阅读说明：本技术 β淀粉样蛋白低聚物的检测方法、检测β淀粉样蛋白低聚物的试剂盒及应用 (Detection method of beta amyloid protein oligomer, kit for detecting beta amyloid protein oligomer and application ) 是由 张文娟 丁海云 李雪驼 刘辰 于 2021-08-19 设计创作，主要内容包括：本发明提供了一种β淀粉样蛋白低聚物的检测方法、检测β淀粉样蛋白低聚物的试剂盒及应用,涉及纳米生物技术领域,本发明提供的β淀粉样蛋白低聚物的检测方法,采用分光光度法检测人体肠道排泄物中β淀粉样蛋白低聚物的含量,该方法操作简单,能够达到定性半定量的有益效果。并且,对于与阿尔兹海默症密切相关的β淀粉样蛋白低聚物,应用本发明提供的检测方法,通过测定人体肠道排泄物样品对阿尔兹海默症的鉴别具有实际意义,对人体无任何损害。(The invention provides a detection method of beta amyloid protein oligomer, a kit for detecting the beta amyloid protein oligomer and application, and relates to the technical field of nano biology. Moreover, for beta amyloid oligomer closely related to the Alzheimer's disease, the detection method provided by the invention has practical significance for identifying the Alzheimer's disease by determining human intestinal excreta samples, and has no damage to human bodies.)

1. A method for detecting amyloid beta oligomer, comprising the steps of:

(a) preparing a standard solution of amyloid beta oligomer;

(b) drawing a standard curve by using the standard solution obtained in the step (a) and the corresponding absorbance ratio;

(c) calculating the content of the beta amyloid oligomer according to the standard curve obtained in the step (b) and the absorbance ratio of the treated sample to be detected;

wherein, the sample to be detected is intestinal excrement, and the amyloid beta oligomer comprises A beta 40 and A beta 42.

2. The detection method according to claim 1, wherein the preparation method of the standard solution of a β 40 comprises:

dissolving and drying the protein peptide by using 1,1,1,3,3, 3-hexafluoroisopropanol, then re-dissolving the dried solid by using dimethyl sulfoxide, and diluting the solid by using PBS buffer solution into Abeta 40 standard solutions with the concentrations of 35nM, 50nM, 100nM, 150nM, 250nM, 300nM and 400nM respectively;

preferably, the concentration of the protein peptide is 1 mg/ml;

preferably, ultrasonic treatment is carried out for 25-35 min after the protein peptide is dissolved, then drying treatment is carried out, preferably ultrasonic treatment is carried out for 30min, and nitrogen is blown for drying;

preferably, ultrasonic treatment is carried out for 25-35 s after redissolution, and then dilution treatment is carried out, preferably ultrasonic treatment is carried out for 30 s;

preferably, the concentration of the PBS buffer is 10mM, and the pH is 7.4;

preferably, the method further comprises the step of incubating for 24h at 37 ℃.

3. The detection method according to claim 1, wherein the preparation method of the standard solution of a β 42 comprises:

dissolving the protein peptide by using 1,1,1,3,3, 3-hexafluoroisopropanol and drying, then re-dissolving the dried solid by using NaOH solution, and diluting the solid by using PBS buffer solution into standard solutions of Abeta 42 with the concentrations of 0 mu M, 0.75 mu M, 1 mu M, 1.5 mu M, 2.5 mu M, 3 mu M and 5 mu M respectively;

preferably, the concentration of the protein peptide is 1 mg/ml;

preferably, ultrasonic treatment is carried out for 25-35 min after the protein peptide is dissolved, then drying treatment is carried out, preferably ultrasonic treatment is carried out for 30min, and nitrogen is blown for drying;

preferably, ultrasonic treatment is carried out for 25-35 s after redissolution, and then dilution treatment is carried out, preferably ultrasonic treatment is carried out for 30 s;

preferably, the concentration of the PBS buffer is 10mM, and the pH is 7.4;

preferably, the method further comprises the step of incubating for 24h at 37 ℃.

