CN107478844A - One kind detection new prawn Serum specificity immunoglobulin E method of knife volume - Google Patents
One kind detection new prawn Serum specificity immunoglobulin E method of knife volume Download PDFInfo
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- CN107478844A CN107478844A CN201710623119.8A CN201710623119A CN107478844A CN 107478844 A CN107478844 A CN 107478844A CN 201710623119 A CN201710623119 A CN 201710623119A CN 107478844 A CN107478844 A CN 107478844A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses one kind to detect the new prawn Serum specificity immunoglobulin E method of knife volume, belongs to material and biomedical crossing domain.The present invention uses carriers of the fluorescent hollow mesoporous silicon oxide FHMNs as anti-IgE antibodies, and coated with silica ferroso-ferric oxide prepares the Fe of gained3O4@SiO2As allergy original vector, two kinds of materials capture the new prawn Serum specificity immunoglobulin E (IgE) of target knife volume simultaneously, utilize Fe3O4@SiO2Magnetic fast enriching and separation target sIgE, utilize the signal of the fluorescence molecule signal amplification low concentration target sIgE in FHMNs, fluorescence intensity is associated with IgE concentration, the specific IgE for detecting the new prawn of knife volume in people's blood of rapid sensitive.The present invention effectively reduces IgE test limits, shortens detection time.
Description
Technical field
The present invention relates to one kind to detect the new prawn Serum specificity immunoglobulin E method of knife volume, belongs to material and biomedicine
Crossing domain.
Background technology
Shrimp, crab are most common crust based foods on China's fishery market, belong to high protein, high nutrition group food.But
It is also simultaneously one of high sensitization food of eight major classes, incidence 15.6%-35%, causes allergic asthma, anaphylactic shock
Etc. the generation of disease.
At present, China is in the starting stage in allergy Related Research Domain, there is no shrimp, the correlation of crab group food allergy to examine
Disconnected reagent.The detection reagent clinically used relies primarily on import, and the Immuno CAP system such as U.S. FDA certification are automatic
Detecting system, its generally acknowledged detection is limited to 0.24ng/mL, and testing cost is costly, and most of autopaths can not bear；Face
Wider ELISA detection kit is used on bed, its test limit is higher, can not precise Identification whether be autopath.And mesh
Anaphylactogen used in the detection of preceding allergy is that biomaterial slightly carries product, slightly carry product include anaphylactogen and unspecific allergic it is former and
, interference to allergy testing result be present, and its sensitization efficiency is unable to reach standardization in not yet qualitatively doubtful anaphylactogen at present.
Meanwhile the generation of anaphylactia, genetic background, the life living environment of patient with individual, allergic effect original species, anaphylactogen connect
It is closely related to touch the factors such as history.Therefore, main allergen, the quick spirit of exploitation in the necessary Species of Crustacea using purifying
Quick detection method.
The content of the invention
To solve the above problems, the present invention provides a kind of allergy detection platform, detected for specific allergy.
First purpose of the present invention is to provide a kind of specific allergy detection method, the allergen detection methods include with
Lower step：
(1) anti-immunoglobulin E antibody is connected as fluorescence probe by the hollow mesoporous silicon oxide for including fluorescence molecule；
(2) Fe of gained is prepared by coated with silica ferroso-ferric oxide3O4@SiO2Anaphylactogen is connected as magnetic probe；
(3) fluorescence probe of step (1) and the magnetic probe of step (2) are added simultaneously and contains specific immunoglobulin
In E serum；
(4) it is incubated, supernatant is removed in centrifugation, and using alkali lye is added after wash buffer, then ultrasound release fluorescence molecule, is detected
Fluorescence intensity.
In one embodiment of the invention, the fluorescence probe is that particle diameter is 190-210nm, internal diameter 120-
140nm；Or particle diameter is 110-120nm, internal diameter 60-70nm.
In one embodiment of the invention, the particle diameter of the magnetic probe is 100-120nm.
In one embodiment of the invention, serum solution pH 10-12 in methods described, incubation time 100-
120min, incubation temperature are 35-38 DEG C.
