CN108072758A - Immuno-chromatographic test paper strip and preparation method thereof is quenched in a kind of quantum dot fluorescence for detecting bisphenol-A - Google Patents
Immuno-chromatographic test paper strip and preparation method thereof is quenched in a kind of quantum dot fluorescence for detecting bisphenol-A Download PDFInfo
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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
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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Abstract
The present invention provides a kind of quantum dot fluorescences for detecting bisphenol-A to be quenched immuno-chromatographic test paper strip, including sample pad 1, glass fibre element film 2, nitrocellulose filter 3, blotting paper 4 and PVC backboards 7, it is characterized in that, it is stained with sample pad 1, glass fibre element film 2, nitrocellulose filter 3, blotting paper 4 successively in order on PVC backboards；The detection line 5 of bisphenol-A antigen and quantum dot chicken ovalbumin conjugate composition and the nature controlling line 6 of quantum dot chicken ovalbumin conjugate composition are coated on the nitrocellulose filter 3 respectively.The invention also discloses the preparation methods of this test strips.Invention has the advantages that following prominent：1st, specificity is high, and sensitivity is good；2nd, testing cost is low.3rd, it is easy to operate.
Description
Technical field
The present invention relates to a kind of methods of bisphenol-A in quick detection food and environment water, particularly a kind of detection bisphenol-A
Quantum dot fluorescence immuno-chromatographic test paper strip and preparation method thereof is quenched.
Background technology
Bisphenol-A is a kind of important intermediate for producing makrolon, epoxy resin, polysulfones and phenol-formaldehyde resin modified etc..
It is a kind of typical incretion interferent, is a kind of different estrogen found in the environment, and bisphenol-A can enter life by food chain
It interacts in object and with estrogen receptor, so as to influence the interior of the functions such as the reproduction of biology, immune nerve and organism
Excretory system.Since the application range of bisphenol-A has the trend gradually widened, therefore will be progressed into production and using process
The health of the mankind is endangered in food and environment, bisphenol-A can not be ignored the potential hazard of ecological environment and human health.
The method of detection bisphenol-A mainly includes chromatogram analysis method and immunoassay method at present.Wherein chromatography side
Method mainly includes thin-layered chromatography (Thin-layer chromatography, TLC), gas chromatography (Gas
Chromatography, GC), high performance liquid chromatography (Liquid chromatography, HPLC) etc., immunoassay method
Mainly include enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), immunochromatographic method
(Immunochromatographic, ICA), chemiluminescence immunoassay (Chemiluminescence immunoassay,
CLIA) etc..Instrument detection method is that have many advantages, such as that accuracy is high, sensitivity is good using most detection methods at present.It but should
The shortcomings that method is that sample handling procedure is cumbersome, it is necessary to which derivative, is returned, it is necessary to which expensive instrument and the personnel of specialty are operated
Yield is low etc., so being not suitable for widely promoting the use of.
Quantum dot (Quantum dots, QDs), also known as semiconductor nano, mainly by II~VI race or III~V
Race's element composition, such as cadmium telluride (CdTe), cadmium selenide (CdSe).Compared with conventional fluorescent material, the advantages of QDs, has：Have
Quantum effect and good photism；With wider excitation wavelength and relatively narrow launch wavelength；Stability is good；With good
Bio-compatibility；Fluorescence lifetime is longer.
It is based on fluorogenic donor (quantum dot) and fluorescent receptor (colloidal gold) that immune series of strata test strips, which are quenched, in quantum dot fluorescence
Resonance energy transfer (FRET) realize.I.e. when the emission spectrum of fluorogenic donor (quantum dot) and fluorescent receptor (colloidal gold)
Absorption spectrum has certain overlapping, (is generally less than when the distance between the two fluorophors is suitable), so that it may it observes glimmering
Light energy from donor to receptor shift the phenomenon that.It is on the basis of immuno analytical method that immunochromatography technique, which is quenched, in quantum dot fluorescence
On, make fluorogenic donor using fluorescence quantum and mixed with envelope antigen, is fixed on NC films, colloidal gold makees fluorescent receptor and antibody
With reference to, when antibody is combined with antigentic specificity, colloidal gold is just marked on determined antigen, under the irradiation of ultraviolet lamp with quantum
Resonance energy transfer occurs for point.Wherein, the presence or absence of nature controlling line (C lines) color decides the validity of test strips, and detection line (T
Line) the presence or absence of then represent the presence or absence of object.The technical operation is simple and quick, result easily judges, safety non-pollution, has
It is widely applied prospect.
