CN109946280A - Method for quantitatively detecting soluble β amyloid protein by using proportional metering type fluorescent probe - Google Patents
Method for quantitatively detecting soluble β amyloid protein by using proportional metering type fluorescent probe Download PDFInfo
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract
The invention discloses a method for quantitatively detecting soluble β amyloid protein (A β) by using a proportional metering type fluorescent probe, belonging to the technical field of medicines, wherein a metal ion proportional metering type fluorescent probe BPNS, a soluble A β -Zn pair, is prepared by coupling 2-pyridyl benzothiazole and dansyl fluorescent group2+Or Cu2+Has high affinity and can chelate metal ions from BPNS-metal ion adductsThe method can realize accurate quantitative detection of the soluble A β aggregate by determining the linear relation between the BPNS dual-wavelength fluorescence intensity ratio and the soluble A β aggregate concentration.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of to use ratio metering type fluorescence probe quantitative detection β starch
The method of sample albumen.
Background technique
Alzheimer disease (Alzheimer ' s disease, AD) is a kind of progressive neurodegenerative diseases, clinically
It is mainly shown as memory and cognitive disorder, currently there is no effective diagnosing and treating method.Although the pathomechanism of AD is still unknown
Really, a large number of studies show that, the major pathologic features of AD are that beta-amyloid protein (A β) assembles and deposit and be formed by senile plaque
(SP) and Hyperphosphorylationof Tau albumen assembles the neurofibrillary tangles to be formed (NFTs).Wherein, A beta-aggregation is considered as AD
The core cause of disease in development process.The polypeptide that A β is made of 39~43 amino acid is amyloid precusor protein in pathology item
Under part as produced by β-and gamma-secretase hydrolysis.A β is easy to assemble, especially easily with metal ion (Zn2+Or Cu2+) coordination at
Core accelerates aggregation to form A beta-aggregation body.Wherein, the neurotoxicity of the soluble A beta oligomers of disease early stage is most strong, can trigger one
The pathogenic access of series, lures nerve cell apoptosis into, further results in hippocampus and Cortical Neurons denaturation and death, causes to remember
Recall dysfunction, finally causes AD.So soluble A beta-aggregation body can be used as biomarker and diagnose for early stage AD.And
, in addition to being also widely present among cerebrospinal fluid and blood other than cerebral deposition, this is early diagnosed for non-invasive rapid disease for they
Or it examines provide possibility in advance.Therefore, the quantitative detecting method for developing solubility A β has important value to AD diagnosis.
A large amount of neuroimaging techniques are detected for brain A β at present, mainly including positron emission computerized tomography (PET),
Single photon emission computerized tomography (SPECT) and Magnetic resonance imaging (MRI) etc., but these imaging methods are mainly used for
Qualitative research to A beta body patch surveys soluble A beta-aggregation physical examination ineffective.It is some to be used for vitro detection solubility A
The method of β so that their influences vulnerable to cross reaction and high non-specific binding, and is used based on immunoassay
Antibody is expensive, the used time is longer and cumbersome.Ratio metering type fluorescence detection has at low cost, quantitative precision height, letter
The advantages that making an uproar than high, Noninvasive has great potential in biomedical context of detection.But do not have the metering of discovery ratio also at present
Type small-molecule fluorescent probe is used for quantitative detection solubility A beta-aggregation body and is developed the application of AD diagnosticum with this.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that provide a kind of proportion of utilization metering type fluorescence response external
The new method of quantitative detection solubility A beta-aggregation body, the exploitation for early diagnosing for AD and examining in advance provide advantageous information.
Purpose to realize the present invention, the present invention will using chromophore 2- pyridyl group benzothiazole as metal ion cheating moiety with
Red sulphonyl fluorophor coupling obtains fluorescence probe BPNS.The architectural characteristic of Bichromophore can make BPNS in bind metal ion
(Zn2+Or Cu2+) afterwards simultaneously regulate and control two fluorophors fluorescence, to realize ratio metering type fluorescence response.It recycles solvable
Property A β is to Zn2+Or Cu2+High-affinity, in the adduction objects system that BPNS and metal ion is added in soluble A β sample, A β energy
Enough chelate metal ion from BPNS- metal ion adduct comes out, to trigger the ratio metering type fluorescence response of BPNS.
