CN106018374A

CN106018374A - Up-conversion fluorescent nanoparticle-based immunochromatographic test paper for rapidly detecting pesticide residues and preparation method for immunochromatographic test paper

Info

Publication number: CN106018374A
Application number: CN201610590578.6A
Authority: CN
Inventors: 华瑞年; 国婷婷; 张伟; 于基成; 那立艳
Original assignee: Dalian Nationalities University
Current assignee: Dalian Minzu University
Priority date: 2016-07-26
Filing date: 2016-07-26
Publication date: 2016-10-12

Abstract

The invention relates to up-conversion fluorescent nanoparticle-based immunochromatographic test paper for rapidly detecting pesticide residues and a preparation method for the immunochromatographic test paper, and belongs to the technical field of chemical analysis. The pesticide test paper comprises a test paper board, a sample dropwise-addition unit, a detection unit and a sample recovery unit, wherein the sample dropwise-addition unit, the detection unit and the sample recovery unit are all connected to the test paper board; the detection unit is positioned in middle of the test paper board, and comprises an antigen coating buffer and confining liquid. The immunochromatographic test paper and the preparation method for the same have the beneficial effects that the test paper has the characteristics of rapidness, convenience, specificity, sensitivity and low cost, particularly the characteristics of rapidness and convenience, is particularly suitable for grassroots units, field operating personnel, large-batch short-time detection and large-scale general survey and the like, has great development potential and broad application prospect, is considered as one of the most promising novel technologies for food safety detection, and becomes the main development trend of food safety detection.

Description

A kind of immunochromatography based on up-conversion fluorescence nanoparticle fast detecting pesticide residue Reagent paper and preparation method thereof

Technical field

The present invention relates to chemical identification technical field, particularly one and quickly detect agriculture based on up-conversion fluorescence nanoparticle Immune chromatography test paper of medicine residual and preparation method thereof.

Background technology

In early 1980s, having risen the technology of a kind of quick detection at western developed country, its principle is to pass through The capillarity of gold conjugation pad, nano material pad, quantum dot pad, fluorescein pad etc., with combination Pad corresponding material to be marked, specific binding at this through celluloid membrane interaction, antibody and antigen, by this technology It is referred to as immune chromatography test paper detection technique.These materials for labelling all have seen from naked eyes or find out under ultraviolet excitation Color or fluorescence that antibody is combined with antigenic specificity manifest.Immuno-chromatographic test paper strip is quick, easy and simple to operate etc. because of it Feature, it uses field wider, and has obtained good development.Material for test strips labelling also gets more and more.

Immune chromatography test paper key component is sample pad, label pad, celluloid (Nitrocellulose, NC) film, T line (detection line), C line (nature controlling line), adsorptive pads, polrvinyl chloride (Polyvinlchloride, PVC) base plate.Such as colloid gold test paper, due to gold colloidal, reagent paper preparation principle is divided into competition Method and sandwich assay.Competition law: when the sample drop being associated with determined antigen is added in sample pad, by capillarity, sample to Front movement, time on gold conjugation pad, the antigen in sample can be combined with antibody specificity.When arriving T line, resisting on T line Know from experience remaining antibody specificity in sample to be combined, redden.Sample is further continued for mobile, when arriving C line, again with on C line Antibody response, reddens.When two red lines occur in test strips, illustrate that the antigenic content in sample, less than detection line, is negative. Otherwise, when the antigenic content in sample is prescribed a time limit higher than detection, on gold conjugation pad, antigen major part is tied with antibody specificity Closing, there is not redness in T line, and C line outlet is red, then be positive.No matter the determined antigen in sample is above or below detection Line, C line all should occur, otherwise test strips is invalid.Experimental result is contrary therewith, occurs that two red lines are positive, and a red line is Negative method is sandwich assay.

Pesticide Residue is as pesticide and produces in a large number and be widely used and produce.Up to the present, change in the world Nearly 2,000,000 tons of medicine annual production of learning to farm, there are about more than 1000 kind of artificial-synthetic compound and is used as insecticide, antibacterial, algicide, removes The class pesticide such as worm agent, defoliant.Pesticide especially organic agricultural chemicals is used in a large number, causes serious Pesticides Residues, becomes right The serious threat of health.

Immune test paper technology is at medical science, Disease Diagnosis of Veterinary, agriculture, biological welfare, food safety, Environmental security, industrial detection, The most emerging molecular diagnosis and nanometer treatment field are all widely used, and have application prospect widely.At food In product safety testing field, it is mainly concerned with veterinary drug, pesticide residues, Illegal addition, biotoxin, heavy metal, environmental toxin Deng, what the most most food safety detection related to can be applied to.Technically, immune test paper detection technique tool There are the feature of " quick, easy, special, sensitive, low cost ", the particularly feature of its " quickly, easy ", it is adaptable to on-the-spot flood tide Sample detection, has huge application prospect it is considered to be one of the most promising new technique of food safety detection, it has also become food The Main Trends of The Development of product safety detection.