In one embodiment of the invention, methods described also includes：
(1) serum solution containing various concentrations Serum specificity immunoglobulin E is taken, it is strong to determine fluorescence using the above method
Degree；
(2) regression equation of Serum specificity immunoglobulin E concentration and fluorescence intensity is built；
(3) serum to be detected is used into identical method fluorescence intensity, the regression equation brought into step (2), meter
Calculate Serum specificity immunoglobulin E concentration in serum to be detected.
Second object of the present invention is to provide application of the allergen detection methods in specific allergy detection.
Third object of the present invention is to provide the allergen detection methods in the detection new prawn specific immunity ball of knife volume
Application in albumen E.
In one embodiment of the invention, the application is to use particle diameter as 190-210nm, internal diameter 120-
Carrier of the 140nm fluorescence probe as anti-immunoglobulin E antibody, particle diameter are 100-120nm magnetic probe Fe3O4@SiO2
As allergy original vector, two kinds of materials capture the new prawn Serum specificity immunoglobulin E of target knife volume simultaneously, utilize Fe3O4@SiO2
Magnetic fast enriching and separation target；The regression equation of the application is：Y=6489-0.02037*208.3-x-
456.7249*0.5113x, R2=0.9959, Serum specificity immunoglobulin E lowest detection is limited to 0.0159ng/mL.
In one embodiment of the invention, the application is to use particle diameter as 110-120nm, internal diameter 60-70nm
Carrier of the fluorescence probe as anti-immunoglobulin E antibody, particle diameter is 100-120nm magnetic probe Fe3O4@SiO2As
Allergy original vector, two kinds of materials capture the new prawn Serum specificity immunoglobulin E of target knife volume simultaneously, utilize Fe3O4@SiO2Magnetic
Property fast enriching and separation target；The regression equation of the application is：Y=2119-12270x, R2=0.9908, specificity is exempted from
Epidemic disease globulin E lowest detections are limited to 0.0180ng/mL.
In one embodiment of the invention, the anaphylactogen is tropomyosin or arginine kinase.
Beneficial effect of the present invention
Using the signal of the fluorescence molecule amplification low concentration target specificity immunoglobulin E in FHMNs, by fluorescence intensity
It is associated with Serum specificity immunoglobulin E concentration, the specific immunity ball egg of the new prawn of knife volume in detection people's blood of rapid sensitive
White E.
Brief description of the drawings
The hollow mesoporous silicon oxide HMNs-A detections figure A of Fig. 1:Transmission electron microscope TEM is detected；B：Scanning electron microscope sem detects；
The hollow mesoporous silicon oxide HMNs-B detections figure A of Fig. 2:Transmission electron microscope TEM is detected；B：Scanning electron microscope sem detects；
Fig. 3 transmission electron microscopes TEM detects A:Fe3O4；B：Fe3O4@SiO2Detection figure；
Fig. 4 testing conditions optimize：ApH；B temperature；The C times；
Fluorescence intensity and various concentrations IgE relation curves when Fig. 5 FHMNs-A are fluorescence probe；
Fluorescence intensity and various concentrations IgE relation curves when Fig. 6 FHMNs-B are fluorescence probe；
Relation between Fig. 7 fluorescence intensities and separate sources IgE serum.
Embodiment
Embodiment 1：It is prepared by different-grain diameter fluorescence probe FHMNs@PDDA@PAA
Cetyl trimethylammonium bromide (CTAB) powder is dissolved in 29mL deionized waters, 12mL ethanol and 1mL ammoniacal liquor
Mixed liquor in, then into reaction solution add polystyrene microsphere dispersion liquid (being scattered in 10ml deionized waters), at room temperature
After magnetic stirring 60min, tetraethyl orthosilicate (TEOS) is added dropwise, keeps certain mixing speed 150rpm/min, at room temperature
Continue to stir 48h.After question response terminates, reaction solution is done for 65 DEG C after being filtered by vacuum repeatedly, wash for several times in vacuum drying chamber
Dry 12h, obtain hud typed PS/SiO2Complex microsphere powder.8h is calcined at 550 DEG C, template polystyrene is removed, obtains hollow two
Silica.0.05g HMNs are taken to be scattered in 0.012g 5 (6)-fluorescein isothiocynate (FITC) in 5ml deionized waters, in room
The lower stirring 48h of temperature, obtained compound is centrifuged, 40 DEG C of intermediary hole silica FHMNs for being dried to obtain fluorescence labeling.Take
10mg FHMNs are dispensed into 0.2% hexadecyltrimethylammonium chloride solution (PDDA), and stirring 30min produces homogeneous suspension liquid,
Compound centrifugation washing nowhere remains PDDA, will precipitation is rapid is scattered in 0.5% polyacrylic acid (PAA) stirring 30min, at a high speed from
The fluorescence molecule that the heart removes residual PAA, polymer P DDA and PAA protection inherence is not compromised, repeats until being produced from group
The FHMNs@PDDA@PAA of dress, as fluorescence probe.
