CN114184561B - Preparation and application of cerium oxide-cobalt hydroxide composite material - Google Patents
Preparation and application of cerium oxide-cobalt hydroxide composite material Download PDFInfo
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- CN114184561B CN114184561B CN202111349848.1A CN202111349848A CN114184561B CN 114184561 B CN114184561 B CN 114184561B CN 202111349848 A CN202111349848 A CN 202111349848A CN 114184561 B CN114184561 B CN 114184561B
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- acetylcholinesterase
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- cerium oxide
- cobalt hydroxide
- hydroxide composite
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- 239000002131 composite material Substances 0.000 title claims abstract description 56
- -1 cerium oxide-cobalt hydroxide Chemical compound 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 108010022752 Acetylcholinesterase Proteins 0.000 claims abstract description 103
- 229940022698 acetylcholinesterase Drugs 0.000 claims abstract description 103
- 238000001514 detection method Methods 0.000 claims abstract description 29
- 230000000694 effects Effects 0.000 claims abstract description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 15
- 238000012216 screening Methods 0.000 claims abstract description 13
- 239000002904 solvent Substances 0.000 claims abstract description 13
- 230000005496 eutectics Effects 0.000 claims abstract description 12
- 239000003112 inhibitor Substances 0.000 claims abstract description 11
- 238000005406 washing Methods 0.000 claims abstract description 11
- 230000000007 visual effect Effects 0.000 claims abstract description 10
- 229930014626 natural product Natural products 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- QGUAJWGNOXCYJF-UHFFFAOYSA-N cobalt dinitrate hexahydrate Chemical compound O.O.O.O.O.O.[Co+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O QGUAJWGNOXCYJF-UHFFFAOYSA-N 0.000 claims abstract description 5
- 102000012440 Acetylcholinesterase Human genes 0.000 claims abstract 20
- 238000002835 absorbance Methods 0.000 claims description 31
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- 238000012360 testing method Methods 0.000 claims description 18
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- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
- 239000008351 acetate buffer Substances 0.000 claims description 10
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- 239000001257 hydrogen Substances 0.000 claims description 9
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 8
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- QQZMWMKOWKGPQY-UHFFFAOYSA-N cerium(3+);trinitrate;hexahydrate Chemical compound O.O.O.O.O.O.[Ce+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O QQZMWMKOWKGPQY-UHFFFAOYSA-N 0.000 claims description 8
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- ZSBXGIUJOOQZMP-JLNYLFASSA-N Matrine Chemical compound C1CC[C@H]2CN3C(=O)CCC[C@@H]3[C@@H]3[C@H]2N1CCC3 ZSBXGIUJOOQZMP-JLNYLFASSA-N 0.000 description 4
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- 235000011164 potassium chloride Nutrition 0.000 description 3
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- 108010053652 Butyrylcholinesterase Proteins 0.000 description 2
- 229910052684 Cerium Inorganic materials 0.000 description 2
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- ADEBPBSSDYVVLD-UHFFFAOYSA-N donepezil Chemical compound O=C1C=2C=C(OC)C(OC)=CC=2CC1CC(CC1)CCN1CC1=CC=CC=C1 ADEBPBSSDYVVLD-UHFFFAOYSA-N 0.000 description 2
- ASUTZQLVASHGKV-JDFRZJQESA-N galanthamine Chemical compound O1C(=C23)C(OC)=CC=C2CN(C)CC[C@]23[C@@H]1C[C@@H](O)C=C2 ASUTZQLVASHGKV-JDFRZJQESA-N 0.000 description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
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- 229960003980 galantamine Drugs 0.000 description 1
- ASUTZQLVASHGKV-UHFFFAOYSA-N galanthamine hydrochloride Natural products O1C(=C23)C(OC)=CC=C2CN(C)CCC23C1CC(O)C=C2 ASUTZQLVASHGKV-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
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- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
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- 229960004136 rivastigmine Drugs 0.000 description 1
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- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229960001685 tacrine Drugs 0.000 description 1
- YLJREFDVOIBQDA-UHFFFAOYSA-N tacrine Chemical compound C1=CC=C2C(N)=C(CCCC3)C3=NC2=C1 YLJREFDVOIBQDA-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Nanotechnology (AREA)
- General Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Molecular Biology (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Manufacturing & Machinery (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of a cerium oxide-cobalt hydroxide composite material, which comprises the steps of sequentially adding cobalt nitrate hexahydrate and sodium hydroxide into a eutectic solvent, reacting for 0.5-3.0 h at the temperature of between room temperature and 70 ℃, centrifuging, washing and drying. The cerium oxide-cobalt hydroxide composite material can be used for acetylcholinesterase activity detection and inhibitor screening. The cerium oxide-cobalt hydroxide composite material can realize quantitative analysis and visual detection of acetylcholinesterase, can be successfully applied to screening of acetylcholinesterase inhibitors in natural products, and has important guiding significance for developing medicines for treating neurodegenerative diseases such as Alzheimer disease. The visual detection method established by the invention can rapidly, sensitively and highly selectively realize the detection of the activity of acetylcholinesterase and the screening of natural product inhibitors. In addition, the preparation process is simple, no modification or marking is needed, the cost is low, and the applicability is strong.
