CN108931646A - A kind of test strips and its preparation method and application detecting safrole - Google Patents
A kind of test strips and its preparation method and application detecting safrole Download PDFInfo
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- CN108931646A CN108931646A CN201810540551.5A CN201810540551A CN108931646A CN 108931646 A CN108931646 A CN 108931646A CN 201810540551 A CN201810540551 A CN 201810540551A CN 108931646 A CN108931646 A CN 108931646A
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- safrole
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
The present invention provides a kind of test strips and its preparation method and application for detecting safrole.The test strips include sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate；Wherein, have on the reaction film and is coated with the detection line of safrole hapten-carrier protein conjugate and is coated with the nature controlling line of sheep anti mouse antiantibody, safrole monoclonal antibody-colloid gold label object is coated in the conjugate release pad, the safrole haptens is to be reacted by 5- (the bromo- 2- propylene -1- base of 2-) -1,3- benzodioxolane with alanine.The present invention also provides the methods of safrole in the above-mentioned test strips test sample of the preparation method and application of safrole test strips.Test strips and detection method provided by the invention have the advantages that easy to operate, high sensitivity, detection fast, at low cost, the not examined equipment limit of speed, can be realized and are used for quickly detecting to the safrole in batch samples and on-site supervision.
Description
Technical field
The present invention relates to a kind of test strips and its preparation method and application for detecting safrole, and in particular to one kind is for examining
The colloidal gold strip of safrole is surveyed, it is especially suitable for the detections of safrole in essence spice for cigarette, tobacco and tobacco product.
Background technique
Safrole is also known as safrole, safrole, 1-ally-3,4-methy-lene dioxy benzene, and colourless or yellowish liquid is not soluble in water, be soluble in chloroform, ether and
Other non-polar organic solvents have camphorwood smell, are naturally present in the plants such as cortex cinnamomi, nutmeg, black pepper and purple perilla, are to be permitted
The main component of such as yellow camphor tree essential oil of more edible natural essential oils, aniseed essential oil and camphorated oil.Due to its comfortable smell, sassafras oil was once
Through being used as flavoring agent and deodorant tune in food, articles for washing and cosmetics.Safrole class compound mainly includes safrole, different
Safrole, dihydrosafrole etc..Safrole is also an important component of illegal production drugs head-shaking pill.Due to testing table
Bright safrole has the effect for causing liver tumour to mouse, and therefore, various countries have carried out limitation regulation to safrole.2001, committee of European Union
Member can provide that safrole and the total of isosafrole are 1 mg/kg in food and beverage using limiting the quantity, and pick-me-up is 5 mg/
kg.2005, safrole is classified as by European commission suspected weak carcinogen, although being not specified by daily limitation, requirement is set
That sets is as low as possible.What China implemented in 2005《Regulation on Management of Drug-Making Chemicals》Safrole is classified as first kind poison easily processed
Chemicals.Essence spice for cigarette is possible to introduce safrole by natural plant essential oils during the manufacturing, and then passes through cigarette
The flavors and fragrances added in silk is present in tobacco product, causes potential hazard to the health of consumer.Therefore, safrole is established
Rapid detection method, it is very necessary that quality control is carried out to flavors and fragrances and tobacco product.
Currently, safrole mainly passes through direx process, ultrasonic extraction method, Simultaneous distillation, steam distillation
Deng extraction, in conjunction with gas chromatography（GC）, gas chromatography-mass spectrography（GC-MS）, gas chromatography tandem mass spectrometry method（GC-MS/
MS）, high performance liquid chromatography（HPLC）, ultra performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）Detection.Instrument analysis side
Though accurate qualitative, quantitative may be implemented in method, instrument and equipment and the professional operator of complex and expensive are needed, in addition before sample
Manage it is cumbersome it is time-consuming, testing cost is high, it is difficult to meet the needs of on-site supervision and great amount of samples screening.Therefore, develop it is a kind of not by
Detection device, which limits and can be realized the product being used for quickly detecting to batch samples and method, becomes in the urgent need to address
The problem of.So far, patent relevant to safrole detection in essence spice for cigarette and tobacco and document report are less, market
On there has been no the appearance of safrole test strips.
