CN105651752B - The detection method of amyloid protein - Google Patents
The detection method of amyloid protein Download PDFInfo
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- CN105651752B CN105651752B CN201610108749.7A CN201610108749A CN105651752B CN 105651752 B CN105651752 B CN 105651752B CN 201610108749 A CN201610108749 A CN 201610108749A CN 105651752 B CN105651752 B CN 105651752B
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The present invention relates to a kind of detection methods of amyloid protein.Specifically, the present invention is to be realized using carbon-based quantum dot as fluorescence probe using the unique structure of carbon-based quantum dot and the characteristics of luminescence.The present invention is not only that amyloid aggregation detection proposes new scheme, and avoids the interference caused by system to be measured in conventional fluorescent detection method.
Description
Technical field
The present invention relates to the detection methods of amyloid protein, more particularly to utilize carbon-based quantum dots characterization amyloid protein
Method is related to protein structure detection field.
Background technique
The generation of amyloidosis is originated from the false folding of protein, and bodily tissue or device are deposited in the form of precipitating
Official, and directly result in the illnesss such as alzheimer's disease, parkinsonism, rabid ox disease (Cell, 2012,148,1188.).Starch
The specific accumulation process of sample albumen is as follows, and false folding occurs for Proteins In Aqueous Solutions, changes to β-lamellar structure, then assembles
At minimum oligomer and the small oligomer of diffusion-type, annular fibrinogen, fibrinogen, fiber with physiological-toxicity, spot is ultimately formed
Block.Current research hotspot such as the change of detecting and tracking amyloid protein structure, currently, thioflavine T and it is Congo red be the most frequently used
Detect the fluorescence probe (Nature.2011,472,226.) of the process.However, these methods in the detection process can be to system
Generate interference.In order to probe into more detection methods, a variety of nano materials such as: gold nano grain, aggregation-induced emission molecule and
Photonic crystal (Chem.Commun., 2015,51,1866.) etc. receives extensive research.Then the preparation process of these materials is numerous
Trivial and biocompatibility is general, brings big inconvenience for clinical application.
Summary of the invention
The object of the present invention is to provide a kind of methods using carbon-based quantum dots characterization amyloid protein, which can
Effectively detection amyloid levels, can also effectively detect amyloid aggregation dynamic behavior.The method of the present invention is easy, at
This low, at low cost and carbon-based quanta point material good biocompatibility；Detection process does not generate interference to system；It solves and examines now
Survey method is single, detection process introduces the technical bottleneck interfered.
To achieve the above object, technical scheme is as follows:
Present invention firstly provides application of the carbon-based quantum dot in amyloid protein detection.
The application includes at least the cohesion conformation for detecting amyloid protein, dynamic for detecting amyloid aggregation
Mechanics, or for detecting amyloid levels etc..
The present invention also provides a kind of methods of cohesion conformation for detecting amyloid protein, which comprises
1) amyloid protein of cohesion conformation to be determined is combined with carbon-based quantum dot, obtains its spectral signature；
2) amyloid protein of known cohesion conformation is combined with carbon-based quantum dot, obtains predetermined spectral signature；
By comparing step 1) and the resulting spectral signature of step 2), the amyloid egg of the cohesion conformation to be determined is determined
White cohesion conformation；Wherein the spectral signature is assessed with fluorescent spectrometry.
Preferably, the known cohesion conformation includes fibrous conformation.
The present invention also provides a kind of dynamic (dynamical) method of detection amyloid aggregation, the method includes by carbon-based quantum
Point is mixed with the amyloid protein solution for being incubated for different time, and according to the variation of carbon-based quantum dot fluorescence intensity, detection is formed sediment
The kinetics of aggregation behavior of powder sample albumen.