The content of the invention
In view of this, the invention is directed to a kind of quantum dot fluorescence quenching for detecting bisphenol-A in food and water body
Immuno-chromatographic test paper strip.
In order to achieve the above objectives, the technical solution of the invention is realized in：
It is a kind of detect bisphenol-A quantum dot fluorescence quenching immuno-chromatographic test paper strip, including sample pad, glass fibre element film,
Nitrocellulose filter, water absorption pad and PVC backboards, are stained with nitrocellulose filter, glass fibre successively in order on PVC backboards
Plain film, sample pad, water absorption pad；Bisphenol-A antigen and quantum dot-chicken ovalbumin are coated on the nitrocellulose filter respectively
The nature controlling line 6 that detection line 5 and quantum dot-chicken ovalbumin conjugate that conjugate is formed are formed, two lines spacing distance are
5mm。
The invention also discloses the preparation method of above-mentioned quantum dot fluorescence quenching immuno-chromatographic test paper strip, including walking as follows
Suddenly：
1. the purifying of bisphenol-A serum
The purifying of antibody purifies antiserum using affinity chromatography principle.Protein A are from staphylococcus aureus
It separates, specific can be combined with the Fc segments of antibody protein IgG heavy chains in (Staphylococcus aureus).When
After antiserum is added to chromatographic column, Protein A and IgG is specifically bound, and is fixed on filler, by washing off other
Impurity changes eluent and elutes again, so that it may obtain destination protein.The step of purifying, is as follows：
(1) the good solution used of configuration in advance, and lid is unscrewed slightly, ultrasound 20 minutes, to remove the bubble in solution,
To prevent bubble is entered purification column, purification effect is influenced.
(2) purification column is balanced：First with ultrapure water pipeline 15min, ensure free from admixture in entire pipeline, then with pH 7.4
Combination buffer (Binding buffer) flushing line 20min, flow velocity is set to 5mL/min.By purification column from refrigerator
It takes out, outwells 1/3rd ethanol solutions, be connected with protein purification instrument, continue to rinse column with the Binding buffer of pH 7.4
Son, flow rate set 1mL/min.Ultraviolet in software and two baseline values of conductance are observed, and are kept for a period of time.
(3) loading：Antiserum is taken out and is restored to room temperature, after the isometric mixed dilutings of Binding buffer, mistake
0.45 μm of moisture film is to remove impurity, loading.Flow velocity is unsuitable too fast during loading, is traditionally arranged to be 0.5mL/min.With antiserum into
Entering into gel, antibody protein can be attached on pillar, and other albumen do not combine, and can be rinsed out with Binding buffer,
At this point, ultraviolet and conductance is present with foreign protein peak, after two baselines again sub-level, kept for a period of time.
(4) elute：Pillar is rinsed with the eluent buffer solution of pH 2.7 (Elution buffer, now with the current), it will be solidifying
Destination protein on glue elutes, flow velocity 0.5mL/min.
(5) destination protein is collected：After adding in Elution buffer, the variation of ultraviolet conductance figure is observed, when baseline is oriented
On walk trend when collect liquid, often pipe collects 1mL, until the curve sub-level again of ultraviolet conductance figure.It often pipe liquid will use
Ultraviolet-visible spectrophotometer measures absorbance in 280nm, retains absorbance>0.2 antibody purification, by all antibody
Mixing adjusts antibody pH to 7.0 with the Tris of 0.1mol/L.
(6) purification column is handled：Pillar 2min, flow rate set 2mL/min are rinsed with 0.1mol/L acetic acid, then is used
Binding buffer rinse pillar half an hour, and the pH value of outflow buffer solution is measured with pH test paper, after pH test paper shows neutrality,
Pillar 20min is rinsed with 20% ethanol solution, pillar is removed, purification devices is closed, the pillar removed need to be molten with 20% ethyl alcohol
Liquid filling is full, 4 DEG C of preservations.
Polyclonal antibody after purifying in 1 × PB solution is dialysed 3 days for 4 DEG C, is stored for future use for 4 DEG C after taking-up.