Summary of the invention
The purpose of the present invention is based on above-mentioned prior art situation and provides a kind of test strips and its system for detecting safrole
Preparation Method and application, present invention application colloidal gold immunity chromatography measure the safrole in essence spice for cigarette and tobacco product,
It is high, easy to operate with accuracy, without professional operation, without other mating detection devices, be convenient for carrying detection, time
Short, the features such as testing cost is low, it is applicable in the screening of a large amount of samples on site.
In order to achieve the object of the present invention, the present invention provides a kind of test strips for detecting safrole, which includes
Sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate；Wherein, have on the reaction film and be coated with safrole
The detection line of hapten-carrier protein conjugate and the nature controlling line for being coated with sheep anti mouse antiantibody, in the conjugate release pad
It is coated with safrole monoclonal antibody-colloid gold label object；The safrole monoclonal antibody is with safrole hapten-carrier
Protein conjugate is prepared as immunogene.The safrole hapten-carrier protein conjugate by safrole haptens with
Carrier protein couplet obtains, and the carrier protein is bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin or human seralbumin egg
White, the safrole haptens is to be reacted by 5- (the bromo- 2- propylene -1- base of 2-) -1,3- benzodioxolane with alanine
It arrives, molecular structural formula is：
The preparation method of the safrole haptens is as follows：
5- (the bromo- 2- propylene -1- base of 2-) -1,3- benzodioxolane, 1.0 g is taken, anhydrous dimethyl sulphoxide is added（DMSO） 50 mL
Dissolution, stirring clarification, adds 0.48 g of potassium hydroxide, adds 0.20 g of anhydrous sodium iodide, after being vigorously stirred, add alanine 0.41
G, 100 DEG C of reaction 4h.Stop reaction, add 50 mL of water, add 1mol/L salt acid for adjusting pH value to 6, add 80 mL of ethyl acetate, extracts
Three times, merge organic phase, revolving is evaporated, upper silicagel column, is 1 with volume ratio:5 ethyl acetate/petroleum ether elutes separation, obtains
Haptens product propionic acid safrole 0.91g.
The sheep anti mouse antiantibody is to carry out immune prepare to goat using source of mouse antibody as immunogene.
The sample absorption pad, conjugate release pad, reaction film, water absorption pad are successively pasted on bottom plate, the conjugate
Release pad 1/3 ~ 1/2 is capped under sample absorption pad.
The bottom plate is PVC bottom plate；The sample absorption pad is suction strainer paper；The conjugate release pad is mineral wool；Institute
Stating water absorption pad is blotting paper；The reaction film is nitrocellulose filter.
It is a further object to provide a kind of methods for preparing above-mentioned test strips, and this approach includes the following steps：
1）Preparation is coated with safrole monoclonal antibody-colloid gold label object conjugate release pad；
2）Preparing has the detection line for being coated with safrole hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate prepared is assembled into test paper
Item.
Specifically, step includes：
1）It is reacted by 5- (the bromo- 2- propylene -1- base of 2-) -1,3- benzodioxolane with alanine, it is anti-to prepare safrole half
It is former；
2）By safrole haptens and carrier protein couplet, safrole hapten-carrier protein conjugate is prepared；
3）Mouse is immunized with safrole hapten-carrier protein conjugate, by mouse boosting cell and myeloma cell by fusion,
Screening obtains the hybridoma cell strain of secretion safrole monoclonal antibody；
4）Mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5）Safrole hapten-carrier protein conjugate and sheep anti mouse antiantibody are coated in the detection line of reaction film respectively（T）
And nature controlling line（C）On；
6）It is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
7）The safrole monoclonal antibody of preparation is added in the colloidal gold of preparation, safrole monoclonal antibody-colloid is obtained
Golden marker；
8）Safrole monoclonal antibody-colloid gold label object is sprayed in conjugate release pad, takes out, is placed in after 37 DEG C of baking 1h
It is saved backup in dry environment；
9）Sample absorption pad is used and contains 0.5% bovine serum albumin(BSA)（Mass fraction）, pH 7.2 0.1mol/L phosphate buffer
2h is impregnated, dries 2h at 37 DEG C；
10）Paste upper sample absorption pad, conjugate release pad, reaction film, water absorption pad, conjugate release pad in order on bottom plate
There is 1/3 region to be absorbed by the sample pad covering from starting point.It is finally cut into the small item of 3mm wide, adds plastic casing, is vacuum-packed, 4 ~ 30
It can be reserved for 12 months under the conditions of DEG C.The 1/3 of conjugate release pad be absorbed by the sample pad covering can extend testing result observation when
Between, sample absorption pad can be made to will test liquid and fully absorb and sufficiently reacted with gold labeling antibody, to reduce error.