By determining that solubility A beta-aggregation body and the fluorescence intensity ratio of two launch wavelengths of response variation have the dense of linear relationship
Range is spent, can be obtained the operation interval of BPNS accurate quantitative analysis detection solubility A beta-aggregation body.
The present invention provides a kind of method with ratio metering type fluorescence probe quantitative detection solubility A beta oligomers, this method
Include:
Step 1: synthesis ratio metering type fluorescence probe BPNS；
Step 2: BPNS is to Zn for assessment2+Or Cu2+Ratio measure fluorescence response performance；
Step 3: A β sample is added in buffer solution, be incubated for respectively at 37 DEG C 0h, for 24 hours, 48h and 72h prepare it is solvable
Property A beta monomers, oligomers, insoluble fibre precursor and insoluble fibre liquid solution；
Step 4: being separately added into solution made from step 3 in BPNS- metal ion adduct solution, test
BPNS- metal measures fluorescence response to the selection sex ratio of soluble A β sample；
Step 5: fluorescence is measured by ratio of the BPNS- metal ion adduct to the soluble A β sample of various concentration
In response to determining that quantitative detection concentration range；
Step 6: the content of solubility A β in BPNS- metal ion adduction analyte detection difference sample is utilized；
Preferably, the ratio metering type fluorescence probe is using chromophore 2- pyridyl group benzothiazole as metal ion chela
It closes group and is coupled red sulphonyl fluorophor formation fluorescence probe BPNS, structural formula is as shown in Fig. 1.BPNS fluorescence has greatly this
Lentor is displaced, to the advantages such as the very high sensibility of environment and VISIBLE LIGHT EMISSION (green light).
Preferably, the metal ion is Zn2+Or Cu2+。
Preferably, the buffer solution is 20mM Tris-HCl, 150mM NaCl, pH 7.4.
Compared with prior art, the invention has the following advantages:
The present invention provides a kind of method with ratio metering type fluorescence probe quantitative detection solubility A beta-aggregation body, the party
Method is based on ratio metering type metal-ion fluorescent probe BPNS, using soluble A β to the strong compatibility of metal ion, by can
Dissolubility A β chelates out the activation ratio metering fluorescence response of the metal ion in conjunction with BPNS, and then quantitative detection solubility A β.It is real
Test the result shows that, BPNS is to metal ion (Zn2+Or Cu2+) apparent ratio metering type change in fluorescence is shown, and combine
Compatibility is both less than solubility A β.Further, BPNS- metal ion adduct can selectively provide soluble A β sample
Ratio metering type fluorescence response, and there is the linear passes of height between fluorescence response Wavelength strength ratio and solubility A β concentration
System, thus illustrate BPNS can in the presence of metal ion quantitative detection solubility A β.And BPNS is easily prepared, testing cost
It is low, accuracy rate is high, selectivity is good.Therefore, ratio metering type fluorescence probe provided by the invention is in quantitative detection solubility A β sample
There is good effect in terms of product, there is preferable AD early diagnosis and examine application prospect in advance.
Detailed description of the invention
Fig. 1 is embodiment ratio metering type fluorescence probe BPNS structural formula；
Fig. 2 is embodiment BPNS to various concentration Zn2+Ratio measure fluorescence response figure；
Fig. 3 is embodiment BPNS-Zn2+Fluorescence response figure is measured to the ratio of various concentration solubility A β；
Fig. 4 is embodiment BPNS-Zn2+To the fluorescence response selective figure of different coherent condition A β；
Fig. 5 is embodiment BPNS-Zn2+To different large biological molecules (A β40Oligomer, human serum albumins (HSA), metal
Sulfoprotein (MT-I), Tau albumen, trypsase (trypsin) and the red albumin of ox blood (BHb)) fluorescence response selective figure；
Fig. 6 is embodiment BPNS-Zn2+Fluorescence intensity ratio and solubility A β at 505nm and 423nm40The linear pass of concentration
System's figure.