Description
Technical Field
The invention relates to preparation and application of a nano material, in particular to preparation of a cerium oxide-cobalt hydroxide composite material, and simultaneously relates to application of the cerium oxide-cobalt hydroxide composite material in acetylcholinesterase detection and inhibitor screening, belonging to the field of nano materials.
Background
The eutectic solvent is a eutectic mixture formed by hydrogen bond donor and hydrogen bond acceptor with certain stoichiometric ratio through hydrogen bond action, has the characteristics of simple preparation, low vapor pressure, good solubility and conductivity, reusability, strong design, biodegradability and the like, and is often used as a novel green solvent. Since being reported for the first time in 2003, it has become popular with researchers. It is worth mentioning that eutectic solvents can regulate the nucleation and growth rate of the material synthesis process, so they are of great interest in the field of material synthesis. According to research, no report is provided on green synthesis of nano materials, especially synthesis of cerium oxide-cobalt hydroxide composite nano materials, by using a eutectic solvent composed of L-proline and cerium nitrate hexahydrate.
Alzheimer's Disease (AD) is a chronic neurodegenerative disease, one of the "ten killers" worldwide. Clinically, global dementia characterized by memory impairment, aphasia, disuse, disrecognition, impairment of visual space skills, executive dysfunction, personality and behavioral changes, etc., has heretofore been unknown in etiology. Studies have shown that the key symptom of the disease is a decrease in the synthesis of the neurochemical transmitter acetylcholine. However, acetylcholinesterase is a key hydrolase of acetylcholine, and abnormal fluctuations in acetylcholinesterase can directly affect the metabolism of acetylcholine, thereby disrupting nerve communication in the brain. Therefore, acetylcholinesterase is currently regarded as an important target for screening anti-Alzheimer's disease drugs, and it is important to see rapid, high-sensitivity and high-selectivity detection of acetylcholinesterase. At the same time, inhibition of excessive acetylcholinesterase activity by the drug would be helpful in treating neurodegenerative disorders represented by Alzheimer's disease. At present, most of the treatment drugs for patients with Alzheimer disease on the market are acetylcholinesterase inhibitors, such as Anapplication, donepezil, galantamine, tacrine, rismin, rivastigmine and the like. Clinically, the enzyme inhibitors can alleviate or relieve symptoms of diseases to a certain extent, but the drugs have the defects of low drug activity, expensive price of imported drugs, obvious side effects, easy occurrence of drug resistance of patients and the like, so that the treatment needs cannot be met. The natural product (such as plants, traditional Chinese medicines and the like) has the characteristics of rich resources, safety, effectiveness, environmental friendliness, small toxic and side effects and the like, and is one of the important sources of natural enzyme inhibitors. Thus, screening for enzyme inhibitors from natural products has become an important strategy for developing new drugs.
In view of the foregoing, it is a need for a method for rapidly, highly sensitively and selectively detecting acetylcholinesterase and natural inhibitor screening by green synthesis of a cerium oxide-cobalt hydroxide composite material in a eutectic solvent.
Disclosure of Invention
The invention aims to provide a preparation method of a cerium oxide-cobalt hydroxide composite material;
the invention also aims to provide the application of the cerium oxide-cobalt hydroxide composite material in acetylcholinesterase activity detection and inhibitor screening.
1. Preparation of cerium oxide-cobalt hydroxide composite material
The invention relates to a preparation method of a cerium oxide-cobalt hydroxide composite material, which comprises the steps of sequentially adding cobalt nitrate hexahydrate and sodium hydroxide into a eutectic solvent, reacting for 0.5-3.0 h at the temperature of between room temperature and 70 ℃, centrifuging, washing and drying to obtain cerium oxide-cobalt hydroxide (CeO) 2 -Co(OH) 2 ) A composite material; the eutectic solvent is prepared by taking L-proline as a hydrogen bond donor, taking cerium nitrate hexahydrate as a hydrogen bond acceptor and heating at 60-80 ℃ until the mixture is clear and transparent. Wherein the mol ratio of the cobalt nitrate hexahydrate to the sodium hydroxide is 1:1-1:4; the molar ratio of the L-proline to the cerium nitrate hexahydrate is 1:4-4:1.
