaction: Add 3,3',5,5'-tetramethylbenzidine (TMB) to the centrifuge tube, cap the centrifuge tube with the pre-filled hydrogel, invert the centrifuge tube to allow the screening reaction system to diffuse into the hydrogel, invert the centrifuge tube at room temperature for 4-6 minutes, then return the centrifuge tube to the upright position and obtain the color information of the hydrogel; if the hydrogel is blue, it indicates that the compound to be screened has acetylcholinesterase inhibitory activity and can be used as an acetylcholinesterase inhibitor. Under LED light source illumination, use a smartphone to take pictures to obtain the color information of the hydrogel. The deeper the blue of the hydrogel, the stronger the acetylcholinesterase inhibitory activity. The concentration of 3,3',5,5'-tetramethylbenzidine (TMB) in the reaction system is 0.1-10 mM.

[0010] This invention discloses a method for rapidly screening acetylcholinesterase inhibitors based on Fe-NC single-atom nanozyme acidic hydrogel. This method can quickly and efficiently screen AChEIs in natural products. The SAzyme-like oxidase activity catalyzes the generation of reactive oxygen species (ROS) from O2, driving a colorimetric reaction; TMB oxidation produces a blue color. Through an SAzyme / AChE cascade system, thiocholine (TCh) generated from AChE hydrolysis specifically inhibits SAzyme activity, leading to a decrease in color intensity. When AChEIs are present, AChE activity is inhibited, hindering TCh generation and restoring color intensity. By constructing an Fe-NC single-atom nanozyme acidic hydrogel, detection only requires diffusing different substances to be screened into the hydrogel for a colorimetric reaction. The color directly indicates the inhibitory effect, enabling rapid and accurate screening of substances with acetylcholinesterase inhibitory activity. This method is low-cost, portable, and easy to operate. During detection, a series of hydrogels can be photographed simultaneously under the same light source, eliminating the influence of light source and shooting angle on the color of different substances to be screened. This allows for rapid screening of substances with acetylcholinesterase (AChE) inhibitory activity or those with the best AChE inhibitory activity. The construction of Fe-NC single-atom nanozyme acidic hydrogels also facilitates further analysis of the hydrogel's RGB values ​​using images captured by a smartphone. The blue depth of the hydrogel is positively correlated with AChE inhibitory activity; combined with smartphone RGB analysis, quantitative output of the inhibition rate can be achieved. This invention constructs a new generation of portable biodetection method, providing an innovative solution for biological sample analysis in complex environments.

[0011] The beneficial effects of this invention are: 1. Cascaded signal amplification strategy to improve sensitivity Fe-NC single-atom nanozymes (Fe-NC SAzyme) are Fe-N nanobiota dispersed at the atomic level. x As the active center, its catalytic efficiency is close to that of natural enzymes, significantly enhancing the sensitivity of colorimetric reactions. By constructing an "AChE-SAzyme" cascade system, a rapid response to AChEIs can be achieved.

[0012] 2. Acidic hydrogel carriers enhance stability and portability. Environmental adaptability optimization: Acidic HAc-NaAc buffer maintains the optimal catalytic activity of Fe-NC SAzyme, and agarose hydrogel immobilizes nanozyme, extending catalytic life and preventing leakage; moreover, the acidic hydrogel of nanozyme can be prepared in batches and used directly for on-site detection, which is portable and inexpensive.

[0013] Enhanced anti-interference capability: The acidic hydrogel detection matrix shields against interference from complex sample matrices, improving stability in complex environments and making it suitable for screening in impure environments such as crude extracts of natural products.

[0014] 3. Integrated detection of "centrifuge tube inversion" No specialized equipment required: The pre-loaded nanozyme hydrogel is solidified onto the tube cap. During the reaction, the centrifuge tube is inverted to allow the liquid to diffuse into the gel. After color development, the tube is returned to the upright position to terminate the reaction. Rapid detection is achieved through the inverted centrifuge tube design and smartphone photography, eliminating the need for specialized equipment.

[0015] High-throughput parallel screening: Multiple samples can be processed simultaneously, eliminating the influence of differences in shooting light source / angle, and capturing all hydrogel colors at once via smartphone. Figure 7 B), which greatly improves screening efficiency.

[0016] Portable field screening: Eliminates the reliance on large instruments (such as ultraviolet spectrophotometers) and is suitable for field or resource-limited scenarios.