4. The detection method according to claim 1, wherein the method for drawing the standard curve of the A beta 40 comprises the following steps:

respectively adding an Alzheimer's disease marker aptamer into Abeta 40 standard solutions with different concentrations, adding nanogold after reaction, adding NaCl solution after the reaction again, then determining absorbance values at 520nm and 725nm wavelengths respectively by using an ultraviolet spectrophotometer, and drawing a standard curve by taking the concentration of the Abeta 40 standard solution as an abscissa and the ratio of the absorbance at 725nm to 520nm as an ordinate;

preferably, the alzheimer's disease marker aptamer comprises Apt at a concentration of 2 μ M;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the concentration of the NaCl solution is 350 mM;

preferably, the volume ratio of the nanogold, the Alzheimer's disease marker aptamer, the A beta 40 standard solution and the NaCl solution is 60:18:9: 40;

preferably, the equation of the resulting standard curve is-0.004 x +1.5829 and the linear coefficient R is 0.9888.

5. The detection method according to claim 1, wherein the method for drawing the standard curve of the A beta 42 comprises the following steps:

respectively adding Alzheimer's disease marker specific binding peptide into Abeta 42 standard solutions with different concentrations, adding nanogold after reaction, respectively measuring absorbance values at 520nm and 615nm wavelength positions by using an ultraviolet spectrophotometer after the reaction again, and drawing a standard curve by taking the concentration of the Abeta 42 standard solution as an abscissa and the ratio of absorbance at 615nm to absorbance at 520nm as an ordinate;

preferably, the Alzheimer's disease marker specific binding peptide comprises PrP at a concentration of 250 ng/mL;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the volume ratio of the nanogold, the A beta 42 standard solution and the Alzheimer disease marker specific binding peptide solution is 2:1: 2;

preferably, the equation of the resulting standard curve is-0.1023 x +0.6693 and the linear coefficient R is 0.9880.

6. The detection method according to claim 1, wherein the method for processing the sample to be detected comprises:

adding each 0.25g of intestinal excrement into 10mL of PBS buffer solution for dissolving, filtering supernate after solid-liquid separation to obtain a treated sample to be detected;

preferably, the concentration of the PBS buffer is 10mM, and the pH is 7.2 or 7.4;

preferably, the solid-liquid separation is carried out by a centrifugal mode, and the centrifugal condition is preferably 10000rpm for 10 min;

preferably, filtration is performed using a 0.22 μm aqueous membrane filter.

7. The method according to any one of claims 1 to 6, wherein the determination of A β 40 in the sample to be tested comprises:

adding an Alzheimer's disease marker aptamer into the treated sample to be detected, adding nanogold after reaction, adding a NaCl solution after reaction again for reaction, and then determining the absorbance value;

preferably, the alzheimer's disease marker aptamer comprises Apt at a concentration of 2 μ M;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the concentration of the NaCl solution is 370 mM;

preferably, the volume ratio of the nanogold, the Alzheimer's disease marker aptamer, the sample to be detected and the NaCl solution is 60:18:9: 40.

8. The method according to any one of claims 1 to 6, wherein the determination of A β 42 in the sample to be tested comprises:

adding the Alzheimer's disease marker specific binding peptide into the treated sample to be detected, adding the nanogold after reaction, and measuring the absorbance value after re-reaction;

preferably, the alzheimer's marker specific binding peptide comprises PrP at a concentration of 5 μ M;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the volume ratio of the nanogold, the sample to be tested and the Alzheimer's disease marker specific binding peptide solution is 2:1: 2.

9. A kit for detecting amyloid beta oligomer, comprising a standard solution of amyloid beta oligomer in the detection method according to any one of claims 1 to 7.

10. Use of the kit for detecting amyloid beta oligomer according to claim 9 for the preparation of a product for detecting alzheimer's disease.

