Hydrogen-bonding-based organic framework nanoscale enzyme and preparation method and application thereof

Abstract

The application discloses a kind of based on hydrogen bond organic framework nano-enzyme and its preparation method and application.Preparation, take PyTTA completely dissolved in DMF, after dissolving the solution is added in stirring water;Under stirring, anhydrous ethanol is added to the solution, after reaction, centrifugal washing, finally dispersed in water, and HOFs solution is prepared;Take ABEI and HAuCl 4• 4H2O after mixing, stirring makes it mix uniformly, then Co (CH3COO) 2 and HOFs solution are added, stirring and washing, and HAACo is prepared.HAACo has multiple enzyme activities, uses the strong and long-life CL phenomenon caused by its confinement effect, and combines the CL real-time detection method constructed by 3D printing device, has the advantages of nearly zero background, portability, high sensitivity and the like, provides a new idea for the application of HOFs in biosensing, simultaneously provides a new method for constructing multi-enzyme activity material, and shows great application potential in the field of in vitro diagnosis, drug screening and the like.

Description

Technical Field

[0001] This invention relates to hydrogen-bonded organic frameworks (HOFs), specifically to the preparation of multi-enzyme-active hydrogen-bonded organic framework nanozymes, and discloses their preparation methods and applications. Background Technology

[0002] Hydrogen-bonded organic frameworks (HOFs) are a new class of porous crystalline materials, typically formed by the self-assembly of small-molecule organic structural units linked by hydrogen bonds. They exhibit well-structured architectures, and the introduction of π-π interactions and electrostatic attraction overcomes the weaknesses and poor directionality of hydrogen bonds, improving the stability of HOFs and enabling the creation of permanent voids. These unique advantages make HOFs a highly versatile platform for exploring the multifunctionality of porous materials.

[0003] Acetylcholinesterase (AChE) is one of the serine hydrolases mainly found in the central nervous system. It rapidly converts the natural substrate acetylcholine (ATCh) into acetate and choline, participating in the regulation of ATCh levels and the termination of neurotransmission. It plays a crucial role in biological signal transduction. Increasing evidence suggests that abnormal AChE expression is closely related to adverse reactions to neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and the development of drug-resistant strains.

[0004] AChE inhibitors can affect AChE activity and are currently considered effective drugs for treating Alzheimer's disease. For early treatment of Alzheimer's disease, scientists have developed many methods for AChE detection and screening of potential inhibitors, such as colorimetry, fluorescence, electrochemistry, and chemiluminescence (CL). Among these, CL has attracted much attention due to its advantages of no background and high signal-to-noise ratio; however, the need for expensive specialized equipment limits its widespread application in AChE detection and inhibitor screening. Furthermore, commonly used CL reaction systems produce flash-type luminescence, lasting only a few minutes or even seconds. Strong and persistent CL, i.e., glow-type CL, exhibits a slow decay curve, or even a constant decay, during analysis, which is crucial for improving the detection accuracy and reproducibility of CL detection methods. Therefore, it is necessary to establish a label-free, non-toxic, economical, and simple method for AChE activity detection and inhibitor screening.

[0005] Among the published patents related to the detection of acetylcholinesterase and its inhibitors, CN114184561A describes the preparation and application of a cerium oxide-cobalt hydroxide composite material. This invention discloses a method for preparing the cerium oxide-cobalt hydroxide composite material, which involves sequentially adding cobalt nitrate hexahydrate and sodium hydroxide to a eutectic solvent, reacting at room temperature to 70°C for 0.5 to 3.0 hours, followed by centrifugation, washing, and drying. This cerium oxide-cobalt hydroxide composite material can be used for the detection of acetylcholinesterase activity and the screening of inhibitors. The cerium oxide-cobalt hydroxide composite material enables quantitative analysis and visual detection of acetylcholinesterase and has been successfully applied to the screening of acetylcholinesterase inhibitors from natural products. This result has important guiding significance for the development of drugs for the treatment of neurodegenerative diseases such as Alzheimer's disease. However, the material synthesis using this method is relatively cumbersome, and the established visual detection method has a detection range of 0-18 mU / mL.