[0017] 4. Highly efficient screening of active ingredients from natural products Rapid identification of alkaloid inhibitors: Successful and efficient screening of AChEIs from natural products, identifying highly active AChEIs (with significant inhibition of berberine hydrochloride) from 11 alkaloids, highlighting their potential for drug development.

[0018] High selectivity: Apart from the target compound, other alkaloids (such as betaine and caffeine) did not show significant inhibition, confirming the excellent selectivity of the method.

[0019] In summary, this invention achieves portable, highly sensitive, and low-cost screening of AChE inhibitors through a comprehensive design that combines the efficient catalysis of Fe-NC single-atom nanozymes, the stable encapsulation of acidic hydrogels, and the inverted centrifuge tube method. Its breakthroughs lie in: ① the innovative combination of nanozyme biomimetic catalysis and biological enzyme inhibition mechanisms to enhance detection sensitivity; ② the use of a hydrogel solid carrier to replace the traditional solution system, enhancing environmental adaptability; and ③ the use of smartphone visualization analysis to drive the transformation from laboratory-dependent technology to on-site, real-time detection. This technology provides an efficient tool for large-scale primary screening of active ingredients in natural drugs and for the research and development of treatments for neurodegenerative diseases, possessing significant scientific value and application potential. Attached Figure Description

[0020] Figure 1 The diagram shows the preparation and characterization of Fe-NC SAzyme, where (A) is a schematic diagram of Fe-NC SAzyme preparation, (B) is a TEM image, (C) is a HAADF-STEM image, (D) is an EDS mapping image, (E) is a Fe K-edge XANES spectrum, (F) is a Fe K-edge XANES Fourier transform spectrum, and (G) is a N 1s high-resolution XPS spectrum.

[0021] Figure 2 (A) is a test of the activity of Fe-NC SAzyme oxidase; (B) is a comparison diagram of the activities of Fe-NC and NC; (C) is an EPR spectrum and a diagram of the catalytic mechanism.

[0022] Figure 3 The UV absorption spectra of different combinations of Fe-NC SAzyme, TMB, AChE, and ATCh after 5 minutes.

[0023] Figure 4 (A) shows the UV absorption spectrum of the Fe-NC SAzyme / AChE cascade catalytic system as a function of AChE concentration; (B) shows the linear relationship between UV absorbance at 652 nm and AChE concentration (inset: linear relationship on a logarithmic scale with base 10).

[0024] Figure 5 (A) Schematic diagram of enzyme inhibitor screening process; (B) Effect of neostigmine bromide on Fe-NC SAzyme / AChE cascade catalytic system; (C) Relationship between the logarithmic concentration of neostigmine bromide and inhibition rate; (D) Inhibition rate of 11 different alkaloids on AChE (1-11 are berberine hydrochloride, betaine, caffeine, camptothecin, evodiamine, matrine, neostigmine bromide, sophoridine, sophoridine, theobromine, and theophylline, respectively).

[0025] Figure 6 (A) The prepared Fe-NC SAzyme acidic hydrogel; (B) Color changes of different concentrations of AChE in the hydrogel catalytic system.

[0026] Figure 7 (A) is a schematic diagram of the process of screening enzyme inhibitors using a single-atom-based hydrogel; (B) shows the screening results of 11 different alkaloids against AChE. Detailed Implementation

[0027] The present invention will be described in detail below with reference to embodiments, so that those skilled in the art can better understand the present invention, but the present invention is not limited to the following embodiments.

[0028] Example 1: Preparation of Fe-NC SAzyme with oxidase-like activity (1) Add 1.5 mmol Zn(NO3)2·6H2O, 0.5 mmol Fe(NO3)3·9H2O and 2 mmol 2-methylimidazole to 50 mL N,N-dimethylformamide and stir magnetically for 30 minutes at room temperature until completely dissolved to form a homogeneous mixed solution.

[0029] (2) Transfer the mixed solution to a 50 mL polytetrafluoroethylene-lined reactor, seal it, and heat it in a 140 °C oven for 24 hours. After the reaction is complete, allow it to cool naturally to room temperature.

[0030] (3) Open the reaction vessel and wash the obtained precipitate three times with methanol to remove unreacted raw materials and solvents. Then dry it at 60 °C for 12 hours to obtain Fe / Zn-ZIF nanomaterials.