Pesticide residue analysis mainly uses Instrumental Analysis and immune analysis method at present.We country most, press If calculating according to test number, it is simply that reagent paper.Other technology certainly the most all with, but the absolute number of test does not all have reagent paper many, Gas-matter chromatograph, liquid-matter chromatograph is difficult to deal with the sample of flood tide.Wherein to have sensitivity, accuracy high and accurate for instrumental method The advantages such as degree is good, but have that sample pre-treatments is loaded down with trivial details, reagent dosage is big, time-consuming, analysis cost is high, need expensive instrument and equipment etc. Shortcoming, is not suitable for field quick detection.Nanometer biotechnology is the new technique that nanotechnology is formed with biotechnology Cross slot interference, Utilize nanometer biotechnology that intraor extracellular species analysis and detection, disease early diagnosis, the trace labelling etc. of biomolecule are ground Study carefully the research emphasis of association area always both at home and abroad.In order to be able to realize the quick detection of food and Environmental Pesticide residual, section Worker begins to focus on conversion (long wavelength light is converted to the photoemissive process of short wavelength) nano-luminescent material.Upper conversion Nano-luminescent material is the material that a class 980nm infrared light does excitation source, and the material light making excitaton source of this exciting light is worn Degree is relatively deep thoroughly, will not be luminous in biological tissue, therefore does not produce background fluorescence, and the damage to biological tissue is almost nil.Synthesis The substance toxicity of such material is less, and has good chemical stability, and luminous intensity is preferable, its absorption and transmitting band Narrow, thus reduce detection background, improving signal to noise ratio, these features cause conversion nano fluorescent material to have extraordinary biology Application prospect.Set up that examine the method that pesticide many (singly) remains soon based on up-conversion nano material the most very important, arrive So far, upper conversion nano fluorescent material is treated in cell marking, biomacromolecule detection, small animal imaging, photodynamics The aspects such as method and medicine conveying are applied.But upper conversion nano fluorescent material is used as label, with immunochromatography technique Combine and carry out the analysis of little molecule pesticide residues and have not been reported.Up-conversion nano material is utilized quickly to detect pesticide many (singly) The analysis method of residual, has important theoretical research and actual application value to developing fast inspection pesticide multi-residues detection technique.

Summary of the invention

It is an object of the invention to overcome the deficiencies in the prior art, produce luminescent material converted in nano level.The present invention Thering is provided the immune chromatography test paper of fast detecting pesticide residue, described reagent paper comprises: test paper plate, sample dropping unit, detector unit And sample recovery unit；Sample dropping unit, detector unit and sample recovery unit are sequentially connected and are all connected to described On test paper plate；Described sample dropping unit and sample recovery unit are positioned at the two ends of described test paper plate, and described detector unit is positioned at The centre of described test paper plate；Described detector unit comprises nanometer particle to mark pad, detection line and nature controlling line；Described detection line Between described nanometer particle to mark pad and described nature controlling line；Described nanometer particle to mark pad comprises antigen coated Liquid and up-conversion fluorescence nanometer particle to mark antibody sample；Described up-conversion fluorescence nanometer particle to mark antibody sample be β- NaYF₄:Yb³⁺/Er³⁺The PBS solution of labelling 2,4-D-IgG.

Preferably, described detection line and nature controlling line are respectively positioned in chromatographic film, nanometer particle to mark pad and described layer Analysis film is connected；Described chromatographic film is NC nitrocellulose filter.

Preferably, β-NaYF in described nanometer particle to mark antibody sample₄:Yb³⁺/Er³⁺The content of labelling 2,4-D-IgG is 0.00666-0.132μg/mL。

Preferably, the label on described detection line is 2,4-D-OVA, and the label of described nature controlling line is goat-anti rabbit two Anti-.

The present invention also protects the preparation method of detection pesticide residual test paper, comprises the steps of S1. and prepares water-solubility rare-earth Up-conversion nano material；S2. prepare pesticide polyclonal antibody and detect the titer of described pesticide polyclonal antibody；S3. utilize Pesticide polyclonal antibody described in Product Labeling prepared by described step S1；S4. described step S3 product preparation detection pesticide is utilized Residual reagent paper.

Preferably, described step S1 comprises:

Rare earth chloride, oleic acid and octadecylene are placed in flask stirring evacuation 30min by S1.1, and rear stopping stirring adding Heat；

S1.2 is by NaOH, NH₄HF₂And methanol is placed in beaker and puts into stirrer, seal up sealed membrane, in magnetic stirring apparatus Upper stirring at normal temperature；

S1.3, by described step S1.1 product heats to 150 DEG C, is incubated 15～20min, closes stirring immediately, closes Heating, is allowed to be reduced to room temperature；

S1.4 drips S1.2 solution in the case of entering air less as far as possible, is passed through N after reaction completely₂, enter during dropping The air entered drains；

System is heated to 75-85 DEG C by S1.5, is incubated 1.5h；

The product of described step S1.5 is warming up to 310 DEG C in 0.5h by S1.6, N₂It is incubated 2h, immediately at N under protection₂Protect Protect and stir down and be cooled to room temperature；

S1.7 washed product；

S1.8 utilizes described step S1.7 product to prepare water-soluble beta-NaYF₄:Yb³⁺,Er³⁺Nanoparticle.