2. the preparation of checking matter antigen (BHPVA-OVA)
6.36mg BHPVA are weighed, 200 μ L DMSO is added in and is allowed to dissolve, 3.20mg NHS are added under stirring,
4.31mg EDC, room temperature are protected from light 2h, obtain solution A.It weighs 20.00mg OVA and is dissolved in 2mL 0.1mol/L NaHCO3 (use
HCl tune pH obtains solution B in 7).It will be added dropwise in protein solution under activated ester solution condition of ice bath, stirred under the conditions of 4 DEG C
Mix reaction overnight.With (bag filter specification of dialysing under the conditions of 4 DEG C of the PB buffer solutions of pH 7.4：Molecular cut off 8000-14000Da)
3 days, 3-4 dialyzate was changed daily.The situation of combining is measured under ultraviolet light, and calculating concentration is 7.50mg/mL, dispenses simultaneously 4 DEG C of guarantors
It deposits, it is spare.
3. for the preparation of quantum dot-chicken ovalbumin conjugate：
Quantum dot-chicken ovalbumin (QDs-OVA) conjugate is prepared using active ester method.
(1) coupling reaction.25 μ L quantum dots are taken in centrifuge tube, 0.3mg chicken ovalbumins is added in and 11.5 μ L EDC is molten
Liquid (10mg/ml), mixing, and liquor capacity is supplemented to 200 μ L with borate buffer solution.Then peace road is wrapped up with aluminium-foil paper
Pipe is protected from light in shaking table and shakes reaction 3h.
(2) centrifugal purification.By reaction solution under the conditions of 4 DEG C, 10000rpm centrifugation 3min are formed with removing in labeling process
Coagulation object；Supernatant is transferred in ultrafiltration membrane, under the conditions of 4 DEG C, 8000rpm centrifugation 3min, remove solution in salt from
Son；Finally filter membrane is upside down in another centrifuge tube, under the conditions of 4 DEG C, 8000rpm centrifugation 3min collect the solution in filter membrane.
4 DEG C be kept in dark place it is spare.
4. the preparation of colloidal gold-bisphenol-A antibody (AuNPs-BPA-Ab) label
(1) the accurate 1mL colloidal gold solutions that pipette are placed in import peace deferent, add in 10 μ L 0.2mol/L K2CO3Solution,
Jog pacifies deferent mixing, adjusts pH value to optimum value.
(2) the bisphenol-A antibody (BPA-Ab) of 15 μ L 3.6mg/mL is added in above-mentioned solution and be uniformly mixed, be placed on 4
1h in DEG C refrigerator.
(3) add in thereto 20 μ L concentration be 20% BSA solution to stablize gold labeling antibody, add 10 μ L's 20%
20000 solution of PEG, other sites that closing gold colloid surface is not connected with antibody, stands 30min at room temperature.
(4) deferent trim will be pacified, 15min is centrifuged under the conditions of 4 DEG C of 2000rpm, the gold particle for being not connected with upper antibody is reunited, shape
Into precipitation, Aspirate supernatant is transferred in another peace deferent.
(5) again with 4 DEG C, the pelleted by centrifugation 30min of 10000rpm, at this moment gold labeling antibody forms precipitation.
(6) supernatant is siphoned away, preserves precipitation, is resuspended with gold mark working solution.
5. the coating of nitrocellulose filter
With the double dimensional planes of Shanghai gold target draw after BPA-OVA prepared by step 3 PBS solution is diluted 12 times by film instrument with use
The QDs-OVA labels that PBS solution is diluted after 5.7 times are coated on nitrocellulose filter 3 as detection line 5, package amount 0.8
μL/cm；It is coated in the QDs-OVA after 5.7 times is diluted by the use of PBS solution on nitrocellulose filter 3 as nature controlling line 6, package amount
For 0.8 μ L/cm, 37 DEG C of drying encapsulate spare.
6. the AuNPs-BPA-Ab labels that 4.5 μ L steps 4 is taken to prepare, make an addition in sample solution；
7. adhering to nitrocellulose filter, glass fibre element film, sample pad, water absorption pad successively in order on PVC backboards, obtain
Immuno-chromatographic test paper strip is quenched to the bisphenol-A quantum dot fluorescence.
