CN105651752A - Detection method of amyloid protein - Google Patents

Abstract

The invention relates to a detection method of amyloid protein. Specifically, the detection method is realized by means of the unique structure and the luminescent characteristic of carbon-based quantum dots serving as fluorescent probes. A novel scheme is provided for aggregation detection of amyloid protein, and interference to a to-be-detected system in a traditional fluorescent detection method is avoided.

Description

Methods for the detection of amyloid

technical field

The invention relates to a method for detecting amyloid, in particular to a method for detecting amyloid by using carbon-based quantum dots, and relates to the field of protein structure detection.

Background technique

The occurrence of amyloidosis originates from the misfolding of proteins, which are deposited in body tissues or organs in the form of precipitates, and directly lead to Alzheimer's disease, Parkinson's syndrome, mad cow disease and other diseases (Cell, 2012, 148, 1188.) . The specific aggregation process of amyloid protein is as follows. The protein in the solution is misfolded and transformed into a β-sheet structure, and then assembled into very small oligomers and diffuse small oligomers, circular fibrils, and fibrils with physiological toxicity. , fibers, and eventually form plaques. For example, detecting and tracking the changes of amyloid structure is a research hotspot today. At present, Thioflavin T and Congo red are the most commonly used fluorescent probes for detecting this process (Nature. 2011, 472, 226.). However, these methods will cause interference to the system during the detection process. In order to explore more detection methods, a variety of nanomaterials such as gold nanoparticles, aggregation-induced luminescent molecules, and photonic crystals (Chem. Commun., 2015, 51, 1866.) have been extensively studied. However, the preparation process of these materials is cumbersome and the biocompatibility is average, which brings great inconvenience to clinical application.

Contents of the invention

The purpose of the present invention is to provide a method for detecting amyloid by using carbon-based quantum dots, which can effectively detect the content of amyloid, and can also effectively detect the dynamic behavior of amyloid aggregation. The method of the invention is simple, low in cost, low in cost, and the carbon-based quantum dot material has good biocompatibility; the detection process does not interfere with the system; and the current technical bottleneck of single detection method and interference introduced in the detection process is solved.

To achieve the above object, the technical scheme of the present invention is as follows:

The present invention firstly provides the application of carbon-based quantum dots in the detection of amyloid protein.

The application includes at least detecting the aggregation conformation of amyloid, detecting amyloid aggregation dynamics, or detecting amyloid content and the like.

The present invention also provides a method for detecting the aggregation conformation of amyloid, the method comprising:

1) Combining the amyloid protein whose cohesive conformation is to be determined with carbon-based quantum dots to obtain its spectral characteristics;

2) Combining amyloid with known cohesive conformation and carbon-based quantum dots to obtain predetermined spectral characteristics;

By comparing the spectral characteristics obtained in step 1) and step 2), the aggregation conformation of the amyloid whose aggregation conformation is to be determined is determined; wherein the spectral characteristics are evaluated by fluorescence spectroscopy.

Preferably, said known condensed conformation comprises a fibrous conformation.

The present invention also provides a method for detecting the aggregation kinetics of amyloid, the method comprising mixing carbon-based quantum dots with amyloid solutions incubated for different times, and detecting amyloid Aggregation kinetics of proteins.

Specifically, the above-mentioned method for detecting amyloid aggregation kinetics includes the following steps:

1) Make amyloid powder into a completely dissolved organic solution, incubate at constant temperature for a period of time, blow dry with nitrogen, disperse in ultrapure water to form a certain concentration of amyloid aqueous solution; after that, place the sample in a shaker, Constant temperature, constant speed aging;

2) Take quantitative samples from the above solution at regular intervals, drop them into cell culture plates (such as 96-well plates), and add carbon-based quantum dot solution, and mix well; preferably at least three groups of parallel samples are taken for experiments each time; After taking the point for the first time, the amyloid protein stock solution was put back into the shaker;

3) Place the mixed solution in the cell culture plate (such as a 96-well plate) in a fluorescence spectrometer, set the excitation and emission wavelengths, and measure the fluorescence intensity of the carbon-based quantum dots at the corresponding time;

4) Taking time as the abscissa and fluorescence intensity as the ordinate, draw the trend graph of the fluorescence intensity of the carbon-based quantum dots changing with time, and fit the data to obtain the kinetic curve of the amyloid aggregation behavior detected by the carbon quantum dots.