Preferably, described step S1.8 comprises: S1.8.1 by the product of described step S1.7, hexamethylene, the tert-butyl alcohol, go from Sub-water and K₂CO₃Mix and blend under solution room temperature；S1.8.2 is by KMnO₄And NaIO₄Aqueous solution is slowly dropped into described step The product of S1.8.1, reacts 48h, is centrifuged to obtain head product by 95% washing with alcohol at 40 DEG C；S1.8.3 head product is dissolved in HCl, Stirring reaction under room temperature, reaction terminates that rear centrifuge washing is centrifugal obtains product.

Preferably, described step S3 comprises: S3.1 is respectively configured the phosphate buffered saline(PBS) of pH=5 and pH=7.4； S3.2 prepares water-soluble beta-NaYF₄: Yb, Er labelling 2,4-D-IgG.

Preferably, described step S3.2 is, and: S3.2.1 learns from else's experience KMnO₄-NaIO₄Oxidation is dissolved in the β-NaYF of water₄: Yb, Er system Become solution A, after adding the PBS of pH=5 prepared by described step S3.1 in described solution A, add EDC and Sulfo- NHS, stirring at normal temperature；S3.2.2 adds 2,4-D-IgG in the product of described step S3.2.1, normal-temperature reaction 2h, centrifugal produces Thing；After described product PBS is washed 2 times in the PBS of the pH=7.4 that constant volume is prepared in described step S3.1.

Advantages of the present invention and good effect be: upper conversion nano fluorescent material have organic dyestuff and quanta point material without Method analogy advantage, as little in toxicity, chemical stability is high, luminous intensity is high, good light stability, absorb and launches carry narrow and fluorescence Life-span length etc.；In addition 980nm iraser makees excitaton source also many advantages, the deepest light penetration depth, to biological group Knit almost without damage, biological tissue will not luminous (without background fluorescence), thus reduce detection background, improve signal to noise ratio, these spies Levy and cause conversion nano fluorescent material to have extraordinary biologic applications prospect.Set up and examine soon based on up-conversion nano material The method of pesticide multi-residues is the most very important, and utilizing up-conversion nano material quickly to detect, pesticide many (singly) remains point Analysis method, has important theoretical research and actual application value to developing fast inspection pesticide multi-residues detection technique.Immune test paper Technology is at medical science, Disease Diagnosis of Veterinary, agricultural, biological welfare, food safety, Environmental security, industrial detection, the most emerging molecule Diagnosis and nanometer treatment field are all widely used, and have application prospect widely.In field of detection of food safety, It is mainly concerned with veterinary drug, pesticide residues, Illegal addition, biotoxin, heavy metal, environmental toxin etc., the most most What food safety detection related to can be applied to.Technically, immune test paper detection technique has " quick, easy, special Different, sensitive, low cost " feature, the particularly feature of its " quickly, easy ", be particularly suitable for vast grass-roots unit, field is made Industry personnel and the detection being pressed for time in high volume and large area generaI investigation etc., show huge development potentiality and application prospect, recognized For being one of the most promising new technique of food safety detection, it has also become the Main Trends of The Development of food safety detection.

Accompanying drawing explanation

Fig. 1 is synthesis β-NaYF₄:Yb³⁺,Er³⁺The scanning electron microscope (SEM) photograph of nanoparticle；

Fig. 2 is β-NaYF₄:Yb³⁺,Er³⁺The transmission electron microscope picture of nanoparticle；

Fig. 3 is water-soluble beta-NaYF₄:Yb³⁺,Er³⁺The TEM figure of up-conversion nanoparticles；

Fig. 4 is water-soluble beta-NaYF₄:Yb³⁺,Er³⁺TEM figure after up-conversion nanoparticles labelling 2,4-D-IgG；

Fig. 5 is under 595nm absorbing wavelength, the concentration of BSA and the relation curve of absorbance；

Fig. 6 is the immune chromatography test paper structure chart of detection pesticide list residual；

Fig. 7 is variable concentrations sample the selection result criterion figure, and A-E is the anti-of the labelling up-conversion nanoparticles of variable concentrations Body sample；

Fig. 8 is the sensitivity schematic diagram of reagent paper, A: negative B: positive T: detection line C: nature controlling line；

Fig. 9 determines ELISA test strip 2,4-D agriculture residual sensitivity results figure；

Figure 10 determines ELISA test strip 2,4-D agriculture residual sensitivity results figure；

Figure 11 is the liquid chromatogram of 2,4-D standard sample；

Figure 12 is the 2,4-D content in sample pears；

Figure 13 is the 2,4-D content in sample Fructus Mali pumilae；

Figure 14 is the 2,4-D content in sample Fructus Cucumidis sativi；

Figure 15 is the 2,4-D content in sample Fructus Lycopersici esculenti；

Figure 16 is the 2,4-D content in sample rice；

Figure 17 is the 2,4-D content in sample Semen setariae；

Figure 18 is test strips the result figure, a: pears, b: Fructus Mali pumilae, c: Fructus Cucumidis sativi, d: Fructus Lycopersici esculenti, e: rice, f: Semen setariae.

Reference:

1-test paper plate, 2-sample dropping unit, 3-nanometer particle to mark pad, 4-detection line,

5-nature controlling line, 6-sample recovery unit, 7-chromatographic film, A-sample flow direction.