In above-mentioned material, water-soluble quantum dot is purchased from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., sample pad and nitre
Acid cellulose film is purchased from Millipore companies of the U.S., and water absorption pad and PVC backboards are purchased from Shanghai Jin Biao biotechnologies company.
The present invention has purified checking matter antibody serum, has obtained checking matter polyclonal antibody, spare.BPA-OVA is prepared to mix
The envelope antigen that QDs-OVA labels are detection line is closed, using QDs-OVA label nature controlling lines.It is detected using competition law double
Phenol A judges whether contain object in sample to be tested according to the depth of detection line band.
Compared with the method for existing domestic and international detection bisphenol-A, the present invention has the advantages that following prominent：1st, amount of the invention
Son point fluorescent quenching immuno-chromatographic test paper strip is realized using the specific reaction of antigen-antibody, therefore specificity is high, sensitive
It spends.2nd, test strip detection time of the invention is quick and any specific apparatus and equipment is not required, and testing cost is low.3、
The test strip of the present invention is easy to operate, is not required to be operated by professional.
Description of the drawings
The attached drawing for forming the part of the invention is used for providing further understanding the invention, present invention wound
The schematic description and description made does not form the improper restriction to the invention for explaining the invention.
In attached drawing：
Fig. 1 is the assembling schematic diagram of test strip of the present invention.
Fig. 2 be bisphenol-A detection limit determine (concentration of bisphenol-A from left to right be respectively 0,10,20,50,100,500,
1000μg/L)。
Fig. 3 is from left to right followed successively by the sample detection result that bisphenol-A is added in Wine Sample.The concentration of every group of bisphenol-A from
Left-to-right is respectively 0,4,10,100 μ g/kg or μ g/L.
Fig. 4 is from left to right followed successively by the sample detection result that bisphenol-A is added in cabbages leaves.The concentration of every group of bisphenol-A from
Left-to-right is respectively 0,4,10,100 μ g/kg or μ g/L.
Fig. 5 is from left to right followed successively by the sample detection result that bisphenol-A is added in grass carp sample.The concentration of every group of bisphenol-A from
Left-to-right is respectively 0,4,10,100 μ g/kg or μ g/L.
Fig. 6 is from left to right followed successively by the sample detection result that bisphenol-A is added in river water sample.The concentration of every group of bisphenol-A from
Left-to-right is respectively 0,4,10,100 μ g/kg or μ g/L.
1 description of symbols of attached drawing：
1st, sample pad
2nd, glass fibre element film
3rd, nitrocellulose filter
4th, blotting paper
5th, detection line
6th, nature controlling line
7th, PVC backboards
Specific embodiment
It should be noted that in the case where there is no conflict, the feature in embodiment and embodiment in the invention can
To be mutually combined.
The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments creates.
Embodiment l (preparation embodiment)
(1) purifying of bisphenol-A serum
(1) the good solution used of configuration in advance, and lid is unscrewed slightly, ultrasound 20 minutes, to remove the bubble in solution,
To prevent bubble is entered purification column, purification effect is influenced.
(2) purification column is balanced：First with ultrapure water pipeline 15min, ensure free from admixture in entire pipeline, then with pH 7.4
Combination buffer (Binding buffer) flushing line 20min, flow velocity is set to 5mL/min.By purification column from refrigerator
It takes out, outwells 1/3rd ethanol solutions, be connected with protein purification instrument, continue to rinse column with the Binding buffer of pH 7.4
Son, flow rate set 1mL/min.Ultraviolet in software and two baseline values of conductance are observed, and are kept for a period of time.
(3) loading：Antiserum is taken out and is restored to room temperature, after the isometric mixed dilutings of Binding buffer, mistake
0.45 μm of moisture film is to remove impurity, loading.Flow velocity is unsuitable too fast during loading, is traditionally arranged to be 0.5mL/min.With antiserum into
Entering into gel, antibody protein can be attached on pillar, and other albumen do not combine, and can be rinsed out with Binding buffer,
At this point, ultraviolet and conductance is present with foreign protein peak, after two baselines again sub-level, kept for a period of time.
(4) elute：Pillar is rinsed with the eluent buffer solution of pH 2.7 (Elution buffer, now with the current), it will be solidifying
Destination protein on glue elutes, flow velocity 0.5mL/min.