2. CeO (CeO) 2 -Co(OH) 2 Structure of composite material
CeO was measured by X-ray diffraction (XRD), scanning Electron Microscope (SEM), transmission Electron Microscope (TEM), energy spectrum (EDX), dark field STEM 2 -Co(OH) 2 The structure, morphology and the like of the composite material are characterized.
FIG. 1 shows CeO obtained in example 1 of the present invention 2 -Co(OH) 2 Composite materialXRD pattern of the material. As can be seen from the figure, the material has CeO present at 2θ=28.7, 33.2, 47.6, 56.6, 59.6 and 69.6 ° 2 (PDF card is 4-593) crystal planes (111), (200), (220), (311), (222) and (400). Meanwhile, co (OH) is present at 2θ=19.0, 32.6, 37.9, 51.5, 57.9 and 61.6° 2 (PDF card is 3-913) crystal planes (001), (100), (101), (102), (110) and (111). The material was confirmed to be CeO 2 -Co(OH) 2 A composite material.
FIG. 2 shows CeO prepared according to the present invention 2 -Co(OH) 2 SEM (a) and TEM images (B) of the composite material. It can be seen that the material is a laminar structure.
FIG. 3 shows CeO obtained in the present invention 2 -Co(OH) 2 EDX plot of composite material. From the energy spectrum, the material consists of three elements of Ce, O and Co.
FIG. 4 shows CeO obtained in the present invention 2 -Co(OH) 2 Dark field STEM diagram (a) of the composite material and element map (B-G) of the corresponding elements in the material. Wherein B: O-K; c: co-K; d: co-L; e: ce-K, F, ce-L and G, ce-M. It can be seen from graphs B-G that the material mainly comprises three elements of Ce, co and O, and the result is completely consistent with that of FIG. 3.
3. CeO (CeO) 2 -Co(OH) 2 Composite material for acetylcholinesterase detection
And (3) incubating the thiocholine compound (thiobutylcholine, thioacetylcholine or thiopropionyl choline) and acetylcholinesterase with different concentrations for 30-60 min at 37 ℃. Then adding a certain volume of acetate buffer solution, 3', 5' -tetramethyl benzidine (TMB) and the prepared CeO 2 -Co(OH) 2 After the composite material is uniformly mixed by vortex, incubating for 5-40 min at room temperature, and recording the absorbance value of the test solution at 652 and nm as A 2 . The control group was replaced with the same volume of phosphate buffer solution without adding acetylcholinesterase, and the absorbance value of the solution at 652 nm was measured under the same conditions and recorded as A 1 . According to the change in absorbance value of the solution at 652 nm (y=a 1 -A 2 ) And (3) withThe linear relation between the concentration of the acetylcholinesterase can be used for quantitatively detecting the acetylcholinesterase.
The concentration of acetate buffer solution (composed of acetic acid and sodium acetate) is 0.1M, and the pH value range is 3.5-5.0; the concentration of the phosphate buffer solution (composed of sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate) is 100 mM, and the pH value range is 7.0-8.0.
FIG. 5 is a graph showing the visible absorption spectrum of the system after the system has been added with acetylcholinesterase at various concentrations. As can be seen from fig. 5, the absorbance value of the system at 652 nm gradually decreased with increasing concentration of acetylcholinesterase (the concentration of acetylcholinesterase was 0.2, 0.5, 1, 2, 5, 6, 8, 10, 12, 14, 16, 18, 20 mU/mL in this order from top to bottom).
FIG. 6 is a graph showing the linear relationship between the change in absorbance value of the system after addition of different concentrations of acetylcholinesterase and the concentration of acetylcholinesterase. As can be seen from fig. 6, there is a good linear relationship between the absorbance intensity variation value of the system at 652 nm and the concentration of acetylcholinesterase (the concentration interval is 0.2-20 μg/mL), and the linear regression equation is: y= 0.0801 x+0.296, r 2 =0.992 (where Y is the absorbance intensity change of the system at 652 nm, X is the acetylcholinesterase concentration).
The detection limit=0.084 mU/mL of the method was calculated with 3 times the standard deviation of the 10 measurement results of the blank solution as the signal to noise ratio, indicating that the method has a wider linear range and a lower detection limit.