[0031] (4) Place the dried Fe / Zn-ZIF in a tube furnace and heat it to 1100 ℃ at a heating rate of 10 ℃ / min under N2 protection. Then, keep it at this temperature for 1 hour and let it cool naturally to room temperature after calcination.

[0032] (5) The calcined black product was dispersed in 3 M HCl solution and acid-washed in a 60°C water bath for 8 hours to remove metal impurities. The solid product was then separated by vacuum filtration and washed three times each with deionized water and anhydrous ethanol. Finally, it was dried in a 60°C oven for 12 hours to obtain Fe-NC SAzyme.

[0033] As a comparative experiment, Zn-ZIFs were prepared by dissolving 2 mmol Zn(NO3)2·6H2O and 2 mmol 2-methylimidazole in 50 mL DMF, following the same steps, and subjected to the same calcination, acid washing, washing and drying treatments to finally obtain undoped Fe NC powder.

[0034] like Figure 1 As shown, characterization using TEM, HRTEM, BF-STEM, SAED, and XRD reveals that the Fe-NC material maintains the elongated framework morphology of the precursor, with no visible nanoparticles on the carbon support, and no characteristic diffraction signals associated with the iron-related phase were observed. These results corroborate each other, indicating that iron is primarily dispersed in an amorphous form as single atoms or ultra-small atomic clusters. Characterization results using HAADF-STEM, EDS, XAS, and XPS demonstrate that the Fe-NC SAzyme possesses atomically dispersed Fe-N x Sites, and exist in a mixed valence state (Fe 2+ / Fe3+ ).

[0035] Example 2: Study on the oxidase-like performance of Fe-NC SAzyme (1) Add 800 μL HAc-NaAc buffer, 100 μL Fe-NC SAzyme suspension and 100 μL TMB solution to a 1.5 mL centrifuge tube, mix thoroughly and incubate at room temperature for 5 minutes.

[0036] (2) The absorbance at 652 nm was measured using a UV-Vis spectrophotometer to quantify the formation of TMB oxidation products.

[0037] (3) To verify the effect of Fe doping on catalytic performance, the oxidase-like activity of undoped NC powder was tested under the same conditions as a control experiment.

[0038] like Figure 2 As shown, the Fe-NC and TMB mixture exhibits a distinct blue color, and the UV-vis spectrum reveals a characteristic absorption peak at 652 nm, indicating that Fe-NC effectively catalyzes the oxidation of TMB to blue TMBox. The NC material without an iron active center shows no significant color reaction, confirming the crucial role of single-atom iron in catalytic activity. EPR technology detected significant signals of superoxide radicals, hydroxyl radicals, and singlet oxygen, indicating that FeN with a single-atom iron center... x The structure can effectively activate dissolved oxygen, triggering the generation of highly active ROS. The generated ROS acts as a strong oxidant, driving TMB to rapidly transform into blue TMBox.

[0039] Example 3: Feasibility Study of Fe-NC SAzyme / Acetylcholinesterase (AChE) Cascade System (1) Preparation of cascade system System 1: Take 0.80 mL of HAc-NaAc buffer, add 0.1 mL of TMB and 0.1 mL of Fe-N-CSAzyme suspension, and then mix the above solution thoroughly; System 2: Mix 10 μL of ATCh with 100 μL of AChE solution thoroughly and react at 37°C for 30 minutes. Then add 0.69 mL of HAc-NaAc buffer, 0.1 mL of TMB and 0.1 mL of Fe-NC SAzyme suspension, and mix the above solution thoroughly. System 3: Take 0.70 mL of HAc-NaAc buffer, add 100 μL of LATCHE, 0.1 mL of TMB and 0.1 mL of Fe-NC SAzyme suspension, and then mix the above solution thoroughly. System 4: Take 0.79 mL of HAc-NaAc buffer, add 10 μL of thioacetylcholine (ATCh), 0.1 mL of TMB and 0.1 mL of Fe-NC SAzyme suspension, and then mix the above solutions thoroughly. System 5: Take 0.89 mL of HAc-NaAc buffer, add 10 μL of ATCh and 100 μL of LATChE, and then mix the above solution thoroughly; (2) React the mixtures obtained from each system in step (1) at room temperature for 5 minutes respectively; (3) The ultraviolet-visible absorption spectrum of the above mixed solution was measured using an ultraviolet-visible absorption spectrophotometer.