Detailed description of the invention

In recent years because of the optical property of rare earth upconversion nano material so that it is the application at biological field has in a large number may be used Potential leaved for development.β-NaYF₄:Yb³⁺,Er³⁺(Tm³⁺) be the material that the upper conversion efficiency found so far is the highest, therefore its Biological detection obtains extensive concern with labelling aspect.But, the rare earth upconversion nano material because preparing is oil-soluble material Material, need to utilize KMnO₄/NaIO₄Oil-soluble nano particles is transformed into water soluble nanometer particles.Utilize high temperature thermal decomposition method herein It is prepared for the β-NaYF of Coated with Oleic Acid₄:Yb³⁺,Er³⁺Nanoparticle.

1, the preparation of rare earth upconversion nano material

(1) experimental apparatus and experiment reagent

Table 1 experimental apparatus catalog

Table 2 chemical reagent catalog

(2) rare earth chloride is prepared

Rare earth oxide 10g is poured in new beaker, adds 5ml distilled water, add the HCl 50-60ml of concentration 37%, Stirring, if the most thick, continuously adds HCl.

After slightly soluble, be placed in be covered with on the electric furnace of asbestos gauge be heated to boiling, if do not clarify always, then add 20-30ml HCl, boiling to surplus solution is to clarify not yet after original half, then continuously add the HCl that concentration is 37%, until clarification.

After clarification solution boiling be evaporated to solution start to occur air pocket time, add distilled water 80ml, rinse wall of cup and Glass rod.Recrystallization quickly stirs into crystalloid, tubulature after being evaporated for the last time for about four times repeatedly.

(3) high temperature thermal decomposition method prepares oil-soluble β-NaYF₄:Yb³⁺,Er³⁺Up-conversion fluorescence nanoparticle

1. by rare earth chloride (2mmol) [YCl₃·6H₂O (78%), YbCl₃.6H₂O (20%), ErCl₃.6H₂O (2%)], 90% oleic acid (12ml), 90% octadecylene (30ml) be placed in there-necked flask, put into stirrer, evacuation, 30min Post-heating.Being placed on during evacuation on thermocouple, only stirring is not heated.

2. by NaOH (5mmol), NH₄HF₂(5mmol), absolute methanol (10ml) be placed in small beaker, put into stirrer, envelope Upper sealed membrane, is placed in stirring at normal temperature on magnetic stirring apparatus.

3. step 1. in evacuation complete, in 15min, solution in there-necked flask is heated to 150 DEG C, insulation 15～ 20min, closes stirring immediately, closes heating, is allowed to be reduced to room temperature.

4. add solution in step 2 in the case of entering air less as far as possible, the most dropwise drip, be allowed to fully react.Drip Start to be passed through N after adding end₂, it is passed through about 30min, the air entered during dropping is drained.

System is heated to after 5. draining air 80 DEG C (about 15-20min rises to 80 DEG C), and range of temperature is at 5 DEG C Within, it is impossible to less than 80 DEG C, it is incubated 1.5h.

6., after insulation 1.5h, in 0.5h, solution is warming up to 310 DEG C, fully reacts, N₂It is incubated 2h under protection.Exist immediately N₂It is cooled to room temperature under protection and stirring.

7. washed product: centrifuge is adjusted to 9000r/min, the time is set as 10min, is washed by product with dehydrated alcohol One time；Hexamethylene: product is washed twice by dehydrated alcohol with the mixing of 1:3 ratio；Product is washed twice by dehydrated alcohol；95% second Product is washed twice by alcohol.Centrifuge tube is put into drying baker desciccate.

(4) water-soluble beta-NaYF₄:Yb³⁺,Er³⁺The preparation of nanoparticle

By the above-mentioned β-NaYF prepared₄:Yb³⁺,Er³⁺(50mg), hexamethylene (50ml), the tert-butyl alcohol (35ml), deionization Water (5ml) and 5wt%K₂CO₃Mix and blend 20min under solution (2.5ml) room temperature.By 10ml KMnO Han 2.85mmol₄With 0.0525mmol NaIO₄Aqueous solution is slowly dropped into.React 48h at 40 DEG C, produce by 95% washing with alcohol centrifugal (9000r/min) Thing obtains head product.Head product is dissolved in the HCl that 12.5ml pH is 4-5, stirring reaction 30min under room temperature.Reaction terminates 9000r/ Min is centrifuged, and finally obtains product with 95% washing with alcohol is centrifugal.Product is scattered in 15ml deionized water stand-by.

(5)β-NaYF₄:Yb³⁺,Er³⁺The sign of nanoparticle

A) sem analysis

From the SEM photograph of Fig. 1 it can be seen that high temperature pyrolytic cracking (HTP) synthesis β-NaYF₄:Yb³⁺,Er³⁺Up-conversion nanoparticles, its Pattern is solid nanosphere, even particle size, and particle diameter is about 30nm, also observes that product thing is mutually pure from SEM picture Only.

B) tem analysis

From the TEM photo of Fig. 2 it can be seen that high temperature pyrolytic cracking (HTP) synthesis β-NaYF₄:Yb³⁺,Er³⁺Nanoparticle, its pattern is Solid nanosphere, even particle size, particle diameter is about 30nm, also observes that product thing is mutually pure from TEM picture.