[0006] CN110408069A describes a molecularly imprinted polymer-coated carbon dot fluorescent probe, its preparation method, and its application. c-dots are prepared via a hydrothermal method, with silica grafted onto their surface, and then sealed in situ within the silica pores using a molecularly imprinted polymer (mip@c-dots). This fluorescent probe has been successfully applied to AChE activity detection, and is of significant importance in the study of Alzheimer's disease pathogenesis, disease diagnosis, and new drug development. However, the material synthesis process is relatively complex and requires specialized equipment and skilled personnel.

[0007] CN108120761A discloses an electrochemical biosensor based on an electrocatalytically active peptide mimic for the detection of acetylcholinesterase. This invention utilizes the active molecule hydrogen peroxide (H2O2) produced by the decomposition of acetylcholine under the action of acetylcholinesterase (AChE) and choline oxidase (ChOx), and then employs the peptide mimic Cys-Ag(I)CP to catalyze the H2O2 system for electrochemical signal detection. This sensor can be used to detect acetylcholinesterase activity and screen for its small-molecule organophosphate inhibitors, exhibiting high specificity, high sensitivity, and accurate and reliable results. However, this method is complex to operate and requires specialized equipment. Summary of the Invention

[0008] This invention addresses the shortcomings of existing technologies by proposing a hydrogen-bonded organic framework nanoenzyme, HAACo, its preparation method, and its applications. In the HAACo prepared by this invention, H represents a hydrogen-bonded organic framework (HOFs) formed by the self-assembly of 1,3,6,8-tetra-(p-aminophenyl)-pyrene (PyTTA), abbreviated as HOF-PyTTA; the first "A" represents gold nanoparticles (Au NPs); the second "A" represents N-(4-aminobutyl)-N-ethylisoluminol (ABEI); and "Co" represents Co. 2+ This material exhibits excellent peroxidase-like, oxidase-like, and catalase-like activities, enabling the luminol-H2O2 chemiluminescence system to demonstrate strong and stable chemiluminescence performance (CL duration up to 1 hour). The further developed chemiluminescence point-of-care testing (C-POCT) method overcomes the limitations of traditional chemiluminescence sensors, which require precision instruments, professional operators, and stringent testing conditions, and has great potential for clinical diagnosis in remote areas.

[0009] The technical solution to achieve the objective of this invention is:

[0010] A method for preparing hydrogen-bonded organic framework nanozymes includes the following steps:

[0011] Step 1: Dissolve 1,3,6,8-tetra-(p-aminophenyl)-pyrene (PyTTA) completely in N,N-dimethylformamide (DMF), and slowly add the dissolved solution dropwise to water under stirring while stirring to react;

[0012] Step 2: While stirring, slowly add anhydrous ethanol to the solution from Step 1, continue stirring the reaction, and after the reaction is complete, wash with anhydrous ethanol and water by centrifugation, and finally disperse in water to obtain HOFs solution.

[0013] Step 3: Mix ABEI and HAuCl4·4H2O, stir to make them evenly mixed, then add Co(CH3COO)2 and the HOFs solution prepared in step 2, continue stirring, and wash the product with water by centrifugation. After washing, disperse it in water to prepare the hydrogen-bonded organic framework nanozyme HAACo.

[0014] Furthermore, in step 1, the ratio of PyTTA to DMF is 1-100 mg: 1-1000 mL;

[0015] The stirring reaction time is 5-20 minutes;

[0016] In step 2, the amount of anhydrous ethanol added is 20-200 mL;

[0017] The stirring reaction time is 5-20 minutes;

[0018] In step 3, the ratio of ABEI to HAuCl4·4H2O is 1 mL: 1-10 mL;

[0019] The ratio of Co(CH3COO)2 to HOFs solution is 1 mL: 1-10 mL.