Fig. 7 is embodiment BPNS in Zn2+In the presence of to A β40Solubility A in self aggregation solution difference brooding time supernatant
β40Quantitative detection figure.
Specific embodiment
The present invention is better illustrated below with reference to the attached drawing in embodiment and embodiment.
Embodiment: ratio metering type fluorescence probe BPNS quantitative detection solubility A beta oligomers:
Step 1: synthesis preferred proportion metering type fluorescence probe BPNS, synthetic route chart are as follows.
Weigh the poly of 2- amino benzenethiol (1mmol, 0.125g), PAS (1mmol, 0.138g) and 50ml
Phosphoric acid (PPA) is in three neck round bottom flask.130 DEG C of reaction 120min, are arrived to the reaction time, add water to stop reaction, system is in clear
Saturation sodium hydroxide solution is added in clear bright yellow solution, pH value 1, and adjusting pH is neutrality, and glassy yellow powder is precipitated, and filters
And filter residue is collected, washing is three times (3 × 100mL).Crude product passes through silicagel column (PE: EA=4: 1) separation again, and it is bright for finally obtaining
The compound 1 (0.184g, yield:70%) of yellow powder.1H NMR(CDCl3, 400MHz, δ, ppm): 8.16 (d, J=
8.5Hz, 1H, benzothiazole), 8.12 (d, J=2.6Hz, 1H, benzothiazole), 8.02 (d, J=8.5Hz, 1H,
Pyridine), 7.91 (dd, J=7.9,0.5Hz, 1H, benzothiazole), 7.50-7.42 (m, 1H, pyridine),
7.40-7.31 (m, 1H, pyridine), 7.07 (dd, J=8.5HZ, 2.8Hz, 1H, benzothiazole).ESI-MS
found(calcd)for C12H9N3S (m/z): 227.05 (227.28) [M+H]+。
Weigh Compound 1 (1mmol, 0.225g) and cesium carbonate (1.8mmol, 0.567g) add in three neck round bottom flask
Acetonitrile after entering 50ml drying, leads to nitrogen, is heated to 85 DEG C, and flow back 2h.It is dry that dansyl chloride (1mmol, 0.270g) is dissolved in 30ml
Acetonitrile after dry, is slowly added to constant pressure funnel into system, and wait be added dropwise, whole system is heated to reflux under nitrogen protection
6h.To the end of reacting, filters and collect filter residue.Crude product passes through silicagel column (PE: EA=4: 1) separation again, and it is light for finally obtaining
The BPNS (0.247g, yield:50%) of yellow powder.1H NMR(CDCl3, 400MHz, δ, ppm): 8.55 (d, J=8.2Hz,
1H, naphthene), 8.33 (d, J=8.7Hz, 1H, naphthene), 8.25 (d, J=7.1Hz, 1H, naphthene),
8.21 (s, 1H, pyridine), 8.17 (d, J=8.6Hz, 1H, benzothiazole), 8.02 (d, J=8.1Hz, 1H,
Benzothiazole), 7.90 (d, J=7.9Hz, 1H, pyridine), 7.64-7.61 (m, 1H, naphthene), 7.60 (d,
J=4.0Hz, 1H, naphthene), 7.47 (t, J=7.9Hz, 2H, benzothiazole), 7.38 (t, J=7.3Hz, 1H,
Naphthene), 7.21 (d, J=7.6Hz, 1H, pyridine), 2.89 (s, 6H ,-CH3).ESI-MS found(calcd)
for C24H20N4O2S2(m/z): 461.33 (460.57) [M+H]+。
Step 2: system be 3mL BPNS Tris buffer solution (20mM Tris-HCl, 150mM NaCl,
PH7.4 in), the ZnCl2 solution (5mM, 24 μ L) of equivalent is instilled, 0.1 equivalent (5mM, 1.2 μ L) are added every time, on luminoscope
Test its fluorescence spectrum.