[0040] like Figure 3 As shown, Figure 3 The five spectra are the UV-Vis absorption spectra corresponding to the five reaction systems. As can be seen from the figure, AChE or ATCh itself may have a slight influence on the nanozyme activity due to its surface groups, but its effect can be completely ignored in the system. However, when AChE and its substrate ATCh are added simultaneously, the hydrolysis product TCh of ATCh significantly inhibits the catalytic color development process of the nanozyme.

[0041] Example 4: Colorimetric Detection Method for Acetylcholinesterase (AChE) Activity Based on Fe-NC SAzyme / AChE Cascade System (1) In a 1.5 mL centrifuge tube, mix 10 μL of thioacetylcholine (ATCh) with 100 μL of AChE solutions of different concentrations (0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, 100, 200 mU / mL).

[0042] (2) Place the mixture in a 37 ℃ constant temperature water bath for 30 minutes to allow AChE to fully catalyze the hydrolysis of ATCh to generate TCh.

[0043] (3) After the reaction is complete, add 690 μL HAc-NaAc buffer, 100 μL Fe-NC SAzyme suspension and 100 μL TMB solution in sequence, mix well and react at room temperature for 5 minutes.

[0044] (4) Observe the color change of the solution, and use a UV-Vis spectrophotometer to measure the absorbance at 652 nm and plot the working curve. The activity of AChE can be indirectly evaluated by quantifying the amount of TMB oxidation products generated.

[0045] like Figure 4As shown in Figure A, with the increase of AChE, the activity of Fe-NC SAzyme is inhibited by the increased generation of TCh, leading to a corresponding decrease in the absorbance value of the system. Based on this, we established a curve showing the relationship between the absorbance of the cascade catalytic system and the activity of AChE (…). Figure 4 B), its detection linear range is 0.019 ~ 20 mU / mL, and its detection limit is 0.01 mU / mL.

[0046] Example 5: AChEIs Colorimetric Screening Method Based on the above AChE colorimetric activity detection method, an AChEIs colorimetric screening method is established, including the following steps: (1) Add 10 μL of a 10 mM alkaloid compound to 100 μL of a 200 mU / mL AChE solution. Incubate the mixture at 37°C for 5 minutes to allow the compound to fully bind with the enzyme.

[0047] (2) Then add 10 μL ATCh (concentration of 10 mM), then gently mix the solution and react again at 37°C for 30 minutes to allow AChE to act on the substrate.

[0048] (3) After the reaction is complete, add 680 μL of HAc-NaAc buffer, 100 μL of Fe-NC SAzyme suspension and 100 μL of TMB solution in sequence.

[0049] (4) Mix the mixture thoroughly and let it stand at room temperature for 5 minutes to develop color. Measure the absorbance (A) at 652 nm using a UV-Vis spectrophotometer. i ).

[0050] (5) Set up two control experiments: one is an experiment without adding any inhibitors, the detection method is the same as in steps (1)-(4), the same volume of HAc-NaAc buffer solution is used instead of alkaloids in the detection, and the absorbance at 652 nm (A) is recorded; the other is an experiment without adding inhibitors and AChE, the detection method is the same as in steps (1)-(4), the same volume of phosphate buffer solution is used instead of alkaloids and AChE in the detection, and the absorbance at 652 nm (A0) is recorded.

[0051] (6) Calculate the inhibition rate of different alkaloid compounds on AChE activity and determine their half-inhibition concentration (IC50). 50 value).

[0052] The inhibition rate was compared with that of neostigmine bromide, a commercially available acetylcholinesterase inhibitor.

