CN113358528B - Method for detecting acetylcholinesterase and inhibitor thereof based on pendant drop method - Google Patents
Method for detecting acetylcholinesterase and inhibitor thereof based on pendant drop method Download PDFInfo
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Abstract
The invention relates to a method for detecting acetylcholinesterase and an inhibitor thereof based on a pendant drop method. The method comprises the following steps: (1) preparing a surfactant solution; (2) preparing an acetylcholinesterase standard solution and an acetylcholinesterase inhibitor standard solution; (3) measuring an acetylcholinesterase standard curve; (4) measuring a standard curve of the acetylcholinesterase inhibitor; (5) and (3) measuring the acetylcholinesterase or acetylcholinesterase inhibitor of the sample to be measured. The method for detecting the acetylcholinesterase and the inhibitor thereof based on the pendant drop method has the advantages of simple operation process, low cost, accurate detection, high sensitivity, quantitative detection limit of the acetylcholinesterase to 0.1mU/mL, and quantitative detection limit of the acetylcholinesterase inhibitor to 1 nM.
Description
Technical Field
The invention provides a method for detecting acetylcholinesterase and an inhibitor thereof based on a pendant drop method, and belongs to the technical field of detection and analysis.
Background
Acetylcholinesterase (AChE), as a primary cholinesterase, is responsible for biological signal transmission in the nervous system, mainly at the postsynaptic neuromuscular junction, especially in muscles and nerves. It can specifically modulate acetylcholine levels by hydrolyzing the neurotransmitter acetylcholine (ACh) to acetate and choline, resulting in termination of neuronal transmission and signaling between synapses. Importantly, Alzheimer's Disease (AD), the so-called senile dementia, is primarily caused by low concentrations of ACh in the cortex. Clinically, the acetylcholinesterase inhibitor is mainly used for treating AD, so a technical means capable of detecting the activity inhibition rate of acetylcholinesterase is needed.
The traditional methods for detecting the activity of the acetylcholinesterase include a colorimetric method, a fluorescence method, an electrochemical method and the like. However, these methods generally require complicated procedures for labeling molecules, synthesizing nanoparticles, expensive instruments, and the like. Therefore, it is important to develop a simple, convenient and low-cost method for real-time and label-free detection of AChE.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting acetylcholinesterase and an inhibitor thereof based on a pendant drop method. The detection method has the advantages of high speed, high efficiency, low cost, sensitivity and the like.
The technical scheme of the invention is as follows:
a method for detecting acetylcholinesterase and an inhibitor thereof based on a pendant drop method comprises the following steps:
(1) preparing a surfactant solution: dissolving a surfactant in a Tris-HCl buffer solution, and carrying out ultrasonic treatment for 25-35 min to uniformly mix to obtain a surfactant solution with the concentration of 0.01-1 mM;
(2) preparing an acetylcholinesterase standard solution and an acetylcholinesterase inhibitor standard solution: respectively dissolving the acetylcholinesterase standard substance and the acetylcholinesterase inhibitor standard substance into a Tris-HCl buffer solution to prepare acetylcholinesterase standard solutions and acetylcholinesterase inhibitor standard solutions with different concentrations;
(3) determination of standard curve of acetylcholinesterase: respectively adding the surfactant solution prepared in the step (1) into the acetylcholinesterase standard solutions with different concentrations prepared in the step (2), and incubating for 25-55 min at 20-60 ℃ to obtain a mixed solution a; then, slowly injecting the oil phase into the mixed solution a by using a hanging drop method, starting to record hanging drop time until the oil phase falls off when the oil phase is completely hung and dropped, and drawing a standard curve a according to the hanging drop time and the corresponding logarithmic value of the concentration of the acetylcholinesterase standard solution;