Specifically, the above-mentioned dynamic (dynamical) method of detection amyloid aggregation, comprising the following steps:
1) amyloid protein powder is made into the organic solution being completely dissolved, constant-temperature incubation for a period of time after, be dried with nitrogen,
It is dispersed in ultrapure water and forms certain density amyloid protein aqueous solution；Later, sample is placed in shaking table, constant temperature, constant speed
Aging；
2) quantitative sample is taken out from above-mentioned solution at regular intervals, is instilled tissue culture plate (such as 96 orifice plates), and is added
Enter carbon-based quantum dot solution, is uniformly mixed；It is preferred that at least taking three groups of parallel samples to be tested every time；After taking a little every time, starch
Sample albumen stoste all puts back to shaking table；
3) mixed liquor in tissue culture plate (such as 96 orifice plates) is placed in Fluorescence Spectrometer, setting excitation and transmitted wave
It is long, measure the fluorescence intensity of corresponding time carbon-based quantum dot；
4) using the time as abscissa, fluorescence intensity is ordinate, and the fluorescence intensity for drawing carbon-based quantum dot changes over time
Tendency chart is fitted data, obtains the amyloid aggregation behavior dynamics curve of carbon quantum dot detection.
The above-mentioned dynamic (dynamical) method of detection amyloid aggregation, wherein
Step 1) the organic solution is hexafluoroisopropanol (HFIP).
Step 1) the amyloid protein concentration of aqueous solution is 5-100 μM.
Step 2) the certain time interval are 10-60min.
The concentration of step 2) the carbon-based quantum dot solution is 0.5-5mg/mL.
The excitation wavelength of the step 3) fluorescence spectrum is in 350nm between 450nm, and launch wavelength is in 450nm to 550nm
Between；Preferably, the excitation wavelength of the fluorescence spectrum is 400nm, launch wavelength 500nm.
The present invention also provides a kind of for detecting the kit of amyloid levels, and the kit includes carbon-based quantum
Point.
Preferably, the kit includes the carbon-based quantum dot solution of 0.5-5mg/mL；Preferably, the kit includes
The carbon-based quantum dot solution of 1-2mg/mL.
The present invention also provides a kind of methods for detecting amyloid levels, and this method is using carbon-based quantum dot as probe
Fluorescence spectrum detection method.
The excitation wavelength of fluorescence spectrum of the present invention is in 350nm between 450nm, and launch wavelength is in 450nm to 550nm
Between.Preferably, the excitation wavelength of the fluorescence spectrum is 400nm, launch wavelength 500nm.
The method of detection amyloid levels of the present invention, the system of drafting, sample prepare liquid including standard curve
Standby and quick detection.
The sample prepare liquid includes cerebrospinal fluid, blood etc..
Specifically, the method for the detection amyloid levels, the drafting of standard curve the following steps are included:
1) the carbon-based quantum dot solution of 1mg/mL is prepared；
2) A β powder is made into the solution of 1mg/mL, solvent is hexafluoroisopropanol (HFIP)；After being incubated for 3 hours in 37 DEG C,
It is dried with nitrogen, with the dimethyl sulfoxide (DMSO) of 1% volume content for cosolvent, being dispersed in formation concentration in ultrapure water is respectively
6 parts of A beta monomers solution of 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 250mg/mL, 300mg/mL；Take above-mentioned 6 parts
It each 100 microlitres of solution, is mixed in 96 orifice plates with the carbon-based quantum dot solution of 100 microlitres of 1mg/mL respectively；Later, it is placed in fluorescence
In spectrometer, the fluorescence intensity of carbon-based quantum dot is measured, sets excitation wavelength as 400nm, launch wavelength 500nm；It is mono- with A β
Body content mg/mL is abscissa, and fluorescence intensity level (a.u.) is ordinate, draws A beta monomers standard curve.
Shown in the A beta monomers standard curve such as Fig. 4 (a) drawn.
Its equation of linear regression is Y=ax+b, and coefficient R 0.9557, wherein x represents the concentration mg/mL of A beta monomers,
Y represents fluorescence intensity level (a.u.)；A=-5.2, b=1829.5.
Specifically, it is described detection amyloid levels method, wherein the preparation of sample prepare liquid the following steps are included:
Sample to be tested (such as cerebrospinal fluid, blood etc.) is appropriately processed, after removing interfering substance, retain amyloid egg
It is white, to obtain sample prepare liquid.