[0053] like Figure 5 As shown in Figure A, the principle of screening AChEIs in this invention is as follows: when AChEIs are present, they can inhibit the activity of AChE, hinder the formation of TCh, and thus cause the solution color to return to blue. To verify the responsiveness of this system to AChEIs, we first evaluated it using the commercially available AChEI neostigmine bromide as a positive control. Figure 5 As shown in Figure B, the addition of neostigmine bromide significantly increased the absorbance of the Fe-NC SAzyme / TMB / AChE / ATCh mixed system at 652 nm. Further analysis of the relationship between its concentration and inhibition rate is shown in Figure B. Figure 5 As shown in C, the IC50 of neostigmine bromide inhibiting AChE was calculated. 50 The value was 4.25 nM, confirming that the method can sensitively characterize the inhibitory effect through colorimetric changes. The method of this invention was applied to screen AChE inhibitors from active ingredients in natural products, and the inhibitory activity of 11 alkaloids (berberine hydrochloride, betaine, caffeine, camptothecin, evodiamine, matrine, neostigmine bromide, sophoridine, sophoridine, theobromine, and theophylline) against AChE was evaluated at a concentration of 100 μM. The results are as follows: Figure 5 As shown in Figure D, among the compounds tested, only berberine hydrochloride exhibited significant and effective inhibitory activity; in contrast, no significant inhibitory effect was observed in the other alkaloids tested. Based on these results, this method can be effectively used to screen compounds with AChE inhibitory activity from natural products.

[0054] Example 6: Rapid detection of acetylcholinesterase activity based on Fe-NC single-atom nanozyme acidic hydrogel To leverage the optimal catalytic activity of Fe-NC SAzyme in acidic environments, an acidic agarose hydrogel was used as a solid support. Fe-NC SAzyme units were pre-loaded and encapsulated within centrifuge tubes to construct a non-fluid dynamics detection platform. The preparation process is as follows: (1) Preparation of acidic hydrogel: Dissolve 50 mg of agarose in 5 mL of HAc-NaAc buffer, heat to 100℃ to form a clear agarose solution, add 5 mg of Fe-NC SAzyme, and mix thoroughly to obtain an integrated hydrogel. Quickly take 20 μL of the hydrogel, place it in the cap of a centrifuge tube, and allow it to cool naturally to solidify. Figure 6 As shown in Figure A.

[0055] (2) Mix 100 μL of AChE solutions of different concentrations (0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, 100, 200 mU / mL) with 10 μL of ATCh (10 mM) and add the mixture into a centrifuge tube. Incubate at 37°C for 30 minutes.

[0056] (3) Add 50 μL TMB (concentration of 1-20 mM) to the centrifuge tube, invert the centrifuge tube to allow the solution to diffuse into the hydrogel, and invert the tube at room temperature for 5 minutes to allow Fe-NC SAzyme to catalyze the oxidation of TMB in the acidic hydrogel matrix.

[0057] (4) After the reaction is complete, straighten the centrifuge tube, open the cap, and observe the color change of the Fe-NC SAzyme hydrogel. Use a smartphone to take a picture of the hydrogel under LED light to obtain its color information. See [link to relevant documentation]. Figure 6 B.

[0058] like Figure 6 As shown in Figure A, the Fe-NC single-atom nanozyme acidic hydrogel after cooling and solidification exhibits excellent physical stability, with no deformation or flow. The color pattern in the hydrogel is consistent with that in the solution; increased AChE activity reduces color intensity, causing the blue color of the hydrogel to fade, as shown in Figure A. Figure 6 As shown in B.

[0059] Example 7: Rapid screening of AChEIs based on Fe-NC SAzyme acidic hydrogel (1) Preparation of acidic hydrogel: Dissolve 50 mg of agarose in 5 mL of HAc-NaAc buffer, heat to 100℃ to form a clear agarose solution, add 5 mg of Fe-NC SAzyme, and mix thoroughly to obtain an integrated hydrogel. Quickly take 20 μL of the hydrogel, place it in the cap of a centrifuge tube, and allow it to cool naturally to solidify. Figure 6 As shown in Figure A.

[0060] (2) Add 100 μL of 200 mU / mL AChE solution and 10 μL of 10 mM 11 alkaloids (berberine hydrochloride, betaine, caffeine, camptothecin, evodiamine, matrine, neostigmine bromide, sophoridine, sophoridine, theobromine, and theophylline) to the centrifuge tubes. Incubate the mixture at 37°C for 5 minutes to allow the compounds to fully bind with the enzyme.

[0061] (3) Then add 10 μL ATCh (concentration of 10 mM), then gently mix the solution and react again at 37°C for 30 minutes to allow AChE to act on the substrate ATCh.

[0062] (4) Add 50 μL of TMB (concentration of 1-20 mM) to the centrifuge tube, cover it with the pre-filled hydrogel cap, and quickly invert the centrifuge tube. This operation brings the reaction mixture inside the tube into contact with the Fe-NC SAzyme hydrogel inside the cap, triggering a cascade colorimetric reaction.