(4) determination of standard curve of acetylcholinesterase inhibitor: respectively adding the acetylcholinesterase standard solution prepared in the step (2) into the acetylcholinesterase inhibitor standard solutions with different concentrations prepared in the step (2), incubating for 25-55 min at 20-60 ℃, continuously adding the surfactant solution prepared in the step (1) after incubation is completed, and incubating for 25-55 min at 20-60 ℃ to obtain a mixed solution b; then, slowly injecting the oil phase into the mixed solution b by using a hanging drop method, starting to record hanging drop time until the oil phase falls off when the oil phase is completely hung and dropping, and drawing a standard curve b according to the hanging drop time and the corresponding logarithm value of the concentration of the standard solution of the acetylcholinesterase inhibitor;
(5) and (3) measuring the acetylcholinesterase of the sample to be detected: dissolving acetylcholinesterase to be detected in a Tris-HCl buffer solution to obtain an acetylcholinesterase solution of a sample to be detected, determining the hanging drop time of the system according to the method in the step (3), and calculating by comparing the determined hanging drop time with a standard curve a to obtain the content of the acetylcholinesterase in the sample to be detected;
determination of acetylcholinesterase inhibitor of sample to be tested: and (3) dissolving the acetylcholinesterase inhibitor of the sample to be detected in a Tris-HCl buffer solution, determining the hanging drop time of the system according to the method in the step (4), and calculating by comparing the determined hanging drop time with a standard curve b to obtain the content of the acetylcholinesterase inhibitor in the sample to be detected.
Preferably, in step (1), the surfactant is lauryl sodium sulfate, myristyl sodium sulfate, cetyl sodium sulfate, lauryl trimethyl sodium bromide, myristyl trimethyl sodium bromide, cetyl trimethyl sodium bromide, formyl choline chloride, acetyl choline chloride or myristyl choline chloride.
Further preferably, the surfactant is myristoylcholine chloride.
Preferably, in step (1), the concentration of the surfactant solution is 0.1 to 1 mM.
Further preferably, the concentration of the surfactant solution is 1 mM.
Preferably, in the step (2), the concentration of acetylcholinesterase in the standard acetylcholinesterase solution is 0.1-1000 mU/mL, and the concentration of acetylcholinesterase inhibitor in the standard acetylcholinesterase inhibitor solution is 10-10000 nM.
Further preferably, the acetylcholinesterase inhibitor is neostigmine bromide.
Preferably, in step (3), the volume ratio of the acetylcholinesterase standard solution to the surfactant solution is 1: 1.
preferably, in step (3), the volume ratio of the oil phase to the mixed solution a is 1: (70-90).
Preferably, in the step (3) and the step (4), the oil phase is a liquid with the density close to that of water, and the density of the oil phase is 0.9-1.2 g/cm3。
More preferably, the oil phase is liquid crystal 5 CB.
According to the invention, in the step (3) and the step (4), the hanging drop method comprises the following specific steps:
and (3) sucking the oil phase by using a micro-syringe, fixing the micro-syringe above the transparent glass bottle by using an iron stand, ensuring that the needle point of the micro-syringe is positioned in the middle of the mixed solution a or the mixed solution b, slowly injecting the oil phase, ensuring that the oil phase does not drop in the injection process, and starting to record the hanging drop time until the oil phase drops when the oil phase is completely hung and dropped on the needle point of the micro-syringe.
Preferably, in step (4), the volume ratio of the standard acetylcholinesterase inhibitor solution to the standard acetylcholinesterase solution is 1: 1.
preferably, in step (4), the volume ratio of the oil phase to the mixed solution b is 1: (70-90).
Preferably, in step (4), the concentration of the standard acetylcholinesterase solution is 10 mU/mL.
Preferably, in step (5), the acetylcholinesterase inhibitor is neostigmine bromide.
According to the invention, the method for detecting acetylcholinesterase and the inhibitor thereof based on the pendant drop method is used for detecting acetylcholinesterase in serum or detecting the acetylcholinesterase inhibitor in a medicine.