The above-mentioned method for detecting amyloid aggregation kinetics, wherein

Step 1) The organic solution is hexafluoroisopropanol (HFIP).

Step 1) The concentration of the amyloid aqueous solution is 5-100 μM.

Step 2) said certain time interval is 10-60min.

Step 2) The concentration of the carbon-based quantum dot solution is 0.5-5mg/mL.

Step 3) The excitation wavelength of the fluorescence spectrum is between 350nm and 450nm, and the emission wavelength is between 450nm and 550nm; preferably, the excitation wavelength of the fluorescence spectrum is 400nm, and the emission wavelength is 500nm.

The present invention also provides a kit for detecting amyloid content, the kit including carbon-based quantum dots.

Preferably, the kit includes 0.5-5 mg/mL carbon-based quantum dot solution; preferably, the kit includes 1-2 mg/mL carbon-based quantum dot solution.

The invention also provides a method for detecting amyloid content, which is a fluorescence spectrum detection method using carbon-based quantum dots as probes.

The excitation wavelength of the fluorescence spectrum in the present invention is between 350nm and 450nm, and the emission wavelength is between 450nm and 550nm. Preferably, the excitation wavelength of the fluorescence spectrum is 400nm, and the emission wavelength is 500nm.

The method for detecting the amyloid content of the present invention includes drawing a standard curve, preparing a sample solution to be tested, and rapidly detecting.

The sample fluid to be tested includes cerebrospinal fluid, blood and the like.

Specifically, the method for detecting amyloid content, wherein the drawing of the standard curve comprises the following steps:

1) Prepare a carbon-based quantum dot solution of 1 mg/mL;

2) Aβ powder was made into a 1 mg/mL solution, and the solvent was hexafluoroisopropanol (HFIP); after incubating at 37°C for 3 hours, blown dry with nitrogen, and mixed with 1% volume content of dimethyl sulfoxide (DMSO) As a co-solvent, disperse in ultrapure water to form 6 parts of Aβ monomer solutions with concentrations of 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 250mg/mL, and 300mg/mL; take the above 6 parts of the solution Each 100 microliters was mixed with 100 microliters of 1mg/mL carbon-based quantum dot solution in a 96-well plate; after that, it was placed in a fluorescence spectrometer to measure the fluorescence intensity of carbon-based quantum dots, and the excitation wavelength was set to 400nm. The emission wavelength is 500nm; the Aβ monomer standard curve is drawn with the Aβ monomer content mg/mL as the abscissa and the fluorescence intensity value (a.u.) as the ordinate.

The drawn Aβ monomer standard curve is shown in Figure 4(a).

Its linear regression equation is Y=ax+b, and the correlation coefficient R is 0.9557, wherein x represents the concentration mg/mL of Aβ monomer, and Y represents the fluorescence intensity value (a.u.); a=-5.2, b=1829.5.

Specifically, the method for detecting amyloid content, wherein the preparation of the sample liquid to be tested comprises the following steps:

The sample to be tested (such as cerebrospinal fluid, blood, etc.) is properly processed to remove the interfering substances and retain the amyloid, so as to obtain the sample to be tested.

