CN114184561A - Preparation and application of a cerium oxide-cobalt hydroxide composite material - Google Patents
Preparation and application of a cerium oxide-cobalt hydroxide composite material Download PDFInfo
- Publication number
- CN114184561A CN114184561A CN202111349848.1A CN202111349848A CN114184561A CN 114184561 A CN114184561 A CN 114184561A CN 202111349848 A CN202111349848 A CN 202111349848A CN 114184561 A CN114184561 A CN 114184561A
- Authority
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- China
- Prior art keywords
- acetylcholinesterase
- composite material
- cerium oxide
- cobalt hydroxide
- hydroxide composite
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 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
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- 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
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- 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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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种氧化铈‑氢氧化钴复合材料的制备方法，是将六水合硝酸钴和氢氧化钠依次加入低共熔溶剂中，在室温~70℃下反应0.5~3.0 h，再经离心、洗涤、干燥即得。该氧化铈‑氢氧化钴复合材料可用于乙酰胆碱酯酶活性检测及抑制剂筛选。氧化铈‑氢氧化钴复合材料可以实现乙酰胆碱酯酶的定量分析和可视化检测，并可成功应用于天然产物中乙酰胆碱酯酶抑制剂的筛选，该结果对于阿尔茨海默病等神经退行性疾病治疗药物的开发具有重要指导意义。本发明建立的可视化检测方法能快速、灵敏、高选择性地实现乙酰胆碱酯酶活性检测及天然产物抑制剂筛选。另外，本发明制备工艺简单、无需任何修饰和标记，成本低，应用性强。The invention discloses a preparation method of a cerium oxide-cobalt hydroxide composite material. The hexahydrate cobalt nitrate and sodium hydroxide are sequentially added to a deep eutectic solvent, reacted at room temperature to 70° C. for 0.5 to 3.0 h, and then subjected to Centrifuge, wash and dry. 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 the quantitative analysis and visual detection of acetylcholinesterase, and can be successfully applied to the screening of acetylcholinesterase inhibitors in natural products. The results are useful for the treatment of neurodegenerative diseases such as Alzheimer's disease. The development of drugs has important guiding significance. The visual detection method established by the invention can realize the detection of acetylcholinesterase activity and the screening of natural product inhibitors rapidly, sensitively and with high selectivity. In addition, the preparation process of the invention is simple, does not need any modification and marking, has low cost and strong applicability.
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 a hydrogen bond donor and a hydrogen bond acceptor in a certain stoichiometric ratio through hydrogen bond action, has the characteristics of simple preparation, low vapor pressure, good solubility and conductivity, reusability, strong designability, biodegradability and the like, and is often used as a novel green solvent. Since the first report in 2003, the Chinese medicinal composition is gradually favored by researchers. It is worth mentioning that the eutectic solvent can adjust the nucleation and growth rate of the material synthesis process, so that it is of great interest in the field of material synthesis. According to investigation, no report related to the synthesis of a nano material, in particular to the synthesis of a cerium oxide-cobalt hydroxide composite nano material, by using a eutectic solvent composed of L-proline and cerium nitrate hexahydrate is found at present.
Alzheimer's Disease (AD) is a chronic degenerative disease of the nervous system, one of the world's ten killers '. Clinically, the overall dementia such as dysmnesia, aphasia, disuse, agnosia, impairment of visual spatial skills, dysfunction in execution, and personality and behavior changes are characterized, and the etiology is unknown. Studies have shown that a key symptom of the disease is a reduction in the synthesis of the neurochemical transmitter acetylcholine. However, acetylcholinesterase is a key hydrolase of acetylcholine, and abnormal fluctuations in acetylcholinesterase can directly affect acetylcholine metabolism, thereby disrupting neurotransmission in the brain. Therefore, acetylcholinesterase is regarded as an important target for screening anti-Alzheimer disease drugs at present, and the rapid, high-sensitivity and high-selectivity detection of acetylcholinesterase is very important. Meanwhile, the use of drugs for inhibiting excessive acetylcholinesterase activity will contribute to the treatment of neurodegenerative diseases represented by alzheimer's disease. Most of the currently available therapeutic drugs for Alzheimer's disease patients are acetylcholinesterase inhibitors, such as aricept, donepezil, galantamine, tacrine, rivastigmine, etc. Although the above enzyme inhibitor drugs can alleviate or relieve the symptoms of diseases to a certain extent clinically, these drugs also have the defects of weak pharmaceutical activity, high price of imported drugs, obvious side effects, easy occurrence of drug resistance of patients and the like, so that the requirements of treatment cannot be met. The natural products (such as plants, traditional Chinese medicines and the like) have the characteristics of rich resources, safety, effectiveness, environmental friendliness, small toxic and side effects and the like, and are one of the important sources of natural enzyme inhibitors. Therefore, screening enzyme inhibitors from natural products has become an important strategy for the development of new drugs.