Advantageous effects
1. The detection method provided by the invention is used for detecting AChE based on different hanging drop times of an oil phase in surfactant solutions with different concentrations in a hanging drop method, the hanging drop time corresponding to a surfactant solution with high concentration is short, the hanging drop time corresponding to a surfactant solution with low concentration is long, when AChE is used for carrying out enzymolysis on the surfactant, the concentration of the surfactant solution can be obviously reduced after the AChE with high concentration is subjected to enzymolysis on the surfactant, the corresponding hanging drop time is long, otherwise, the concentration of the surfactant solution is not obviously reduced after the AChE with low concentration is subjected to enzymolysis on the surfactant, the corresponding hanging drop time is short, and further the AChE is detected, so that a development prospect is provided for later clinical diagnosis and application.
2. The method for detecting the acetylcholinesterase and the inhibitor thereof based on the pendant drop method has the advantages of simple operation process, low cost, accurate detection, high sensitivity, quantitative detection limit of the acetylcholinesterase to 0.1mU/mL, and quantitative detection limit of the acetylcholinesterase inhibitor to 1 nM.
Drawings
FIG. 1 is a diagram of an apparatus used in the pendant drop method in the example.
FIG. 2 is a schematic diagram showing the hanging drop time of the oil phase in solutions of myristoylcholine chloride (Myr) of different concentrations in example 1.
FIG. 3 is a standard curve of the dependence of the logarithmic value of the concentration of acetylcholinesterase standard solution on the hanging drop time in example 2.
FIG. 4 is a standard curve of the log of neostigmine bromide standard solution concentration versus hanging drop time in example 3.
FIG. 5 is a standard curve of the log of the concentration of standard solutions of acetylcholinesterase in serum versus hanging drop time in example 4.
In the figure: 1. iron stand table, 2, micro-syringe, 3, transparent glass bottle.
Detailed Description
The technical solutions of the present invention are further described below with reference to specific embodiments, but the scope of the present invention is not limited thereto. Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
In the following examples, myristylcholine chloride (Myr) is available from Shanghai Arlatin Biotechnology Ltd. The micro-injector is sold by Shanghai Pigeon worker and trade company Limited.
An experimental apparatus used in the pendant drop method in the examples is shown in fig. 1, and includes an iron support 1, a micro syringe 2, and a transparent glass bottle 3, the micro syringe 2 being fixed to the iron support 1, the transparent glass bottle 3 being located below the micro syringe 2.
Example 1 screening of a suitable concentration of tetradecanoylcholine chloride solution
0.035g of myristylcholine chloride (Myr) was weighed out and dissolved in 10mL of Tris-HCl buffer, and sonicated for 30min to give a 10mM Myr solution, which was then diluted to 1mM, 0.1mM, 0.01 mM.
Respectively sucking 800 mu L of Myr solutions with concentrations of 10mM, 1mM, 0.1mM and 0.01mM, transferring the Myr solutions into a 1mL glass bottle, sucking 10mu L of liquid crystal 5CB by using a micro syringe, fixing the micro syringe above the glass bottle by using an experimental device, ensuring that the needle point of the micro syringe is in the middle position of the solution, slowly injecting the liquid crystal 5CB, ensuring that the liquid crystal 5CB does not fall off in the injection process, and starting to record the hanging drop time until the liquid crystal 5CB falls off when the liquid crystal 5CB is completely hung and dropped on the needle point of the micro syringe, wherein the result is shown in FIG. 2.
As can be seen from FIG. 2, the concentration of liquid crystal 5CB in a 10mM Myr solution is too high to cause the hanging drop phenomenon; the liquid crystal 5CB is excessively dripped in the Myr solution with the concentration of 0.01mM, and the liquid crystal 5CB is dripped in the Myr solution with the concentration of 1mM and 0.1mM for a proper time, so that the liquid crystal 5CB can be used for detecting the content of acetylcholinesterase.