It is a further object to provide a kind of method using safrole in above-mentioned test strips test sample, the party
Method includes the following steps：
（1）Pre-treatment is carried out to sample；
（2）It is detected with test strips；
（3）Analysis detection result.
The test strips of detection safrole of the invention are reacted using antibody antigen high degree of specificity and immunochromatographiassays assays skill
Safrole monoclonal antibody-colloid gold label object is fixed in conjugate release pad by art, and the safrole in sample is flowing over
Cheng Zhong forms safrole-antibody-colloid in conjunction with safrole monoclonal antibody-colloid gold label object in conjugate release pad
Golden marker.The safrole hapten-carrier protein conjugate competitive binding on safrole and reaction film detection line in sample
Safrole monoclonal antibody-colloid gold label object judges whether contain in analyte sample fluid according to the detection line red stripes depth
There is safrole residual.
When detection, sample is instilled after processing in test strips card hole, when the concentration of safrole in the sample is lower than detection limit
Or when being zero, monoclonal antibody-colloid gold label object meeting and the safrole haptens-being fixed on reaction film in chromatography process
Carrier protein couplet object combines, in detection line（T）And nature controlling line（C）Locate one red stripes of each appearance, and the colour developing of T line is than C line
Colour developing is deep or consistent with the colour developing of C line；If the concentration of safrole in the sample is equal to or higher than detection limit, monoclonal antibody-glue
Body gold marker can all be combined with safrole, thus because competitive reaction will not be with safrole hapten-carrier egg at T line
White conjugate is combined without red stripes occur or developing the color than C line shallow.
It is negative：Work as nature controlling line（C）Show red stripes, detection line（T）Red stripes, and T line face are also showed that simultaneously
When color is close or is deeper than C line, it is judged to feminine gender.
It is positive：Work as nature controlling line（C）Show red stripes, and detection line（T）It does not develop the color or when T line color is shallower than C line, sentences
For the positive.
In vain：Work as nature controlling line（C）Do not show red stripes, then no matter detection line（T）It, should whether showing red stripes
Test strips are judged in vain.
It there is no the test strips for detecting safrole in essence spice for cigarette and tobacco product at present, the present invention has filled up this
Blank.Haptens of the invention has appropriate terminal reactive group, and decorating site and spacer length selection are suitable, and can be maximum
Degree simulates the molecular structure of safrole, and the test strips developed based on this haptens have high sensitivity, high specificity
Feature.Test strips of the invention simultaneously are at low cost, easy to operate, detection time is short, not examined equipment limit, is suitble to respectively
The advantages of kind unit uses, storage is simple, long shelf-life.To sum up, the method for detecting safrole with test strips of the present invention, easy,
Quickly, intuitively, accurately, applied widely, at low cost, easy popularization and use.
Detailed description of the invention
Fig. 1 is test strips the schematic diagram of the section structure, in figure：1, sample absorption pad；2, conjugate release pad；3, reaction film；
4, water absorption pad；5, detection line；6, nature controlling line；7, bottom plate；
Fig. 2 is test strips testing result process decision chart；
Fig. 3 is safrole hapten synthesis figure（The figure is as Figure of abstract）.