FIG. 7 is a graph showing absorbance intensity of the system after addition of acetylcholinesterase or other interferents. The numbers 1 to 17 in the figure are in turn: control, 10 mU/mL acetylcholinesterase, 100 mU/mL α -chymotrypsin, 100 mU/mL trypsin, 100 mU/mL lipase, 100 mU/mL pepsin, 100 mU/mL lysozyme, 100 mU/mL uricase, 100 mU/mL glucose oxidase, 0.1 mg/mL bovine serum albumin, 0.1M glucose, 10 mM histidine, 10 mM D-phenylalanine, 10 mM serine, 0.1M potassium ion, 0.1M calcium ion, and 0.1M magnesium ion. From the figure, only in the presence of acetylcholinesterase, the absorbance value of the system is obviously reduced, and other interferents do not influence the detection of acetylcholinesterase. The invention has good selectivity in detecting acetylcholinesterase.
FIG. 8 is a visual examination of acetylcholinesterase and preparation of paper-based acetylcholinesterase sensors. CeO is first prepared 2 -Co(OH) 2 The composite material was immobilized on a filter paper as a test paper, which was seen to be almost colorless. However, immediately after the TMB solution was applied as an ink drop to the test paper, the test paper turned dark blue (fig. 8A). Subsequently, a volume of the analyte (containing acetylcholinesterase, thioacetylcholine and phosphate buffer solution) was dropped onto the above test paper, and it was seen that the color of the test paper was gradually changed from dark blue to light blue as the concentration of acetylcholinesterase was increased, thereby constructing a paper-based acetylcholinesterase sensor, and visual detection of acetylcholinesterase was achieved (fig. 8B).
CeO 2 -Co(OH) 2 The mechanism of the composite material for acetylcholinesterase detection: ceO (CeO) 2 -Co(OH) 2 The composite material has oxidase-like activity, and can catalyze hydrogen peroxide to oxidize 3,3', 5' -tetramethyl benzidine (TMB) to generate blue oxidation state TMB (ox-TMB). However, when acetylcholinesterase and thiocholine are present in the system, ceO is produced due to the production of thiocholine 2 -Co(OH) 2 The oxidase activity of (a) is inhibited, resulting in gradual fading of the color of the system with increasing concentration of acetylcholinesterase. Based on the above, quantitative analysis and visual detection of acetylcholinesterase can be realized.
4. CeO (CeO) 2 -Co(OH) 2 Composite material for screening acetylcholinesterase inhibitor
Positive group: and incubating acetylcholinesterase, thiocholine and 6 alkaloids (commercial acetylcholinesterase inhibitor-neostigmine bromide is selected as verification, and the other 5 alkaloids are active components derived from natural products, namely berberine hydrochloride, caffeine, camptothecine, evodiamine and matrine) at 37 ℃ for 30-60 min. Then adding a certain volume of acetate buffer solution and 3,3', 5' -tetramethyl biphenylAmine (TMB) and CeO 2 -Co(OH) 2 A composite material. After vortex mixing, incubating for 5-40 min at room temperature, and recording the absorbance value of the test solution at 652 and nm as Ai.
Blank group: the same volume of phosphate buffer was used instead of the alkaloid, and other experimental conditions were exactly identical to those of the positive group, and the absorbance value of the solution at 652 nm was measured and noted as a.
Negative group: the phosphate buffer solution was used instead of alkaloid and acetylcholinesterase, other experimental conditions were completely consistent with the positive group, and the absorbance value of the solution at 652 nm was measured and recorded as A 0 。
The inhibition ratio (inhibition ratio=) of six alkaloids was calculated) Half maximal Inhibitory Concentration (IC) 50 ）。
Table 1 shows the inhibition of six alkaloids. From the table it can be seen that the commercial inhibitor neostigmine bromide has a remarkable inhibitory effect on acetylcholinesterase and is comparable to the values reported in the literature (ACS appl. Mat. Interfaces 2013, 5, 3275-3280.). The berberine hydrochloride in the other five natural alkaloids has similar inhibition effect on acetylcholinesterase as neostigmine bromide, while the caffeine, camptothecine, evodiamine and matrine have relatively weak inhibition effect on acetylcholinesterase. The above results indicate that berberine hydrochloride can be used as an inhibitor of acetylcholinesterase.
。
FIG. 9 shows inhibition curves of different concentrations of neostigmine bromide versus berberine hydrochloride. Wherein the concentration corresponding to 50% inhibition percentage is half Inhibition Concentration (IC) of neostigmine bromide and berberine hydrochloride 50 ) IC of neostigmine bromide and berberine hydrochloride is obtained 50 2.68 nM and 0.94 μm respectively.