Example 2 detection of acetylcholinesterase
A method for detecting acetylcholinesterase based on a pendant drop method comprises the following steps:
(1) preparing a tetradecanoylcholine chloride solution: weighing 0.0035g of myristoyl choline chloride (Myr) to be dissolved in 10mL of Tris-HCl buffer solution, and carrying out ultrasonic treatment for 30min to uniformly mix to obtain 1mM myristoyl choline chloride solution;
(2) preparation of acetylcholinesterase standard solution: respectively dissolving the standard acetylcholinesterase products in Tris-HCl buffer solution to obtain standard acetylcholinesterase solutions with the concentrations of 0.1mU/mL, 1mU/mL, 10mU/mL, 100mU/mL and 1000 mU/mL;
(3) determination of standard curve of acetylcholinesterase: respectively adding 400 mu L of the tetradecanoylcholine chloride solution prepared in the step (1) into 400 mu L of the acetylcholinesterase standard solution with different concentrations prepared in the step (2), and incubating for 40min at 30 ℃ to obtain a mixed solution; then, slowly injecting 10mu L of liquid crystal 5CB into the mixed solution by using a hanging drop method, starting to record hanging drop time until the liquid crystal 5CB falls off when the liquid crystal 5CB is completely hung and then drawing a standard curve according to the hanging drop time and a corresponding logarithmic value of the concentration of the acetylcholinesterase standard solution, wherein the standard curve is shown in figure 3;
(4) and (3) measuring the acetylcholinesterase of the sample to be detected: and (3) dissolving the acetylcholinesterase to be detected in a Tris-HCl buffer solution to obtain an acetylcholinesterase solution of a sample to be detected, determining the hanging drop time of the system according to the method in the step (3), and calculating by comparing the determined hanging drop time with a standard curve to obtain the content of the acetylcholinesterase in the sample to be detected.
Example 3 detection of neostigmine Bromide
A method for detecting acetylcholinesterase inhibitor based on a pendant drop method comprises the following steps:
(1) preparing a tetradecanoylcholine chloride solution: weighing 0.0035g of Myr, dissolving in 10mL of Tris-HCl buffer solution, and uniformly mixing by ultrasonic treatment for 30min to obtain 1mM tetradecanoylcholine chloride solution;
(2) preparing an acetylcholinesterase standard solution and an acetylcholinesterase inhibitor standard solution: respectively dissolving the standard acetylcholinesterase products in Tris-HCl buffer solution to obtain standard acetylcholinesterase solutions with the concentrations of 0.1mU/mL, 1mU/mL, 10mU/mL, 100mU/mL and 1000 mU/mL; respectively dissolving the neostigmine bromide standard substances in Tris-HCl buffer solution to prepare neostigmine bromide standard solutions with the concentrations of 10nM, 50nM, 100nM, 1000nM and 10000nM respectively;
(3) determination of the standard curve of neostigmine bromide: adding 200 mU L of acetylcholinesterase standard solution with the concentration of 10mU/mL prepared in the step (2) into 200 mU L of neostigmine bromide standard solution with different concentrations prepared in the step (3), incubating for 40min at 30 ℃, continuing adding 400 mU L of tetradecanoylcholine chloride solution prepared in the step (1) after incubation is finished, and incubating for 40min at 30 ℃ to obtain a mixed solution; finally, slowly injecting 10mu L of liquid crystal 5CB into the mixed solution by using a hanging drop method, starting to record hanging drop time until the liquid crystal 5CB falls off when the liquid crystal 5CB is completely hung and then drawing a standard curve according to the hanging drop time and the corresponding logarithm value of the concentration of the neostigmine bromide standard solution, as shown in figure 4;
(4) determination of the sample to be tested for neostigmine bromide: and (3) dissolving the neostigmine bromide of the sample to be detected in a Tris-HCl buffer solution, determining the hanging drop time of the system according to the method in the step (3), and calculating by comparing the determined hanging drop time with a standard curve to obtain the neostigmine bromide content in the sample to be detected.