In summary, the problem to be solved by the skilled person is how to provide a method for green synthesis of cerium oxide-cobalt hydroxide composite material in eutectic solvent and for rapid, highly sensitive and highly selective detection of acetylcholinesterase and screening of natural inhibitors.
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 the detection of acetylcholinesterase activity and the screening of an inhibitor.
Preparation of cerium oxide-cobalt hydroxide composite material
The preparation method of the cerium oxide-cobalt hydroxide composite material comprises the steps of sequentially adding cobalt nitrate hexahydrate and sodium hydroxide into a eutectic solvent, reacting for 0.5-3.0 hours at room temperature-70 ℃, centrifuging, washing and drying to obtain cerium oxide-cobalt hydroxide (CeO)2-Co(OH)2) A composite material; the eutectic solvent is obtained by taking L-proline as a hydrogen bond donor and cerium nitrate hexahydrate as a hydrogen bond acceptor, and heating the eutectic solvent to be clear and transparent at the temperature of 60-80 ℃. Wherein the molar ratio of the cobalt nitrate hexahydrate to the sodium hydroxide is 1: 1-1: 4; the molar ratio of the L-proline to the cerous nitrate hexahydrate is 1: 4-4: 1.
II, CeO2-Co(OH)2Structure of composite material
X-ray diffraction (XRD), Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), energy spectrum (EDX) and dark-field STEM are used to treat CeO2-Co(OH)2The structure, morphology and the like of the composite material are characterized.
FIG. 1 shows CeO obtained in example 1 of the present invention2-Co(OH)2XRD pattern of the composite. From the figureIt can be seen that the material has CeO present at 2 θ = 28.7, 33.2, 47.6, 56.6, 59.6 and 69.6 °2(PDF cards: 4-593) crystal planes (111), (200), (220), (311), (222) and (400). Meanwhile, co (oh) exists at 2 θ =19.0, 32.6, 37.9, 51.5, 57.9 and 61.6 °2(PDF cards: 3-913) of (001), (100), (101), (102), (110) and (111). The material was confirmed to be CeO2-Co(OH)2A composite material.
FIG. 2 shows CeO prepared by the present invention2-Co(OH)2SEM (A) and TEM images (B) of the composite material. It can be seen that the material is a sheet structure.
FIG. 3 shows CeO obtained by the present invention2-Co(OH)2EDX picture of composite material. As can be seen from the energy spectrum, the material consists of three elements, namely Ce, O and Co.
FIG. 4 shows CeO obtained by the present invention2-Co(OH)2Dark field STEM maps (a) of the composite material and elemental maps (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. From the graphs B-G, it can be seen that the material mainly comprises three elements Ce, Co and O, and this result is completely consistent with fig. 3.
III, CeO2-Co(OH)2Composite material for detecting acetylcholinesterase
And (3) incubating the thiocholine compound (sulfobutylcholine, thioacetyl choline or thiopropionyl choline) and acetylcholinesterase with different concentrations for 30-60 min at 37 ℃. Then adding a certain volume of acetate buffer solution, 3',5,5' -Tetramethylbenzidine (TMB) and the prepared CeO2-Co(OH)2After the composite material is uniformly mixed by vortex, incubating for 5-40 min at room temperature, and testing the absorbance value of the solution at 652 nm, and recording as A2. In the control group, no acetylcholinesterase was added, the same volume of phosphate buffer solution was used instead, and the other conditions were unchanged, and the absorbance value at 652 nm of the solution was measured and recorded as A1. Value of change in absorbance at 652 nm according to solution (Y = A)1-A2) And acetylcholinesterase concentrationThe linear relation of (A) 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 (consisting 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 acetylcholinesterase was added to the system at different concentrations. As can be seen from FIG. 5, the absorbance value of the system at 652 nm gradually decreased with the increase of the concentration of acetylcholinesterase (from top to bottom, the concentrations of acetylcholinesterase are 0.2, 0.5, 1, 2, 5, 6, 8, 10, 12, 14, 16, 18, 20 mU/mL).