Technical Field

The invention relates to the technical field of nano biology, in particular to a detection method of beta amyloid oligomer, a kit for detecting the beta amyloid oligomer and application of the kit.

Background

Alzheimer's Disease (AD) is a rapid and common neurodegenerative disease characterized by decreased cognitive ability, memory impairment, dyskinesia, etc., which severely affects the quality of life of patients. Now, there are about two thousand four million patients worldwide, and the incidence rate thereof is higher and higher with the progress of aging of society, and it is expected that the number of patients will rise to about 1 hundred million after thirty years, which will cause a heavy burden on society. The pathogenesis of the AD is very complex, and the cause of the AD needs to be further determined. Currently, there are hypotheses about the pathogenic mechanism of AD disease mainly including abnormal deposition of β -amyloid, hyperphosphorylation of Tau protein, oxidative stress, inflammatory reaction, cholinergic theory, etc., among which the abnormal deposition of β -amyloid is widely spotlighted.

Beta-amyloid (beta) is Amyloid Precursor Protein (APP) produced by a complex enzymatic cleavage of beta, gamma-secretase. Clinical studies have shown that abnormal levels of β are closely associated with the progression of the disease course of AD. Beta exists in a variety of forms, the monomeric form of beta is not neurotoxic, whereas oligomers (a β) and fibrils formed by a nucleation-dependent complex process exhibit neurotoxicity. Among them, β 42 and β 40 can aggregate into a β oligomers (a β 42 or a β 40), then aggregate into fibrils, and finally cause plaque formation, and a β can cause AD disease. Therefore, most people identify AD by detecting either a β 42 or a β 40.

At present, a main reference sample for research based on Abeta is cerebral medullary fluid, so that cerebrospinal fluid of a patient needs to be obtained if AD needs to be detected, a regular hospital needs to be obtained when the cerebrospinal fluid is extracted, the patient is anesthetized after corresponding examination, and the cerebrospinal fluid is extracted, so that certain physical trauma to the patient is inevitably caused.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The main object of the present invention is to provide a method for detecting amyloid beta oligomers, which alleviates at least one of the technical problems of the prior art.

The second purpose of the invention is to provide a kit for detecting beta amyloid oligomer.

The third purpose of the invention is to provide the application of the kit for detecting the beta amyloid oligomer.

The invention provides a detection method of beta amyloid oligomer, which comprises the following steps:

(a) preparing a standard solution of amyloid beta oligomer;

(b) drawing a standard curve by using the standard solution obtained in the step (a) and the corresponding absorbance ratio;

(c) calculating the content of the beta amyloid oligomer according to the standard curve obtained in the step (b) and the absorbance ratio of the treated sample to be detected;

wherein, the sample to be detected is intestinal excrement, and the amyloid beta oligomer comprises A beta 40 and A beta 42.

Further, the preparation method of the standard solution of the Abeta 40 comprises the following steps:

dissolving and drying the protein peptide by using 1,1,1,3,3, 3-hexafluoroisopropanol, then re-dissolving the dried solid by using dimethyl sulfoxide, and diluting the solid by using PBS buffer solution into Abeta 40 standard solutions with the concentrations of 35nM, 50nM, 100nM, 150nM, 250nM, 300nM and 400nM respectively;

preferably, the concentration of the protein peptide is 1 mg/ml;

preferably, ultrasonic treatment is carried out for 25-35 min after the protein peptide is dissolved, then drying treatment is carried out, preferably ultrasonic treatment is carried out for 30min, and nitrogen is blown for drying;

preferably, ultrasonic treatment is carried out for 25-35 s after redissolution, and then dilution treatment is carried out, preferably ultrasonic treatment is carried out for 30 s;

preferably, the concentration of the PBS buffer is 10mM, and the pH is 7.4;

preferably, the method further comprises the step of incubating for 24h at 37 ℃.