[0020] The concentration of ABEI was 0.00001-0.04 mol / L;

[0021] The concentration of HAuCl4·4H2O is 0.00001-0.05 mol / L;

[0022] The concentration of the HOFs solution was 0.0001-0.25 g / mL;

[0023] The concentration of Co(CH3COO)2 is 0.00001-1 mol / L;

[0024] After mixing ABEI and HAuCl4·4H2O, stir for 0.5-6 hours;

[0025] Add Co(CH3COO)2 and HOFs solution, and continue stirring for 0.5-16 hours;

[0026] In steps 1, 2 and 3, the stirring speed is 20-2000 rpm / min.

[0027] During preparation, each raw material can be added according to the multiple of the raw material ratio in the preparation method.

[0028] The hydrogen-bonded organic framework nanozyme HAACo prepared using the above method exhibits a micron-sized strip-like morphology, indicating that the growth of Au NPs did not affect the morphology of the HOFs. Energy-dispersive X-ray spectroscopy (EDXPS) revealed that HAACo mainly contains C, N, Co, and Au elements. The distribution of Au elements is consistent with that of Au NPs in the TEM image, while Co elements are uniformly distributed on the material surface. This demonstrates the successful growth of Au NPs and the doping of Co on the HOFs. 2+ The success.

[0029] The present invention further provides the application of the prepared hydrogen-bonded organic framework nanozyme HAACo in the in vitro detection of acetylcholinesterase in human serum samples.

[0030] Specifically, the hydrogen-bonded organic framework nanozyme HAACo was used in combination with chemiluminescence to detect acetylcholinesterase in human serum samples in vitro. The detection method included the following steps:

[0031] Step A: Mix AChE, ATCh and Tris-HCl with different activities in a series of centrifuge tubes and incubate at 30-40℃ for 20-45 min;

[0032] Step B: Add HAACo to the 24-well plate, then add the AChE and ATCh reaction mixture;

[0033] Step C: Place the 24-well plate in the 3D printing device, turn on the mobile phone recording function, add luminol and H2O2 to the well plate, and record the chemiluminescence intensity changes within 10 minutes;

[0034] Step D: After converting the video to R, G, B values ​​using a color signal converter, use the difference between the G+B values ​​of the experimental group and the control group and the concentration of AChE to perform a linear regression curve.

[0035] The AChE concentration in the control group was 0 U / mL;

[0036] The specific steps for determining AChE in human serum are as follows: human serum and N-ethylmaleimide (NEM) are vortex-mixed and then diluted. The AChE solution in step B is replaced with the treated serum, and other operations remain unchanged.

[0037] Furthermore, in step A of the detection method, the volume of AChE is 1-1000 μL;

[0038] The volume of ATCh is 1-1000 μL, and the concentration is 0.00001-0.08 mol / L;

[0039] The volume of Tris-HCl is 1-3000 μL, and the concentration is 0.00001-10 mol / L;

[0040] In step B, the volume of HAACo is 1-3000 μL and the concentration is 0.001-10 mg / mL;

[0041] The volume of the AChE and ATCh reaction mixture is 1-3000 μL;

[0042] In step C, the volume of luminol is 0.001-5 mL, and the concentration is 0.0001-1 mol / L;

[0043] The volume of H2O2 is 0.001-5 mL.

[0044] The present invention further provides the application of the prepared hydrogen-bonded organic framework nanozyme HAACo in the screening of acetylcholinesterase inhibitor tacrine.

