CN105974109B

CN105974109B - A kind of fluorescence immune chromatography test paper for detecting ochratoxin A

Info

Publication number: CN105974109B
Application number: CN201610427117.7A
Authority: CN
Inventors: 李靖靖; 职爱民; 闵玉涛; 程丽英; 郭林; 王岚; 张小梅; 聂卉; 赵国欣; 张晓宇; 杨联芝
Original assignee: Zhongzhou University
Current assignee: Zhongzhou University
Priority date: 2016-06-14
Filing date: 2016-06-14
Publication date: 2017-08-04
Anticipated expiration: 2036-06-14
Also published as: CN105974109A

Abstract

The invention discloses a kind of fluorescence immune chromatography test paper for detecting ochratoxin A, including supporter and the adsorption layer being fixed on supporter, adsorption layer is followed successively by adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer and absorbent material layer since test lead, and described cellulose film layer is provided with the stealthy detection trace printed with coupling OTA carrier protein solution and the stealthy control trace printed with goat anti-mouse igg, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution；Described fluorescence antibody fibrous layer is made of the glass fibre cotton of absorption fluorescence antibody, and fluorescence antibody is graphene oxide fluorescent nano material, NaYF₄:Yb, Tm nano particle or NaGd (WO₄)₂:Eu³⁺The OTA monoclonal antibodies or polyclonal antibody of nanoparticle label.The test strips of the present invention have that high specificity, sensitivity are high, stability is high, security is good, easy, it is quick the characteristics of, in situ quantitation detection can be achieved under Portable fluorescence readout instrument, the need for different levels personnel can be met.

Description

A kind of fluorescence immune chromatography test paper for detecting ochratoxin A

Technical field

The present invention relates to a kind of immune chromatography test paper, more particularly to a kind of fluorescence immunoassay layer for detecting ochratoxin A Analyse test paper.

Background technology

Ochratoxin is to tell after mould toxin another mycotoxin for causing world's extensive concern after yellow.It is by aspergillus One group of important, contaminated food products mycotoxin that 7 kinds of aspergillus of category and 6 kinds of Penicillium notatums of Penicillium are produced, it includes 7 knots The similar compound of structure, its Poisoning is maximum, distribution is most wide, production poison amount highest, most heavy to the pollutions of agricultural product, with human health It is ochratoxin A (Ochratoxin A, OTA) that relation is most close.OTA mainly poisons the kidney and liver of animal, kidney It is the first target organ, with stronger teratogenesis, carcinogenic, mutagenesis.In China's GB2761-2011 national food safety standard Regulation, beans and and its product, cereals and its to roll in product OTA limit standards be 5 μ g/kg；On July 5th, 2012, European Union The committee issues (EU) No594/2012 regulations, revises (EC) No. 1881/2006 regulation, ochratoxin A is in bread basket In MRL be 3.0 μ g/kg；MRL in pepper is 15 μ g/kg；Maximum residual in capsicum Limitation was adjusted to 15 μ g/kg (standard for continuing to use 30 μ g/kg before this) from 1 day January in 2015；In wheat gluten most Big residue limits are 8.0 μ g/kg.

Conventional OTA method for detecting residue is at present：Microbial method, chromatography and immunoassay.Microbial method is simple, Economy, but susceptibility is not high：Chromatography is standard method that is qualitative and quantitatively detecting, with higher accuracy, accuracy and Sensitiveness, but its flow is loaded down with trivial details, and equipment costliness is, it is necessary to which professional and technical personnel, detection speed is slow；Immunization is simple to operate, cost It is low, and have preferable accuracy and sensitivity, qualitative and quantitative detection can be also carried out, is suitable for the examination to a large amount of samples. Immune test paper method has sxemiquantitative and certain quantitation capabilities, can provide the preliminary information of determinand, and the method sensitivity is high, point Analysis process is simple, has unique advantage as examination, is the detection technique for needing to first develop.

Unmodified graphene oxide shows very weak fluorescence, does not observe substantially with the naked eye under ultraviolet light Fluorescence.This is due to that stannic oxide/graphene nano piece surface has epoxy bond and hydroxyl on many oxy radicals, such as nanometer sheet surface Base, the carboxyl of nanometer sheet side, the non-radiative recombination in these groups generally energy photoinduced electron-hole pair, so as to cause to aoxidize stone The fluorescence of black alkene is very weak.After n-butylamine is modified, the epoxy bond and carboxyl on stannic oxide/graphene nano piece surface are all reacted Fall, this will greatly reduce its non-radiative recombination ability.Therefore after modifying, graphene oxide shows very strong fluorescence can conduct Novel markings thing is in field of biological detection application.Although colloid gold test paper is in this regard using relatively extensively, it can not accomplish quantitative Detection, as it is more sensitive, stably, flexibly, safety graphene oxide fluorescent chromatographic test paper research, be colloid gold immune detection The strong supplement of technology.

Upper conversion fluorescent nano particle is after surface modification and activation, as novel markings thing in fields such as biological detections Applied.Because of its unique physical arrangement and optical characteristics, make up-conversion fluorescence immune test paper method and Physico-chemical tests and micro- life Quality testing survey technology is compared, high with sensitivity, and operating process is simple, the characteristics of cost is low and can carry out mass field detection； Compared with colloid gold test paper, possess the characteristic of quantitative detection, as it is more sensitive, stably, flexibly, safety UPT-LF research, It is the strong supplement of colloid gold immune detection technique.But the preparation of upconverting fluorescent material at present and the research of test paper are still suffered from Luminous efficiency not enough, the defect, the research and discussion of assistant officer's this aspect to be strengthened such as fluorescent material species is few.

Down-conversion fluorescent nano particle has high sensitivity, is a kind of completely inert phosphor, with uniqueness Physical arrangement and optical characteristics, can be applied in field of biological detection as novel markings thing.By itself and biology, immunology, material Material learns the down-conversion fluorescent nanoparticle label immune chromatography method for OTA quick detections that is combined and develops, it is sensitive, Stable, flexible aspect shows excellent characteristic, is the favourable supplement of colloid gold immune detection technique.

The content of the invention

The technical problems to be solved by the invention are to provide a kind of fluorescence immune chromatography test paper for detecting ochratoxin A, The features such as test paper has special, sensitive, quick, easy, can quantify the ochratoxin in detection food, feed, Chinese herbal medicine A。

To achieve these goals, the technical solution adopted in the present invention is：

A kind of fluorescence immune chromatography test paper for detecting ochratoxin A, including supporter and the suction being fixed on supporter Attached layer, adsorption layer is followed successively by adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer and absorbent material since test lead Layer, described cellulose film layer detects trace and small with goat-anti provided with the stealthy of the carrier protein solution printing with coupling OTA Mouse IgG, rabbit anti-mouse IgG or the stealthy control trace of goat anti-rabbit igg antibody solution printing；Described fluorescence antibody fibrous layer is adopted It is made of the glass fibre cotton of absorption fluorescence antibody, fluorescence antibody is graphene oxide fluorescent nano material, NaYF₄:Yb, Tm receive Rice grain or NaGd (WO₄)₂：Eu³⁺The OTA monoclonal antibodies or polyclonal antibody of nanoparticle label.