FIG. 6 is a linear relationship diagram between the variation of absorbance value of the system after adding acetylcholinesterase of different concentrations and the concentration of acetylcholinesterase. As can be seen from FIG. 6, there is a good linear relationship between the variation of the absorbance intensity at 652 nm of the system and the concentration of acetylcholinesterase (the concentration interval is 0.2-20 μ g/mL), and the linear regression equation is: y = 0.0801X + 0.296, R2= 0.992 (where Y is the change in the intensity of the absorbance of the system at 652 nm and X is the acetylcholinesterase concentration).
The detection limit =0.084 mU/mL of the method is calculated by taking 3 times of the standard deviation of the blank solution in 10 times as the signal-to-noise ratio, which shows that the method has a wider linear range and a lower detection limit.
FIG. 7 is a graph showing the absorbance intensity of the system after addition of acetylcholinesterase or other interferents. In the figure, the numbers 1 to 17 are as follows: control group, 10 mU/mL acetylcholinesterase, 100 mU/mL alpha-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. It can be seen from the figure that the absorbance value of the system is obviously reduced only in the presence of acetylcholinesterase, and other interferents do not influence the detection of acetylcholinesterase. The invention is proved to have good selectivity when detecting the acetylcholinesterase.
FIG. 8 is the preparation of paper-based acetylcholinesterase sensor and the visual detection of acetylcholinesterase. Firstly, CeO is added2-Co(OH)2The composite was fixed to a filter paper as a test paper, which was seen to be almost colorless. However, the test paper turned dark blue immediately after the TMB solution was applied as an ink drop to the test paper (fig. 8A). Then, a certain volume of analysis solution (containing acetylcholinesterase, thioacetylcholine and phosphate buffer solution) is dropped on the test paper, and it can be seen that the color of the test paper gradually changes from dark blue to light blue along with the increase of the concentration of acetylcholinesterase, so that a paper-based acetylcholinesterase sensor is constructed, and the visual detection of acetylcholinesterase can be realized (fig. 8B).
CeO2-Co(OH)2The mechanism of the composite material for detecting acetylcholinesterase is as follows: CeO (CeO)2-Co(OH)2The composite material has oxidase-like activity and can catalyze hydrogen peroxide to oxidize 3,3',5,5' -Tetramethylbenzidine (TMB) to generate blue oxidation state TMB (ox-TMB). However, when acetylcholinesterase and thioacetylcholine are present in the system, CeO is generated due to generation of thiocholine2-Co(OH)2The oxidase activity of (a) is inhibited, resulting in a gradual fading of the system colour with increasing acetylcholinesterase concentration. Based on the method, quantitative analysis and visual detection of acetylcholinesterase can be realized.
Tetra, CeO2-Co(OH)2Composite material for screening acetylcholinesterase inhibitor
Positive group: acetylcholinesterase, thioacetylcholine and 6 alkaloids (commercial acetylcholinesterase inhibitor-neostigmine bromide is selected as verification, and the other 5 alkaloids are derived from active ingredients of natural products, namely berberine hydrochloride, caffeine, camptothecin, evodiamine and matrine respectively) are incubated at 37 ℃ for 30-60 min. Then adding a certain volume of acetate buffer solution, 3',5,5' -tetramethyl benzidine (TMB) and CeO2-Co(OH)2A composite material. And after vortex mixing, incubating at room temperature for 5-40 min, and testing the absorbance value of the solution at 652 nm and recording as Ai.
Blank group: the same volume of phosphate buffer solution was used instead of alkaloid, other experimental conditions were identical to those of the positive group, and the absorbance value of the solution at 652 nm was measured and recorded as A.