Specifically, the method for detecting amyloid content, wherein rapid detection includes the following steps:

1) Prepare a carbon-based quantum dot solution of 1 mg/mL;

2) Mix 100 microliters of the above-mentioned sample solution to be tested with 100 microliters of 1 mg/mL carbon-based quantum dot solution in a 96-well plate; after that, place it in a fluorescence spectrometer to measure the fluorescence intensity of the carbon-based quantum dots, set The excitation wavelength is 400nm, and the emission wavelength is 500nm; at the same time, a blank control test is performed, and the Aβ monomer content in the sample to be tested is calculated according to the following formula:

x=(II ₀ )/a

Wherein, x is the Aβ monomer content in the sample to be tested, I is the fluorescence intensity value of the sample to be tested, _I0 is the fluorescence intensity value of the blank quantum dots, and a is the slope value of the standard curve -5.2.

3) Comparing the Aβ monomer content in the normal sample with the obtained Aβ monomer content in the sample to be tested to obtain the content of abnormal amyloid in the sample to be tested (the abnormal amyloid is, for example, fibrous amyloid); Specifically, the Aβ monomer content x ₀ in the normal sample is obtained by step 2), compared with the Aβ monomer content x in the sample to be tested, and the abnormal amyloid protein content in the sample to be tested is obtained as x ₀ −x.

In the method for detecting the amyloid content described above, the Aβ monomer content in the normal sample can also be obtained by existing techniques.

The present invention is generally based on the discovery that carbon-based quantum dots can be used as fluorescent probes to combine with amyloid proteins. Using the unique structure and luminescent properties of carbon-based quantum dots, amyloid proteins in different conformations (such as monomeric or fibrous) Carbon-based quantum dots are achieved by varying the fluorescence intensity.

Carbon-based quantum dots are a new type of quasi-zero-dimensional nanomaterials, which are composed of amorphous carbon particles or several layers of graphene sheets smaller than 100nm (Science, 2008, 320, 356.). The movement of electrons in carbon-based quantum dots is restricted in all directions, and the quantum confinement effect is remarkable, which has unique properties in electronics, optoelectronics, and electromagnetism. The abundant functional groups on the surface of carbon-based quantum dots endow them with the ability to interact with amyloid (electrostatic interaction, hydrogen bond, π-π interaction, etc.). ) The strength of the force changes with the change of protein conformation. The difference in these interaction forces will be reflected in the fluorescence intensity of carbon-based quantum dots. Therefore, the conformational characteristics of amyloid can be judged by measuring the fluorescence intensity characteristics of carbon-based quantum dots.

The carbon-based quantum dots of the present invention can be prepared by existing technologies, or can be prepared by themselves.

For example, the preparation method of the carbon-based quantum dots includes: taking a certain amount of carbon-based materials, mixing with a strong oxidant, and after the reaction, filtering, centrifuging and dialysis of the obtained product to obtain carbon-based quantum dots.

Preferably, the carbon-based material is natural graphite or carbon fiber.

Preferably, the strong oxidizing agent is sodium nitrate, concentrated sulfuric acid.

Preferably, the effective concentration of carbon-based quantum dots is 0.5-5 mg/mL.

A preparation method of carbon-based quantum dots is given in the following examples.

The amyloid in the present invention includes β-amyloid (Aβ), amylin, α-synaptic protein, polyglutamine, prion protein, TDP43 protein, insulin, αB-crystallin, lysozyme and the like. Preferably, the amyloid is amyloid beta (Aβ). Further preferably, the amyloid is β-amyloid 1-42 (Aβ ₄₂ )

The sequence of the amyloid Aβ ₄₂ is: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA.

Compared with prior art, the beneficial effect of the present invention is:

The carbon-based quantum dot material used in the method for detecting the amyloid aggregation behavior of the present invention has low cost and good biocompatibility, which provides the possibility of application in living bodies. In addition, the present invention is different from the traditional detection method, and the aggregation system of the amyloid protein is not affected during the detection.

Description of drawings

FIG. 1 is a transmission electron microscope (TEM) photo of carbon-based quantum dots prepared in Example 1 of the present invention.

Fig. 2 is the fluorescence spectrum of the carbon-based quantum dots prepared in Example 1 of the present invention.