Described supporter includes being arranged on the bottom of adsorption layer bottom surface and is arranged on the surface layer of adsorption layer top surface.

Described supporter includes transparent hollow tube, and adsorption layer is filled in inside hollow tube, and adsorption layer is from test End starts to be followed successively by adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer and absorbent material layer.

The upper end of described hollow tube is equipped with connector, and connector upper end is provided with auxiliary adsorbent equipment, and described is auxiliary Helping adsorbent equipment includes with the connector being in the adapter sleeve being tightly connected and the air bag for being arranged on the adapter sleeve top, air bag Inlet channel of the gas outlet successively through adapter sleeve top, the inner chamber of connector be connected with the upper oral part of hollow tube, air inlet Be provided with adapter sleeve side wall where passage can only outwards be vented can not inside air-breathing unidirectional air-out apparatus.

In the ventilation lumen of adapter sleeve side wall of the described unidirectional air-out apparatus where including being arranged on inlet channel, ventilation lumen It is provided with the ball sealer of spheroidal, the bottom surface of ventilation lumen and is provided with first gas outlet corresponding with ball sealer, the first gas outlet It is connected through outlet passageway with inlet channel, ventilation lumen side is provided with the second gas outlet being connected with ambient atmosphere, constitutes Can only outwards be vented, the unidirectional air outlet structure without normal direction inlet channel air-breathing.

The first described gas outlet is circle, and its diameter is less than the diameter of ball sealer.

Described connector is the hollow structure of upper and lower opening, and the upper end of its lower end and hollow tube is in being tightly connected, even External screw thread is provided with the outer wall on joint top, the bottom of adapter sleeve is provided with the blind hole corresponding with connector, blind hole inwall On be provided with the internal thread corresponding with external screw thread, connector is mounted in the blind hole of adapter sleeve bottom by external screw thread and internal thread rotation Interior, being provided between the top of connector and blind hole top on adapter sleeve prevents the sealing ring of gas leakage.

Described stealthy detection trace and stealthy control trace are in the alternate surface for being arranged on cellulose film layer, and spacing is 5- 8mm。

The inner chamber of described hollow tube is rectangle.

Described graphene oxide fluorescent nano material be one kind by matrix of graphene oxide by acyl chloride reaction with Alkylamine is come after modifying, the fluorescent nano material obtained after being characterized with silver nano-grain.

The OTA monoclonal antibodies of described graphene oxide fluorescent nano material mark or the preparation side of polyclonal antibody Method, comprises the following steps：

(1) graphene oxide fluorescent material is modified：

Take 20mg graphite oxides to grind, be added in 5mL DMF, after ultrasound is mixed, add at 20mL thionyl chlorides, 80 DEG C It is heated to reflux centrifuging after 48h, obtains intermediate acid chloride graphene oxide, after being washed twice with tetrahydrofuran, dries；Taking out again Under conditions of vacuum nitrogen protection, chloride graphene oxide and 1mL n-butylamines are mixed, 60 DEG C of reaction 72h are cooled to room Temperature, obtains the amine-modified graphene oxide of alkyl, is scattered in 20mL distilled waters, and 8000r/min centrifugations, removing is unreacted just Butylamine, remaining reaction solution is evaporated by Rotary Evaporators, and the dry after being evaporated is dispersed in distilled water again, is configured to Concentration is the graphene oxide fluorescent material aqueous solution of 1mg/mL modification；

(2) preparation of surface markers OTA antibody silver nanoparticle solution：

(a) 100mg silver nitrates are dissolved in 250mL ultra-pure waters, ebuillition of heated, add the lemon of 10mL mass fractions 1% Lemon acid sodium solution, 80 DEG C are heated to reflux after 1h stirring under the conditions of 0.5h, 4 DEG C overnight；Take 1mL stay overnight after solution, add 1mM Centrifuged after the μ L of TGA 10, stirring 24h, remove unreacted TGA, milli-Q water 3 times is steamed with Rotary Evaporators It is dry, plus it is the silver nanoparticle solution that 1mg/mL TGAs are coated that ultra-pure water, which is configured to concentration,；

(b) 0.1mM EDC 10 μ L and 0.1mM the μ L of NHS 10 are taken, the Yin Na of 1mL TGAs cladding is added step-wise to In rice grain solution, 1h is reacted, the silver nanoparticle solution of activated carboxylic is obtained；

(c) 0.1mM OTA monoclonal antibodies or the μ L of polyclonal antibody 10 is taken, the silver nano-grain of activated carboxylic is added to In solution, 4 DEG C of reactions are stayed overnight, and obtain surface markers OTA antibody silver nanoparticle solutions；

(3) mark of graphene oxide fluorescent nano material OTA antibody：

The graphene oxide fluorescent material aqueous solution 5mL of modification is taken, 2mL glutaraldehydes are added, 3h is reacted at room temperature, centrifugation is removed Unreacted glutaraldehyde is removed, remaining reaction solution is dispersed in 0.01mol/L, pH7.4 PBS cushioning liquid, surface is added OTA antibody silver nanoparticle solutions are marked, 4 DEG C of reaction 3h, through centrifugation, collect graphene oxide fluorescent nano material mark OTA monoclonal antibodies or polyclonal antibody, 0.01mol/L, pH7.4 PBS washing, are saved backup.

The NaYF₄:Yb, Tm nano particle are with NaYF₄For matrix, Yb³⁺For sensitizer, formed by Yb and Tm codopes A diameter of 60~80nm hair blue-fluorescence nano particle.

Described NaYF₄:The OTA monoclonal antibodies or the preparation method of polyclonal antibody of Yb, Tm nanoparticle label, Comprise the following steps：

(1) NaYF is prepared using hydrothermal synthesis method₄:Yb, Tm nano particle：

Y (the NO that concentration is 0.5mol/L are taken respectively₃)₃Solution 5.5mL, Yb (NO₃)₃Solution 0.5mL and Tm (NO₃)₃It is molten Liquid 0.5mL, is well mixed, and lucifuge fully reacts 1h under the conditions of adding 0.4mol/L sodium citrate solution 2mL, 25 DEG C；Plus Enter 1.5mol/L NH₄F solution 7mL, lucifuge stirring 0.5h under the conditions of 25 DEG C, uses HNO₃PH value is adjusted to 7.4,0.5h is stood, plus Distilled water is diluted to 30mL；24h is reacted at 220 DEG C again, room temperature is cooled to, through filtering, distilled water washing, dries, is sent out The NaYF of blue light₄:Yb, Tm fluorescent nano particle；