Negative group: using phosphate buffer solution to replace alkaloid and acetylcholinesterase, the other experimental conditions are completely consistent with those of the positive group, and measuring the absorbance value of the solution at 652 nm, which is marked as A0。
The inhibition rates of six alkaloids were calculated (inhibition rate = inhibition rate)
) And median Inhibitory Concentration (IC)50）。
Table 1 shows the inhibition rates of six alkaloids. It can be seen from the table that the commercial inhibitor neostigmine bromide has a significant inhibitory effect on acetylcholinesterase and is comparable to the value reported in the literature (ACS appl. mater. Interfaces 2013, 5, 3275-3280.). In addition, berberine hydrochloride in the five natural alkaloids has similar inhibitory effect on acetylcholinesterase to neostigmine bromide, and caffeine, camptothecin, evodiamine and matrine have relatively weak inhibitory effect on acetylcholinesterase. The results show that the berberine hydrochloride can be used as an inhibitor of acetylcholinesterase.
FIG. 9 is a graph of inhibition curves corresponding to different concentrations of neostigmine bromide and berberine hydrochloride. Wherein the concentration corresponding to 50% inhibition is the half Inhibition Concentration (IC) of neostigmine bromide and berberine hydrochloride50) To obtain the IC of neostigmine bromide and berberine hydrochloride502.68 nM and 0.94. mu.M, respectively.
Inhibition of acetylcholinesterase by alkaloids: three alkaloids of camptothecin, berberine hydrochloride and evodiamine with relatively high inhibition rate are taken as representatives, and the intermolecular binding mode of the three alkaloids and acetylcholinesterase is analyzed in detail through calculation. FIG. 10 shows acetylcholinesterase crystal protein (RCSB PDB ID: 1DX4), and it can be seen that the crystal protein 1DX4 contains small molecules, so the Binding pocket (Binding pocket) of the protein is clear and the position of the Binding pocket is reliable. Subsequently, molecular docking calculation of acetylcholinesterase and three alkaloids is carried out through molecular dynamics software Amber14 software, and the binding energy is obtained. FIG. 11 is the electrostatic surface focusing diagram (A-C) and the partial diagram (D-F) of the binding pocket of acetylcholinesterase and three small-molecule alkaloids of camptothecin, berberine hydrochloride and evodiamine. Of these, camptothecin, which has the smallest volume among the three alkaloids, can be inserted into proteins but does not fill the entire space, has the weakest binding ability, i.e., -7.65 kcal/mol (FIG. 11A, D). The berberine hydrochloride and the evodiamine have larger volume, can be inserted into a protein binding pocket and occupy most of the binding cavity space, and further form a better geometric matching and physicochemical property binding mode with a plurality of amino acids of the protein conservative binding pocket. Wherein, when the small molecular berberine hydrochloride is combined with the protein combination cavity, the small molecular berberine hydrochloride can form better hydrogen bond action and hydrophobic property matching with the surrounding amino acid, wherein the hydrophobic property combination action is taken as the main action and plays a leading role in the protein combination process, and the combination energy is-9.74 kcal/mol (figure 11B, E). Compared with berberine hydrochloride, evodiamine has no too many atomic systems, and only Y162 can form less polar hydrogen bonding action with the berberine hydrochloride, so that the binding energy is obviously reduced to-8.69 kcal/mol (FIG. 11C, F). In conclusion, the interaction between berberine hydrochloride and acetylcholinesterase is strongest, so that the inhibition effect of berberine hydrochloride is best.
The method is used for detecting the acetylcholinesterase, but the reaction mechanism and the enzyme action substrate of the butyrylcholinesterase are similar to those of the acetylcholinesterase, so the method is also suitable for detecting the butyrylcholinesterase and screening inhibitors.
In conclusion, the invention has the beneficial effects and advantages that:
the detection method established by the invention can realize the detection of the activity of the acetylcholinesterase and the screening of the natural product inhibitor rapidly, sensitively and selectively, and has important guiding significance for the development of medicaments for treating neurodegenerative diseases such as Alzheimer disease and the like. In addition, the invention has simple preparation process, no need of any modification and marking, low analysis cost and strong applicability.
Drawings
FIG. 1 shows CeO2-Co(OH)2XRD pattern of the composite.