Specifically, it is described detection amyloid levels method, wherein quickly detection the following steps are included:
1) the carbon-based quantum dot solution of 1mg/mL is prepared；
2) 100 microlitres of above-mentioned sample prepare liquid and the carbon-based quantum dot solution of 100 microlitres of 1mg/mL is taken to mix in 96 orifice plates
It closes；Later, it is placed in Fluorescence Spectrometer, measures the fluorescence intensity of carbon-based quantum dot, set excitation wavelength as 400nm, transmitted wave
A length of 500nm；Blank control test is done simultaneously, A beta monomers content in measuring samples is calculated as follows:
X=(I-I0)/a
Wherein, x is A beta monomers content in sample to be tested, and I is the florescent intensity value of sample to be tested, I0For blank quantum dot
Florescent intensity value, a are 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, is obtained to sample
The content of abnormal amyloid in product (abnormal amyloid is, for example, fibrillar amyloid)；Specifically, lead to
It crosses step 2) method and obtains A beta monomers content x in normal specimens0, compare, obtained to sample with A beta monomers content x in measuring samples
The content of abnormal amyloid is x in product0-x。
The method of detection amyloid levels described above, wherein A beta monomers content can also be by existing in normal specimens
Technology obtains.
The present invention is generally based on the discovery that carbon-based quantum dot can be used as fluorescence probe and combine with amyloid protein, utilizes
The unique structure of carbon-based quantum dot and the characteristics of luminescence, tripe systems are as amyloid protein (such as monomer or threadiness) is to carbon-based quantum dot
Fluorescence intensity it is different and realize.
Carbon-based quantum dot is a kind of novel quasi-zero dimension nano material, it is by agraphitic carbon particle or several layers of to be less than
The graphene film of 100nm constitutes (Science, 2008,320,356.).Movement of the carbon-based quantum dot internal electron in all directions
All limited to, quantum confinement effect is significant, electronics, photoelectricity and in terms of have peculiar property.Carbon-based quantum
Point surface functional group abundant imparts it, and (electrostatic interaction, hydrogen bond, π-π interact with amyloid protein interaction
Deng) ability, in addition, these functional groups and tripe systems are as the active force power of amyloid protein (monomer or fiber) is with albumen structure
The change of elephant and change.The difference of these interaction forces can be embodied in the fluorescence intensity of carbon-based quantum dot.Therefore, pass through survey
The fluorescence intensity feature of fixed carbon-based quantum dot can determine whether amyloid protein conformational characteristic.
Carbon-based quantum dot of the present invention can also can voluntarily be prepared by prior art preparation.
Such as the preparation method of the carbon-based quantum dot includes: to take a certain amount of carbon-based material, is mixed with strong oxidizer, instead
Ying Hou will obtain product filtering, centrifugation and dialysis, can be obtained carbon-based quantum dot.
Preferably, the carbon-based material is natural graphite, carbon fiber.
Preferably, the strong oxidizer is sodium nitrate, the concentrated sulfuric acid.
Preferably, carbon-based quantum dot effective concentration is 0.5~5mg/mL.
A kind of preparation method of carbon-based quantum dot is given in the examples below.
Amyloid protein of the present invention includes amyloid beta (A β), amylin, alpha-synapse albumen, poly glutamy
Amine, PrPC, TDP43 albumen, insulin, α B- crystallin, lysozyme etc..Preferably, the amyloid protein is the shallow lake β
Powder sample albumen (A β).It is further preferred that the amyloid protein is amyloid beta 1-42 (A β42)
The amyloid A β42, sequence are as follows: DAEFR HDSGY EVHHQ KLVFF AEDVG SNKGA IIGLM
VGGVV IA。
Compared with prior art, the invention has the benefit that
Carbon-based quanta point material used in the method for detection amyloid aggregation behavior of the present invention is at low cost, biology
Compatibility is good, provides possibility for application in vivo.In addition, the present invention is different from traditional detection method, to shallow lake in detection
The aggregation system of powder sample albumen does not have an impact.
Detailed description of the invention
Fig. 1 is transmission electron microscope (TEM) photo of carbon-based quantum dot prepared by the embodiment of the present invention 1.
Fig. 2 is the fluorescence spectrum of carbon-based quantum dot prepared by the embodiment of the present invention 1.
Fig. 3 is the size distribution of carbon-based quantum dot prepared by the embodiment of the present invention 1.
Fig. 4 is detection of the carbon-based quantum dot to amyloid beta monomer and fiber.