[0063] (5) After incubating upside down for 5 minutes, return the centrifuge tube to its upright position and use gravity to separate the liquid from the gel, thus precisely terminating the color development reaction. At this time, the hydrogel in the tube cap will show a significant color change due to the color development reaction. Place the hydrogel in the 11 tube caps on a platform and use a smartphone to take pictures under LED light to obtain the color information of the hydrogel. Figure 7 (B) As shown in the figure, Fe-NC SAzyme acidic hydrogel was applied to screen 11 alkaloids for AChE inhibitory activity. The inhibitory effect was highly consistent with the solution phase detection results, enabling rapid and accurate screening of 11 alkaloids with acetylcholinesterase inhibitory activity. Except for the positive control (neostigmine bromide), only berberine hydrochloride showed significant inhibitory activity, indicating that this method has good selectivity. This result effectively verifies the feasibility of hydrogel screening for AChEIs.

[0064] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made using the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for rapid screening of acetylcholinesterase inhibitors based on Fe-NC single-atom nanozyme acidic hydrogel, characterized in that, Includes the following steps: (1) Preparation of Fe-NC single-atom nanozyme acidic hydrogel: Dissolve agarose in HAc-NaAc buffer, heat to 80-120℃ to form a transparent and clear agarose solution, add Fe-NC single-atom nanozyme and mix thoroughly, take the mixed solution and place it in the cap of a centrifuge tube to cool naturally into a gel, and obtain Fe-NC single-atom nanozyme acidic hydrogel. (2) Preparation of screening reaction system: Add acetylcholinesterase AChE solution and the substance to be screened into the centrifuge tube, incubate at 35-40℃ to allow the substance to be screened to fully combine with acetylcholinesterase AChE, then add acetylthiocholine ATCh, and react at 35-40℃ for 25-35 minutes. (3) Color reaction: Add 3,3',5,5'-tetramethylbenzidine (TMB) to the centrifuge tube, cover the centrifuge tube with the pre-filled hydrogel, invert the centrifuge tube to allow the screening reaction system to diffuse into the hydrogel, invert the centrifuge tube at room temperature for 4-6 minutes, then turn the centrifuge tube back to the right side and obtain the color information of the hydrogel; the hydrogel is blue, indicating that the substance to be screened has acetylcholinesterase inhibitory activity.

2. The method according to claim 1, characterized in that: In step (1), the pH of the HAc-NaAc buffer is 3.0-5.0; the mass ratio of agarose to Fe-NC single-atom nanozyme is 8:1-12:

1.

3. The method according to claim 1, characterized in that: In step (1), the preparation method of Fe-NC single-atom nanozyme includes the following steps: (1) Add Zn(NO3)2·6H2O, Fe(NO3)3·9H2O and 2-methylimidazole to N,N-dimethylformamide and stir until completely dissolved to form a homogeneous mixed solution. React the mixed solution at 135-145 °C for 20-25 hours. After the reaction is completed, cool naturally to room temperature, wash and dry to obtain Fe / Zn-ZIF nanomaterials; the molar ratio of Fe(NO3)3·9H2O, Zn(NO3)2·6H2O and 2-methylimidazole is 1:(2-4):(3-5). (2) Fe / Zn-ZIF was heated to 1000-1200℃ at a heating rate of 10℃ / min under N2 protection and calcined at a constant temperature for 1-2 hours. After calcination, it was naturally cooled to room temperature, and then acid-washed, filtered, washed and dried to obtain Fe-NC single-atom nanozyme Fe-NC SAzyme.

4. The method according to claim 1, characterized in that: In step (2), the concentration of acetylcholinesterase AChE in the reaction system is 1-50 mU / mL, the concentration of the substance to be screened in the reaction system is 0.01-1 mM, and the concentration of acetylthiocholine in the reaction system is 0.01-1 mM. In step (3), the concentration of 3,3',5,5'-tetramethylbenzidine TMB in the reaction system is 0.1-10 mM.

5. The method according to claim 1, characterized in that: In step (3), the hydrogel color information is obtained by taking a picture of the hydrogel color under the LED light source with a smartphone.

6. The method according to claim 1, characterized in that: In step (3), the blue color depth is positively correlated with the inhibitory activity.

7. The method according to claim 1, characterized in that: The substances to be screened are alkaloid compounds.

Images