[0045] Specifically, the hydrogen-bonded organic framework nanozyme HAACo was used to screen for the acetylcholinesterase inhibitor tacrine. The screening method included the following steps:

[0046] Step a: Mix different concentrations of tacrine with AChE, then add ATCh and Tris-HCl, and incubate at 30-40℃ for 20-40 min;

[0047] Step b: Add HAACo to a 24-well plate, then add the AChE and ATCh reaction mixture;

[0048] Step c: Place the 24-well plate in the 3D printing device, turn on the mobile phone recording function, add luminol and H2O2 to the well plate, and record the chemiluminescence intensity changes within 10 minutes;

[0049] The inhibition rate initially increased rapidly with increasing tacrine concentration from 0 to 0.78 μmol / L, then changed slowly, and can be expressed by the formula IE% = (GB... i -GB b ) / (GB0-GB b The result is calculated by multiplying GB by 100%. i GB represents the G+B values ​​for different concentrations of inhibitor. b For G+B values ​​without AChE, according to IE-C Tacrine The IC50 value of Taklin was calculated from the graph.

[0050] Furthermore, in step a of the screening method, the volume of tacrine is 1-1000 μL;

[0051] The volume of AChE is 1-1000 μL, and the concentration is 0.0001-2 U / mL;

[0052] The volume of ATCh is 1-1000 μL, and the concentration is 0.0001-2 mol / L;

[0053] The volume of Tris-HCl is 1-5000 μL, and the concentration is 0.00001-10 mol / L;

[0054] In step b, the volume of HAACo is 1-3000 μL and the concentration is 0.001-10 mg / mL;

[0055] The volume of the AChE and ATCh reaction mixture is 1-3000 μL;

[0056] In step c, the volume of luminol is 0.001-5 mL and the concentration is 0.0001-1 mol / L;

[0057] The volume of H2O2 is 0.001-5 mL.

[0058] The beneficial effects of this invention are as follows:

[0059] The HAACo prepared by this invention has multiple enzyme activities. The strong and long-lived CL phenomenon induced by its confinement effect, combined with the CL instant detection method constructed by 3D printing device, has the advantages of near-zero background, portability, and high sensitivity. It provides a new idea for the application of HOFs in biosensing and a new method for constructing multi-enzyme active materials, showing great application potential in in vitro diagnostics, drug screening and other fields. Attached Figure Description

[0060] Figure 1 This is a schematic diagram of the process for preparing HAACo in Example 1;

[0061] Figure 2 Transmission electron microscopy (TEM) image and energy-dispersive X-ray spectroscopy (EDS-mapping) of HAACo prepared in Example 1;

[0062] in, Figure 2 AB is the TEM image of HAACo. Figure 2 CG is the EDS-mapping plot of HAACo;

[0063] Figure 3 This is a schematic diagram of the in vitro detection of AChE using HAACo combined with CL in Example 2;

[0064] Figure 4 The graphs shown in Example 2 are: AChE concentration versus chemiluminescence intensity (4A), AChE concentration versus Δ(G+B) value (4B), and standard working curve (4C) of Δ(G+B) value versus AChE concentration.

[0065] Figure 5 This is a schematic diagram of the application of HAACo to screen the AChE inhibitor tacrine in Example 3;

[0066] Figure 6 The graphs shown in Example 3 are: 6A (chemiluminescence intensity) versus tacrine concentration and 6B (inhibition efficiency) versus tacrine concentration. Detailed Implementation

[0067] The present invention will be further described below with reference to the embodiments and accompanying drawings, but this is not intended to limit the scope of the invention.

[0068] Example 1

[0069] Reference Figure 1 A method for preparing hydrogen-bonded organic framework nanozymes includes the following steps:

[0070] Step 1: Dissolve 100 mg of PyTTA completely in 15 mL of DMF, and slowly add the dissolved solution dropwise to the water under stirring. Stir for 10 min.

[0071] Step 2: While stirring at 1500 rpm / min, slowly add 100 mL of anhydrous ethanol to the solution from Step 1, stir for 10 min, and after the reaction is complete, wash the solution 6 times with anhydrous ethanol and water respectively by centrifugation, and finally disperse it in 40 mL of water to obtain the HOFs solution.