Fig. 5 is detection of the carbon-based quantum dot to amyloid beta difference fiber content.
Fig. 6 is detection of the carbon-based quantum dot to amyloid beta kinetics of aggregation.
Fig. 7 is detection of the carbon-based quantum dot to amyloid beta kinetics of aggregation.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment.Particular technique is not specified in embodiment
Or condition person, according to the literature in the art described technology or conditions, or carried out according to product description.Agents useful for same
Or production firm person is not specified in instrument, is conventional products that can be commercially available by regular channel.
Unless specifically stated otherwise, the solvent of aqueous solution used is sterile ultra-pure water solution in following embodiment.
Unless specifically stated otherwise, reagent used in following embodiment is analytical reagents.
Embodiment 1: the preparation of carbon-based quantum dot
1) 1g graphite powder is mixed with the 200mL concentrated sulfuric acid and 40g sodium nitrate, is cooled to zero degree.Then, it is slowly added to 4g Gao Meng
Sour potassium, temperature are maintained at 10 DEG C, are vigorously stirred.
2) said mixture after mixing evenly, is transferred in 30 DEG C of oil baths, is reacted 50 minutes.And then, temperature is increased
To 140 DEG C, then react 20h.
3) after reacting, product is cooled to room temperature, is diluted with 500mL deionized water, and adjusts pH value to 3 left sides with Na2CO3
It is right.By 0.22 μm of membrane filtration, supernatant is taken.It is placed in a centrifuge, 10000rpm is centrifuged 10 minutes.Finally, with retention
The bag filter of molecular weight 1000Da is dialysed 3 days, and final quantum point is obtained.
Transmission electron microscope can intuitively reflect the size characteristics of carbon-based quantum dot, and Fig. 1 is that embodiment 1 is made carbon-based
Transmission electron microscope (TEM) photo of quantum dot.Fig. 3 is the size point of carbon-based quantum dot prepared by the embodiment of the present invention 1
Cloth.It can be seen from the figure that the carbon-based quantum dot being prepared is the nanoscale twins of 5nm size.
Fluorescence spectrum can reflect the fluorescent characteristic of material, as shown in Fig. 2, the fluorescence spectrum of obtained carbon-based quantum dot.
Embodiment 2: detection of the carbon-based quantum dot to amyloid beta monomer and fiber
1) detection of the carbon-based quantum dot to amyloid beta monomer
A β powder is made into hexafluoroisopropanol (HFIP) solution of 1mg/mL, 37 DEG C after being incubated for 3 hours, are dried with nitrogen, with
The dimethyl sulfoxide (DMSO) of 1% volume content is cosolvent, and being dispersed in ultrapure water and forming concentration is respectively 50mg/mL,
6 parts of A beta monomers solution of 100mg/mL, 150mg/mL, 200mg/mL, 250mg/mL, 300mg/mL.Take above-mentioned 6 parts of solution each
It 100 microlitres, is mixed in 96 orifice plates with the carbon-based quantum dot solution of 100 microlitres of 1mg/mL respectively.Later, it is placed in Fluorescence Spectrometer
In, the fluorescence intensity of carbon-based quantum dot is measured, sets excitation wavelength as 400nm, launch wavelength 500nm.Obtain carbon-based quantum
The fluorescence intensity of point is with A beta monomers concentration curve.Shown in curve such as Fig. 4 (a), carbon-based quantum dot fluorescence intensity is with A beta monomers
The trend of linear reduction is presented in the increase of concentration.
2) detection of the carbon-based quantum dot to amyloid beta fiber
A β powder is made into hexafluoroisopropanol (HFIP) solution of 1mg/mL, 37 DEG C after being incubated for 3 hours, are dried with nitrogen, with
The dimethyl sulfoxide (DMSO) of 1% volume content is cosolvent, is dispersed in the A β solution for forming that concentration is 20 μM in ultrapure water.Its
For A beta monomers dispersion liquid.
A β powder is made into hexafluoroisopropanol (HFIP) solution of 1mg/mL, 37 DEG C after being incubated for 3 hours, are dried with nitrogen, with
The dimethyl sulfoxide (DMSO) of 1% volume content is cosolvent, is dispersed in the A β solution for forming that concentration is 20 μM in ultrapure water.It
Afterwards, sample is placed in shaking table, 37 DEG C of constant temperature, 150rpm constant speed aging, is incubated for 7 days, obtains A beta dispersion liquid.