(2) surface siliconization of fluorescent nano particle：

By NaYF₄:Yb, Tm fluorescent nano particle are added in 0.01mol/L, pH7.4 PBS, are configured to concentration For 1mg/mL NaYF₄:Yb, Tm fluorescent nano particle solution, then 5mL concentrated ammonia liquors are instilled into 5mL NaYF₄:Yb, Tm fluorescence nano In particle solution, it is sufficiently stirred for reacting 20min, adds under the conditions of 2.5mL tetraethyl orthosilicates, 4 DEG C of lucifuges and react 4h； 6000r/min centrifuges 10min, obtains the fluorescent nano particle of surface siliconization, is washed with ethanol 4 times, disperses in methyl alcohol, to prepare Into the fluorescent nano particle methanol solution that concentration is 10mg/mL；

(3) surface amination of fluorescent nano particle：

Take 3mL fluorescent nano particle methanol solutions, add 5mL acetone, magnetic agitation 20min after sealing, then with ultrasound Ripple is dispersed, adds 3 μ L APTSA, and 30min is reacted at 40 DEG C after sealing, then in 70 DEG C of water-baths 1h, 6000r/min 5min is centrifuged, the fluorescent nano particle of surface amination is obtained, with ethanol cyclic washing 4 times, after vacuum drying, 4 DEG C of sealings are protected Deposit；

(4) mark of OTA antibody：

The fluorescent nano particle of surface amination is dissolved with 0.01mol/L, pH7.4 PBS cushioning liquid, is configured to dense Spend the fluorescent nano particle solution of the surface amination for 10mg/mL；Take the fluorescent nano particle solution of 10mL surface aminations Mixed with 5.6mg NHS and 15mg EDC, room temperature lucifuge reaction 3h, 6000r/min centrifugation 5min takes supernatant；By 1mg/mL OTA monoclonal antibodies or polyclonal antibody add supernatant in, OTA monoclonal antibodies or polyclonal antibody and supernatant Volume ratio be 1:100,4 DEG C of lucifuges react 4h, and centrifugation is dispersed in PBS cushioning liquid after distilled water washing, under the conditions of 4 DEG C Dialyse 3d, and centrifugation collects precipitation, obtains NaYF₄:The OTA monoclonal antibodies or polyclonal antibody of Yb, Tm nanoparticle label, It is placed in PBS cushioning liquid, 4 DEG C of preservations.

Described NaGd (WO₄)₂：Eu³⁺Nano particle is with gadolinium oxide (Gd₂O₃), sodium tungstate (Na₂WO₄·2H₂O) it is base Matter, europium ion (Eu³⁺) it is sensitizer, 100~200nm nano particle is formed under conditioned response.

Described NaGd (WO₄)₂：Eu³⁺The OTA monoclonal antibodies of nanoparticle label or the preparation side of polyclonal antibody Method, comprises the following steps：

(1) NaGd (WO are prepared using hydrothermal synthesis method₄)₂：Eu³⁺Nano particle

Weigh 7g Gd₂O₃With 7g Eu₂O₃, it is added separately to 50mL HNO₃In, 80 DEG C are heated to, insulation is concentrated into whole Crystallization, is made Gd (NO₃)₃With Eu (NO₃)₃；By Na₂WO₄·2H₂O is dissolved in deionized water, and it is 0.2mol/L's to be configured to concentration Na₂WO₄Solution；By obtained Gd (NO₃)₃With Eu (NO₃)₃With deionized water dissolving, the Gd that mass fraction is 80% is prepared respectively (NO₃)₃Solution and the Eu (NO that mass fraction is 15%₃)₃Solution, draws the 5mL Gd (NO prepared₃)₃Solution, 10mL Na₂WO₄ Solution and 5mL Eu (NO₃)₃Solution, is added in reactor, adjusts pH=5 with dust technology, sodium hydroxide, stirs, closes After reactor, 200 DEG C of heated at constant temperature 24h, room temperature is down to naturally；Solution in reactor is poured out into standing, treats that powder sinks in solution Behind shallow lake, supernatant is removed, powder is washed with distilled water 3 times, 3h is stood every time；Washed powder is heated to 80 DEG C of dry 12h, Obtain NaGd (WO₄)₂：Eu³⁺Nano particle；

(2) mark of OTA antibody

By OTA monoclonal antibodies or Anti-TNF-α liquid solution 10000r/min centrifugation 30min, impurity is removed；Use deionization Water dissolving NaGd (WO₄)₂：Eu³⁺Nano particle, is configured to the NaGd (WO that concentration is 0.1mg/mL₄)₂：Eu³⁺Nanoparticles solution, Then 0.1mol/L K are used₂CO₃Solution regulation NaGd (WO₄)₂：Eu³⁺Nanoparticles solution is to pH 8.2；

Take 10mL NaGd (WO₄)₂：Eu³⁺Nanoparticles solution, under stirring, adds OTA monoclonal antibodies or many μ l, the OTA monoclonal antibodies of clonal antibody solution 100 or polyclonal antibody solution concentration 1mg/mL, are mixed, incubation at room temperature 30min, 4 DEG C, 10000r/min centrifugation 30min, abandon supernatant, precipitation are used into 0.02mol/L Na₂B₄O₇Solution is diluted to 10mL, 10000r/ Min centrifuges 30min, and precipitation uses 0.02mol/L Na₂B₄O₇Solution is diluted to 1mL, and 4 DEG C save backup.

Described adsorbing fiber layer is made up of glass fibre cotton, nylon membrane, PVDF membrane or polyester film.

Described absorbent material layer is made up of absorbent filter.

Described cellulose film layer is made up of nitrocellulose filter, pure cellulose film or carboxylated cellulose film.

Described coupling OTA carrier protein is bovine serum albumin(BSA), chicken egg white or hemocyanin.

The stealthy detection trace and stealthy control trace can also be for " 10 " font arrangement trace, " ┬ ┬ " fonts are arranged Print mark, " ┴ ┴ " fonts arrangement trace, " ├ ├ " fonts arrange trace or " ┤ ┤ " fonts arrange trace.

Described surface layer is covered on adsorbing fiber layer, fluorescence antibody fibrous layer and absorbent material layer, in adsorbing fiber layer Sample mark line is printed with surface layer corresponding with fluorescence antibody fibrous layer intersection, sample mark line deviation adsorbing fiber layer 0.3~0.7cm of side.

The test strips of the present invention have high specificity, sensitivity height, stability is high, security is good, easy, quick, result The characteristics of display is vivid, directly perceived, applied widely, easy to carry.In situ quantitation inspection can be achieved under Portable fluorescence readout instrument Survey.The need for different levels personnel can be met, including professional chemical examination, customs quarantine control, health quarantine, quality-monitoring, livestock products add Work, raiser and consumer individual etc..The present invention has of crucial importance in terms of ensuring food safety, protecting consumer health Meaning, will have obvious economic benefit and social benefit.

Brief description of the drawings

Fig. 1 is the front view of the test paper of embodiment 4, and in figure, 2 be adsorbing fiber layer, and 3 be fluorescence antibody fibrous layer, and 4 be fibre The plain film layer of dimension, 7 be absorbent material layer, and 14 be surface layer, and 15 be bottom.