FIG. 2 shows CeO2-Co(OH)2SEM (A) and TEM (B) images of the composite material.
FIG. 3 shows CeO2-Co(OH)2EDX picture of composite material.
FIG. 4 shows CeO2-Co(OH)2Dark field STEM maps (a) of the composite material and elemental maps (B-G) of the corresponding elements in the material.
FIG. 5 is a graph showing the visible absorption spectrum of the system after acetylcholinesterase was added to the system at different concentrations.
FIG. 6 is a linear relationship diagram between the absorbance intensity change value of the system and the concentration of acetylcholinesterase after acetylcholinesterase with different concentrations is added.
FIG. 7 is a bar graph of the absorbance intensity of the system after addition of acetylcholinesterase or other interferents.
FIG. 8 is the preparation of paper-based acetylcholinesterase sensor and the visual detection of acetylcholinesterase.
FIG. 9 is a graph showing inhibition curves for different concentrations of neostigmine bromide (A) and berberine hydrochloride (B).
FIG. 10 shows the binding pocket of acetylcholinesterase crystallin molecules.
FIG. 11 is the electrostatic surface focusing diagram (A-C) and the partial diagram (D-F) of the binding pocket of acetylcholinesterase and three small-molecule alkaloids of camptothecin, berberine hydrochloride and evodiamine.
Detailed Description
The following embodiments are combinedEXAMPLES on CeO of the present invention2-Co(OH)2The preparation and use of the composite material are described in further detail.
Example 1 CeO2-Co(OH)2Preparation of composite materials
First, 0.658 g of L-proline and 8.685 g of cerium nitrate hexahydrate (molar ratio: 1: 3.5) were heated at 60 ℃ for 1 hour to obtain a clear and transparent solution. Subsequently, 0.02 mol of Co (NO) was added successively3)2 .6H2O (5.82 g) and 7 mLNaOH (5 mol).L-1) Adding the obtained product into the eutectic solvent, reacting for 2 h in an oil bath at 40 ℃, centrifuging, washing (firstly washing with water and then washing with ethanol), and drying at 60 ℃ to obtain CeO2-Co(OH)2A composite material. The characterization results show that the XRD, SEM, TEM, EDX and other characterization results of the material are similar to those of fig. 1 to 4.
Example 2 CeO2-Co(OH)2Preparation of composite materials
First, 4.605 g of L-proline and 17.369 g of cerous nitrate hexahydrate (molar ratio: 1) were heated at 80 ℃ for 1 hour to obtain a clear and transparent solution. Subsequently, 0.02 mol of Co (NO) was added successively3)2 .6H2O (5.82 g) and 7 mLNaOH (10 mol).L-1) Adding the obtained product into the eutectic solvent, reacting for 1 h in an oil bath at 65 ℃, centrifuging, washing (firstly washing with water and then washing with ethanol), and drying at 60 ℃ to obtain CeO2-Co(OH)2A composite material. The characterization results show that the XRD, SEM, TEM and EDX characterization results of the material are completely consistent with fig. 1 to 4.
Example 3 CeO2-Co(OH)2Preparation of composite materials
Firstly, 9.210 g of L-proline and 8.685 g of cerous nitrate hexahydrate (molar ratio of 4: 1) are heated at 70 ℃ for 2 h to obtain a clear and transparent solution. Subsequently, 0.02 mol of Co (NO) was added successively3)2 .6H2O (5.82 g) and 7 mLNaOH (7 mol).L-1) Oil added to the eutectic solvent at 25 deg.CReacting in bath for 2.5 h, centrifuging, washing (water washing and ethanol washing), and drying at 60 deg.C to obtain CeO2-Co(OH)2A composite material. The characterization results show that the XRD, SEM, TEM and EDX characterization results of the material are completely consistent with fig. 1 to 4.