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not intended to limit the scope of the invention.In addition, the range that those skilled in the art limits in the appended claims
Interior to carry out various changes or modification to the present invention, these changes or modification should equally fall into protection scope of the present invention.
Embodiment 1 detects the preparation of the test strips of safrole
The preparation method of the test strips mainly includes the following steps that：
1）Preparation is coated with safrole monoclonal antibody-colloid gold label object conjugate release pad；
2）Preparing has the detection line for being coated with safrole hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, water absorption pad and the PVC bottom plate prepared is assembled into examination
Paper slip.
Substep narration in detail below：
1, the synthesis of safrole haptens（Synthetic route is shown in attached drawing 3）And identification
5- (the bromo- 2- propylene -1- base of 2-) -1,3- benzodioxolane, 1.0 g is taken, anhydrous 50 mL of DMSO is added to dissolve, is stirred clear
Clearly, add 0.48 g of potassium hydroxide, add 0.20 g of anhydrous sodium iodide, after being vigorously stirred, add 0.41 g of alanine, 100 DEG C of reactions 4
h.Stop reaction, add 50 mL of water, adds 1mol/L salt acid for adjusting pH value to 6, add 80 mL of ethyl acetate, extraction three times, is associated with
Machine phase, revolving are evaporated, upper silicagel column, ethyl acetate/petroleum ether（V/v, 1/5）Elution separation, it is yellow to obtain haptens product propionic acid
Camphor tree element 0.91g, yield 87.37%.
Above-mentioned haptens is taken to identify through nuclear magnetic resonance spectroscopy,1H-NMR（CDCl3, 300MHz）δ：11.0（1H, -COOH）,
6.90（1H, ArH, J=6.864）, 6.68（1H, ArH）, 6.76（1H, ArH）, 6.07（2H, s, -CH2-）, 4.16
（2H, s, -CH2-）, 3.75（1H, dd, =CH2）, 3.12（2H, t, -CH2-）, 2.49（2H, t, -CH2-）, 2.0
（1H, s, -NH-）.Chemical shift δ=11.0 are the resonance absorbing peak of carboxyl hydrogen in map, and δ=3.12,2.49 are interval
The resonance absorbing peak of methylene hydrogen on arm, the presence at these peaks, it was demonstrated that spacerarm is coupled successfully, and safrole haptens structure is just
Really.
2, the synthesis and identification of safrole coupled antigen
Immunogene preparation --- safrole haptens and bovine serum albumin(BSA)（BSA）Coupling obtains immunogene.
9 mg of safrole haptens is taken, 0.3 mL of n,N-Dimethylformamide is added to dissolve, clarification adds carbodiimides
（EDC）8.3 mg stir 20 min, add 7 mg of N- succinimide, continue to stir 30 min, obtain haptens activating solution A liquid；
50 mg of BSA is taken, adds 0.8% sodium-chloride water solution, 4 mL to dissolve, obtains B liquid；A liquid is slowly dropped in B liquid, 4 DEG C of stirrings 4
H, 0.02 mol/L PB buffer dialysis purification 3 days, obtains safrole-BSA immunogene, and packing, -20 DEG C save backup.
Coating antigen preparation --- safrole haptens and ovalbumin（OVA）Coupling obtains coating antigen.
6 mg of safrole haptens is taken, adds 0.2 mL of dimethyl sulfoxide to dissolve, adds 20 μ L of triethylamine, chlorination isobutyl formate
18 μ L of ester, mixes, and 4 DEG C of 30 min of reaction obtain haptens activating solution A liquid；50 mg of OVA is taken, 0.8% sodium-chloride water solution is added
4 mL dissolution, obtains B liquid；A liquid is slowly dropped in B liquid, 4 DEG C of 4 h of stirring, 0.02 mol/L PB buffer dialysis purification 3
It, obtains safrole-OVA coating antigen, and packing, -20 DEG C save backup.
3, the preparation of safrole monoclonal antibody
（1）Animal immune
In the immunogene injection Balb/c Mice Body that step 1 is obtained, immunizing dose is 150 μ g/, its is made to generate antiserum.