Further, the preparation method of the standard solution of the A beta 42 comprises the following steps:

dissolving the protein peptide by using 1,1,1,3,3, 3-hexafluoroisopropanol and drying, then re-dissolving the dried solid by using NaOH solution, and diluting the solid by using PBS buffer solution into standard solutions of Abeta 42 with the concentrations of 0 mu M, 0.75 mu M, 1 mu M, 1.5 mu M, 2.5 mu M, 3 mu M and 5 mu M respectively;

preferably, the concentration of the protein peptide is 1 mg/ml;

preferably, ultrasonic treatment is carried out for 25-35 min after the protein peptide is dissolved, then drying treatment is carried out, preferably ultrasonic treatment is carried out for 30min, and nitrogen is blown for drying;

preferably, ultrasonic treatment is carried out for 25-35 s after redissolution, and then dilution treatment is carried out, preferably ultrasonic treatment is carried out for 30 s;

preferably, the concentration of the PBS buffer is 10mM, and the pH is 7.4;

preferably, the method further comprises the step of incubating for 24h at 37 ℃.

Further, the method for drawing the A beta 40 standard curve comprises the following steps:

respectively adding an Alzheimer's disease marker aptamer into Abeta 40 standard solutions with different concentrations, adding nanogold after reaction, adding NaCl solution after the reaction again, then determining absorbance values at 520nm and 725nm wavelengths respectively by using an ultraviolet spectrophotometer, and drawing a standard curve by taking the concentration of the Abeta 40 standard solution as an abscissa and the ratio of the absorbance at 725nm to 520nm as an ordinate;

preferably, the alzheimer's disease marker aptamer comprises Apt at a concentration of 2 μ M;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the concentration of the NaCl solution is 350 mM;

preferably, the volume ratio of the nanogold, the Alzheimer's disease marker aptamer, the A beta 40 standard solution and the NaCl solution is 60:18:9: 40;

preferably, the equation of the resulting standard curve is-0.004 x +1.5829 and the linear coefficient R is 0.9888.

Further, the method for drawing the standard curve of the A beta 42 comprises the following steps:

respectively adding Alzheimer's disease marker specific binding peptide into Abeta 42 standard solutions with different concentrations, adding nanogold after reaction, respectively measuring absorbance values at 520nm and 615nm wavelength positions by using an ultraviolet spectrophotometer after the reaction again, and drawing a standard curve by taking the concentration of the Abeta 42 standard solution as an abscissa and the ratio of absorbance at 615nm to absorbance at 520nm as an ordinate;

preferably, the Alzheimer's disease marker specific binding peptide comprises PrP at a concentration of 250 ng/mL;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the volume ratio of the nanogold, the A beta 42 standard solution and the Alzheimer disease marker specific binding peptide solution is 2:1: 2;

preferably, the equation of the resulting standard curve is-0.1023 x +0.6693 and the linear coefficient R is 0.9880.

Further, the method for processing the sample to be tested comprises the following steps:

adding each 0.25g of intestinal excrement into 10mL of PBS buffer solution for dissolving, filtering supernate after solid-liquid separation to obtain a treated sample to be detected;

preferably, the concentration of the PBS buffer is 10mM, and the pH is 7.2 or 7.4;

preferably, the solid-liquid separation is carried out by a centrifugal mode, and the centrifugal condition is preferably 10000rpm for 10 min;

preferably, filtration is performed using a 0.22 μm aqueous membrane filter.

Further, the method for determining the Abeta 40 in the sample to be detected comprises the following steps:

adding an Alzheimer's disease marker aptamer into the treated sample to be detected, adding nanogold after reaction, adding a NaCl solution after reaction again for reaction, and then determining the absorbance value;

preferably, the alzheimer's disease marker aptamer comprises Apt at a concentration of 2 μ M;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the concentration of the NaCl solution is 370 mM;

preferably, the volume ratio of the nanogold, the Alzheimer's disease marker aptamer, the sample to be detected and the NaCl solution is 60:18:9: 40.