Fig. 3 is the size distribution of carbon-based quantum dots prepared in Example 1 of the present invention.

Figure 4 is the detection of β-amyloid monomers and fibers by carbon-based quantum dots.

Figure 5 is the detection of carbon-based quantum dots on different fiber contents of β-amyloid protein.

Figure 6 is the detection of carbon-based quantum dots on the aggregation kinetics of β-amyloid protein.

Figure 7 is the detection of carbon-based quantum dots on the aggregation kinetics of β-amyloid protein.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through regular channels.

Unless otherwise specified, the solvents of the aqueous solutions used in the following examples are sterile ultrapure aqueous solutions.

Unless otherwise specified, the reagents used in the following examples are analytical reagents.

Embodiment 1: Preparation of carbon-based quantum dots

1) Mix 1g of graphite powder with 200mL of concentrated sulfuric acid and 40g of sodium nitrate, and cool to zero. Subsequently, 4 g of potassium permanganate was slowly added, the temperature was kept at 10° C., and the mixture was vigorously stirred.

2) After the above mixture was stirred evenly, it was transferred to an oil bath at 30°C and reacted for 50 minutes. Immediately afterwards, the temperature was raised to 140° C., and the reaction was continued for 20 h.

3) After the reaction, the product was cooled to room temperature, diluted with 500 mL of deionized water, and adjusted to about pH 3 with Na2CO3. After filtering through a 0.22 μm membrane filter, take the supernatant. Place in a centrifuge and centrifuge at 10,000 rpm for 10 minutes. Finally, the final quantum dots were obtained by dialysis for 3 days with a dialysis bag with a molecular weight cut-off of 1000 Da.

The transmission electron microscope can intuitively reflect the size characteristics of the carbon-based quantum dots. FIG. 1 is a transmission electron microscope (TEM) photo of the carbon-based quantum dots prepared in Example 1. Fig. 3 is the size distribution of carbon-based quantum dots prepared in Example 1 of the present invention. It can be seen from the figure that the prepared carbon-based quantum dots are nanosheets with a size of 5 nm.

The fluorescence spectrum can reflect the fluorescence characteristics of the material, as shown in Figure 2, the fluorescence spectrum of the obtained carbon-based quantum dots.

Example 2: Detection of β-amyloid monomers and fibers by carbon-based quantum dots

1) Detection of β-amyloid monomer by carbon-based quantum dots

Aβ powder was formulated into 1 mg/mL hexafluoroisopropanol (HFIP) solution, incubated at 37°C for 3 hours, blown dry with nitrogen, and dispersed in Six Aβmonomer solutions with concentrations of 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 250mg/mL and 300mg/mL were formed in ultrapure water. Take 100 microliters of each of the above six solutions and mix them with 100 microliters of 1 mg/mL carbon-based quantum dot solution in a 96-well plate. Afterwards, place in a fluorescence spectrometer to measure the fluorescence intensity of the carbon-based quantum dots, set the excitation wavelength to 400nm, and the emission wavelength to 500nm. The curve of the fluorescence intensity of the carbon-based quantum dots changing with the concentration of the Aβ monomer was obtained. As shown in the curve in Figure 4(a), the fluorescence intensity of carbon-based quantum dots showed a linear decrease trend with the increase of Aβ monomer concentration.

2) Detection of β-amyloid fibrils by carbon-based quantum dots

Aβ powder was formulated into 1 mg/mL hexafluoroisopropanol (HFIP) solution, incubated at 37°C for 3 hours, blown dry with nitrogen, and dispersed in An Aβ solution with a concentration of 20 μM was formed in ultrapure water. It is an Aβ monomer dispersion.

Aβ powder was formulated into 1 mg/mL hexafluoroisopropanol (HFIP) solution, incubated at 37°C for 3 hours, blown dry with nitrogen, and dispersed in An Aβ solution with a concentration of 20 μM was formed in ultrapure water. Afterwards, the sample was placed in a shaker, aged at a constant temperature of 37° C. and at a constant speed of 150 rpm, and incubated for 7 days to obtain an Aβ fiber dispersion.