From figure 3, it can be seen that the pattern of β-NaYF4:Yb, Er nanoparticle after Yang Hua and grain size all change Become, be still the particle diameter spherical particle at about 30nm.

2, the preparation of pesticide polyclonal antibody and titer

(1) preparation of 2,4-D pesticide polyclonal antibody (IgG) and titer

The normal saline solution of a certain amount of immunizing antigen (2,4-D-BSA) is mixed with the Freund's complete adjuvant of equivalent, fills Point emulsifying, 2 New Zealand white rabbit (2-2.5kg) of immunity, subcutaneous inoculation 400 μ g/ time, 2-3 week immunity once, immune 4 times.Often Within after secondary booster immunization the 10th day, take a blood sample in ear vein, centrifugal acquisition serum, measure serum titer with ELISA method, until titer More than 1:50,000 carries out final blood sampling prepares antiserum, uses the method for Protein A affinity purification to obtain many grams of high-titer Grand antibody (IgG).

Table 3 antibody indirect Elisa result

(2) water-soluble beta-NaYF₄:Yb³⁺,Er³⁺Nanometer particle to mark pesticide polyclonal antibody (2,4-D-IgG)

A) water-soluble beta-NaYF₄: Yb, Er labelling 2,4-D-IgG

I. the preparation (0.01M) of phosphate buffered saline(PBS) (PBS)

By NaCl (0.8g), KCl (0.002g), Na₂HPO₄(0.144g)、KH₂PO₄(0.024g) it is settled to water 100ml, i.e. can get the PBS solution of pH=7.4.

The PBS solution of preparation pH=5, is adjusted to 5.0 with 30%HCl by above-mentioned pH value of solution.

Ii. water-soluble beta-NaYF₄:Yb³⁺,Er³⁺The method of labelling 2,4-D-IgG

Learn from else's experience KMnO₄-NaIO₄Oxidation is dissolved in the β-NaYF of 15ml water₄:Yb³⁺,Er³⁺, in above-mentioned solution, add 15mL After PBS (pH=5) buffer, add 0.01152g (0.000384 × 30ml) EDC Yu 0.03257g Sulfo-NHS (0.00108565 × 30ml), stirring at normal temperature 30min, activated carboxyl.Add 50 μ L (C=260mg/ml) IgG, normal-temperature reaction 2h.9000rpm is centrifuged out product, product PBS (pH=7.4) wash 2 times after constant volume in 15ml PBS (pH=7.4).

As can be seen from Figure 4, β-NaYF₄:Yb³⁺,Er³⁺Coupling 2, after 4-D-IgG, compared with the oxidation product before coupling (see Fig. 4), therefore its pattern there occurs change, and the surface of nanoparticle covers layer of substance, and particle diameter is about about 50nm.

B) content of Coomassie Brilliant Blue quantitative analysis of protein

1. preparation Bradford's reagent: take Coomassie brilliant G-250 50mg and be placed in 200ml beaker, adds 25ml's 95% ethanol makes it dissolve, and is subsequently added the phosphoric acid of 60ml 85% (w/v), is transferred to 500ml volumetric flask, dilute with deionized water Release to 500ml.Solution needs now with the current.

2. configuration BSA concentration is respectively 0, each 1.00mL of titer of 0.02,0.04,0.06,0.08 and 0.10mg/mL.

3. the BSA solution configured in taking 2. is respectively put in appointment test tube, is separately added into 5.00mL according to order Bradford's reagent, rock uniformly, after reaction 2min, as liquid to be measured.Set the absorbing wavelength of ultraviolet spectrophotometer At 595nm, carry out absorbance measurement with liquid to be measured.

The measurement result obtained according to order is as follows: 0,0.164,0.349,0.508,0.648,0.813.By concentration and Absorbance mapping understands the two following linear relationship.Seeing Fig. 5, regression equation is: Y=8.108X+0.0089 (R²= 0.998)

X is BSA concentration, and Y is absorbance.After measured, sample β-NaYF₄:Yb³⁺,Er³⁺The absorbance of labelling 2,4-D-IgG It is 0.195, calculates β-NaYF according to protein standard curve₄: the 2,4-D-IgG coupling on Yb, Er labelling 2,4-D-IgG surface Rate is 2.67% (containing 2,4-D-IgG 23.1 μ g/mL).This result further demonstrates that, 2,4-D-IgG by β-NaYF₄:Yb, Er nanoparticle pass flag.

3, the preparation of pesticide list residue detection immune chromatography test paper

Table 4 chemical material catalog

Table 5 experimental apparatus catalog

(1) assembling of immuno-chromatographic test paper strip

The structure of pesticide list residue detection immuno-chromatographic test paper strip is as shown in Figure 6.Test strips comprises test paper plate 1, sample drop Add unit 2, detector unit and sample recovery unit 6.

Sample dropping unit 2, detector unit and sample recovery unit 6 are sequentially connected and are all connected to described test paper plate 1 On；Described test paper plate 1 is PVC offset plate.Described sample dropping unit 2 is positioned at the two of described test paper plate 1 with sample recovery unit 6 End, described detector unit is positioned at the centre of described test paper plate 1.