Morphology characterization：The hollow mesoporous silicon oxide HMNs prepared is taken and is scattered in right amount in ethanol solution, after ultrasound
7ul is added dropwise to be detected with transmission electron microscope TEM after copper mesh drying；Scanning electron microscope sem detects.The HMNs- of preparation is found out by detection Fig. 1
A particle diameters about 200n, internal diameter about 130nm, pattern is complete, the HMNs-B particle diameters about 105nm of preparation, internal diameter about 65nm, and pattern is complete.
Embodiment 2：Magnetic probe Fe3O4@SiO2It is prepared by@PAA nano-particles
Weigh 300mgFe3O4It is scattered in the mixed liquor of 40mL ethanol and 4mL deionized waters, after ultrasonic 15min, keeps certain
Mixing speed, add 5mL ammoniacal liquor, be slowly added to 2mLTEOS, react 12h at room temperature, it is molten with 0.1moL/L hydrochloric acid after Magneto separate
Liquid, deionized water are respectively washed 3 times, and 40 DEG C of dry 12h, it is ferroso-ferric oxide/silica shell core nanometer to obtain reddish-brown precipitation
Particle.By obtained Fe3O4@SiO2It is scattered in dimethylformamide (DMF), with the 33ml DMF dissolved with 2g polyacrylic acid (PAA)
Mixing, ultrasonic 30min, mixture are stirred vigorously and are heated to 110 DEG C, add dissolved with DMAP 3.3mLDMF and
Dissolved with N, the 6.6mLDMF of N'- dicyclohexylcarbodiimides (DCC), after mixture keeps 110 DEG C of stirring 12h, product Magneto separate,
Washed 3 times with ethanol, 60 DEG C of dry 12h obtain magnetic probe Fe3O4@SiO2@PAA。
Morphology characterization：By Fe3O4With the Fe prepared3O4@SiO2Take and be scattered in right amount in ethanol solution respectively, dripped after ultrasound
7uL is added to be detected after copper mesh drying with transmission electron microscope TEM.Fe is found out by detection Fig. 33O4Particle diameter 7-20nm, success coated Si O2Shape
Fe is formed into core-shell structure3O4@SiO2, particle diameter about 100nm.
Embodiment 3pH optimization process, time-optimized process, temperature optimization process provide pH optimizations respectively
Take appropriate Fe3O4@SiO2It is positive that@PAA-antigens and FHMNs@PDDA@PAA-Ab2 add the new prawn of knife volume
IgE (Serum specificity immunoglobulin E), after being incubated at a certain temperature, Magneto separate collects precipitation, uses phosphate buffer
(pH7.4) wash 3 times, Magneto separate collects precipitation, adds 3mL differences pH sodium hydroxide solution ultrasound 20min, dissolves hollow Jie
Hole shell, fluorescence molecule is discharged, fluorescence intensity, as a result as shown in Figure 4 A, in pH 11, fluorescence intensity is maximum, so selection
PH 11 sodium hydroxide solution is detection solution.
Temperature optimization
Take appropriate Fe3O4@SiO2It is positive that@PAA-antigens and FHMNs@PDDA@PAA-Ab2 add the new prawn of knife volume
IgE, respectively at 25 DEG C, 37 DEG C, 45 DEG C, after 60 DEG C are incubated certain time, Magneto separate collects precipitation, with phosphate buffer (pH
7.4) wash 3 times, Magneto separate collects precipitation, adds the sodium hydroxide solution ultrasound 20min of 3mL certain pHs, and dissolving is hollow mesoporous
Shell, fluorescence molecule is discharged, fluorescence intensity, as a result as shown in Figure 4 B, fluorescence intensity is maximum at 37 DEG C, so 37 DEG C of selection
It is incubated.