Further, the method for determining the A beta 42 in the sample to be detected comprises the following steps:

adding the Alzheimer's disease marker specific binding peptide into the treated sample to be detected, adding the nanogold after reaction, and measuring the absorbance value after re-reaction;

preferably, the alzheimer's marker specific binding peptide comprises PrP at a concentration of 5 μ M;

preferably, the reaction is carried out under the condition of keeping out of the sun, and the reaction time is 3-7 min, preferably 5 min;

preferably, the volume ratio of the nanogold, the sample to be tested and the Alzheimer's disease marker specific binding peptide solution is 2:1: 2.

The invention also provides a kit for detecting the beta amyloid oligomer, which comprises the standard solution of the beta amyloid oligomer in the detection method.

In addition, the invention also provides application of the kit for detecting the beta amyloid protein oligomer in preparation of a product for detecting Alzheimer's disease.

Compared with the prior art, the invention has the following beneficial effects:

the detection method of the amyloid beta oligomer provided by the invention adopts a spectrophotometry method to detect the content of the amyloid beta oligomer in human intestinal excreta, is simple to operate, and can achieve the beneficial effects of qualitative and semi-quantitative. Moreover, for beta amyloid oligomer closely related to Alzheimer's disease, the detection method provided by the invention is applied, and the identification of Alzheimer's disease by determining the human intestinal tract excreta sample has practical significance and no damage to human body.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the concentration of A β 40 and A provided in example 1 of the present invention_725nm/A_520nmA standard working curve of absorbance ratio;

FIG. 2 shows the concentration of A β 42 and A provided in example 2 of the present invention_615nm/A_520nmStandard working curve of absorbance ratio.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the present invention, there is provided a method for detecting amyloid beta oligomer, comprising the steps of:

(a) preparing a standard solution of amyloid beta oligomer;

(b) drawing a standard curve by using the standard solution obtained in the step (a) and the corresponding absorbance ratio;

(c) calculating the content of the beta amyloid oligomer according to the standard curve obtained in the step (b) and the absorbance ratio of the treated sample to be detected;

wherein, the sample to be detected is intestinal excrement, and the amyloid beta oligomer comprises A beta 40 and A beta 42.

The detection method of the amyloid beta oligomer provided by the invention adopts a spectrophotometry method to detect the content of the amyloid beta oligomer in human intestinal excreta, is simple to operate, and can achieve the beneficial effects of qualitative and semi-quantitative. Moreover, for beta amyloid oligomer closely related to Alzheimer's disease, the detection method provided by the invention is applied, and the identification of Alzheimer's disease by determining the human intestinal tract excreta sample has practical significance and no damage to human body.

In some preferred embodiments, the standard solution of a β 40 is prepared by a method comprising:

the protein peptide was dissolved and dried with 1,1,1,3,3, 3-hexafluoroisopropanol, and then the dried solid was redissolved with dimethyl sulfoxide and diluted with PBS buffer to standard solutions of a β 40 at concentrations of 35nM, 50nM, 100nM, 150nM, 250nM, 300nM and 400nM, respectively.

The protein peptide is dissolved by using 1,1,1,3,3, 3-hexafluoroisopropanol, and the characteristics of high polarity, easy mixing with water and a plurality of organic solvents, good thermal stability and the like of Hexafluoroisopropanol (HFIP) are utilized, so that the HFIP can become an ideal solvent in a plurality of polymerization systems. Can make beta amyloid protein in uniform dispersion state.

The concentration of the protein peptide may be, for example, but not limited to, 1 mg/ml.

Preferably, ultrasonic treatment is carried out for 25-35 min after the protein peptide is dissolved, and then drying treatment is carried out.

The protein peptide can be dissolved more fully by ultrasonic treatment, for example, but not limited to, 25min, 28min, 30min, 32min or 35min, preferably 30min, and dried by nitrogen blowing to avoid oxidation and other unnecessary reactions;

preferably, ultrasonic treatment is carried out for 25-35 s after redissolution, and then dilution treatment is carried out.