(5) destination protein is collected：After adding in Elution buffer, the variation of ultraviolet conductance figure is observed, when baseline is oriented
On walk trend when collect liquid, often pipe collects 1mL, until the curve sub-level again of ultraviolet conductance figure.It often pipe liquid will use
Ultraviolet-visible spectrophotometer measures absorbance in 280nm, retains absorbance>0.2 antibody purification, by all antibody
Mixing adjusts antibody pH to 7.0 with the Tris of 0.1mol/L.
(6) purification column is handled：Pillar 2min, flow rate set 2mL/min are rinsed with 0.1mol/L acetic acid, then is used
Binding buffer rinse pillar half an hour, and the pH value of outflow buffer solution is measured with pH test paper, after pH test paper shows neutrality,
Pillar 20min is rinsed with 20% ethanol solution, pillar is removed, purification devices is closed, the pillar removed need to be molten with 20% ethyl alcohol
Liquid filling is full, 4 DEG C of preservations.
(7) polyclonal antibody after purifying in 1 × PB solution is dialysed 3 days for 4 DEG C, is stored for future use for 4 DEG C after taking-up.
(2) preparation of checking matter antigen (BHPVA-OVA)
6.36mg BHPVA are weighed, 200 μ L DMSO is added in and is allowed to dissolve, 3.20mg NHS are added under stirring,
4.31mg EDC, room temperature are protected from light 2h, obtain solution A.It weighs 20.00mg OVA and is dissolved in 2ml 0.1mol/L NaHCO3 (use
HCl tune pH obtains solution B in 7).It will be added dropwise in protein solution under activated ester solution condition of ice bath, stirred under the conditions of 4 DEG C
Mix reaction overnight.With (bag filter specification of dialysing under the conditions of 4 DEG C of the PB buffer solutions of pH 7.4：Molecular cut off 8000-14000Da)
3 days, 3-4 dialyzate was changed daily.The situation of combining is measured under ultraviolet light, and calculating concentration is 7.50mg/ml, dispenses simultaneously 4 DEG C of guarantors
It deposits, it is spare.
It is mixed using the antigen with quantum dot-chicken ovalbumin QDs-OVA, the detection line being coated on nitrocellulose filter.
Embodiment 2 (preparation embodiment)
Assembling and the preparation method of immuno-chromatographic test paper strip is quenched in quantum dot fluorescence
1st, test strips assemble：
The test strips composition of the present invention is as follows：Sample pad, glass fibre element film, nitrocellulose filter, water absorption pad and the PVC back ofs the body
Plate is stained with nitrocellulose filter, glass fibre element film, sample pad, water absorption pad successively in order on PVC backboards；Described
Be coated with respectively on nitrocellulose filter 3 detection line 5 that antigen and quantum dot-chicken ovalbumin QDs-OVA mixtures form and
The nature controlling line 6 that quantum dot-chicken ovalbumin QDs-OVA is formed.
2nd, the preparation of standby quantum dot-chicken ovalbumin conjugate：
Quantum dot-chicken ovalbumin (QDs-OVA) conjugate is prepared using active ester method.
(1) coupling reaction.25 μ L quantum dots are taken in centrifuge tube, 0.3mg chicken ovalbumins is added in and 11.5 μ L EDC is molten
Liquid (10mg/ml), mixing, and liquor capacity is supplemented to 200 μ L with borate buffer solution.Then peace road is wrapped up with aluminium-foil paper
Pipe is protected from light in shaking table and shakes reaction 3h.
(2) centrifugal purification.By reaction solution under the conditions of 4 DEG C, 10000rpm centrifugation 3min are formed with removing in labeling process
Coagulation object；Supernatant is transferred in filter membrane, under the conditions of 4 DEG C, 8000rpm centrifugation 3min remove the salt ion in solution；
Finally filter membrane is upside down in another centrifuge tube, under the conditions of 4 DEG C, 8000rpm centrifugation 3min collect 4 DEG C of solution in filter membrane
It saves backup.
3rd, the preparation of colloidal gold-bisphenol-A antibody (AuNPs-BPA-Ab) label
(1) the accurate 1mL colloidal gold solutions that pipette are placed in import peace deferent, add in 10 μ L 0.2mol/L K2CO3Solution,
Jog pacifies deferent mixing, adjusts pH value to optimum value.
(2) the bisphenol-A antibody (BPA-Ab) of 15 μ L 3.6mg/mL is added in above-mentioned solution and be uniformly mixed, be placed on 4
1h in DEG C refrigerator.