Fig. 2 is the top view of the test paper of embodiment 4, and in figure, 4 be cellulose film layer, and 5 be stealth for stealthy detection trace, 6 Trace is compareed, 14 be surface layer, and 15 be bottom, and 16 be sample mark line.

Fig. 3 is the front view of the test paper of embodiment 5, and in figure, 1 is hollow tube, and 2 be adsorbing fiber layer, and 3 be fluorescence antibody Fibrous layer, 4 be cellulose film layer, and 5 be stealthy detection trace, and 6 be stealthy control trace, and 7 be absorbent material layer, and 8 be connector, 9 be the inner chamber of connector, and 10 be sealing ring, and 11 be unidirectional air-out apparatus, and 12 be air bag, and 13 be adapter sleeve.

Fig. 4 for Fig. 3 A-A to sectional view, in figure, 1 is hollow tube, and 4 be cellulose film layer, and 6 be stealthy control trace.

Fig. 5 is the front view of the connector in Fig. 3, and in figure, 1 is hollow tube, and 8 be connector.

Fig. 6 is the top view of the connector in Fig. 3, and in figure, 8 be connector, and 9 be the inner chamber of connector.

Fig. 7 is the sectional view of the adapter sleeve in Fig. 3, and in figure, 10 be sealing ring, and 12 be air bag, and 13 be adapter sleeve, and 111 are Ventilation lumen, 112 be the second gas outlet, and 113 be ball sealer, and 114 be the first gas outlet, and 115 be outlet passageway, and 131 be blind hole, 132 be inlet channel.

Embodiment

The embodiment to the present invention is described in further detail with reference to embodiments.

Embodiment 1

The preparation of the fluorescence immune chromatography test paper of ochratoxin A is detected, is mainly included：The preparation of OTA artificial antigens, The preparation of OTA monoclonal antibodies or polyclonal antibody, the preparation of graphene oxide fluorescent nano material mark OTA antibody, fiber The step such as the preparation of plain film layer and the assembling of immune chromatography test paper.

1st, it is coupled the preparation of OTA carrier proteins

Using active ester method, OTA is coupled with carrier protein, artificial antigen is prepared.

It is concretely comprised the following steps：Accurately weigh 1.3mg OTA, 2mg dicyclohexylcarbodiimide (DCC) and 1.2mg N- hydroxyls Succinimide (NHS) is successively dissolved in 1.0mL methanol solutions, room temperature lucifuge stirring 2h, is sufficiently chemically reacted, named For A liquid.Weigh bovine serum albumin(BSA) (BSA) 5mg to be dissolved in 2mL PBS (0.01M, pH7.4) buffer solution, be named as B liquid.In room Under warm magnetic agitation, A liquid is slowly added dropwise in B liquid, under the conditions of 4 DEG C of lucifuge overnight, with PBS 4 days, 6 times/day of liquid is changed. After the completion of dialysis, 4000r/min centrifugation 5min take supernatant, are stored in -20 DEG C, standby.

2nd, the preparation of anti-OTA antibody

(1) preparation of monoclonal antibody

Animal immune：6~8 week old are immunized with 30 μ g~50 μ g/ consumption with the OTA carrier protein couplets thing of preparation Balb/C mouse 3~4 times, each 3~5 weeks immunization interval time, determine that antibody titer is carried out after meeting the requirements superpower immune, and Its suppression valency is detected before fusion.

Cell fusion：It is superpower immune latter 3~4 days, sinus under immune mouse socket of the eye is taken a blood sample, positive serum is separated；De- neck is lethal, Body surface is sterilized with 75% alcohol-pickled 5~10min of mouse, it is sterile to take its spleen, spleen is shredded and ground, through 120 mesh Buddhist nuns Imperial filtered through gauze, 1000r/min centrifugation 10min, collects splenocyte.By 1 × 10⁸Splenocyte and NS₀Myeloma cell presses 10:1 Ratio mixing, 1000r/min centrifugations 10min abandons supernatant, cell pellet is slowly added into 0.7 in 37 DEG C of water-baths~ Added in 1.0mL 50%PEG4000,1min, preceding 30s adds 0.1~0.3mL, middle 15s adds 0.2~0.4mL, last 15s adds It is complete；Then the culture medium 15mL of serum-free 1640 is slowly added into, to terminate PEG effect, 37 DEG C of water-baths 5~10min, 1000r/ Min centrifugations 10min abandons supernatant, and cell pellet is resuspended in HAT Selective agar mediums, and is added (8 in 96 porocyte culture plates ~10 pieces), the μ L/ holes of 100 μ L~200 are placed in 37 DEG C, 5% CO₂Cultivated in incubator.

The screening of monoclonal antibody：Culture 10~14 days, positive hole sizer is carried out with indirect elisa method and is selected, selection strong positive, The hole that inhibiting rate is high, cell growth is vigorous carries out 3~6 limited dilution clonings, and (until cell clone is monoclonal, detection is each Individual clone hole potency, suppression valency are basically identical), then expand culture, set up hybridoma cell strain.Prepared hybridoma The monoclonal antibody of secretion specifically can react with OTA, and affinity constant reaches 10¹⁰~10¹², light chain subtype is κ or λ, heavy chain Hypotype is IgG₁、IgG_2a、IgG_2b、IgG₃, for the monoclonal antibody of OTA specific epitopes, for preparing graphite oxide The glass fibre cotton of alkene fluorescent nano material labelled antibody.

(2) preparation of polyclonal antibody

NZw is immunized with OTA carrier protein couplets thing, immunizing dose is 200 μ g~500 μ g/ times, dorsal sc Divide 4~6 points of injections.Head exempts from, and the OTA carrier protein couplet things prepared is dissolved with sterile PBS, with equivalent Freund's complete adjuvant (FCA) mix, it is fully emulsified；Booster immunization, dissolves OTA carrier protein couplet things with sterile PBS, is not exclusively helped with equivalent Freund Agent (FIA) is mixed, fully emulsified, is carried out within 2~3 weeks after head exempts from, continuous immunity 4~5 times, every minor tick 2~3 weeks, last time 10~15 days after immune, it is surveyed with ELISA method determine potency and reach 10⁵During the above, take a blood sample and separate and collect hyper-immune serum.

IgG antibody is extracted by salting out method with saturation sulfuric acid, that is, takes 1 portion of hyper-immune serum plus 2 parts of PBS (pH7.2) to mix, plus etc. Volume saturation sulfuric acid is mixed by solution, under the conditions of putting 4 DEG C after static 12h, is centrifuged 15min with 2500r/min, is abandoned supernatant, plus PBS (pH7.2) dissolving precipitation, puts and PBS (pH7.2) dialysis 72h is used in 4 DEG C of refrigerators, and liquid is changed for several times in centre, 12000r/min centrifugations 15min, collects supernatant, the anti-OTA polyclonal antibodies of purifying is obtained, -20 DEG C freeze, for preparing graphene oxide fluorescence nano material Expect the glass fibre cotton of labelled antibody.