100 microlitres of the carbon-based quantum dot (1.5mg/mL) synthesized in Example 1, respectively with 100 microlitres of above-mentioned A beta monomers
After the mixing of A beta dispersion liquid, the fluorescence spectra of above two mixed liquor and carbon-based quantum dot is measured.It obtains corresponding
Shown in fluorescence spectrum such as Fig. 4 (b).Illustrate, carbon-based quantum dot goes out difference to the detectability of A beta monomers and A beta, and A beta monomers can
It is substantially reduced carbon-based quantum dot fluorescence intensity, and A beta has no significant effect carbon-based quantum dot fluorescence intensity.
Embodiment 3: detection of the carbon-based quantum dot to amyloid beta fiber content
The preparation method of A beta monomers solution and A beta solution: with embodiment 2.
1) the A beta monomers being prepared and fiber solution are mixed from 0 to 100% according to the mass percent of A beta, is obtained
To A beta monomers/fiber mixed liquor of different proportion.
2) it takes 100 microlitres of above-mentioned solution to mix with 100 microlitres of carbon-based quantum dots (0.8mg/mL) respectively, 96 orifice plates is added
In, set excitation wavelength as 400nm, launch wavelength 450nm.It measures carbon-based in A beta monomers/fiber mixed liquor of different proportion
The fluorescence intensity of quantum dot.As a result as shown in Figure 5.The result shows that the fluorescence intensity of carbon-based quantum dot contains with fiber in mixed liquor
The linear growth trend of the increase of amount.
Embodiment 4: the detection for the amyloid beta kinetics of aggregation that carbon-based quantum dot is 20 μM to concentration
1) A β powder is made into hexafluoroisopropanol (HFIP) solution of 1mg/mL to be dried with nitrogen after 37 DEG C are incubated for 6 hours,
With the dimethyl sulfoxide (DMSO) of 1% volume content for cosolvent, it is dispersed in the A β solution for forming that concentration is 20 μM in ultrapure water.
Later, sample is placed in shaking table, 37 DEG C of constant temperature, 150rpm constant speed aging.
2) 100 microlitres of solution were taken out from above-mentioned solution every 30 minutes, are added in 96 orifice plates, and it is 1mg/ that concentration, which is added,
The carbon-based quantum dot solution of mL is uniformly mixed.Three groups of parallel samples are taken to be tested every time.After taking a little every time, A β stoste is all put
Shaking table is returned to continue to be incubated for.
3) mixed liquor in 96 orifice plates is placed in Fluorescence Spectrometer, measures the fluorescence intensity of carbon-based quantum dot, setting swashs
Hair wavelength is 450nm, launch wavelength 500nm.
4) using the time as abscissa, fluorescence intensity is ordinate, and the fluorescence intensity for drawing carbon-based quantum dot changes over time
Tendency chart is fitted data, obtains the amyloid aggregation behavior dynamics curve of carbon quantum dot detection.Curve is as schemed
Shown in 6.The result shows that the growth kinetics curve of amyloid beta fiber can be successfully obtained using carbon-based quantum dot as probe, it is fine
Dimension reaches the growth platform phase after 12h.
Embodiment 5: the detection for the amyloid beta kinetics of aggregation that carbon-based quantum dot is 50 μM to concentration
1) A β powder is made into hexafluoroisopropanol (HFIP) solution of 1mg/mL to be dried with nitrogen after 37 DEG C are incubated for 6 hours,
With the dimethyl sulfoxide (DMSO) of 1% volume content for cosolvent, it is water-soluble to be dispersed in the A β that formation concentration is 50 μM in ultrapure water
Liquid.Later, sample is placed in shaking table, 37 DEG C of constant temperature, 150rpm constant speed aging.
2) 100 microlitres of solution were taken out from above-mentioned solution every 20 minutes, are added in 96 orifice plates, and it is 2mg/ that concentration, which is added,
The carbon-based quantum dot solution of mL is uniformly mixed.Three groups of parallel samples are taken to be tested every time.After taking a little every time, A β stoste is all put
Return shaking table.