Described detector unit comprises nanometer particle to mark pad 3, detection line 4 and nature controlling line 5；Described nanometer particle to mark Pad 3 comprises antigen coated liquid and up-conversion fluorescence nanometer particle to mark antibody sample；Described up-conversion fluorescence nanoparticle Sub-traget antibody sample is β-NaYF₄:Yb³⁺/Er³⁺The PBS solution of labelling 2,4-D-IgG.Described antigen coated liquid and described Up-conversion fluorescence nanometer particle to mark antibody sample can be prepared by aforementioned preparation process.

Detection line 4 and nature controlling line 5 are respectively positioned in chromatographic film 7.Nanometer particle to mark pad 3 is connected with chromatographic film 7, layer Analysis film 7 is NC nitrocellulose filter.

I () utilizes water solublity green β-NaYF₄:Yb³⁺,Er³⁺Nanometer particle to mark 2,4-D-IgG detects 2,4-D immunity layer The preparation method of analysis reagent paper

A) selection of antigen coated liquid

Determination for pesticides test paper detection line (T line).Accurately weigh 2,4-D 250mg, add 10mL DMF (N, N-bis- Methylformamide) dissolve, then in this solution, it is sequentially added into 50.0 μ L 99% tri-n-butylamines and 30 μ L 98% chloro-butyric acid isobutyls Ester, room temperature magnetic stirrer over night；Again this solution is slowly dropped to dissolved with 450mg OVA's under stirring In the carbonic acid buffer of 20mL0.05mol/L pH9.6, stirring reaction 24h；This reactant liquor at 4 DEG C to 0.01mol/L (dialysing 6 times, each 6 hours, 500ml dialysis solution, bag filter is with needing before with boiling water boiling 10 in the phosphate buffer dialysis of pH7.4 Minute, need to put into during dialysis in 4 DEG C of refrigerators), dialysis solution 3500rpmn is centrifuged 10min, supernatant lyophilization, must synthesize anti- Former.Take 10mg synthetic antigen constant volume in 50mL volumetric flask, be made into 0.2mg/mL and be coated liquid.

B) selection of nanometer particle to mark antibody concentration:

The determination of standby test strips.The antibody sample of the labelling up-conversion nanoparticles of preparation variable concentrations, at 980nm light Exciting under, observe the luminous intensity rule and select optimum concentration.

A:200 μ L contains 0.0330 μ g/ml β-NaYF4:Yb³⁺/Er³⁺The PBS solution of labelling 2,4-D-IgG；

B:200 μ L contains 0.1665 μ g/ml β-NaYF4:Yb³⁺/Er³⁺The PBS solution of labelling 2,4-D-IgG；

C:200 μ L contains 0.3330 μ g/ml β-NaYF4:Yb³⁺/Er³⁺The PBS solution of labelling 2,4-D-IgG；

D:200 μ L contains 0.4995 μ g/ml β-NaYF4:Yb³⁺/Er³⁺The PBS solution of labelling 2,4-D-IgG；

E:200 μ L contains 0.6600 μ g/ml β-NaYF4:Yb³⁺/Er³⁺The PBS solution of labelling 2,4-D-IgG.

Experimental result is as shown in Fig. 7 and table 6:

Table 6 ("+", "-" represent fluorescence intensity)

Sample	A	B	C	D	E
						No. 1 fluorescent brightness	+	++	+++	+	+
No. 2 fluorescent brightness	+	++	+++	+	+

As it is shown in fig. 7, carry out result judgement at 980nm wavelength light irradiation NC film.By being coated liquid the selection result (table 6 and figure 7) understanding, the antibody sample C:200 μ L of the labelling up-conversion nanoparticles of variable concentrations contains 0.3330 μ g/ml β-NaYF₄:Yb³⁺/ Er³⁺The PBS solution of labelling IgG, the line fluorescence intensity on NC film is the strongest, the reagent paper of other concentration nanometer particle to mark antibody samples Bar fluorescence intensity is the most weak.From said structure, when the antibody sample of the labelling up-conversion nanoparticles of variable concentrations contains C:200 μ L contains 0.3330 μ g/ml β-NaYF₄:Yb³⁺/Er³⁺Labelling 2, the best results during PBS solution of 4-D-IgG, detects line (T Line) it is 2,4-D envelope antigen, nature controlling line (C line) is that goat-anti rabbit two resists, and is dripped in sample pad by this sample, can make after drying For detecting the standby test strips of 2,4-D content.