3rd, the preparation of the OTA monoclonal antibodies of graphene oxide fluorescent nano material mark or polyclonal antibody

(1) graphene oxide fluorescent material is modified：

Take 20mg graphite oxides to grind, be added in 5mL dimethylformamides (DMF), after ultrasound is mixed, add 20mL bis- Chlorine sulfoxide, is heated to reflux at 80 DEG C centrifuging after 48h, obtains intermediate acid chloride graphene oxide, washed twice with tetrahydrofuran Afterwards, dry；Again under conditions of nitrogen protection is vacuumized, chloride graphene oxide and 1mL n-butylamines are mixed, 60 DEG C of reactions 72h, is cooled to room temperature, obtains the amine-modified graphene oxide of alkyl, is scattered in 20mL distilled waters, 8000r/min centrifugations, removes Unreacted n-butylamine is removed, remaining reaction solution is evaporated by Rotary Evaporators, the dry after being evaporated is dispersed in double again Steam in water, be configured to the graphene oxide fluorescent material aqueous solution for the modification that concentration is 1mg/mL；

(2) preparation of surface markers OTA antibody silver nanoparticle solution：

(b) 0.1mM 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC) 10 μ L and 0.1mM NHS10 μ are taken In L, the silver nanoparticle solution for being added step-wise to 1mL TGAs cladding, 1h is reacted, the silver nano-grain of activated carboxylic is obtained Solution；

(3) mark of graphene oxide fluorescent nano material OTA antibody：

4th, the preparation of fluorescence antibody fibrous layer

By 1:100~1:The OTA antibody of the graphene oxide fluorescent nano material mark of 500 dilutions is adsorbed in processed glass In cellucotton, 4 DEG C of low-temperature vacuum dryings prepare the glass fibre cotton of graphene oxide fluorescent nano material labelled antibody.

5th, the preparation of adsorptive cellulose film layer：

Cellulose film layer is made up of nitrocellulose, and the diverse location with point sample instrument in cellulose film layer distinguishes specking OTA Artificial antigen detects trace and rabbit anti-mouse igg antibody (or sheep anti-mouse igg antibody, goat anti-rabbit igg antibody) control trace, in 37 DEG C Dry for standby.

6th, the assembling of immune chromatography test paper

Adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer, absorbent material layer are attached to successively since test lead On bottom with adhesive, then in covering upper layer, and it is cut into the wide test paper of 3-4cm.

7th, reaction principle is detected

After test paper test lead inserts testing sample solution, under siphonage drive, OTA and fluorescence are anti-in solution to be measured Fluorescence antibody in body fibrous layer spreads to cellulose film layer together, until arriving absorbent material layer.

In diffusion process, OTA antibody-silver nano-grain that OTA to be measured can be with absorption on graphene oxide is combined, Because of Ag-Ab effect and electrostatic attraction effect, cause OTA antibody-silver nano-grain to be stripped out from graphene oxide, enter And the fluorescence that graphene oxide is distributed is discharged, and combined with coupling OTA carrier protein.And sheep anti-mouse antibody then can be with OTA Antigen binding, excites lower stealthy detection trace line (T lines) and stealthy control trace line (C lines) all to occur in 350nm ultraviolets Absworption peak, the content in testing sample in OTA is quantitatively detected by fluorescence reading bar instrument reading T lines peak value.Conversely, nothing in sample During OTA, then T lines and C lines are not in ultraviolet absorption peak.

Embodiment 2

The preparation of immune chromatography test paper and embodiment 1 are essentially identical in embodiment 2, are in place of main difference：Fluorescence resists Body is NaYF₄:The OTA monoclonal antibodies or polyclonal antibody of Yb, Tm nanoparticle label.

(2) surface siliconization of fluorescent nano particle：

(3) surface amination of fluorescent nano particle：

Take 3mL fluorescent nano particle methanol solutions, add 5mL acetone, magnetic agitation 20min after sealing, then with ultrasound Ripple is dispersed, adds 3 μ L N- (2- aminoethyls) -3- aminopropyl trimethoxysilanes (APTSA), in 40 DEG C of reactions after sealing 30min, then in 70 DEG C of water-bath 1h, 6000r/min centrifugation 5min, obtain the fluorescent nano particle of surface amination, use second Alcohol cyclic washing 4 times, after vacuum drying, 4 DEG C of sealing preserves；

(4) mark of OTA antibody：

Detect reaction principle

In diffusion process, OTA to be measured can be combined with fluorescence antibody, and then close the antigen knot of OTA on fluorescence antibody Chalaza, prevents fluorescence antibody from being combined with the detection trace of the OTA artificial antigens on cellulose membrane, it is impossible to display detection trace, and Sheep or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody can then be combined with fluorescence antibody, and bar is read by fluorescence under infrared excitation Would not occur that absworption peak occurs at absworption peak, C lines at instrument T lines.Otherwise when in sample solution without OTA, then it can not prevent glimmering Photoactivated antibody is combined with being coupled the detection trace of OTA carrier proteins on cellulose membrane, reads that suction just occurs at bar instrument T lines by fluorescence Peak is received, same goat-anti or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody can also be combined with fluorescent labeled antibody, read by fluorescence Absworption peak also occurs at bar instrument C lines.If there is no T lines and C line absorptions peak on cellulose membrane, show that test strips have failed.

Embodiment 3

The preparation of immune chromatography test paper and embodiment 1 are essentially identical in embodiment 3, are in place of main difference：Fluorescence resists Body is NaGd (WO₄)₂：Eu³⁺The OTA monoclonal antibodies or polyclonal antibody of nanoparticle label.

(2) mark of OTA antibody

Detect reaction principle

After test paper test lead inserts testing sample solution, solution to be measured drives OTA to be measured and fluorescence by siphonage Down-conversion fluorescent nano particle NaGd (WO in nanoparticle label antibody glass fibre cotton₄)₂：Eu³⁺Labelled antibody together to Cellulose film layer spreads, and eventually penetrates the absorbent material layer of handle end.In diffusion process, OTA to be measured can be with fluorescence nano Down-conversion fluorescent nanoparticle label antibody in particle marker antibody fibrous layer is combined, and then closes down-conversion fluorescent nanometer OTA antigen-combining site on particle marker antibody, prevents down-conversion fluorescent nanoparticle label antibody with being coupled on cellulose membrane The detection traces of OTA carrier proteins is combined, and can not show detection trace on test paper, and sheep or rabbit anti-mouse IgG (or goat-anti rabbit IgG) antibody then can read bar instrument T lines under infrared excitation with down-conversion fluorescent nanoparticle label antibody binding by fluorescence Place would not occur that absworption peak occurs at absworption peak, C lines.If otherwise without OTA in sample solution, lower conversion can not be prevented glimmering Light nanoparticle label antibody is combined with being coupled the detection trace of OTA carrier proteins on cellulose membrane, and bar instrument T lines are read by fluorescence Absworption peak just occurs in place, and same goat-anti or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody also can be with down-conversion fluorescent nanometers Grain labelled antibody is combined, and reads that absworption peak also occurs at bar instrument C lines by fluorescence.If there is no T lines and C lines to inhale on cellulose membrane Peak is received, then shows that test strips have failed.