Example 4 quantitative detection of Acetylcholinesterase Activity
1. Quantitative detection of acetylcholinesterase activity in buffer
Test group: first, 20. mu.L, 5 mM thiocholine and 100. mu.L of acetylcholinesterase at various concentrations (2, 5, 10, 20, 50, 60, 80, 100, 120, 140, 160, 180, 200 mU/mL) were incubated at 37 ℃ for 30 min. Then 680. mu.L of acetate buffer (0.1M, pH 4.0.0), CeO were added2-Co(OH)2After the composite material (100. mu.L, 1 mg/mL) and 3,3',5,5' -tetramethylbenzidine (TMB, 100. mu.L, 8 mM) were vortexed and mixed, the mixture was incubated at room temperature for 10 min, the visible spectrum of the test solution was recorded, and the absorbance value at 652 nm of the solution was recorded as A1. It can be seen that the absorbance value of the solution at 652 nm gradually decreased with increasing concentration of acetylcholinesterase (FIG. 5).
Control group: without addition of acetylcholinesterase, the same volume of phosphate buffer (100 mM, pH 8.0, composed of sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate) was used instead of the buffer, and the absorbance value at 652 nm of the solution was measured and recorded as A2。
Variation in absorbance intensity at 652 nm (Y = A) for the system1-A2) And the concentration of the acetylcholinesterase has a good linear relation (figure 6) with the concentration range of 0.2-20 mu g/mL, and the linear regression equation is as follows: y = 0.0801X + 0.296, R2= 0.992 (where Y is the change in the intensity of the absorbance of the system at 652 nm and X is the acetylcholinesterase concentration). The detection limit of the method is 0.084 mU/mL by taking 3 times of standard deviation of the blank solution in 10 times of measurement results as the 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
The same volume of serum sample was taken and tested according to the procedure for detecting acetylcholinesterase activity in the buffer, and the results, recovery and relative standard deviation were calculated and shown in table 2:
3. selective assay for 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 an interfering substance (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 CaCl2、1 M MgCl2) Incubate at 37 ℃ for 30 min. Then 700. mu.L acetate buffer (0.1M, pH 4.0.0), 100. mu.L CeO were added2-Co(OH)2After vortexing the composite (1 mg/mL) and 100. mu.L TMB (8 mM) and incubation at room temperature for 10 min, the solution was tested for visible spectra and the absorbance at 652 nm of the solution was recorded and plotted. The results are shown in FIG. 7, which shows that the method established by the present invention has good selectivity.
4. Preparation of paper-based acetylcholinesterase sensor and visual detection of acetylcholinesterase
Test paper: soaking filter paper in 1 mg/mL CeO2-Co(OH)2After being dried in the solution for 5 min, the solution is used as a test paper, and the test paper is almost colorless. Subsequently, 100. mu.L of TMB (8 mM) was applied drop-wise to the strip and the strip was seen to turn dark blue immediately (FIG. 8A).
Analysis solution: mu.L of phosphate buffered solution (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 μ L of acetate buffer (0.1M, pH 4.0) was then added.
Preparation of a paper-based acetylcholinesterase sensor and visual detection of acetylcholinesterase: when 200. mu.L of the analyte was dropped onto the above test paper, it was observed that the color of the test paper gradually changed from deep blue to light blue as the concentration of acetylcholinesterase increased (0, 2, 10, 14, 18 mU/mL) (FIG. 8B).
Example 5 screening of acetylcholinesterase inhibitors
1. Inhibition rate 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. 680 μ L of acetate buffer (0.1M, pH 4.0.0), 100 μ L of CeO were then added2-Co(OH)2Composite (1 mg/mL) and 100. mu.L TMB (8 mM). Vortex and mix well, incubate for 10 min at room temperature, test solution at 652 nm absorbance value, mark Ai.
Blank group: the absorbance value of the solution at 652 nm was measured as A using 10. mu.L of phosphate buffer solution instead of alkaloid and other experimental conditions were identical to those of the positive group.
Negative group: 110. mu.L of phosphate buffer solution was used instead of alkaloid and acetylcholinesterase, 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 recorded as A0。
According to the formula: inhibition rate =
The inhibition rates of six alkaloids were calculated, as shown in table 1, the commercial inhibitor neostigmine bromide showed the best inhibition rate, and berberine hydrochloride among the other five natural alkaloids had similar inhibition effect to neostigmine bromide, while caffeine, camptothecin, evodiamine and matrine had relatively weak inhibition effect to acetylcholinesterase.