Get 100 microliters of carbon-based quantum dots (1.5 mg/mL) synthesized in Example 1, mix them with 100 microliters of the above-mentioned Aβ monomer and Aβ fiber dispersion respectively, and measure the above-mentioned two kinds of mixed solutions and carbon-based quantum dots. Fluorescence spectrum graph. The corresponding fluorescence spectrum obtained is shown in Fig. 4(b). It shows that carbon-based quantum dots have different detection capabilities for Aβ monomers and Aβ fibers. Aβ monomers can significantly reduce the fluorescence intensity of carbon-based quantum dots, while Aβ fibers have no significant effect on the fluorescence intensity of carbon-based quantum dots.

Example 3: Detection of carbon-based quantum dots on the content of β-amyloid fibers

The preparation method of Aβ monomer solution and Aβ fiber solution: same as Example 2.

1) Mixing the prepared Aβ monomer and fiber solution according to the mass percentage of Aβ fiber from 0 to 100% to obtain Aβ monomer/fiber mixed solution with different proportions.

2) Mix 100 microliters of the above solution with 100 microliters of carbon-based quantum dots (0.8mg/mL), add them into a 96-well plate, set the excitation wavelength to 400nm, and the emission wavelength to 450nm. The fluorescence intensity of carbon-based quantum dots in different proportions of Aβ monomer/fiber mixture was measured. The result is shown in Figure 5. The results show that the fluorescence intensity of carbon-based quantum dots increases linearly with the increase of fiber content in the mixture.

Example 4: Detection of aggregation kinetics of β-amyloid protein with a concentration of 20 μM by carbon-based quantum dots

1) Prepare Aβ powder into 1mg/mL hexafluoroisopropanol (HFIP) solution, incubate at 37°C for 6 hours, blow dry with nitrogen, and use 1% volume content of dimethyl sulfoxide (DMSO) as co-solvent, Disperse in ultrapure water to form an Aβ solution with a concentration of 20 μM. Afterwards, the sample was placed in a shaker, aged at a constant temperature of 37°C and at a constant speed of 150rpm.

2) Take 100 microliters of the solution from the above solution every 30 minutes, add it to a 96-well plate, and add a carbon-based quantum dot solution with a concentration of 1 mg/mL, and mix well. Three sets of parallel samples were taken for each experiment. After each point was taken, the Aβ stock solution was returned to the shaker to continue incubation.

3) Put the mixed solution in the 96-well plate in a fluorescence spectrometer, measure the fluorescence intensity of the carbon-based quantum dots, set the excitation wavelength to 450nm, and the emission wavelength to 500nm.

4) Taking time as the abscissa and fluorescence intensity as the ordinate, draw the trend graph of the fluorescence intensity of the carbon-based quantum dots changing with time, and fit the data to obtain the kinetic curve of the amyloid aggregation behavior detected by the carbon quantum dots. The curve is shown in Figure 6. The results showed that the growth kinetic curve of β-amyloid fibers could be successfully obtained by using carbon-based quantum dots as probes, and the fibers reached the growth plateau after 12 hours.

Example 5: Detection of aggregation kinetics of β-amyloid protein with a concentration of 50 μM by carbon-based quantum dots

1) Prepare Aβ powder into 1mg/mL hexafluoroisopropanol (HFIP) solution, incubate at 37°C for 6 hours, blow dry with nitrogen, and use 1% volume content of dimethyl sulfoxide (DMSO) as co-solvent, Disperse in ultrapure water to form an Aβ aqueous solution with a concentration of 50 μM. Afterwards, the sample was placed in a shaker, aged at a constant temperature of 37°C and at a constant speed of 150rpm.