(3) add in thereto 20 μ L concentration be 20% BSA solution to stablize gold labeling antibody, add 10 μ L's 20%
20000 solution of PEG, other sites that closing gold colloid surface is not connected with antibody, stands 30min at room temperature.
(4) deferent trim will be pacified, 15min is centrifuged under the conditions of 4 DEG C of 2000rpm, the gold particle for being not connected with upper antibody is reunited, shape
Into precipitation, Aspirate supernatant is transferred in another peace deferent.
(5) again with 4 DEG C, the pelleted by centrifugation 30min of 10000rpm, at this moment gold labeling antibody forms precipitation.
(6) supernatant is siphoned away, preserves precipitation, is resuspended with gold mark working solution.
4th, the coating of nitrocellulose filter
The bisphenol-A antigen after 12 times will be diluted with using PBS solution with PBS solution by drawing film instrument with the double dimensional planes of Shanghai gold target
Mixing is coated on nitrocellulose filter 3 as detection line 5 QDs-OVA after 5.7 times of dilution in equal volume, and package amount is 0.8 μ L/
cm；It is as nature controlling line 6, package amount using diluting the QDs-OVA after 5.7 times by the use of PBS solution and being coated on nitrocellulose filter 3
0.8 μ L/cm, 37 DEG C of drying, encapsulation are spare.
5th, the assembling of test strips
Nitrocellulose filter 3, glass fibre element film 2, sample pad 1, the order of water absorption pad 4 as shown in Figure 1 are adhered to successively
In PVC board 7, the small item of 3.7mm wide, Vacuum Package are cut into.
Embodiment 3 (Application Example)
1st, quantum dot-labeled immunochromatographyassay assay test-strip application method
The pretreatment of sample
For white wine and Haihe River water both fluid samples, draw respectively after 5mL samples add in 5mL dichloromethane thereto
Vortex oscillation, fully extracts 5min, after stratification, takes out lower floor's organic phase into teat glass.Supernatant liquor is repeated to carry
It takes once, after merging supernatant 2mL is taken to dry up.Spare loading after being redissolved with 400 μ L PBS.For this kind of solid sample of Chinese cabbage,
After vortex oscillation extraction, the centrifugal treating 15min of 8000r/min is carried out so that solid, moisture and organic phase can be effective
Quickly separation takes out organic phase and repeats the above steps.
2nd, detecting step
100 μ L measuring samples extracting solutions are drawn in pacify in deferent with liquid-transfering gun, and add in 4.5 μ L AuNPs-BPA-Ab, it is mixed
It after even, is added drop-wise in the well of test strips, observes result after ten minutes.
3rd, result judgement
If sample to be tested ELISA test strip line fluorescence disappears, it is judged as negative sample, i.e., without bisphenol-A；If treat test sample
Product ELISA test strip line fluorescence be shallower than Quality Control line color or with nature controlling line solid colour, then be judged as positive, that is, treat test sample
Contain bisphenol-A in product；Positive findings and negative findings, the aobvious green fluorescence band of nature controlling line, if nature controlling line green fluorescence band
It disappears, then ELISA test strip fails.
Embodiment 4 (Application Example)
The application effect citing of the present invention
Signified quantum dot fluorescence quenching immuno-chromatographic test paper strip detection method is with reference to described in embodiment 3 in the present embodiment
Operating procedure, testing result are as follows.
1st, sensitivity test
As shown in Fig. 2, it is tested by 3 the method for embodiment.When bisphenol-A standard concentration is less than 20 μ g/L, examination
Paper slip detection line unstressed configuration band, colour developing result have significant difference compared with being estimated with Quality Control line color；When bisphenol-A standard concentration
For 20 μ g/L when, there are fluorescent bands in ELISA test strip line, and colour developing result difference compared with the range estimation of Quality Control line color is smaller；When double
Phenol A standard concentrations continue to increase, and detection line gradually brightens.It is thus determined that the visual detection of method is limited to 20 μ g/L.
2nd, the detection of mark-on sample
As shown in figures 3 to 6, bisphenol-A standard items are added into sample, ultimate density is 0,4,10,100 μ g/kg in sample
Or μ g/L, it is tested by 3 the method for embodiment, testing result such as Fig. 3-Fig. 6, detection limit is 4 μ g/kg or μ g/L.