(2) water solublity green β-NaYF₄:Yb³⁺,Er³⁺Nanometer particle to mark 2,4-D-IgG detects 2,4-D immunity-chromatography test The sensitivity of paper determines

Carried out next step by the standby test strips prepared in (1) to operate.Competition is had during by immune chromatography test paper technology for detection Method and two kinds of methods of sandwich assay, because of 2,4-D belongs to micromolecular compound, i.e. detects with competition law.From test strips principle, Label on T line is 2,4-D-OVA, and the label of C line is that goat-anti rabbit two resists, prepare containing 2, the sample of 4-D antigen Product are added drop-wise in the sample pad of test strips, and sample can move forward along with the nanoparticle pad of test strips, NC film, if sample 2 contained in product, 4-D is less than the detection limit of test strips, when arriving T line, can be with 2 on line, 4-D-OVA reacts, after reaction T line under the exciting of 980nm light it can be seen that a green fluorescence line.Then sample continues to move along, when arriving C line, with C Label goat-anti rabbit two anti-reflective of line is answered, and reacted C line can also see that a green fluorescence line under the exciting of 980nm light. If there are two green fluorescence lines simultaneously, then explanation sample is negative (such as Fig. 8-A).On the contrary, if 2 in sample, 4-D is higher than During detection limit concentration, when sample arrives nanoparticle pad, major part first can be reacted with antigen, and remaining a small amount of part continues Moving forward, antigen now is less, 2 not and on T line, and 4-D-OVA reacts, so under the exciting of 980nm light, It cannot be seen that green fluorescence.Then sample continues to move along, and when arriving C line, answers with label goat-anti rabbit two anti-reflective of C line, Reacted C line can also see that a green fluorescence line under the exciting of 980nm light.When an only green fluorescence line (as Fig. 8-B), sample is positive.If two lines all occurs without, test strips failure is described.

(3) confirmation of 2,4-D Detecting Pesticide limit

The mensuration (500～1ng) of table 7 sensitivity

(remarks: "+", "-" represent positive and negative respectively)

The mensuration (10～1ng) of table 8 sensitivity

(remarks: "+", "-" represent positive and negative respectively)

From table 7,8 and Fig. 9,10, ELISA test strip content is the 2 of more than 10ng/mL, and during 4-D titer, result is all For the positive.To this concentration further experiment, find when 2,4-D that detectable concentration is 5ng/mL, can observe sample test strips T Line.Therefore, the detection sensitivity of test strips is defined as 5ng/mL.

Can be obtained by above-mentioned experiment, because determining 2,4-D is coated the concentration of OVA, and detection line is scheduled on 5ng/mL.When suitable The sensitivity that 2,4-D is coated the concentration scalable test strips of OVA is regulated in the range of when.Detection line is variable.

(4) immuno-chromatographic test paper strip using method

Appropriate amount of sample extracting solution is added drop-wise on NC film, after 10min, ties with 980nm wavelength light irradiation NC film position Fruit judges:

1. identical fluorescence occur such as T line and C line, then be negative, i.e. pesticide concentration is limited the quantity less than design detection, see Fig. 7- B。

2. not producing fluorescence such as T line, C first has fluorescence, then be positive, and i.e. pesticide concentration is higher than design detection limitation, sees figure 7-A。

3. first the most do not produce fluorescence such as T line and C, illustrate to detect unsuccessfully.

(5) contrast with instrumental method based on the up-conversion nanoparticles immune chromatography test paper detection residual content of agriculture

1) the 2,4-D content in liquid-phase chromatographic analysis water fruits and vegetables and raw grain and up-conversion nanoparticles immunity-chromatography test Paper detection pesticide concentration contrast

Table 9 chemical reagent catalog

Experimental apparatus

Be equipped with the Shimadzu high performance liquid chromatograph of Nacalai Tesque company of Germany COSMOSIL-C18 chromatographic column, 0.45 μm filter membrane, 20 μ L injectors, Rotary Evaporators and pulverizer.

2) sample pre-treatments

1. cross rice and each 20g of Semen setariae sample of 20 mesh sieves after weighing pears, Fructus Mali pumilae, Fructus Cucumidis sativi, Fructus Lycopersici esculenti and pulverizing, add respectively Add the 2,4-D standard substance of 2mg.

In above six kinds of samples, add 5mL 30% methanol-PBS solution the most respectively, grind.

3. the product after grinding is centrifuged, and washs 2-3 time by 30% methanol-PBS solution, each consumption 5mL.

4. decompression distillation and concentration extracting solution is to 2.0mL (1mg/mL).Prepare six kinds of samples are made into 250ng/ respectively The solution of mL.

5. the filter membrane that aperture is 0.45 μm is crossed to be measured.

4) experiment condition

1. flow phase: methanol-phosphate aqueous solution=70%:30% (PH=3.0)；

2. retention time: 20min；

3. flow velocity: 1mL/min；

4. wavelength: 278nm is detected；

5. sample size: 20 μ L.

5) experimental result and analysis

1. the actual calculating adding 2,4-D mass in sample:

X = \frac{A_{2} \cdot m_{1}}{A_{1} \cdot m_{2}}

Wherein, A₁The meansigma methods of 2,4-D peak area in standard solution；A₂2,4-D peak area in sample solution Meansigma methods；m₁The quality (g) of 2,4-D in standard specimen；m₂The quality (g) of sample.

The 2,4-D content of 2,4-D standard sample is shown in Figure 11；Each sample a: pears, b: Fructus Mali pumilae, c: Fructus Cucumidis sativi, d: Fructus Lycopersici esculenti, e: big Rice, the 2 of f: Semen setariae, 4-D content is shown in Figure 12-17 and table 10.