Likewise, the solid can be further sufficiently dissolved in dimethyl sulfoxide using sonication for a period of time such as, but not limited to, 25s, 28s, 30s, 32s or 35s, preferably 30 s.

Preferably, the method further comprises the step of incubating for 24h at 37 ℃.

A beta is one of normal products of human metabolism, and the generation and degradation of A beta are in dynamic balance under normal conditions, but when the A beta is in pathological conditions, the generation and degradation of the A beta are increased, so that a large amount of A beta is deposited, and the A beta is automatically aggregated into soluble oligomers by monomers. Incubation at 37 ℃ for 24h was a condition that mimics aggregation of a β in vivo.

In some preferred embodiments, the standard solution of a β 42 is prepared by a method comprising:

dissolving the protein peptide by using 1,1,1,3,3, 3-hexafluoroisopropanol and drying, then re-dissolving the dried solid by using NaOH solution, and diluting the solid by using PBS buffer solution into standard solutions of Abeta 42 with the concentrations of 0 mu M, 0.75 mu M, 1 mu M, 1.5 mu M, 2.5 mu M, 3 mu M and 5 mu M respectively;

the concentration of the protein peptide may be, for example, but not limited to, 1 mg/ml.

Redissolving with a specific alkaline solution NaOH solution can provide better polymerization of a β 42, and the polymerization effect is not satisfactory without using NaOH solution.

Preferably, ultrasonic treatment is carried out for 25-35 min after the protein peptide is dissolved, and then drying treatment is carried out.

The protein peptide can be dissolved more fully by ultrasonic treatment, for example, but not limited to, 25min, 28min, 30min, 32min or 35min, preferably 30min, and dried by nitrogen blowing to avoid oxidation and other unnecessary reactions;

preferably, ultrasonic treatment is carried out for 25-35 s after redissolution, and then dilution treatment is carried out.

Likewise, the solid can be further sufficiently dissolved in dimethyl sulfoxide using sonication for a period of time such as, but not limited to, 25s, 28s, 30s, 32s or 35s, preferably 30 s.

Preferably, the method further comprises the step of incubating for 24h at 37 ℃.

In some preferred embodiments, the method of plotting a β 40 standard curve comprises:

respectively adding an Alzheimer's disease marker aptamer into Abeta 40 standard solutions with different concentrations, adding nanogold after reaction, adding NaCl solution after the reaction again, then measuring absorbance values at 520nm and 725nm respectively by using an ultraviolet spectrophotometer, and drawing a standard curve by taking the concentration of the Abeta 40 standard solution as an abscissa and the ratio of the absorbance at 725nm to 520nm as an ordinate.

The Alzheimer disease marker aptamer can perform specific reaction with the Abeta 40 and is adsorbed on the surface of the nanogold, so that the nanogold is prevented from being aggregated under a high-salt condition, and the content of the Abeta 40 in a sample can be accurately reflected through the detection of absorbance. Based on this, it is preferable to specifically react with a β 40 using Apt as an alzheimer's disease marker aptamer at a concentration of 2 μ M.

In order to sufficiently perform the reaction, the time required for the reaction is preferably 3 to 7min, and may be, for example, but not limited to, 3min, 4min, 5min, 6min or 7 min. When the reaction time is 5min, the time cost can be saved on the basis of sufficient reaction.

Preferably, the volume ratio of the nanogold, the Alzheimer's disease marker aptamer, the A beta 40 standard solution and the NaCl solution is 60:18:9: 40.

The equation of the standard curve drawn based on the above method is y-0.004 x +1.5829, and the linear coefficient R is 0.9888.