（2）Cell fusion and cloning
It takes the Balb/c mouse boosting cell for generating specific antibody to merge with myeloma cell SP20, is exempted from using indirect competitive enzyme-linked
Epidemic disease analysis method measures cell supernatant, screens positive hole.Cloning is carried out to positive hole using limiting dilution assay, obtains and builds
The vertical hybridoma cell strain for producing monoclonal antibody.
（3）Cell cryopreservation and recovery
It takes the hybridoma in logarithmic growth phase that cell suspension is made with frozen stock solution, is sub-packed in cryopreservation tube, it is long in liquid nitrogen
Phase saves.Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and after centrifugation removal frozen stock solution, is moved into culture bottle
Culture.
（4）The preparation and purification of monoclonal antibody
Using method is induced in vivo, by Balb/c mouse（8 week old）Intraperitoneal injection sterilizing paraffin oil, is injected intraperitoneally hybridization after 7 ~ 14 days
Oncocyte acquired ascites after 7 ~ 10 days.Ascites purifying is carried out through octanoic acid-saturated ammonium sulfate method, purity is reflected through SDS-PAGE electrophoresis
It is fixed, bottle packing, -20 DEG C of preservations.
（5）The measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(50000~100000).
Indirect competitive ELISA method：With safrole haptens-OVA conjugate coated elisa plate, safrole standard items are added
The sheep anti mouse antiantibody solution of solution, safrole monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions 30
Min pours out liquid in hole, is washed 3 ~ 5 times with cleaning solution, is patted dry with blotting paper；Substrate developing solution, 25 DEG C of 15 min of reaction are added
Afterwards, terminate liquid is added and terminates reaction；Setting microplate reader measures every hole absorbance value at 450 nm of wavelength.
（6）The measurement of monoclonal antibody specificity
Antibody specificity refer to the ability of its homospecificity antigen binding compared with such antigen-analogues ability, often
Use cross reacting rate as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
Yellow camphor tree analog is serially diluted by this experiment, carries out indirect competitive ELISA, production mark with monoclonal antibody respectively
Directrix curve, analysis obtain IC50, cross reacting rate is then calculated as follows：
The cross reacting rate of the antibody and safrole analog the results are shown in Table 1.
The test of 1 cross reacting rate of table
| Compound | Cross reacting rate（%） |
| Safrole | 100 |
| Isosafrole | 13.2 |
| Dihydrosafrole | 6.7 |
| Cumarin | < 1 |
| Vanillic aldehyde | < 1 |
| Damascenone | < 1 |
As it can be seen from table 1 safrole monoclonal antibody and isosafrole, dihydrosafrole, cumarin, vanillic aldehyde, Tujue's alkene
The equal no cross reaction of ketone.
4, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized using source of mouse antibody as immunogene, obtains sheep anti mouse antiantibody.
5, safrole monoclonal antibody-colloid gold label object preparation
（1）The preparation of colloidal gold
It takes 0.01% aqueous solution of chloraurate, 100 mL to be heated to boiling, stirs lower accurate 1% trisodium citrate aqueous solution 4 that preheating is added
ML, golden yellow aqueous solution of chloraurate become grey in 2 min, then become claret from ash, continue stirring and boil 8
Min uses distilled water constant volume to 100 mL, 4 DEG C of preservations after cooling.It is clear and transparent for preparing good colloidal gold and detecting by an unaided eye
, without muddiness, liquid surface is without floating material, and the color of observing colloid gold is claret in the sunlight.
（2）The preparation of safrole monoclonal antibody-colloid gold label object
Under magnetic stirring, with the pH value of 0.2 mol/L solution of potassium carbonate tune colloidal gold to 7.2（The pH of different antibodies marks model
It is trapped among between 7 ~ 8, can change）, by the standard of 20 ~ 50 μ g antibody is added in every milliliter of colloidal gold solution to colloidal gold solution
It is middle that above-mentioned safrole monoclonal antibody is added, it stirs and evenly mixs, is stored at room temperature 10 min, 10% BSA, which is added, keeps it molten in colloidal gold
Whole mass fraction in liquid is 1%, stands 10 min.12000 r/min, 4 DEG C of 40 min of centrifugation abandon supernatant, precipitating redissolution
Buffer washes twice, with the redissolution buffer that volume is initial colloid gold volume 1/10 will precipitating be resuspended, set 4 DEG C it is spare.