[0072] Step 3: Take 1 mL of 0.01 mol / L ABEI and 3 mL of 0.02 mol / L HAuCl4·4H2O.

[0073] After mixing, the mixture was stirred at 1500 rpm / min for 2 hours to ensure uniform mixing. Then, 4 mL of 0.01 g / mL HOFs solution and 1 mL of 0.04 mol / L Co(CH3COO)2 were added, and stirring was continued for 12 hours. After stirring, the product was washed 6 times by centrifugation with water and dispersed in 5 mL of water to obtain HAACo.

[0074] Reference Figure 2 Transmission electron microscopy image of HAACo, as shown Figure 2 As shown in Figure AB, HAACo exhibits a micron-sized strip-like morphology, indicating that the growth of Au NPs did not affect the morphology of HOFs. From the energy-dispersive X-ray spectroscopy (EDX) spectrum... Figure 2 The CG image shows that HAACo mainly contains C, N, Co, and Au elements. The distribution of Au is consistent with the distribution of Au NPs in the TEM image, while Co is uniformly distributed on the material surface. This proves that Au NPs can be grown on HOF and Co is doped. 2+ The success.

[0075] Example 2

[0076] Reference Figure 3 The prepared hydrogen-bonded organic framework nanozyme HAACo was used to detect AChE in human serum samples in vitro. The detection method included the following steps:

[0077] Step A: Mix 10 μL of AChE with different activities, 10 μL of ATCh (0.01 mol / L) and 30 μL of Tris-HCl (0.01 mol / L) in a series of centrifuge tubes and incubate at 37 °C for 40 min;

[0078] Step B: Add 25 μL of HAACo (0.04 mg / mL) to a 24-well plate, followed by 25 μL of the AChE and ATCh reaction mixture;

[0079] Step C: Place the 24-well plate in the 3D printing device, turn on the mobile phone recording function, add 0.5 mL of luminol (0.1 mol / L) and 0.45 mL of H2O2 to the well plate, and record the changes in chemiluminescence intensity within 10 min;

[0080] Step D: After converting the video to R, G, B values ​​using a color signal converter, use the difference between the experimental group (G+B) and the control group (AChE concentration of 0 U / mL) to plot a linear regression curve with the concentration of AChE.

[0081] When measuring AChE in human serum: human serum and N-ethylmaleimide (NEM) are vortexed for 5 min and then diluted to 50 mL. The AChE solution in step B is replaced with the treated serum, and other operations remain unchanged.

[0082] The CL method based on HAACo peroxidase mimicry activity was used to detect AChE activity, such as... Figure 4 As shown, the CL phenomenon in the detection system weakens with increasing AChE concentration, as... Figure 4 As shown in Figure A, its Δ(G+B) value increases with increasing AChE concentration, eventually reaching a plateau with a response range of 0.001-100 mU / mL. Figure 4 As shown in B. Meanwhile, the Δ(G+B) value exhibits good linearity within the concentration range of 0.001-40 mU / mL, with the regression equation being Δ(G+B) = 407.21logC. [AChE] +1893.25(R 2 =0.9976, n=3), the calculated detection limit is 0.00057 mU / mL, as follows. Figure 4 As shown in C. Compared with other previously reported AChE activity assays, it has a relatively wide linear range and a lower detection line.

[0083] Example 3

[0084] Reference Figure 5 The hydrogen-bonded organic framework nanozyme HAACo was used to screen for the acetylcholinesterase inhibitor tacrine. The screening method included the following steps:

[0085] Step a: Mix 10 μL of different concentrations of tacrine with 10 μL of AChE (0.02 U / mL), then add 10 μL of ATCh (0.01 mol / L) and 50 μL of Tris-HCl (0.01 mol / L), and incubate at 37 °C for 30 min;

[0086] Step b: Add 25 μL of HAACo (0.04 mg / mL) to a 24-well plate, and add 25 μL of the AChE and ATCh reaction mixture;

[0087] Step c: Place the 24-well plate in the 3D printing device, turn on the mobile phone recording function, add 0.5 mL of luminol (0.1 mol / L) and 0.45 mL of H2O2 to the well plate, and record the chemiluminescence intensity changes within 10 min.