2) Take 100 microliters of the solution from the above solution every 20 minutes, add it to a 96-well plate, and add a carbon-based quantum dot solution with a concentration of 2 mg/mL, and mix well. Three sets of parallel samples were taken for each experiment. After each point was taken, the Aβ stock solution was returned to the shaker.

3) Put the mixed solution in the 96-well plate in a fluorescence spectrometer, measure the fluorescence intensity of the carbon-based quantum dots, set the excitation wavelength to 350nm, and set the emission wavelength to 450nm.

4) Taking time as the abscissa and fluorescence intensity as the ordinate, draw the trend graph of the fluorescence intensity of the carbon-based quantum dots changing with time, and fit the data to obtain the kinetic curve of the amyloid aggregation behavior detected by the carbon quantum dots. The curve is shown in Figure 7. The results showed that the growth kinetic curve of β-amyloid fibers could be successfully obtained by using carbon-based quantum dots as probes, and the fibers reached the growth plateau after 12 hours.

Example 6

A method for detecting Aβ aggregation behavior, said method comprising the following steps:

1) Dissolve Aβ powder at 1 mg/mL in hexafluoroisopropanol (HFIP), incubate at 37°C for 3 hours, blow dry with nitrogen, and use 1% dimethyl sulfoxide (DMSO) as a co-solvent , dispersed in ultrapure water. Afterwards, the sample was placed in a shaker, aged at a constant temperature of 37°C and at a constant speed of 150rpm.

2) Take 100 microliters of the solution from the above solution every 30 minutes, add it to a 96-well plate, and add a carbon-based quantum dot solution with a concentration of 1 mg/mL, and mix well. Three sets of parallel samples were taken for each experiment. After each point was taken, the Aβ stock solution was returned to the shaker.

3) Put the mixed solution in the 96-well plate in a fluorescence spectrometer, measure the fluorescence intensity of the carbon-based quantum dots, set the excitation wavelength to 400nm, and the emission wavelength to 500nm.

4) Taking time as the abscissa and fluorescence intensity as the ordinate, draw the trend graph of the fluorescence intensity of the carbon-based quantum dots changing with time, and fit the data to obtain the kinetic curve of the amyloid aggregation behavior detected by the carbon quantum dots.

Example 7

A method for detecting amyloid content, comprising the drawing of a standard curve, the preparation of a sample solution to be tested and rapid detection; details are as follows:

The drawing of described standard curve comprises the following steps:

1) Prepare a carbon-based quantum dot solution of 1 mg/mL;

2) Aβ powder was made into a 1mg/mL solution, and the solvent was hexafluoroisopropanol (HFIP). After incubation at 37°C for 3 hours, the solution was blown dry with nitrogen, and dimethyl sulfoxide (DMSO) with 1% volume content was used as the solution. Co-solvent, dispersed in ultrapure water to form 6 parts of Aβ monomer solutions with concentrations of 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 250mg/mL, and 300mg/mL; 100 microliters were mixed with 100 microliters of 1mg/mL carbon-based quantum dot solution in a 96-well plate; after that, placed in a fluorescence spectrometer to measure the fluorescence intensity of carbon-based quantum dots, set the excitation wavelength to 400nm, and emitted The wavelength is 500nm; the Aβ monomer standard curve is drawn with the Aβ monomer content mg/mL as the abscissa and the fluorescence intensity value (a.u.) as the ordinate.

The drawn Aβ monomer standard curve is shown in Figure 4(a).

Its linear regression equation is Y=ax+b, and the correlation coefficient R is 0.9557. Where x represents the concentration of Aβ monomer in mg/mL, Y represents the fluorescence intensity value (a.u.); a=-5.2, b=1829.5.

The preparation of the sample liquid to be tested comprises the following steps:

The sample to be tested (such as cerebrospinal fluid, blood, etc.) is properly processed to remove the interfering substances and retain the amyloid, so as to obtain the sample to be tested.