Experiment show the present invention test strips accuracy is good, high sensitivity, and sample-pretreating method is simple, entire to examine
Survey process is no more than 20min, can be as being effective screening hand that bisphenol-A quickly detects suitable for the quick screening of a large amount of samples
Section.
The foregoing is merely the preferred embodiments of the invention, are not intended to limit the invention creation, all at this
Within the spirit and principle of innovation and creation, any modifications, equivalent replacements and improvements are made should be included in the invention
Protection domain within.
Claims (3)
1. immuno-chromatographic test paper strip is quenched in a kind of quantum dot fluorescence for detecting bisphenol-A, including sample pad, glass fibre element film, nitre
Acid cellulose film, water absorption pad and PVC backboards, which is characterized in that be stained with nitrocellulose successively in order on PVC backboards
Film, glass fibre element film, sample pad, water absorption pad；Bisphenol-A antigen and quantum are coated on the nitrocellulose filter respectively
The nature controlling line C that detection line T and quantum dot-chicken ovalbumin conjugate that point-chicken ovalbumin conjugate is formed are formed, two lines
Spacing distance is 5mm.
2. immuno-chromatographic test paper strip is quenched in a kind of quantum dot fluorescence for detecting bisphenol-A according to claim 1, feature exists
In for detecting bisphenol-A.
3. the preparation method of immuno-chromatographic test paper strip is quenched in a kind of quantum dot fluorescence for detecting bisphenol-A described in claim 1,
It is characterized in that, includes the following steps：
(1) purifying of the antibody serum containing bisphenol-A, obtains the polyclonal antibody of checking matter；
(2) checking matter antigen is prepared；
(3) quantum dot-chicken ovalbumin conjugate is prepared using active ester method；
(4) colloidal gold-antibody label is prepared；
(5) checking matter colloidal gold-antibody label that appropriate step (4) is taken to prepare, makes an addition in sample solution；
(6) checking matter that checking matter quantum dot-chicken ovalbumin label prepared by appropriate step (3) is taken to be prepared with step (2)
Antigen mixing, which is coated on nitrocellulose filter, forms detection line, takes checking matter quantum dot-ovum gallinaceum prepared by appropriate step (3) white
Protein marker, which is coated on nitrocellulose filter, forms nature controlling line；
(7) nitrocellulose filter, glass fibre element film, sample pad, water absorption pad are adhered to successively in order on PVC backboards, is obtained
Immuno-chromatographic test paper strip is quenched in the quantum dot fluorescence of the bisphenol-A.
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| CN201710860470.9A CN108072758A (en) | 2017-09-21 | 2017-09-21 | Immuno-chromatographic test paper strip and preparation method thereof is quenched in a kind of quantum dot fluorescence for detecting bisphenol-A |
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| CN201710860470.9A CN108072758A (en) | 2017-09-21 | 2017-09-21 | Immuno-chromatographic test paper strip and preparation method thereof is quenched in a kind of quantum dot fluorescence for detecting bisphenol-A |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298188A (en) * | 2018-11-13 | 2019-02-01 | 广东众尔健生物科技有限公司 | One heavy metal species and creatinine combined detection test paper and the preparation method and application thereof |
| WO2023059283A1 (en) * | 2021-10-08 | 2023-04-13 | Marmara Universitesi Rektorlugu Ozel Kalem Birimi | A lateral flow test strip for detection and/or measurement of bisphenol a in breast milk |
-
2017
- 2017-09-21 CN CN201710860470.9A patent/CN108072758A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| WEI SHENG，ET AL.: "Lateral Flow Quantum-Dot-Based Immunochromatographic Assay and Fluorescence Quenching Immunochromatographic Assay with Quantum Dots as Fluorescence Donors to Visually Detect Bisphenol A in Food and Water Samples", 《FOOD ANAL. METHODS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298188A (en) * | 2018-11-13 | 2019-02-01 | 广东众尔健生物科技有限公司 | One heavy metal species and creatinine combined detection test paper and the preparation method and application thereof |
| WO2023059283A1 (en) * | 2021-10-08 | 2023-04-13 | Marmara Universitesi Rektorlugu Ozel Kalem Birimi | A lateral flow test strip for detection and/or measurement of bisphenol a in breast milk |
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