Inhibition of acetylcholinesterase by alkaloids: the intermolecular binding mode of the three alkaloids and acetylcholinesterase is analyzed in detail through calculation by taking three alkaloids of camptothecine, berberine hydrochloride and evodiamine with relatively high inhibition rate as representatives. FIG. 10 shows an acetylcholinesterase crystal protein (RCSB PDB ID: 1DX 4), and it can be seen that the crystal protein 1DX4 contains small molecules, so that the Binding pocket (Binding pocket) of the protein is clear and the position of the docking pocket is reliable. Then, molecular docking calculation of acetylcholinesterase and three alkaloids is carried out by molecular dynamics software Amber14 software, and the binding energy is obtained. FIG. 11 shows electrostatic surface focusing (A-C) and local binding phase (D-F) of binding pocket of acetylcholinesterase with three small molecular alkaloids, camptothecine, berberine hydrochloride and evodiamine. Among these, camptothecin has the smallest volume and can be inserted into the protein but cannot cover the whole space, so that the camptothecin has the weakest binding capacity and the binding capacity of-7.65 kcal/mol (FIG. 11, A, D). The berberine hydrochloride and the evodiamine have larger volumes, can be inserted into a protein binding pocket and occupy most of the binding cavity space, thereby forming better geometric matching and physicochemical property binding modes with a plurality of amino acids of the protein conserved binding pocket. Wherein, when the small-molecule berberine hydrochloride is combined with the protein binding cavity, the small-molecule berberine hydrochloride can form better hydrogen bond action and hydrophobicity with the surrounding amino acid to be matched with each other, wherein the hydrophobic binding action is mainly used, the main action is played in the protein binding process, and the binding energy is-9.74 kcal/mol (figure 11B, E). In contrast, the evodiamine has no much atomic system compared with berberine hydrochloride, and only Y162 can form less polar hydrogen bond action with the evodiamine, so that the binding energy is obviously reduced and reduced to-8.69 kcal/mol (figure 11C, F). In conclusion, the interaction between the berberine hydrochloride and acetylcholinesterase is strongest, so that the inhibition effect of the berberine hydrochloride is best.
The invention is used for detecting acetylcholinesterase, but because the reaction mechanism and enzyme action substrate of the butyrylcholinesterase are similar to those of the acetylcholinesterase, the invention is also applicable to detection of the butyrylcholinesterase and screening of inhibitors.
In summary, the invention has the following beneficial effects and advantages:
the detection method established by the invention can rapidly, sensitively and highly selectively realize the detection of the activity of acetylcholinesterase and the screening of natural product inhibitors, and has important guiding significance for the development of medicines for treating neurodegenerative diseases such as Alzheimer disease. In addition, the preparation process is simple, no modification or marking is needed, the analysis cost is low, and the application is strong.
Drawings
FIG. 1 shows CeO 2 -Co(OH) 2 XRD pattern of the composite material.
FIG. 2 is CeO 2 -Co(OH) 2 SEM (a) and TEM (B) images of the composite.
FIG. 3 is CeO 2 -Co(OH) 2 EDX plot of composite material.
FIG. 4 is CeO 2 -Co(OH) 2 Dark field STEM diagram (a) of the composite material and element map (B-G) of the corresponding elements in the material.
FIG. 5 is a graph showing the visible absorption spectrum of the system after the system has been added with acetylcholinesterase at various concentrations.
FIG. 6 is a graph showing the linear relationship between the change in absorbance intensity of the system and the concentration of acetylcholinesterase after adding acetylcholinesterase at various concentrations.
FIG. 7 is a bar graph of absorbance intensity of the system after addition of acetylcholinesterase or other interferents.
FIG. 8 is a visual examination of acetylcholinesterase and preparation of paper-based acetylcholinesterase sensors.
FIG. 9 shows inhibition curves of different concentrations of neostigmine bromide (A) and berberine hydrochloride (B).
FIG. 10 shows the binding pocket of acetylcholinesterase crystallin molecules.
FIG. 11 shows electrostatic surface focusing (A-C) and local binding phase (D-F) of binding pocket of acetylcholinesterase with three small molecular alkaloids, camptothecine, berberine hydrochloride and evodiamine.
Detailed Description
The following description will be made with reference to specific embodimentsCeO of the invention 2 -Co(OH) 2 The preparation and application of the composite material are described in further detail.