Described rapid detection comprises the following steps:

1) Prepare a carbon-based quantum dot solution of 1 mg/mL;

2) Take 100 microliters of the above-mentioned sample solution to be tested and 100 microliters of 1 mg/mL carbon-based quantum dot solution and mix them in a 96-well plate; after that, place them in a fluorescence spectrometer to measure the fluorescence intensity of the carbon-based quantum dots, set The excitation wavelength is 400nm, and the emission wavelength is 500nm; at the same time, a blank control test is performed, and the Aβ monomer content in the sample is calculated according to the following formula:

x=(II ₀ )/a

Wherein, x is the Aβ monomer content in the sample to be tested, I is the fluorescence intensity value of the sample to be tested, _I0 is the fluorescence intensity value of the blank quantum dots, and a is the slope value of the standard curve -5.2.

3) Comparing the Aβ monomer content in the normal sample with the obtained Aβ monomer content in the sample to be tested to obtain the content of fibrillar amyloid in the sample to be tested;

Specifically, the Aβ monomer content x ₀ in the normal sample is obtained by the above method, compared with the Aβ monomer content x in the sample to be tested, and the fibrous amyloid protein content in the sample to be tested is obtained as x ₀ -x.

Example 8

A method of detecting the cohesive conformation of amyloid, the method comprising:

1) Combining the amyloid protein whose cohesive conformation is to be determined with carbon-based quantum dots to obtain its spectral characteristics;

2) Combining amyloid with known cohesive conformation and carbon-based quantum dots to obtain predetermined spectral characteristics;

By comparing the spectral features obtained in step 1) and step 2), the aggregation conformation of the amyloid whose aggregation conformation is to be determined is determined.

The applicant declares that the present invention illustrates the process method of the present invention through the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. carbon back quantum dot application on amyloid detects.

2. application according to claim 1, it is characterised in that at least include the cohesion conformation for detecting amyloid, be used for detecting amyloid aggregation kinetics, or be used for detecting amyloid levels.

3. the method for the cohesion conformation detecting amyloid, it is characterised in that described method includes:

1) amyloid of cohesion conformation to be determined is combined with carbon back quantum dot, it is thus achieved that its spectral signature;

2) amyloid of known cohesion conformation is combined with carbon back quantum dot, it is thus achieved that predetermined spectral signature;

By comparison step 1) and step 2) spectral signature of gained, it is determined that the cohesion conformation of the amyloid of described cohesion conformation to be determined;

Wherein, described spectral signature fluorescent spectrometry assessment; And/or

Preferably, described known cohesion conformation includes threadiness conformation.

4. the detection dynamic (dynamical) method of amyloid aggregation, it is characterized in that, described method includes mixing carbon back quantum dot with the amyloid solution hatching different time, the change according to carbon back quantum dot fluorescence intensity, the kinetics of aggregation behavior of detection amyloid.

5. method according to claim 4, it is characterised in that comprise the following steps:

1) amyloid powder being made into consoluet organic solution, after constant-temperature incubation a period of time, nitrogen dries up, and is dispersed in ultra-pure water and forms certain density amyloid aqueous solution; Afterwards, being placed in shaking table by sample, constant temperature, constant speed are aging;

2) from above-mentioned solution, take out quantitative sample at regular intervals, instill Tissue Culture Plate, and add carbon back quantum dot solution, mix homogeneously;

3) mixed liquor in Tissue Culture Plate is placed in fluorescence spectrophotometer, sets and excite and launch wavelength, measure the fluorescence intensity of corresponding time carbon back quantum dot;

4) with the time for abscissa, fluorescence intensity is vertical coordinate, and the fluorescence intensity drawing carbon back quantum dot changes over trendgram, and data are fitted, and obtains the amyloid aggregation behavior dynamics curve of carbon quantum dot detection;

Preferably, step 1) described organic solution is hexafluoroisopropanol; And/or

Step 1) described amyloid concentration of aqueous solution is 5-100 ��M; And/or

Step 2) described certain time is spaced apart 10-60min; And/or

Step 2) concentration of described carbon back quantum dot solution is 0.5-5mg/mL; And/or

Step 3) excitation wavelength of described fluorescence spectrum between 350nm to 450nm, launch wavelength between 450nm to 550nm; It is further preferred that the excitation wavelength of described fluorescence spectrum is 400nm, transmitting wavelength is 500nm.