3) mixed liquor in 96 orifice plates is placed in Fluorescence Spectrometer, measures the fluorescence intensity of carbon-based quantum dot, setting swashs
Hair wavelength is 350nm, launch wavelength 450nm.
4) using the time as abscissa, fluorescence intensity is ordinate, and the fluorescence intensity for drawing carbon-based quantum dot changes over time
Tendency chart is fitted data, obtains the amyloid aggregation behavior dynamics curve of carbon quantum dot detection.Curve is as schemed
Shown in 7.The result shows that the growth kinetics curve of amyloid beta fiber can be successfully obtained using carbon-based quantum dot as probe, it is fine
Dimension reaches the growth platform phase after 12h.
Embodiment 6
A method of detection A beta-aggregation behavior the described method comprises the following steps:
1) A β powder is made into 1mg/mL to be dissolved in hexafluoroisopropanol (HFIP), 37 DEG C after being incubated for 3 hours, and nitrogen is blown
It is dry, with the dimethyl sulfoxide (DMSO) of 1% volume content for cosolvent, it is dispersed in ultrapure water.Later, sample is placed in shaking table
In, 37 DEG C of constant temperature, 150rpm constant speed aging.
2) 100 microlitres of solution were taken out from above-mentioned solution every 30 minutes, are added in 96 orifice plates, and it is 1mg/ that concentration, which is added,
The carbon-based quantum dot solution of mL is uniformly mixed.Three groups of parallel samples are taken to be tested every time.After taking a little every time, A β stoste is all put
Return shaking table.
3) mixed liquor in 96 orifice plates is placed in Fluorescence Spectrometer, measures the fluorescence intensity of carbon-based quantum dot, setting swashs
Hair wavelength is 400nm, launch wavelength 500nm.
4) using the time as abscissa, fluorescence intensity is ordinate, and the fluorescence intensity for drawing carbon-based quantum dot changes over time
Tendency chart is fitted data, obtains the amyloid aggregation behavior dynamics curve of carbon quantum dot detection.
Embodiment 7
A method of detection amyloid levels, the preparation of drafting, sample prepare liquid including standard curve and fast
Speed detection；It is specific as follows:
The drafting of the standard curve the following steps are included:
1) the carbon-based quantum dot solution of 1mg/mL is prepared；
2) A β powder is made into the solution of 1mg/mL, solvent is hexafluoroisopropanol (HFIP), and 37 DEG C after being incubated for 3 hours, nitrogen
Air-blowing is dry, and with the dimethyl sulfoxide (DMSO) of 1% volume content for cosolvent, being dispersed in formation concentration in ultrapure water is respectively
6 parts of A beta monomers solution of 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 250mg/mL, 300mg/mL；Take above-mentioned 6 parts
It each 100 microlitres of solution, is mixed in 96 orifice plates with the carbon-based quantum dot solution of 100 microlitres of 1mg/mL respectively；Later, it is placed in fluorescence
In spectrometer, the fluorescence intensity of carbon-based quantum dot is measured, sets excitation wavelength as 400nm, launch wavelength 500nm；It is mono- with A β
Body content mg/mL is abscissa, and fluorescence intensity level (a.u.) is ordinate, draws A beta monomers standard curve.
Shown in the A beta monomers standard curve such as Fig. 4 (a) drawn.
Its equation of linear regression is Y=ax+b, coefficient R 0.9557.Wherein x represents the concentration mg/mL of A beta monomers,
Y represents fluorescence intensity level (a.u.)；A=-5.2, b=1829.5.
The preparation of the sample prepare liquid the following steps are included:
Sample to be tested (such as cerebrospinal fluid, blood etc.) is appropriately processed, after removing interfering substance, retain amyloid egg
It is white, to obtain sample prepare liquid.