Step 3: by the A β of purchase40Freeze-drying sample (4.0mg) is dissolved in NaOH (750 μ L, 20mM), then water bath sonicator
Handle 30s.It is ultrasonically treated 30s after this solution is diluted with 750 μ L deionized waters, then adjusts pH to about 7.4 with 0.5M HCl,
It is finally filtered with 0.22 μm of filter (Millipore), and then measures A β with BCA Protein Assay Kit (Pierce)40
The concentration of stock solution.Take calibrated A β40(20mM Tris-HCl, 150mM NaCl, pH7.4) is added in buffer solution,
37 DEG C be incubated for respectively 0h, for 24 hours, 48h and 72h prepare A β40Monomer, oligomer, fiber precursor and fiber liquid solution.
Step 4: in BPNS-Zn2+Solution made from step 3, test b PNS-Zn are separately added into solution2+It is poly- to difference
Collection state A β40The fluorescence response of sample.
Step 5: the soluble A β of same volume various concentration is pipetted40Sample is to BPNS-Zn2+In solution, 505nm is collected
With the ratio of fluorescence intensity at 423nm, itself and soluble A β are determined40There are linear relationship ranges for stock sample solution concentration.
Step 6: preparation A β40Self aggregation solution, respectively the centrifuging and taking supernatant when hatching 0h, 48h and 72h.It draws certain
Amount supernatant is added to BPNS-Zn2+In solution, by the ratio of fluorescence intensity at measurement 505nm and 423nm, step 5 is utilized
The linear relationship provided calculates solubility A β content in supernatant.
Fig. 1 is the BPNS structural formula figure that embodiment step one synthesizes, and as can be seen from Fig., BPNS is by chromophore 2- pyridyl group
Benzothiazole is connected with dansyl Cl fluorogen.
Fig. 2 is embodiment BPNS to various concentration Zn2+Fluorescence response figure.BPNS at the 505nm and at 423nm there are two
Fluorescence emission peak.With Zn2+There is apparent reduction in the increase of concentration, BPNS fluorescence intensity at 505nm, and in 423nm
Place's fluorescence intensity gradually rises, and the two forms isoelectric point at 442nm.Illustrate BPNS to Zn2+Show ratio metering type fluorescence
Response, and and Zn2+Form BPNS-Zn2+Adduct.
Fig. 3 is embodiment BPNS-Zn2+To the fluorescence response figure of various concentration solubility A β.When soluble A β be added to
BPNS-Zn2+In system, with the raising of A β concentration, fluorescence intensity of the BPNS at 505nm is gradually recovered, small at 423nm
Peak dies down.Illustrate in Zn2+In the presence of, BPNS shows ratio metering type fluorescence response to soluble A β.
Fig. 4 is embodiment BPNS-Zn2+To the fluorescence response selective figure of different coherent condition A β.In identical concentration item
Under part, since soluble A β has higher Zn2+Binding affinity, BPNS-Zn2+To soluble A beta monomers and oligomer
Fluorescence response is substantially better than insoluble fiber precursor and fiber.This illustrates BPNS-Zn2+There is selectivity to soluble A β
Recognition capability.
Fig. 5 is embodiment BPNS-Zn2+To different large biological molecules (A β40Oligomer, HSA, MT-I, Tau, trypsin and
BHb) fluorescence response selective figure.The experimental results showed that under identical concentration conditions, BPNS-Zn2+To other biological macromolecular
Almost without fluorescence response, this explanation compares other biological macromolecular, BPNS-Zn2+There is height to the fluorescence identifying of soluble A β
Selectivity, and be BPNS-Zn2+Application in biological complex system provides possibility.
Fig. 6 is embodiment BPNS-Zn2+Fluorescence intensity ratio and solubility A β at 505nm and 423nm40The linear pass of concentration
System's figure.To BPNS-Zn2+Same volume various concentration solubility A β is separately added into solution40Oligomer is incubated for 37 DEG C altogether after mixing
After changing 30min, the ratio of BPNS fluorescence intensity at 505nm and 423nm is collected to soluble A β40Oligomer Stock concentrations are made
Figure.The experimental results showed that the ratio of fluorescence intensity and solubility A β at 505nm and 423nm40It is deposited between oligomer Stock concentrations
In highly linear relationship (R2=0.998).Linear relationship expression formula is obtained by linear fit, quantitatively to examine in actual sample
Survey solubility A β40It lays a good foundation.