2,4-D measurement result in table 10 water fruits and vegetables and raw grain

(6) experimental result and test strips Comparative result

Use the test strips the result prepared of the present invention as shown in figure 18 (a: pears, b: Fructus Mali pumilae, c: Fructus Cucumidis sativi, d: Fructus Lycopersici esculenti, E: rice, f: Semen setariae).

From table 10 and Figure 18, during liquid chromatographic detection 2,4-D addition is 5ng, test strips can detect that sample in Feminine gender, coincidence detection result.

Claims

1. detection pesticide residual test paper, it is characterised in that described reagent paper comprises: test paper plate, sample dropping unit, detector unit with And sample recovery unit；

Sample dropping unit, detector unit and sample recovery unit are sequentially connected and are all connected on described test paper plate；

Described sample dropping unit and sample recovery unit are positioned at the two ends of described test paper plate, and described detector unit is positioned at described examination The centre of cardboard；

Described detector unit comprises nanometer particle to mark pad, detection line and nature controlling line；Described detection line is positioned at described nanometer Between particle label pad and described nature controlling line；Described nanometer particle to mark pad comprises antigen coated liquid and upper conversion Fluorescent nanometer particle to mark antibody sample；Described up-conversion fluorescence nanometer particle to mark antibody sample is β-NaYF₄:Yb³⁺/Er³⁺ The PBS solution of labelling 2,4-D-IgG.

Detection pesticide residual test paper the most according to claim 1, it is characterised in that described detection line and the equal position of nature controlling line In chromatographic film, nanometer particle to mark pad is connected with described chromatographic film；Described chromatographic film is NC nitrocellulose filter.

Detection pesticide residual test paper the most according to claim 1, it is characterised in that described nanometer particle to mark antibody sample Middle β-NaYF₄:Yb³⁺/Er³⁺The content of labelling 2,4-D-IgG is 0.00666-0.132 μ g.

Detection pesticide residual test paper the most according to claim 1, it is characterised in that the label on described detection line is 2, 4-D-OVA, the label of described nature controlling line is that goat-anti rabbit two resists.

5. detect the preparation method of pesticide residual test paper, it is characterised in that comprise the steps of

S1. rare earth upconversion nano material is prepared；

S2. prepare pesticide polyclonal antibody and detect the titer of described pesticide polyclonal antibody；

S3. pesticide polyclonal antibody described in Product Labeling prepared by described step S1 is utilized；

S4. described step S3 product preparation detection pesticide residual test paper is utilized.

The preparation method of detection pesticide residual test paper the most according to claim 5, it is characterised in that described step S1 bag Contain:

Rare earth chloride, oleic acid and octadecylene are placed in flask stirring evacuation 30min by S1.1, stop agitating heating afterwards；

S1.2 is by NaOH, NH₄HF₂And methanol is placed in beaker and puts into stirrer, seal up sealed membrane, on magnetic stirring apparatus often Temperature stirring；

S1.3, by described step S1.1 product heats to 150 DEG C, is incubated 15～20min, closes stirring immediately, and closedown adds Heat, is allowed to be reduced to room temperature；

S1.4 drips solution described in S1.2 in the case of entering air less as far as possible, is passed through N after reaction completely₂, will will enter during dropping The air entered drains；

System is heated to 75-85 DEG C by S1.5, is incubated 1.5h；

The product of described step S1.5 is warming up to 310 DEG C in 0.5h by S1.6, N₂It is incubated 2h, immediately at N under protection₂Protection and It is cooled to room temperature under stirring；

S1.7 washed product；

S1.8 utilizes described step S1.7 product to prepare water-soluble beta-NaYF₄: Yb, Er nanoparticle.

The preparation method of detection pesticide residual test paper the most according to claim 6, it is characterised in that described step S1.8 bag Contain:

S1.8.1 is by product, hexamethylene, the tert-butyl alcohol, deionized water and the K of described step S1.7₂CO₃Under solution room temperature, mixing is stirred Mix；

S1.8.2 is by KMnO₄And NaIO₄Aqueous solution is slowly dropped into the product of described step S1.8.1, reacts 48h, use at 40 DEG C 95% washing with alcohol is centrifuged to obtain head product；

S1.8.3 head product is dissolved in HCl, stirring reaction under room temperature, and reaction terminates that rear centrifuge washing is centrifugal obtains product.

The preparation method of detection pesticide residual test paper the most according to claim 5, it is characterised in that described step S3 bag Contain:

S3.1 is respectively configured the phosphate buffered saline(PBS) of pH=5 and pH=7.4；

S3.2 prepares water-soluble beta-NaYF₄: Yb, Er labelling 2,4-D-IgG.

The preparation method of detection pesticide residual test paper the most according to claim 8, it is characterised in that described step S3.2 For:

S3.2.1 learns from else's experience KMnO₄-NaIO₄Oxidation is dissolved in the β-NaYF of water₄: Yb, Er make solution A, add in described solution A After the PBS of pH=5 prepared by described step S3.1, add EDC and Sulfo-NHS, stirring at normal temperature；

S3.2.2 adds 2,4-D-IgG in the product of described step S3.2.1, and normal-temperature reaction 2h is centrifuged out product；By described Product PBS wash 2 times after in the PBS of pH=7.4 prepared in described step S3.1 of constant volume.