In some preferred embodiments, the method of plotting a β 42 standard curve comprises:

respectively adding Alzheimer's disease marker specific binding peptide into Abeta 42 standard solutions with different concentrations, adding nanogold after reaction, respectively measuring absorbance values at 520nm and 615nm wavelength positions by using an ultraviolet spectrophotometer after the reaction again, and drawing a standard curve by taking the concentration of the Abeta 42 standard solution as an abscissa and the ratio of absorbance at 615nm to absorbance at 520nm as an ordinate;

the Alzheimer disease marker specific binding peptide PrP can specifically react with A beta 42, and after the nanogold is added, the A beta 42 and the nanogold competitively bind with the PrP, so that the aggregation of the nanogold is prevented, and the content of the A beta 42 in a sample can be accurately reflected by detecting the absorbance. Based on this, it is preferred to use PrP as the Alzheimer's disease marker specific binding peptide at a concentration of 250ng/mL to react specifically with A β 42.

The reaction time is optionally 3-7 min, for example, but not limited to, 3min, 4min, 5min, 6min or 7 min. When the reaction time is 5min, the time cost can be saved on the basis of sufficient reaction.

Preferably, the volume ratio of the nanogold, the A beta 42 standard solution and the Alzheimer's disease marker specific binding peptide solution is 2:1: 2.

The equation of the standard curve drawn based on the above method is-0.1023 x +0.6693, and the linear coefficient R is 0.9880.

In some preferred embodiments, the method for processing the sample to be tested comprises:

and adding 10mL of PBS buffer solution into every 0.25g of intestinal excrement for dissolving, and filtering the supernatant after solid-liquid separation to obtain the treated sample to be detected.

Specifically, the PBS buffer was used at a concentration of 10mM and at a pH of 7.2 or 7.4.

In order to further reduce impurities in the test sample and avoid interference with the test result, it is preferable to filter the supernatant with a 0.22 μm aqueous filter.

When the detection method provided by the invention is used for detecting the A beta 40 and/or the A beta 42 in the sample to be detected, the sample to be detected is brought in according to the method for making the standard curve.

The method obtains the Alzheimer's disease marker Abeta oligomer by performing ultrasonic treatment, nitrogen blowing, redissolution and culture on beta 40 and beta 42 through 1,1,1,3,3, 3-Hexafluoroisopropanol (HFIP). Respectively combining the A beta oligomer with an Alzheimer's disease marker aptamer modified on the surface of the nano-gold, then respectively reacting with NaCl solution, establishing a standard curve of ultraviolet absorption peak value ratio of the A beta oligomer concentration value corresponding to the aggregation state and the dispersion state of the nano-gold, drawing the standard curve by an ultraviolet absorption method, and carrying out qualitative and semi-quantitative analysis on beta amyloid protein oligomer in intestinal excreta. The method is suitable for detecting the A beta 40 and the A beta 42 in the adult intestinal excreta, has high qualitative accuracy, simple process and easy operation, and has practical detection significance.

According to another aspect of the present invention, there is also provided a kit for detecting amyloid beta oligomer, comprising a standard solution of amyloid beta oligomer of the present invention.

The present invention will be described with reference to specific examples.

Example 1 precision and recovery determination of the content of A.beta.40 in adult human intestinal excreta

(1) Preparation of a standard solution: dissolving 1mg of protein peptide with 1,1,1,3,3, 3-hexafluoroisopropanol to obtain 1mg/mL solution, and performing ultrasonic treatment for 30min to blow nitrogen to near dryness. The solid sample after nitrogen blowing was dissolved in dimethyl sulfoxide, sonicated for 30 seconds, diluted with 10mM PBS buffer pH 7.4 to prepare 35nM, 50nM, 100nM, 150nM, 250nM, 300nM and 400nM standard solutions of Abeta 40, and incubated at 37 ℃ in an incubator for 24 h.