2. Inhibition curves 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. 680 μ L of acetate buffer (0.1M, pH 4.0.0), 100 μ L of CeO were then added2-Co(OH)2Composite (1 mg/mL) and 100. mu.L TMB (8 mM). Vortex and mix well, incubate for 10 min at room temperature, test solution at 652 nm absorbance value, mark Ai.
Blank group: the absorbance value of the solution at 652 nm was measured and recorded as A by using 10. mu.L of phosphate buffer solution instead of neostigmine bromide or berberine hydrochloride and other experimental conditions were identical to those of the positive group.
Negative group: using 110 μ L phosphate buffer solution instead of neostigmine bromide and acetylcholinesterase or berberine hydrochloride and acetylcholinesterase, the other experimental conditions are completely consistent with those of the positive group, and measuring the absorbance value of the solution at 652 nm, and marking as A0。
The inhibition rates of neostigmine bromide or berberine hydrochloride at different concentrations are calculated, and inhibition curves corresponding to neostigmine bromide and berberine hydrochloride at different concentrations are drawn (fig. 9). Wherein the concentration corresponding to 50% inhibition is the half Inhibition Concentration (IC) of neostigmine bromide and berberine hydrochloride50) To obtain the IC of neostigmine bromide and berberine hydrochloride502.68 nM and 0.94. mu.M, respectively.
Claims (10)
1. A preparation method of a cerium oxide-cobalt hydroxide composite material is characterized by comprising the following steps: sequentially adding cobalt nitrate hexahydrate and sodium hydroxide into a eutectic solvent, reacting for 0.5-3.0 hours at room temperature-70 ℃, centrifuging, washing and drying to obtain a cerium oxide-cobalt hydroxide composite material; the eutectic solvent is obtained by taking L-proline as a hydrogen bond donor and cerium nitrate hexahydrate as a hydrogen bond acceptor, and heating the eutectic solvent to be clear and transparent at the temperature of 60-80 ℃.
2. The method of claim 1, wherein the cerium oxide-cobalt hydroxide composite material comprises: the molar ratio of the cobalt nitrate hexahydrate to the sodium hydroxide is 1: 1-1: 4.
3. The method of claim 1, wherein the cerium oxide-cobalt hydroxide composite material comprises: the molar ratio of the L-proline to the cerous 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 in the detection of acetylcholinesterase activity, wherein: incubating a thiocholine compound and acetylcholinesterase with different concentrations for 30-60 min at 37 ℃; adding acetate buffer solution, 3',5,5' -tetramethyl benzidine and cerium oxide-cobalt hydroxide composite material, uniformly mixing by vortex, incubating at room temperature for 5-40 min, and testing the absorbance value of the solution at 652 nm, which is marked as A2(ii) a Without adding acetylcholinesterase, the same volume of phosphate buffer solution was used instead of acetylcholinesterase, other conditions were unchanged, and the absorbance value at 652 nm of the solution was measured and recorded as A1(ii) a Change value of absorbance value Y = A at 652 nm of solution1-A2And the linear relation between the concentration of the acetylcholinesterase and the concentration of the acetylcholinesterase is used for 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 the acetylcholinesterase is within the range of 0.2-20 mU/mL, a good linear relation exists between the variation value of the absorbance value of the system at 652 nm and the concentration of the acetylcholinesterase, and the linear equation is as follows: y = 0.0801X + 0.296, correlation coefficient R2And = 0.992, wherein Y is the change value of the absorbance value of the system at 652 nm, and X is the concentration of acetylcholinesterase.
7. The use of the cerium oxide-cobalt hydroxide composite material of claim 5 in acetylcholinesterase detection, wherein: the thiocholine compound is any one of sulfobutylcholine, sulfoacetylcholine and sulfopropionyl choline.
8. The use of the cerium oxide-cobalt hydroxide composite material of 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 in the detection of 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 the test paper immediately turns into dark blue after 3,3',5,5' -tetramethylbenzidine solution is dripped on the test paper; the acetylcholinesterase, the thioacetylcholine and the phosphate buffer solution are dropped on the test paper, and the color of the test paper is gradually changed from dark blue to light blue, so that the paper-based acetylcholinesterase sensor is constructed, and the visual detection of the acetylcholinesterase is realized.
10. Use of the cerium oxide-cobalt hydroxide composite material prepared according to the method of claim 1 in screening of natural product inhibitors.
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