The quick detection the following steps are included:
1) the carbon-based quantum dot solution of 1mg/mL is prepared；
2) 100 microlitres of above-mentioned sample prepare liquid and the carbon-based quantum dot solution of 100 microlitres of 1mg/mL is taken to mix in 96 orifice plates
It closes；Later, it is placed in Fluorescence Spectrometer, measures the fluorescence intensity of carbon-based quantum dot, set excitation wavelength as 400nm, transmitted wave
A length of 500nm；Blank control test is done simultaneously, A beta monomers content in sample is calculated as follows:
X=(I-I0)/a
Wherein, x is A beta monomers content in sample to be tested, and I is the florescent intensity value of sample to be tested, I0For blank quantum dot
Florescent intensity value, a are 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, is obtained to sample
The content of fibrillar amyloid in product；
Specifically, A beta monomers content x in normal specimens is obtained by the above method0, with A beta monomers content x in measuring samples
Comparison, the content for obtaining fibrillar amyloid in measuring samples is x0-x。
Embodiment 8
A method of the cohesion conformation of detection amyloid protein, which comprises
1) amyloid protein of cohesion conformation to be determined is combined with carbon-based quantum dot, obtains its spectral signature；
2) amyloid protein of known cohesion conformation is combined with carbon-based quantum dot, obtains predetermined spectral signature；
By comparing step 1) and the resulting spectral signature of step 2), the amyloid egg of the cohesion conformation to be determined is determined
White cohesion conformation.
The Applicant declares that the present invention illustrates the process method of the present invention through the above embodiments, but the present invention not office
It is limited to above-mentioned processing step, that is, does not mean that the present invention must rely on the above process steps to be carried out.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to raw material selected by the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (3)
1. a kind of method for detecting amyloid levels, which is characterized in that this method is using carbon-based quantum dot as probe
Fluorescence spectrum detection method；The preparation of drafting, sample prepare liquid including standard curve and quickly detection；
The drafting of the standard curve the following steps are included:
1) the carbon-based quantum dot solution of 1 mg/mL is prepared；
2) A β powder is made into the solution of 1mg/mL, solvent is hexafluoroisopropanol, and 37 DEG C after being incubated for 3 hours, are dried with nitrogen, with 1%
The dimethyl sulfoxide of volume content is cosolvent, and being dispersed in ultrapure water and forming concentration is respectively 50 mg/mL, 100 mg/mL,
6 parts of A beta monomers solution of 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL；Take 6 parts of A beta monomers solution each 100
Microlitre, it is mixed in 96 orifice plates with the carbon-based quantum dot solution of 100 microlitre of 1 mg/mL respectively；Later, it is placed in Fluorescence Spectrometer
In, the fluorescence intensity of carbon-based quantum dot is measured, sets excitation wavelength as 400 nm, launch wavelength is 500 nm；Contained with A beta monomers
Amount mg/mL is abscissa, and fluorescence intensity level is ordinate, draws A beta monomers standard curve；
Its equation of linear regression is Y=ax+b, and coefficient R 0.9557, wherein x represents the concentration mg/mL, Y of A beta monomers
Represent fluorescence intensity level；A=- 5.2, b=1829.5；
The preparation of the sample prepare liquid the following steps are included:
Sample to be tested is appropriately processed, after removing interfering substance, retain amyloid protein, to obtain sample prepare liquid；
The quick detection the following steps are included:
1) the carbon-based quantum dot solution of 1 mg/mL is prepared；
2) 100 microlitres of above-mentioned sample prepare liquid and the carbon-based quantum dot solution of 100 microlitre of 1 mg/mL is taken to mix in 96 orifice plates
It closes；Later, it is placed in Fluorescence Spectrometer, measures the fluorescence intensity of carbon-based quantum dot, set excitation wavelength as 400 nm, transmitted wave
A length of 500 nm；Blank control test is done simultaneously, A beta monomers content in measuring samples is calculated as follows:
x=(I-I0)/a
Wherein, x is A beta monomers content in sample to be tested, and I is the florescent intensity value of sample to be tested, I0For blank quantum dot fluorescence
Strength values, a are 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, is obtained in measuring samples
The content of abnormal amyloid.
2. the method according to claim 1, wherein step 3) includes being obtained in normal specimens by the above method
A beta monomers content x0, compared with A beta monomers content x in measuring samples, the content for obtaining abnormal amyloid in measuring samples is
x0-x。
3. method according to claim 1 or 2, which is characterized in that the sample prepare liquid includes cerebrospinal fluid, blood.
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