Fig. 7 is embodiment BPNS in Zn2+In the presence of to A β40Solubility A in self aggregation solution difference brooding time supernatant
β40Quantitative detection figure.By 40 μM of A β40Hatch different time at 37 DEG C, supernatant is collected by centrifugation respectively, is examined by BPNS
Discovery is surveyed, as the increase of brooding time generates more and more insoluble aggregates since A beta-aggregation degree is constantly reinforced,
So that soluble aggregate content declines in supernatant.Supernatant by 36.7 ± 3.7 μM of 0h be reduced to 48h 27.7 ±
2.5 μM, become 26.7 ± 1.3 μM after 72h.A β is in 48h mainly by monomer aggregation through soluble oligomeric to insoluble fibre
The process of precursor, thus quantitative detection result reduction amplitude is larger.And if the insoluble A beta precursor of 48h to 72h is to insoluble
Property fiber aggregation, so solubility A β changes of contents amplitude relative decrease in supernatant, testing result and A β self aggregation process
It is consistent completely.The result shows that BPNS is in Zn2+In the presence of the quantitative detection that can be used for solubility A β in actual sample.
From above-described embodiment as can be seen that ratio metering type fluorescence probe provided by the invention being capable of quantitative detection solubility
Aβ40Aggregation.Chromophore 2- pyridyl group benzothiazole plays key effect, it is not only able to induce with metal ion-chelant
BPNS ratio measures change in fluorescence, and since the binding ability with metal ion is weaker than soluble A β, and soluble A β can be made logical
It crosses coordination and takes metal ion in BPNS out by force, the ratio metering fluorescence response of BPNS is activated to realize that quantitative detection is solvable again
Property A β40Aggregation.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Claims (4)
1. a kind of method with ratio metering type fluorescence probe quantitative detection solubility amyloid beta, which is characterized in that the party
Method includes:
Step 1: the ratio metering type fluorescence probe of synthesis such as flowering structure:
Step 2: ratio metering type fluorescence probe is to Zn made from appraisal procedure one2+Or Cu2+Ratio measure fluorescence response
Energy；
Step 3: amyloid beta sample is added in buffer solution, 37 DEG C respectively be incubated for 0h, for 24 hours, 48h and 72h system
Standby solubility amyloid beta monomer, oligomers, insoluble fibre precursor and insoluble fibre liquid solution；
Step 4: in the adduct solution that the ratio metering type fluorescence probe bind metal ion made from step 1 is formed respectively
Solution made from step 3 is added, test adduct rings the selection sex ratio metering fluorescence of soluble amyloid beta sample
It answers；
Step 5: the adduct that the ratio metering type fluorescence probe as made from step 1 and metal ion are formed is to various concentration
The step of three made from the ratio metering fluorescence response of solubility amyloid beta oligomer determine quantitative detection concentration model
It encloses；
Step 6: the adduction analyte detection difference sample formed using ratio metering type fluorescence probe made from step 1 and metal ion
The content of soluble amyloid beta in product.
2. according to claim 1 a kind of with ratio metering type fluorescence probe quantitative detection solubility amyloid beta
Method, which is characterized in that the ratio metering type fluorescence probe is using chromophore 2- pyridyl group benzothiazole as metal ion chela
The red sulphonyl fluorophor of group coupling is closed to be made.
3. according to claim 1 a kind of with ratio metering type fluorescence probe quantitative detection solubility amyloid beta
Method, which is characterized in that the metal ion is Zn2+Or Cu2+。
4. according to claim 1 a kind of with ratio metering type fluorescence probe quantitative detection solubility amyloid beta
Method, which is characterized in that the buffer solution is 20mM Tris-HCl, 150mM NaCl, pH 7.4.
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