It is time-optimized
Take appropriate Fe3O4@SiO2It is positive that@PAA-antigens and FHMNs@PDDA@PAA-Ab2 add the new prawn of knife volume
IgE, after being incubated 30-150min respectively at a certain temperature, Magneto separate collects precipitation, is washed with phosphate buffer (pH 7.4)
3 times, Magneto separate collects precipitation, adds the sodium hydroxide solution ultrasound 20min of 3mL certain pHs, dissolves hollow mesoporous shell, and release is glimmering
Optical molecule, fluorescence intensity, as a result as shown in Figure 4 C, when reacting 120min, fluorescence intensity is maximum, so selection 120min
Incubation time.
Embodiment 4：The foundation of allergy detection architecture magnetic fluorescence sensing platform (EMFP)
(1) the FHMNs@PDDA@PAA that 50mg is prepared are taken through 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
(EDC) carboxyl in/n-hydroxysuccinimide (NHS) activation PAA, the FHMNs@PDDA@PAA activated in right amount are taken to add certain
The anti-IgE antibodies (Ab2) of concentration, 37 DEG C of incubation 2h, centrifugation are removed supernatant, washed 3 times with PBS, add 37 DEG C of closing 1h of confining liquid,
Supernatant is removed in centrifugation, collects the FHMNs@PDDA@PAA-Ab prepared24 DEG C are scattered in PBS to save backup.
(2) Fe that will be prepared3O4@SiO2@PAA nano particles, the carboxyl in PAA is activated through EDC/NHS, adds purifying
The new prawn main allergen tropomyosin of knife volume and arginine kinase, 37 DEG C of incubation 120min, Magneto separate abandons supernatant, uses
PBS washs 3 times, adds 37 DEG C of closing 1h of confining liquid, and centrifugation removes supernatant, collects the Fe prepared3O4@SiO2@PAA-
Antigens is scattered in PBS 4 DEG C and saved backup.
Embodiment 5：The application of allergy detection architecture magnetic fluorescence sensing platform (EMFP)
Take clinical detection for knife volume newly to the serum of shrimp allergen IgE strong positives, standard is used as using the IgE in people's blood source
Product, it is configured to the dilution (a) of various concentrations：0.0125ng/mL, (b)：0.0159ng/mL, (c)：0.02ng/mL, (d)：
0.025ng/mL, (e)：0.03333ng/mL, (f)：0.05ng/mL, (g)：0.10ng/mL, (h)：0.50ng/mL, (i)：
1ng/mL, (j)：2ng/mL, (k)：5ng/mL, (l)：9.895ng/mL, 100uL serum dilutions are taken, by the blood of various concentrations
Clear dilution adds 100uL Fe3O4@SiO2@PAA-antigens, while 150uL FHMNs@PDDA@PAA-Ab2 are added,
120min is incubated, centrifugation is removed supernatant, washed 3 times with PBS (pH 7.4) buffer solution, adds 3m L, 0.1mol/LNaOH (pH 11)
Solution, ultrasonic 20min, FITC fluorescence molecules are discharged, with luminoscope fluorescence intensity.
As a result as shown in figure 5, using FHMNs-A as fluorescence probe detect target IgE, its fluorescence curve more than
Good linear relation is presented in 0.0125ng/mL, and as the increase of target IgE contents, fluorescence intensity gradually increase, regression equation：y
=6489-0.02037*208.3-x-456.7249*0.5113x, R2=0.9959, IgE lowest detection are limited to 0.0159ng/mL.