Respectively adding 2 mu M of Alzheimer's disease marker aptamer (Apt) into Abeta 40 standard solutions with different concentrations, reacting for 5min under the condition of keeping out of the sun, adding nanogold, reacting for 5min under the condition of keeping out of the sun, adding 350mM NaCl solution, and adding the nanogold, the Alzheimer's disease marker aptamer, Abeta 40 and the NaCl solution according to the volume ratio of 60:18:9: 40. The absorbance values were measured at 520nm and 725nm wavelength respectively with an ultraviolet spectrophotometer. A standard curve (fig. 1) was plotted with the concentration of a β 40 as the abscissa and the ratio of absorbance at 725nm to 520nm as the ordinate, and it was determined that the equation of the standard curve was-0.005 x +0.9886 and the linear coefficient R was 0.9907.

(2) And (3) processing of a sample: accurately weighing 0.25g intestinal tract excreta sample, adding 10mL PBS buffer solution with concentration of 10mM and pH of 7.4 for dissolution, centrifuging for 10min under 10000rpm, taking supernatant, filtering with 0.22 μm water system filter membrane, and testing the sample solution.

(3) And (3) determination of a sample: adding 2 mu M of aptamer (Apt) into the sample solution, reacting for 5min in a dark condition, then adding AuNPs, reacting for 5min in a dark condition, adding 370mM NaCl solution, reacting for 5min in a dark condition, using a reagent blank solution as reference at 520nm and 725nm respectively by using a spectrophotometer, measuring the absorbance value, finding out the content of Abeta 40 from a standard curve, and calculating the content in the sample.

Three parallel samples were obtained in the same treatment, analyzed and calculated for content, with the following specific results:

example 2 precision and recovery determination of the content of Abeta 42 in adult intestinal excreta

(1) Preparation of a standard solution: dissolving 1mg of protein peptide with 1,1,1,3,3, 3-hexafluoroisopropanol to obtain 1mg/mL solution, and ultrasonically blowing nitrogen to near dryness for 30 min. Then, the solid was dissolved in 2mM NaOH solution, sonicated for 30 seconds, diluted with 10mM PBS buffer solution having pH of 7.2 to prepare standard solutions of A.beta.42 at 0. mu.M, 0.75. mu.M, 1. mu.M, 1.5. mu.M, 2.5. mu.M, 3. mu.M and 5. mu.M, and incubated in an incubator at 37 ℃ for 24 hours.

Respectively adding 250ng/mL polypeptide (PrP) into Abeta 42 standard solutions with different concentrations, fully reacting for 5min, adding nanogold, reacting for 5min under the condition of keeping out of the sun, wherein the volume ratio of the added nanogold, Abeta 42 and the polypeptide solution is 2:1: 2. The absorbance values were measured with an ultraviolet spectrophotometer at 520nm and 615m wavelength, respectively. A standard curve (fig. 2) was plotted with the concentration of a β 42 as the abscissa and the ratio of absorbance at 615nm to absorbance at 520nm as the ordinate, and was determined to have an equation of-0.1004 x +0.7818 and a linear coefficient R of 0.9893.

(2) And (3) processing of a sample: accurately weighing 0.25g intestinal tract excreta sample, adding 10mL PBS buffer solution with concentration of 10mM and pH of 7.2 respectively for dissolving, centrifuging for 10min under 10000rpm, taking supernatant, filtering with 0.22 μm water system filter membrane, and testing the sample solution.

(3) And (3) determination of a sample: adding 5 μ M polypeptide (PrP) into the sample solution, reacting for 5min in the dark, adding AuNPs, reacting for 5min in the dark, measuring the absorbance value with spectrophotometer at 520nm and 615nm with reagent blank solution as reference, finding out the content of Abeta 42 from the standard curve, and calculating the content of the sample.

Three parallel samples were obtained in the same treatment, analyzed and calculated for content, with the following specific results:

the results show that one of ordinary skill in the art can spectrophotometrically determine the levels of a β 40 and a β 42 in human intestinal fecal samples. The recovery rate of the A beta 40 measured by the method is 104-112%, the RSD is 0.75-3.9%, the recovery rate of the A beta 42 measured by the method is 83.0-95.9%, and the RSD is 1.6-5.0%, which shows that the method has higher accuracy and precision.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.