Redissolve buffer：Mass fraction containing BSA be the mass fraction of 0.1% ~ 0.3%, Tween-80 be 0.05% ~
0.2%, the 0.02 mol/L phosphate buffer of pH7.2.
6, the preparation of conjugate release pad
Conjugate release pad is soaked in containing 0.5% BSA（Mass fraction）, pH 7.2 0.5 mol/L phosphate buffer in,
1 h uniformly is soaked, 37 DEG C of 3 h of baking are spare.Film instrument is sprayed by the safrole prepared monoclonal antibody-colloidal gold mark with Bio dot
Object even application is remembered in conjugate release pad, and every 1cm conjugate release pad sprays 0.01 mL safrole monoclonal antibody-glue
After body gold marker, it is placed in 37 DEG C of environment（Humidity < 20%）It is taken out after 60 min, is placed in dry environment（Humidity < 20%）In
It saves backup.
7, the preparation of reaction film
Detection line will be constituted in safrole haptens-ovalbumin conjugate coating to reaction film, sheep anti mouse antiantibody is coated with
Nature controlling line is constituted on reaction film.
Coating process：Safrole haptens-ovalbumin conjugate is diluted to 1 mg/mL with phosphate buffer, uses Bio
Dot point film instrument is coated in the detection line on nitrocellulose filter（T）, package amount is 1.0 μ L/cm；With 0.01 mol/L,
Sheep anti mouse antiantibody is diluted to 200 μ g/mL by the phosphate buffer that pH value is 7.4, is coated with Bio dot point film instrument
In the nature controlling line on nitrocellulose filter（C）, package amount is 1.0 μ L/cm.The reaction film being coated with is placed under the conditions of 37 DEG C
Dry 2 h, it is spare.
8, the preparation of sample absorption pad
Sample absorption pad is used and contains 0.5% bovine serum albumin(BSA)（Mass fraction）, pH 7.2 0.1 mol/L phosphate buffer
2 h are impregnated, it is spare that 2 h are dried at 37 DEG C.
9, the assembling of test strips
Sample absorption pad, conjugate release pad, reaction film, water absorption pad are successively pasted on PVC bottom plate in order；Conjugate is released
Put pad has 1/3 region to be absorbed by the sample pad covering from starting point, and the end of conjugate release pad and the beginning of reaction film connect, instead
The end of film is answered to be connected with the beginning of water absorption pad, the beginning of sample absorption pad is aligned with the beginning of PVC bottom plate, the end of water absorption pad
It is aligned with the end of PVC bottom plate；There are detection line and nature controlling line, detection line on the reaction film（T）And nature controlling line（C）It is in and institute
State the perpendicular strip tape of the length of test strips；Detection line is located at the side close to the end of conjugate release pad；Nature controlling line is located at
The side of end far from conjugate release pad；Test strips are cut into the small item of 3 mm wide with machine, mounted in special plastics system
In card, it can be reserved for 12 months under the conditions of 4 ~ 30 DEG C.
The detection of safrole in 2 sample of embodiment
1, the pre-treatment of sample
The tobacco sample of 1.0 ± 0.05 g crushing is weighed into polystyrene centrifuge tube；It is water-soluble that 10 mL, 50% methanol is added
It is sufficiently smashed with refiner, obtains sample liquid by liquid；It pipettes to be checked after 50 μ L sample liquid are mixed with 450 μ L deionized waters.
2, it is detected with test strips
100 μ L of sample to be examined solution is taken vertically to drip in well with pipettor, liquid starts timing, reaction 10 when flowing
Min determines result.
3, analysis detection result
It is negative（-）：The colour developing of T line develops the color deep or develops the color unanimously with C line than C line, indicates that safrole concentration is limited lower than detection in sample,
Such as Fig. 2 a, 2b.