Example 4 detection of acetylcholinesterase in serum by the method of the invention
A method for detecting acetylcholinesterase in serum based on a pendant drop method comprises the following steps:
(1) diluting normal human serum by 500 times in Tris-HCl buffer solution, and then adding acetylcholinesterase standard substance to obtain serum standard substances with concentrations of 0.1mU/mL, 1mU/mL, 10mU/mL, 100mU/mL and 1000 mU/mL;
(2) weighing 0.0035g of myristylcholine chloride (Myr), dissolving in 10mL of Tris-HCl buffer solution, and performing ultrasonic treatment for 30min to uniformly mix to obtain a myristylcholine chloride solution with the concentration of 1 mM;
(3) respectively adding 400 mu L of the tetradecanoylcholine chloride solution prepared in the step (2) into 400 mu L of the acetylcholinesterase standard solution with different concentrations prepared in the step (1), and incubating for 40min at 30 ℃ to obtain a mixed solution; then, 10. mu.L of liquid crystal 5CB was slowly injected into the mixed solution by the hanging drop method, when the liquid crystal 5CB was completely hung, the hanging drop time was recorded until the liquid crystal 5CB dropped, and then a standard curve was plotted based on the hanging drop time and the corresponding logarithmic value of the acetylcholinesterase concentration in the serum standard, as shown in FIG. 5.
And (3) taking serum standard substances of 0.1mU/mL, 1mU/mL and 10mU/mL as samples to be detected, measuring the hanging drop time according to the method in the step (3), averagely carrying out three times of each group of tests, taking the average values for comparison, and then calculating the content of acetylcholinesterase in the serum samples according to the standard curve in the embodiment. The calculation results are shown in table 1.
As can be seen from Table 1, the method of the present invention can rapidly and sensitively detect the ethylene cholinesterase in the serum effectively.
The foregoing is considered as illustrative only of the embodiments of the invention. The scope of the present invention is not limited thereto, and any changes or substitutions that can be easily made by those skilled in the art within the technical scope of the present invention will be covered by the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (14)
1. A method for detecting acetylcholinesterase and an inhibitor thereof based on a pendant drop method is characterized by comprising the following steps:
(1) preparing a surfactant solution: dissolving a surfactant in a Tris-HCl buffer solution, and uniformly mixing by ultrasonic treatment for 25-35 min to obtain a surfactant solution with the concentration of 0.01-1 mM;
(2) preparing an acetylcholinesterase standard solution and an acetylcholinesterase inhibitor standard solution: respectively dissolving the acetylcholinesterase standard substance and the acetylcholinesterase inhibitor standard substance into a Tris-HCl buffer solution to prepare acetylcholinesterase standard solutions and acetylcholinesterase inhibitor standard solutions with different concentrations;
(3) determination of standard curve of acetylcholinesterase: respectively adding the surfactant solution prepared in the step (1) into the acetylcholinesterase standard solutions with different concentrations prepared in the step (2), and incubating for 25-55 min at 20-60 ℃ to obtain a mixed solution a; then, slowly injecting the oil phase into the mixed solution a by using a hanging drop method, starting to record hanging drop time until the oil phase falls off when the oil phase is completely hung and dropped, and drawing a standard curve a according to the hanging drop time and the corresponding logarithmic value of the concentration of the acetylcholinesterase standard solution;
(4) determination of standard curve of acetylcholinesterase inhibitor: respectively adding the acetylcholinesterase standard solution prepared in the step (2) into the acetylcholinesterase inhibitor standard solutions with different concentrations prepared in the step (2), incubating for 25-55 min at 20-60 ℃, continuously adding the surfactant solution prepared in the step (1) after incubation is completed, and incubating for 25-55 min at 20-60 ℃ to obtain a mixed solution b; then, slowly injecting the oil phase into the mixed solution b by using a hanging drop method, starting to record hanging drop time until the oil phase falls off when the oil phase is completely hung and dropping, and drawing a standard curve b according to the hanging drop time and the corresponding logarithm value of the concentration of the standard solution of the acetylcholinesterase inhibitor;
(5) and (3) measuring the acetylcholinesterase of the sample to be detected: dissolving acetylcholinesterase to be detected in a Tris-HCl buffer solution to obtain an acetylcholinesterase solution of a sample to be detected, determining the hanging drop time of the system according to the method in the step (3), and calculating by comparing the determined hanging drop time with a standard curve a to obtain the content of the acetylcholinesterase in the sample to be detected;
determination of acetylcholinesterase inhibitor of sample to be tested: and (3) dissolving the acetylcholinesterase inhibitor of the sample to be detected in a Tris-HCl buffer solution, determining the hanging drop time of the system according to the method in the step (4), and calculating by comparing the determined hanging drop time with a standard curve b to obtain the content of the acetylcholinesterase inhibitor in the sample to be detected.