EXAMPLE 1 CeO 2 -Co(OH) 2 Preparation of composite materials
First, 0.658. 0.658 g L-proline and 8.685 g cerium nitrate hexahydrate (molar ratio: 1:3.5) were heated at 60℃to give a clear and transparent solution of 1. 1 h. Subsequently, 0.02 mol of Co (NO 3 ) 2 . 6H 2 O (5.82 g) and 7 mLNaOH (5 mol) . L -1 ) Adding into the eutectic solvent, reacting in 40 deg.C oil bath for 2 h, centrifuging, washing (water washing and ethanol washing), and drying at 60deg.C to obtain CeO 2 -Co(OH) 2 A composite material. Characterization results show that XRD, SEM, TEM and EDX and other characterization results of the materials are similar to those of fig. 1-4.
EXAMPLE 2 CeO 2 -Co(OH) 2 Preparation of composite materials
First, 4.605. 4.605 g L-proline and 17.369 g cerium nitrate hexahydrate (molar ratio: 1:1) were heated at 80℃for 1:1 h to give a clear and transparent solution. Subsequently, 0.02 mol of Co (NO 3 ) 2 . 6H 2 O (5.82 g) and 7 mLNaOH (10 mol) . L -1 ) Adding into the eutectic solvent, reacting in 65 deg.C oil bath for 1 h, centrifuging, washing (water washing and ethanol washing), and drying at 60deg.C to obtain CeO 2 -Co(OH) 2 A composite material. Characterization results show that XRD, SEM, TEM and EDX and other characterization results of the material completely coincide with those of fig. 1-4.
EXAMPLE 3 CeO 2 -Co(OH) 2 Preparation of composite materials
First, the clear and transparent solution is obtained by heating L-proline of 9.210 g and 8.685 g cerium nitrate hexahydrate (molar ratio 4:1) at 70deg.C for 2 h. Subsequently, 0.02 mol of Co (NO 3 ) 2 . 6H 2 O (5.82 g) and 7 mLNaOH (7 mol) . L -1 ) Adding into the eutectic solvent, and adding oil at 25deg.CReacting in bath 2.5. 2.5 h, centrifuging, washing (washing with water and ethanol), and drying at 60deg.C to obtain CeO 2 -Co(OH) 2 A composite material. Characterization results show that XRD, SEM, TEM and EDX and other characterization results of the material completely coincide with those of fig. 1-4.
EXAMPLE 4 quantitative detection of acetylcholinesterase Activity
1. Quantitative detection of acetylcholinesterase activity in buffer
Test group: first, 20. Mu.L, 5 mM of thiocholine and 100. Mu.L of different concentrations (2, 5, 10, 20, 50, 60, 80, 100, 120, 140, 160, 180, 200 mU/mL) of acetylcholinesterase were incubated at 37℃for 30 min. Then 680. Mu.L of acetate buffer (0.1M, pH 4.0), ceO were added 2 -Co(OH) 2 After vortexing the composite (100. Mu.L, 1 mg/mL) and 3,3', 5' -tetramethylbenzidine (TMB, 100. Mu.L, 8 mM), incubation was performed at room temperature for 10 min, the solution was tested for a visible spectrum, the absorbance of the solution at 652 nm was recorded and recorded as A 1 . It can be seen that the absorbance value of the solution at 652 nm gradually decreased as the concentration of acetylcholinesterase increased (fig. 5).
Control group: instead of adding acetylcholinesterase, the same volume of phosphate buffer solution (consisting of sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate, 100 mM, pH 8.0) was used, the other conditions were unchanged, and the absorbance value of the solution at 652 nm was measured and recorded as A 2 。
Absorbance intensity change value of system at 652 nm (y=a 1 -A 2 ) A good linear relationship exists between the concentration of acetylcholinesterase (the concentration interval is 0.2-20 mug/mL) (figure 6), and the linear regression equation is: y= 0.0801 x+0.296, r 2 =0.992 (where Y is the absorbance intensity change of the system at 652 nm, X is the acetylcholinesterase concentration). The detection limit of the method is 0.084 mU/mL by taking 3 times of standard deviation of a 10-time measurement result of a blank solution as a signal to noise ratio, and the result shows that the method has a wider linear range and a lower detection limit.