[0088] The application of the present invention HAACo in the screening of AChE inhibitors was verified by using tacrine as an experimental model. Figure 6 As shown, the CL intensity gradually increases with increasing tacrine concentration, as... Figure 6 As shown in Figure A.

[0089] The inhibition rate can be expressed by the formula IE% = (GB) i -GB b ) / (GB0-GB b The result is calculated by multiplying GB by 100%. i GB represents the (G+B) values ​​for different concentrations of inhibitor. b This is the (G+B) value without AChE. For example... Figure 6 As shown in B, IE% initially increases rapidly and then changes slowly with increasing tacrine concentration from 0 to 0.78 μmol / L, according to IE-C. Tacrine The IC50 value of tacrine was calculated to be 21.9 nmol / L.

Claims

1. A method for preparing hydrogen-bonded organic framework nanozymes, characterized in that, Includes the following steps: Step 1: Dissolve PyTTA completely in DMF, and slowly add the dissolved solution dropwise to water while stirring to react; Step 2: While stirring, slowly add anhydrous ethanol to the solution from Step 1, continue stirring the reaction, and after the reaction is complete, wash with anhydrous ethanol and water by centrifugation, and finally disperse in water to obtain HOFs solution. Step 3: Mix ABEI and HAuCl4·4H2O, stir until homogeneous, then add Co(CH3COO)2 and the HOFs solution prepared in Step 2, continue stirring, and wash the product by centrifugation with water. After washing, disperse in water to prepare the hydrogen-bonded organic framework nanozyme HAACo. Here, H represents the hydrogen-bonded organic framework HOFs formed by the self-assembly of 1,3,6,8-tetra-(p-aminophenyl)-pyrene PyTTA, abbreviated as HOF-PyTTA; the first "A" represents gold nanoparticles Au NPs; the second "A" represents N-(4-aminobutyl)-N-ethylisoluminol ABEI; and "Co" represents Co. 2+ .

2. The method for preparing hydrogen-bonded organic framework nanozymes according to claim 1, characterized in that: In step 1, the ratio of PyTTA to DMF is 1-100 mg: 1-1000 mL; The stirring reaction time is 5-20 minutes; In step 2, the amount of anhydrous ethanol added is 20-200 mL; The stirring reaction time is 5-20 minutes; In step 3, the ratio of ABEI to HAuCl4·4H2O is 1 mL: 1-10 mL; The ratio of Co(CH3COO)2 to HOFs solution is 1 mL: 1-10 mL. The concentration of ABEI was 0.01-0.04 mol / L; The concentration of HAuCl4·4H2O is 0.02-0.05 mol / L; The concentration of the HOF solution was 0.01-0.25 g / mL; The concentration of Co(CH3COO)2 is 0.04-1 mol / L; After mixing ABEI and HAuCl4·4H2O, stir for 0.5-6 hours; Add Co(CH3COO)2 and HOFs solution, and continue stirring for 0.5-16 hours; In steps 1, 2 and 3, the stirring speed is 20-2000 rpm / min.

3. The hydrogen-bonded organic framework nanozyme prepared by the preparation method according to any one of claims 1-2, characterized in that: The prepared hydrogen-bonded organic framework nanozyme HAACo exhibits a micron-sized strip-like morphology, indicating that the growth of Au NPs did not affect the morphology of the HOFs. Energy-dispersive X-ray spectroscopy (EDXPS) revealed that HAACo mainly contains C, N, Co, and Au elements. The distribution of Au is consistent with that of Au NPs in the TEM image, while Co is uniformly distributed on the material surface. This demonstrates the successful growth of Au NPs and Co doping on the HOFs. 2+ The success.

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