It is positive（+）：The colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color, and indicates that safrole concentration is equal to or higher than in sample
Detection limit, such as Fig. 2 c, 2d.
In vain：Do not occur C line, shows the deterioration failure of incorrect operating process or test strips, such as Fig. 2 e, 2f.Herein
In the case of, specification should be read over again, and is retested with new test strips.
3 sample detection example of embodiment
1, detection limit test
Take blank tobacco sample, wherein respectively addition safrole to final concentration of 0.1,0.2,0.4 mg/kg, take test strips into
Row detection, each sample are repeated three times.
When detecting tobacco sample with test strips, when wherein safrole addition concentration is 0.1 mg/kg, shown in test strips
The colour developing of T line develops the color deep or develops the color unanimously with C line than C line out, is negative；When wherein safrole addition concentration is 0.2 and 0.4 mg/
It shows that the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color when kg, in test strips, is positive, shows this test strips in tobacco
The detection of safrole is limited to 0.2 mg/kg.
2, false positive rate, false negative rate test
Take known safrole content greater than 20 parts of tobacco positive sample of 0.2 mg/kg, it is known that the tobacco without safrole is negative
It 20 parts of sample, is detected with three batches of test strips, calculates its yin and yang attribute rate.
The result shows that：When detecting positive sample with the test strips of 3 batch productions, as a result it is all positive, it is known that positive sample
Product coincidence rate is 100%, false negative rate 0；When detecting negative sample, as a result it is all negative, it is known that negative sample coincidence rate is
100%, false positive rate 0.Illustrate that the test strips of detection safrole of the invention can quickly examine the safrole in tobacco
It surveys.
3, specific test
Isosafrole, dihydrosafrole, cumarin, vanillic aldehyde, the Damascenone of 500 μ g/L are detected with safrole test strips.Knot
Fruit shows that fluorescence detector is detected as feminine gender.Illustrate this test strips to isosafrole, dihydrosafrole, the tonka-bean of 500 μ g/L
Element, vanillic aldehyde, Damascenone no cross reaction.
Claims (7)
1. a kind of test strips for detecting safrole, including sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate；
It is characterized in that, there is the detection line for being coated with safrole hapten-carrier protein conjugate on the reaction film and be coated with
The nature controlling line of sheep anti mouse antiantibody is coated with safrole monoclonal antibody-colloid gold label object in the conjugate release pad；Institute
Stating safrole monoclonal antibody is prepared using safrole hapten-carrier protein conjugate as immunogene；The Huang camphor tree
Plain hapten-carrier protein conjugate is obtained by safrole haptens with carrier protein couplet, and the carrier protein is cow's serum
Albumin, ovalbumin, keyhole limpet hemocyanin or human serum albumins, the safrole haptens are by 5- (the bromo- 2- third of 2-
Alkene -1- base) -1,3- benzodioxolane reacts with alanine, and molecular structural formula is：
。
2. test strips according to claim 1, it is characterised in that：The preparation reaction process of the safrole haptens is such as
Under：
。
3. test strips according to claim 1, which is characterized in that the sample absorption pad, conjugate release pad, reaction
Film, water absorption pad are successively pasted on bottom plate.
4. according to claim 1 or 3 described in any item test strips, which is characterized in that 1/3 ~ 1/2 quilt of conjugate release pad
It is covered under sample absorption pad.
5. test strips according to claim 1, which is characterized in that it is immune that the sheep anti mouse antiantibody, which is with source of mouse antibody,
Original carries out immune prepare to goat.
6. a kind of method for preparing any one of claim 1-5 test strips, which is characterized in that this method includes following step
Suddenly：
1）Preparation is coated with safrole monoclonal antibody-colloid gold label object conjugate release pad；
2）Preparing has the detection line for being coated with safrole hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate prepared is assembled into test paper
Item.
7. a kind of method using safrole in any one of the claim 1-5 test strips test sample, which is characterized in that should
Method includes the following steps：
1）Pre-treatment is carried out to sample；
2）Sample is detected with the test strips；
3）Analysis detection result.
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