2. Quantitative detection of acetylcholinesterase activity in complex samples
Serum samples of the same volume were taken and tested according to the test procedure for acetylcholinesterase activity in buffer, and the results, recovery and relative standard deviation were calculated as shown in table 2:
3. selective testing of acetylcholinesterase
mu.L of phosphate buffer (100 mM, pH 8.0) was mixed with 10. Mu.L of thioacetylcholine (10 mM) or 10. Mu.L of interferents (100 mU/mL acetylcholinesterase, 1U/mL. Alpha. -chymotrypsin, 1U/mL trypsin, 1U/mL lipase, 1U/mL pepsin, 1U/mL lysozyme, 1U/mL uricase, 1U/mL glucose oxidase, 1 mg/mL BSA, 1M glucose, 100 mM histidine, 100 mM D-phenylalanine, 100 mM serine, 1M KCl, 1M CaCl) 2 、1 M MgCl 2 ) Incubate at 37℃for 30 min. Then 700. Mu.L of acetate buffer (0.1, M, pH 4.0) and 100. Mu.L of CeO were added 2 -Co(OH) 2 After vortexing the composite (1 mg/mL) and 100. Mu.L TMB (8 mM), incubation was performed at room temperature for 10 min, the visible spectrum of the solution was tested, the absorbance of the solution at 652 nm was recorded, and plotted. The results are shown in FIG. 7, which demonstrate that the established method of the present invention has good selectivity.
4. Preparation of paper-based acetylcholinesterase sensor and visual detection of acetylcholinesterase
Test paper: soaking the filter paper in CeO 1 mg/mL 2 -Co(OH) 2 The test paper is dried after 5 min in the solution, and is almost colorless. 100 μl of TMB (8 mM) was then applied drop-wise to the test paper, which was seen to immediately turn deep blue (fig. 8A).
Analytical solution: 10. mu.L of phosphate buffer (100 mM, pH 8.0), 10. Mu.L of thioacetylcholine (10 mM) and 100. Mu.L of acetylcholinesterase (0, 2, 10, 14, 18 mU/mL) were incubated at 37℃for 30 min. 780. Mu.L of acetate buffer (0.1M, pH 4.0) was then added.
Preparation of paper-based acetylcholinesterase sensor and visual detection of acetylcholinesterase: 200. Mu.L of the analyte was dropped onto the above test paper, and it was found that the test paper was gradually changed from dark blue to light blue in color as the concentration of acetylcholinesterase increased (0, 2, 10, 14, 18 mU/mL) (FIG. 8B).
EXAMPLE 5 screening for acetylcholinesterase inhibitors
1. Inhibition of six alkaloids
Positive group: mu.L of acetylcholinesterase (100 mU/mL), 10. Mu.L of thioacetylcholine (10 mM) and 10. Mu.L of 6 alkaloids (neostigmine bromide, berberine hydrochloride, caffeine, camptothecin, evodiamine and matrine, 10 mM) were incubated at 37℃for 30 min, respectively. Subsequently 680. Mu.L acetate buffer (0.1M, pH 4.0) and 100. Mu.L CeO were added 2 -Co(OH) 2 Composite (1 mg/mL) and 100. Mu.L TMB (8 mM). Vortex mixing, incubation for 10 min at room temperature, absorbance value of test solution at 652 nm, noted Ai.
Blank group: the absorbance of the solution at 652 nm was measured and was designated as a using 10 μl of phosphate buffer solution instead of the alkaloid, and other experimental conditions were fully consistent with the positive group.
Negative group: using 110. Mu.L of phosphate buffer solution instead of alkaloid and acetylcholinesterase, other experimental conditions were completely consistent with the positive group, and the absorbance value of the solution at 652 nm was measured and recorded as A 0 。
According to the formula: inhibition ratio =The inhibition rates of six alkaloids were calculated, as shown in table 1, commercial inhibitors, neostigmine bromide, showed the best inhibition rate, berberine hydrochloride in the other five natural alkaloids had similar inhibition effects on acetylcholinesterase as neostigmine bromide, while caffeine, camptothecin, evodiamine and matrine had relatively weak inhibition effects on acetylcholinesterase.
2. Inhibition curve of neostigmine bromide and berberine hydrochloride
Positive group: mu.L of acetylcholinesterase (100 mU/mL), 10. Mu.L of thioacetylcholine (10 mM) and 10. Mu.L (10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 50. Mu.M, 100. Mu.M) were incubated at 37℃for 30 min, respectively. Subsequently 680. Mu.L acetate buffer (0.1M, pH 4.0) and 100. Mu.L CeO were added 2 -Co(OH) 2 Composite (1 mg/mL) and 100. Mu.L TMB (8 mM). Vortex mixing, incubation for 10 min at room temperature, absorbance value of test solution at 652 nm, noted Ai.
Blank group: using 10. Mu.L of phosphate buffer solution instead of neostigmine bromide or berberine hydrochloride, other experimental conditions were completely identical to those of the positive group, and the absorbance value of the solution at 652 nm was measured and noted as A.