6. the test kit being used for detecting amyloid levels, it is characterised in that described test kit includes carbon back quantum dot.

7. test kit according to claim 6, it is characterised in that described test kit includes the carbon back quantum dot solution of 0.5-5mg/mL; Preferably, described test kit includes the carbon back quantum dot solution of 1 2mg/mL.

8. the method detecting amyloid levels, it is characterised in that the method is the fluorescence spectrum detection method using carbon back quantum dot as probe;

Preferably, the excitation wavelength of described fluorescence spectrum, between 350nm to 450nm, launches wavelength between 450nm to 550nm; It is further preferred that the excitation wavelength of described fluorescence spectrum is 400nm, transmitting wavelength is 500nm.

9. method according to claim 8, it is characterised in that include the drafting of standard curve, the preparation of sample liquid to be measured and quickly detect;

Preferably, the method for described detection amyloid levels, the drafting of its standard curve comprises the following steps:

1) the carbon back quantum dot solution of 1mg/mL is prepared;

2) A �� powder is made into the solution of 1mg/mL, solvent is hexafluoroisopropanol, 37 DEG C hatch 3 hours after, nitrogen dries up, with the dimethyl sulfoxide of 1% volume content for cosolvent, it is dispersed in ultra-pure water and forms concentration respectively 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 6 parts of A beta monomers solution of 250mg/mL, 300mg/mL; Take above-mentioned each 100 microlitres of 6 parts of solution, mix in 96 orifice plates with the carbon back quantum dot solution of 100 microlitre 1mg/mL respectively; Afterwards, being placed in fluorescence spectrophotometer, measure the fluorescence intensity of carbon back quantum dot, set excitation wavelength as 400nm, transmitting wavelength is 500nm; With A beta monomers content mg/mL for abscissa, fluorescence intensity level (a.u.) is vertical coordinate, draws A beta monomers standard curve;

Its equation of linear regression is Y=ax+b, and coefficient R is 0.9557, and wherein x represents concentration mg/mL, the Y of A beta monomers and represents fluorescence intensity level (a.u.); A=-5.2, b=1829.5;

And/or, the preparation of described sample liquid to be measured comprises the following steps:

Testing sample is appropriately processed, after removing interfering material, retain amyloid, thus obtaining sample liquid to be measured;

And/or, described quick detection comprises the following steps:

1) the carbon back quantum dot solution of 1mg/mL is prepared;

2) the carbon back quantum dot solution taking above-mentioned sample liquid 100 to be measured microlitre and 100 microlitre 1mg/mL mixes in 96 orifice plates; Afterwards, being placed in fluorescence spectrophotometer, measure the fluorescence intensity of carbon back quantum dot, set excitation wavelength as 400nm, transmitting wavelength is 500nm; Do blank test simultaneously, be calculated as follows in measuring samples A beta monomers content:

X=(I-I₀)/a

Wherein, x is A beta monomers content in testing sample, and I is the florescent intensity value of testing sample, I₀For blank quantum dot fluorescence strength values, a is the slope numerical value-5.2 of standard curve;

3) A beta monomers content in normal specimens is compared with A beta monomers content in gained measuring samples, it is thus achieved that the content of abnormal amyloid in measuring samples;

Preferably, A beta monomers content x in normal specimens is obtained by said method₀, compare with A beta monomers content x in measuring samples, obtaining the content of abnormal amyloid in measuring samples is x₀-x��

10. method according to claim 9, it is characterised in that described sample liquid to be measured includes cerebrospinal fluid, blood.