As shown in fig. 6, detecting target IgE by fluorescence probe of FHMNs-B, its fluorescence curve is in 0.02-0.1042ng/mL
Good linear relationship is presented, and with the increase of target IgE contents, fluorescence intensity gradually reduces, because fluorescence molecule
FITC has aggregation inducing quenching effect, and target IgE contents are more, and the fluorescence molecule of aggregation is more, FITC is produced aggregation and lures
Effect is led, its fluorescence intensity reduces, regression equation：Y=2119-12270x, R2=0.9908, IgE lowest detection are limited to
0.0180ng/mL。
Embodiment 6：The new prawn specific IgE detection selectivity analysis of knife volume
Clinical diagnosis is taken as Eriocheir sinensis, mud crab, the new prawn strong positive IgE of knife volume and its mixing IgE and is free of
IgE blank control, same concentrations are taken to add EMFP detection architectures, the fluorescence intensity (Fig. 7) under the conditions of 5 kinds of detection, knot respectively
Fruit shows EMFP detecting systems, only the serum containing the new prawn IgE of knife volume is presented compared with high fluorescent, with blank control phase
Than the serum containing Eriocheir sinensis, mud crab specific IgE shows negligible fluorescence intensity, to sum up result table
It is bright：The EMFP detecting systems, there is higher specificity, detection time 135min to the new prawn specific IgE of knife volume
(120min incubation times, 15min detection times), compared with traditional technique in measuring at least needs 240min, when shortening detection
Between, simplify operating procedure.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (10)
1. a kind of specific allergy detection method, it is characterised in that the allergen detection methods comprise the following steps：
(1) anti-immunoglobulin E antibody is connected as fluorescence probe by the hollow mesoporous silicon oxide for including fluorescence molecule；
(2) Fe of gained is prepared by coated with silica ferroso-ferric oxide3O4@SiO2Anaphylactogen is connected as magnetic probe；
(3) fluorescence probe of step (1) and the magnetic probe of step (2) are added simultaneously and contains Serum specificity immunoglobulin E
In serum；
(4) it is incubated, supernatant is removed in centrifugation, and using alkali lye is added after wash buffer, then ultrasound release fluorescence molecule, detects fluorescence
Intensity.
2. method according to claim 1, it is characterised in that the fluorescence probe is that particle diameter is 190-210nm, and internal diameter is
120-140nm；Or particle diameter is 110-120nm, internal diameter 60-70nm.
3. method according to claim 1, it is characterised in that the particle diameter of the magnetic probe is 100-120nm.
4. method according to claim 1, it is characterised in that serum solution pH 11, incubation time are in methods described
120min, incubation temperature are 37 DEG C.
5. method according to claim 1, it is characterised in that methods described also includes：
(1) serum solution containing various concentrations Serum specificity immunoglobulin E is taken, is determined using claim 1 methods described glimmering
Luminous intensity；
(2) regression equation of Serum specificity immunoglobulin E concentration and fluorescence intensity is built；
(3) serum to be detected is used into identical method fluorescence intensity, the regression equation brought into step (2), calculating is treated
Detect Serum specificity immunoglobulin E concentration in serum.
6. application of claim 1 methods described in specific allergy detection.
7. application of claim 1 methods described in the new prawn Serum specificity immunoglobulin E of knife volume is detected.
8. apply according to claim 7, it is characterised in that the application is to use particle diameter internal diameter is for 190-210nm
Carrier of the 120-140nm fluorescence probe as anti-immunoglobulin E antibody, particle diameter are 100-120nm magnetic probe Fe3O4@
SiO2As allergy original vector, two kinds of materials capture the new prawn Serum specificity immunoglobulin E of target knife volume simultaneously, utilize Fe3O4@
SiO2Magnetic fast enriching and separation target；The regression equation of the application is：Y=6489-0.02037*208.3-x-
456.7249*0.5113x, R2=0.9959, Serum specificity immunoglobulin E lowest detection is limited to 0.0159ng/mL.
9. apply according to claim 7, it is characterised in that the application is to use particle diameter internal diameter is for 110-120nm
Carrier of the 60-70nm fluorescence probe as anti-immunoglobulin E antibody, particle diameter are 100-120nm magnetic probe Fe3O4@
SiO2As allergy original vector, two kinds of materials capture the new prawn Serum specificity immunoglobulin E of target knife volume simultaneously, utilize Fe3O4@
SiO2Magnetic fast enriching and separation target；The regression equation of the application is：Y=2119-12270x, R2=0.9908, it is special
Specific immunological globulin E lowest detections are limited to 0.0180ng/mL.
10. apply according to claim 7, it is characterised in that the anaphylactogen is tropomyosin or arginine kinase.
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