The structure and detection method of immune chromatography test paper are illustrated below in conjunction with other embodiment

Embodiment 4

The fluorescence immune chromatography test paper of the detection ochratoxin A of the present embodiment, reference picture 1-2, including supporter and solid The adsorption layer on supporter is scheduled on, adsorption layer is followed successively by adsorbing fiber layer 2, fluorescence antibody fibrous layer 3, fiber since test lead Plain film layer 4 and absorbent material layer 7, described cellulose film layer are provided with the stealth printed with coupling OTA carrier protein solution Detect trace 5 and the stealthy control trace 6 printed with goat anti-mouse igg, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution；Institute The fluorescence antibody fibrous layer stated is made of the glass fibre cotton of absorption fluorescence antibody, and fluorescence antibody is that graphene oxide fluorescence is received The OTA monoclonal antibodies or polyclonal antibody of rice material marking.

Described supporter includes being arranged on the bottom 15 of adsorption layer bottom surface and is arranged on the surface layer 14 of adsorption layer top surface.

Described surface layer is covered on adsorbing fiber layer, fluorescence antibody fibrous layer and absorbent material layer, in adsorbing fiber layer Sample mark line 16 is printed with surface layer corresponding with fluorescence antibody fibrous layer intersection, sample mark line deviation adsorbing fiber Layer 0.3~0.7cm of side.

For superior technique effect, described stealthy detection trace 5 and stealthy control trace 6 are arranged on fiber in alternate The surface of plain film layer 4, i.e., two trace bands it is arranged in parallel into | | ", spacing be 5~8mm.

The surface layer being covered in above adsorbing fiber layer and fluorescence antibody fibrous layer is white, is covered in above absorbent material layer Surface layer be other colors (such as yellow), test sample mark line be located at adsorbing fiber layer with fluorescence antibody fibrous layer intersection pair At the white surface layer deviation adsorbing fiber layer side about 0.5cm answered, arrow and max printed words are being printed on the right side of mark line on surface layer.

(1) preparation of testing sample and detecting step：

Detect corn-like：By sample comminution, 1 is made with absolute ethyl alcohol dilution:10 (m/v) sample suspension.

Operating method：OTA test paper test lead is inserted in testing sample, insertion depth is no more than mark line, about 10~20 Second test paper is taken out, fluorescence is put into after 5min and reads the direct readings of bar instrument.

Result judgement：

(a) it is positive to judge：Read all occur absworption peak at bar instrument T lines and C lines by fluorescence, it is the positive to represent testing result, Illustrate to contain OTA in testing sample；

(b) it is negative to judge：Read all to occur without absworption peak at bar instrument T lines and C lines by fluorescence, it is the moon to represent testing result Property, illustrate to be free of OTA in testing sample；

(c) fail-ure criterion：Occurred without by fluorescence reading bar instrument T lines and occur occurring at absworption peak, or T lines at absworption peak, C lines Absworption peak is occurred without at absworption peak, C lines, then shows that test paper has failed.

(2) sensitivity of the present embodiment immune chromatography test paper, specific detection

The detection of sensitivity：Be respectively configured with absolute ethyl alcohol concentration for 8,4,2,1,0.5,0.25,0.125,0ng/mL OTA standard items, after the μ L of the present embodiment test paper loading 100, reaction 5min, read bar instrument by fluorescence and directly read peak figure.With peak Value or peak area are ordinate, and the logarithm value using different OTA concentration draws standard suppression curve as abscissa, carries out related return Return analysis, calculate IC of the test paper to OTA₅₀And minimum detection limit.After measured, the test paper is to OTA Regression Equations：y =-0.2356x+0.7659, coefficient correlation is R²=0.9889, IC of the test paper to OTA is gone out according to regression equation calculation₅₀For 13.44ng/mL, the lowest detection of the test paper is limited to 0.27ng/mL, shows that immune chromatography test paper has to OTA higher sensitive Degree.

Specific detection：With aflatoxin B₁, zearalenone, vomitoxin, patulin, ergotoxin make For competitor, it is 1mg/mL to configure above-mentioned mark product concentration, and its inhibiting rate is detected with graphene oxide fluorescence immune chromatography test paper, Cross reacting rate is calculated according to formula.

Measurement result see the table below 1, and the specificity of the graphene oxide-silver nanoparticle fluorescence immune chromatography test paper is preferable, with it The equal no cross reaction of his toxin.

The cross reaction of the immune chromatography test paper of table 1 and other mycotoxins

The test strips of the present embodiment have advantages below：

(1) high specificity, sensitivity is high.The test strips of the present embodiment are marked with graphene oxide fluorescent nano material It is made based on OTA antibody, because the graphene oxide fluorescent material through modified has outstanding optical characteristics and good life Thing compatibility, simultaneous oxidation graphene electron transmission efficiency is high so that this novel immune method sensitivity is high, detectable limit Low, minimum is only 0.27ng/mL.

(2) stability is high, and security is good.The OTA of the test strips graphene oxide fluorescent nano material mark of the present embodiment It is made based on antibody, nano-Ag particles surface carries opposite charge groups, the strong bonded because of Electrostatic Absorption with protein； Graphene specific surface area is big, can be combined with multiple proteins biomolecule by chemically reacting, and on the influence of its bioactivity very Small, stability is high, and flexibility is good.

(3) the graphene oxide fluorescence immune chromatography test paper of the present embodiment can be detected to corn, DDGS, feed etc.. By silver nanoparticle and graphene oxide collective effect, signal enhancing is detected, antibody consumption is reduced, antibody cost is saved.

Embodiment 5

The fluorescence immune chromatography test paper of the detection ochratoxin A of the present embodiment, reference picture 3-7, including supporter and solid The adsorption layer on supporter is scheduled on, described supporter includes transparent hollow tube 1, and adsorption layer is filled in hollow tube Portion, adsorption layer is followed successively by adsorbing fiber layer 2, fluorescence antibody fibrous layer 3, cellulose film layer 4 and absorbent material since test lead Layer 7, described cellulose film layer is provided with the stealthy detection trace 5 printed with coupling OTA carrier protein solution and uses goat-anti Mouse IgG, rabbit anti-mouse IgG or the stealthy control trace 6 of goat anti-rabbit igg antibody solution printing；Described fluorescence antibody fiber Layer is made of the glass fibre cotton of absorption fluorescence antibody, and fluorescence antibody is NaYF₄:The OTA Dan Ke of Yb, Tm nanoparticle label Grand antibody or polyclonal antibody.