2. The method for detecting acetylcholinesterase and its inhibitor according to claim 1, wherein in step (1), the surfactant is sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium dodecyl trimethyl bromide, sodium tetradecyl trimethyl bromide, sodium hexadecyl trimethyl bromide, formyl choline chloride, acetyl choline chloride or tetradecyl choline chloride.
3. The method for detecting acetylcholinesterase and its inhibitor according to claim 2, wherein the surfactant is myristoylcholine chloride.
4. The method for detecting acetylcholinesterase and its inhibitor according to claim 1, wherein in step (1), the concentration of the surfactant solution is 0.1-1 mM.
5. The method for detecting acetylcholinesterase and its inhibitor according to claim 4, wherein the concentration of the surfactant solution is 1 mM.
6. The method for detecting acetylcholinesterase and its inhibitor according to claim 1, wherein in step (2), the concentration of acetylcholinesterase in the standard acetylcholinesterase solution is 0.1-1000 mU/mL, and the concentration of acetylcholinesterase inhibitor in the standard acetylcholinesterase inhibitor solution is 10-10000 nM.
7. The method for detecting acetylcholinesterase and its inhibitor according to claim 6, wherein the acetylcholinesterase inhibitor is neostigmine bromide.
8. The method for detecting acetylcholinesterase and its inhibitor based on the pendant drop method according to claim 1, wherein in step (3), the volume ratio of the standard acetylcholinesterase solution to the surfactant solution is 1: 1; the volume ratio of the oil phase to the mixed solution a is 1: (70-90).
9. The method for detecting acetylcholinesterase and its inhibitor according to claim 1, wherein in steps (3) and (4), the oil phase is a liquid with a density close to that of water, and the density of the oil phase is 0.9-1.2 g/cm3。
10. The method for detecting acetylcholinesterase and its inhibitor according to claim 9, wherein the oil phase is liquid crystal 5 CB.
11. The method for detecting acetylcholinesterase and its inhibitor according to claim 1, wherein the method of pendant drop method is as follows, in steps (3) and (4):
and (3) sucking the oil phase by using a micro-syringe, fixing the micro-syringe above the transparent glass bottle by using an iron stand, ensuring that the needle point of the micro-syringe is positioned in the middle of the mixed solution a or the mixed solution b, slowly injecting the oil phase, ensuring that the oil phase does not drop in the injection process, and starting to record the hanging drop time until the oil phase drops when the oil phase is completely hung and dropped on the needle point of the micro-syringe.
12. The method for detecting acetylcholinesterase and its inhibitor according to claim 1, wherein in step (4), the volume ratio of the standard solution of acetylcholinesterase inhibitor to the standard solution of acetylcholinesterase is 1: 1; the volume ratio of the oil phase to the mixed solution b is 1: (70-90); the concentration of the standard acetylcholinesterase solution is 10 mU/mL.
13. The method for detecting acetylcholinesterase and its inhibitor according to claim 1, wherein in step (5), the acetylcholinesterase inhibitor is neostigmine bromide.
14. A method for detecting acetylcholinesterase and its inhibitor based on the hanging drop method as claimed in any one of claims 1-13, which is used for detecting acetylcholinesterase in serum or acetylcholinesterase inhibitor in medicine.
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