Negative group: using 110. Mu.L of phosphate buffer solution instead of neostigmine bromide and acetylcholinesterase or berberine hydrochloride and acetylcholinesterase, other experimental conditions were completely consistent with the positive group, and the absorbance value of the solution at 652 nm was measured and recorded as A 0 。
The inhibition rate of neostigmine bromide or berberine hydrochloride at different concentrations was calculated, and an inhibition curve corresponding to neostigmine bromide and berberine hydrochloride at different concentrations was plotted (fig. 9). Wherein the concentration corresponding to 50% inhibition percentage is half Inhibition Concentration (IC) of neostigmine bromide and berberine hydrochloride 50 ) IC of neostigmine bromide and berberine hydrochloride is obtained 50 2.68 nM and 0.94 μm respectively.
Claims (10)
1. A preparation method of a cerium oxide-cobalt hydroxide composite material is characterized in that: sequentially adding cobalt nitrate hexahydrate and sodium hydroxide into a eutectic solvent, reacting for 0.5-3.0 h at the temperature of between room temperature and 70 ℃, and centrifuging, washing and drying to obtain a cerium oxide-cobalt hydroxide composite material; the eutectic solvent is prepared by taking L-proline as a hydrogen bond donor, taking cerium nitrate hexahydrate as a hydrogen bond acceptor and heating at 60-80 ℃ until the mixture is clear and transparent.
2. The method for preparing the cerium oxide-cobalt hydroxide composite material according to claim 1, wherein the method comprises the following steps: the molar ratio of the cobalt nitrate hexahydrate to the sodium hydroxide is 1:1-1:4.
3. The method for preparing the cerium oxide-cobalt hydroxide composite material according to claim 1, wherein the method comprises the following steps: the molar ratio of the L-proline to the cerium nitrate hexahydrate is 1:4-4:1.
4. The use of the cerium oxide-cobalt hydroxide composite material prepared according to the method of claim 1 in acetylcholinesterase detection.
5. The use of the cerium oxide-cobalt hydroxide composite material according to claim 4 for detecting acetylcholinesterase activity, wherein: incubating the thiocholine compound and acetylcholinesterase with different concentrations for 30-60 min at 37 ℃; then adding acetate buffer solution, 3', 5' -tetramethyl benzidine and cerium oxide-cobalt hydroxide composite material, mixing uniformly by vortex, incubating for 5-40 min at room temperature, and measuring the absorbance value of the solution at 652 nm, which is marked as A 2 The method comprises the steps of carrying out a first treatment on the surface of the Instead of adding acetylcholinesterase, the same volume of phosphate buffer solution was used to replace acetylcholinesterase, the other conditions were unchanged, and the absorbance value of the solution at 652 nm was measured and recorded as A 1 The method comprises the steps of carrying out a first treatment on the surface of the Change in absorbance value of the solution at 652 nm y=a 1 -A 2 And the linear relation with the concentration of the acetylcholinesterase, and quantitatively detecting the acetylcholinesterase.
6. The use of the cerium oxide-cobalt hydroxide composite material according to claim 5 in the detection of acetylcholinesterase activity, wherein: when the concentration of acetylcholinesterase is in the range of 0.2-20 mU/mL, a good linear relation exists between the change value of the absorbance value of the system at 652 nm and the concentration of acetylcholinesterase, and the linear equation is as follows: y= 0.0801 x+0.296, correlation coefficient R 2 =0.992, wherein Y is the change in absorbance value of the system at 652 nmThe value, X, is the acetylcholinesterase concentration.
7. The use of the cerium oxide-cobalt hydroxide composite material according to claim 5 in acetylcholinesterase detection, wherein: the thiocholine compound is any one of thiocholine, thiocetylcholine and thiopropionyl choline.
8. The use of the cerium oxide-cobalt hydroxide composite material according to claim 5 in acetylcholinesterase detection, wherein: the concentration of the acetate buffer solution is 0.1M, and the pH value range is 3.5-5.0; the concentration of the phosphate buffer solution is 100 mM, and the pH value range is 7.0-8.0.
9. The use of the cerium oxide-cobalt hydroxide composite material according to claim 4 for detecting acetylcholinesterase activity, wherein: fixing the cerium oxide-cobalt hydroxide composite material on filter paper to serve as test paper, wherein the test paper is colorless, and immediately changing the test paper into dark blue after 3,3', 5' -tetramethyl benzidine solution is dripped on the test paper; and dripping acetylcholinesterase, thiocholine and phosphate buffer solution onto the test paper, wherein the color of the test paper is gradually changed from deep blue to light blue, so that a paper-based acetylcholinesterase sensor is constructed, and visual detection of acetylcholinesterase is realized.
10. Use of a ceria-cobalt hydroxide composite prepared according to the method of claim 1 in the screening of natural product inhibitors.
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