The upper end of described hollow tube 1 is equipped with connector 8, and the upper end of connector 8 is provided with auxiliary adsorbent equipment, described It is in the adapter sleeve 13 being tightly connected and the air bag for being arranged on the adapter sleeve top that auxiliary adsorbent equipment, which is included with the connector, 12, inlet channel 132, the inner chamber 9 of connector 8 and the hollow tube 1 of the gas outlet of air bag 12 successively through the top of adapter sleeve 13 Upper oral part is connected, be provided with the adapter sleeve side wall where inlet channel can only outwards be vented can not inwardly air-breathing unidirectionally go out Device of air 11.

The ventilation lumen 111 of adapter sleeve side wall of the described unidirectional air-out apparatus where including being arranged on inlet channel, ventilation It is provided with chamber 111 on the ball sealer 113 of spheroidal, the bottom surface of ventilation lumen 111 and is provided with first outlet corresponding with ball sealer Mouth 114, the first gas outlet is connected through outlet passageway 115 with inlet channel 132, and the side of ventilation lumen 111 is provided with big with the external world Second gas outlet 112 of gas phase connection, composition can only be outwards vented, the unidirectional air outlet structure without normal direction inlet channel air-breathing.

For superior technique effect, the first described gas outlet 114 is circle, and its diameter is less than the diameter of ball sealer. Such setting can make the even closer combination of the ball sealer of the first gas outlet and spheroidal, it is ensured that the sealing in space.

Described connector 8 is the hollow structure of upper and lower opening, and the upper end of its lower end and hollow tube 1 is in be tightly connected, It is provided with external screw thread on the outer wall on connector top, the bottom of adapter sleeve 13 is provided with corresponding with connector blind hole 131, blind The internal thread corresponding with external screw thread is provided with the inwall of hole 131, connector is mounted in adapter sleeve by external screw thread and internal thread rotation In the blind hole 131 of bottom, being provided between the top of connector and blind hole top on adapter sleeve prevents the sealing ring 10 of gas leakage.

Described stealthy detection trace 5 and stealthy control trace 6 are in the alternate surface for being arranged on cellulose film layer 4, spacing For 5~8mm.

The inner chamber of described hollow tube 1 is rectangle.

Test sample mark line is located at adsorbing fiber layer clear hollow body corresponding with fluorescence antibody fibrous layer intersection At upper deviation adsorbing fiber layer side about 0.5cm, arrow and max printed words are printed on the clear hollow body on the right side of mark line.

In use, adapter sleeve and connector are linked together by internal and external threads, gently extruding gasbag, the gas in air bag Body is discharged by the ventilation lumen of inlet channel, the inner chamber of connector and adapter sleeve side wall, now the spheroidal in ventilation lumen The gas that ball sealer is squeezed out from air bag is lifted, and the gas in air bag is smoothly discharged by the first gas outlet；Will examination In paper insertion testing sample solution, air bag is then unclamped, now, the inner chamber of hollow tube, the inner chamber of connector and inlet channel Interior generation negative pressure, under the attraction of negative pressure, ball sealer is tightly adsorbed on the first gas outlet on the bottom surface of ventilation lumen, by its envelope Close, so as to help sample solution more quickly to infiltrate test paper, so as to reduce the time that sample solution infiltrates test strips, improve inspection Survey efficiency.

By above-mentioned situation it should be apparent that the present embodiment structure novel and unique, advantages of simple, by the way that supporter is whole Body is revised as closed structure, while increasing air bag at the top of supporter, makes absorption rapider, so as to effectively improve detection Speed and the degree of accuracy, while cell parts and support body portion are sealing reassembling type connection, detection only need to change support after finishing Body portion, convenient disassembly is easy to operate, and using effect is good, is the innovation on Test paper.

(1) preparation of testing sample and detecting step：

Detect DDGS samples：By sample comminution, 1 is made with absolute ethyl alcohol dilution:10 (m/v) sample suspension.

Operating method：Extruding gasbag, the gas in air bag is discharged, and OTA test paper test lead is inserted in testing sample, is inserted Enter depth no more than mark line, unclamp air bag, take out within about 3-5 seconds and the fluorescence reading direct readings of bar instrument is put into after test paper, 4min.

Result judgement：

(a) it is positive to judge：Read to occur without at bar instrument T lines by fluorescence and occur absworption peak at absworption peak, C lines, represent detection As a result it is the positive, illustrates to contain ochratoxin A in testing sample；

(b) it is negative to judge：Read all occur absworption peak at bar instrument T lines and C lines by fluorescence, it is feminine gender to represent testing result, Illustrate to be free of ochratoxin A in testing sample；

(c) fail-ure criterion：Read all to occur without absworption peak at bar instrument T lines and C lines by fluorescence, then show that test paper has failed.

The detection of sensitivity：Be respectively configured with absolute ethyl alcohol concentration for 8,4,2,1,0.5,0.25,0.125,0ng/mL OTA standard items, after the μ L of the present embodiment test paper loading 100, reaction 4min, read bar instrument by fluorescence and directly read peak figure.With peak Value or peak area are ordinate, and the logarithm value using different OTA concentration draws standard suppression curve as abscissa, carries out related return Return analysis, calculate IC of the test paper to OTA₅₀And minimum detection limit.After measured, the test paper is to OTA Regression Equations：y =-0.4319x+0.9367, coefficient correlation is R²=0.9689, IC of the test paper to OTA is gone out according to regression equation calculation₅₀For 10.26ng/mL, the lowest detection of the test paper is limited to 0.21ng/mL, shows that immune chromatography test paper has to OTA higher sensitive Degree.

Specific detection：With aflatoxin B₁, zearalenone, vomitoxin, patulin, ergotoxin make For competitor, it is 1mg/mL to configure above-mentioned mark product concentration, uses NaYF₄:Yb, Tm nano fluorescent immune chromatography test paper detect that it presses down Rate processed, cross reacting rate is calculated according to formula.

Measurement result see the table below 2, the NaYF₄:The specificity of Yb, Tm nano fluorescent immune chromatography test paper is preferable, with other The equal no cross reaction of toxin.

The cross reaction of the immune chromatography test paper of table 2 and other mycotoxins

Compound	Half-inhibition concentration (ng/mL)	Cross reacting rate (%)
			OTA	10.26	100
Zearalenone	＞ 1.0 × 10⁵	＜ 0.010
			Vomitoxin	＞ 1.0 × 10⁵	＜ 0.010
Aflatoxin B₁	＞ 1.0 × 10⁵	＜ 0.010
			Patulin	＞ 1.0 × 10⁵	＜ 0.010
Ergotoxin	＞ 1.0 × 10⁵	＜ 0.010

The test strips of the present embodiment have advantages below：

(1) high specificity, sensitivity is high.The present embodiment up-conversion fluorescence immune chromatography test paper with the color spectrum that turns blue upper turn Change fluorescent nano material NaYF₄:It is made based on the OTA antibody of Yb, Tm mark, due to NaYF₄:The luminous effect of Yb, Tm nano particle Rate is high, can effectively eliminate sample detection bias light, sensitivity is greatly improved, and lowest detection is limited to 0.21ng/mL.

(2) stability is high, and security is good.Upconversion fluorescence nano material NaYF in the test paper of the present embodiment₄:Yb, Tm energy Effectively launch blue color spectrum, it is entirely avoided light and be quenched caused by other conditions.NaYF₄:Yb, Tm material have inorganic lazy Property, infrared ray excited, VISIBLE LIGHT EMISSION the characteristics of, detected using the test paper for surrounding people and environment without any danger Evil.

(3) it is easy, quick.Read bar instrument using fluorescence, the test paper can direct readings, realize scene Quantitative detection.Only Result can be read after test paper is inserted into test sample 3~5 seconds, 4 minutes, test sample is represented without absworption peak at T lines In contain OTA；Conversely, without OTA.It is visual result, accurate, it is simple and clear, artificial erroneous judgement is avoided to greatest extent.

(4) when prepared by the test paper of the present embodiment, fluorescence antibody uses NaYF₄:The OTA Dan Ke of Yb, Tm nanoparticle label Grand antibody or polyclonal antibody, i.e., by amidized NaYF₄:Yb, Tm nano particle are directly connected with antibody, labeling process letter Single, Conjugate ratio is high, so as to effectively improve based on NaYF₄:The sensitivity of the immune test paper of Yb, Tm material.

Embodiment 6

The structure be the same as Example 5 of the fluorescence immune chromatography test paper of the detection ochratoxin A of the present embodiment, difference It is NaGd (WO in the fluorescence antibody of, the present embodiment₄)₂：Eu³⁺The OTA monoclonal antibodies or Anti-TNF-α of nanoparticle label Body.

(1) preparation of testing sample and detecting step：

Result judgement：

The detection of sensitivity：Be respectively configured with absolute ethyl alcohol concentration for 8,4,2,1,0.5,0.25,0.125,0ng/mL OTA standard items, after the μ L of the present embodiment test paper loading 100, reaction 4min, read bar instrument by fluorescence and directly read peak figure.With peak Value or peak area are ordinate, and the logarithm value using different OTA concentration draws standard suppression curve as abscissa, carries out related return Return analysis, calculate IC of the test paper to OTA₅₀And minimum detection limit.After measured, the test paper is to OTA Regression Equations：y =-0.4154x+1.1123, coefficient correlation is R²=0.9779, IC of the test paper to OTA is gone out according to regression equation calculation₅₀For 9.83ng/mL, the lowest detection of the test paper is limited to 1.07ng/mL, shows that immune chromatography test paper has to OTA higher sensitive Degree.

Specific detection：With aflatoxin B₁, zearalenone, vomitoxin, patulin, ergotoxin make For competitor, it is 1mg/mL to configure above-mentioned mark product concentration, with NaGd (WO₄)₂：Eu³⁺Nano fluorescent immune chromatography test paper is detected Its inhibiting rate, cross reacting rate is calculated according to formula.

Measurement result see the table below 3, the NaGd (WO₄)₂：Eu³⁺The specificity of nano fluorescent immune chromatography test paper is preferable, with it The equal no cross reaction of his toxin.

The cross reaction of the immune chromatography test paper of table 3 and other mycotoxins

Compound	Half-inhibition concentration (ng/mL)	Cross reacting rate (%)
			OTA	9.83	100
Zearalenone	＞ 1.0 × 10⁵	＜ 0.098
			Vomitoxin	＞ 1.0 × 10⁵	＜ 0.098
Aflatoxin B₁	＞ 1.0 × 10⁵	＜ 0.098
			Patulin	＞ 1.0 × 10⁵	＜ 0.098
Ergotoxin	＞ 1.0 × 10⁵	＜ 0.098

The test strips of the present embodiment have advantages below：

(1) high specificity, sensitivity is high.The present embodiment down-conversion fluorescent nanoparticle label immune chromatography test paper is with oxygen Change gadolinium (Gd₂O₃), sodium tungstate (Na₂WO₄·2H₂O) it is matrix, europium ion (Eu³⁺) it is 100~200nm nanometers that sensitizer is formed Particle, with good optical characteristics, makes OTA detection eliminate the interference of bias light, clever lightness is greatly improved, minimum to examine Measure 1.07ng/mL.

(2) stability is high.NaGd(WO₄)₂：Eu³⁺Nano particle belongs to completely inert phosphor, these properties So that it has extraordinary photostability, do not occur photobleaching under the irradiation of uviol lamp so that readings is stable.

(3) it is easy, quick.Bar instrument can be read using the present embodiment test paper with fluorescence to be used in combination, direct readings realizes scene Quantitative detection, test strips need to only be inserted in test sample 3-5 seconds, testing result is can determine that in 4 minutes after taking-up.

(4) result display is vivid, directly perceived, accurate.The present embodiment fluorescent nano particle labeled immunochromatographyassay assay test is with T lines Whether absworption peak is occurred as the positive and negative standard of detection.At the T lines without absworption peak if represent to contain in test sample OTA, it is on the contrary then represent test sample in be free of OTA.It is visual result, accurate, it is less prone to false positive and false negative etc. artificially by mistake Sentence.

Claims

1. a kind of fluorescence immune chromatography test paper for detecting ochratoxin A, including supporter and the absorption being fixed on supporter Layer, adsorption layer is followed successively by adsorbing fiber layer (2), fluorescence antibody fibrous layer (3), cellulose film layer (4) and inhaled since test lead Water material layer (7), it is characterised in that described cellulose film layer, which is provided with, uses the hidden of the carrier protein solution printing for being coupled OTA Shape detection trace (5) and the stealthy control trace printed with goat anti-mouse igg, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution (6)；Described fluorescence antibody fibrous layer is made of the glass fibre cotton of absorption fluorescence antibody, and fluorescence antibody is graphene oxide Fluorescent nano material, NaYF₄:Yb, Tm nano particle or NaGd (WO₄)₂：Eu³⁺The OTA monoclonal antibodies of nanoparticle label or Person's polyclonal antibody；

Described supporter includes transparent hollow tube (1), and adsorption layer is filled in inside hollow tube, and adsorption layer is from test lead Start to be followed successively by adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer and absorbent material layer；

The upper end of described hollow tube (1) is equipped with connector (8), and connector (8) upper end is provided with auxiliary adsorbent equipment, described Auxiliary adsorbent equipment include being in the adapter sleeve (13) that is tightly connected with the connector and being arranged on the adapter sleeve top Air bag (12), inlet channel (132) successively through adapter sleeve (13) top of the gas outlet of air bag (12), the inner chamber of connector (8) (9) upper oral part with hollow tube (1) is connected, and nothing can only be outwards vented by being provided with the adapter sleeve side wall where inlet channel The unidirectional air-out apparatus (11) of air-breathing in normal direction；

The ventilation lumen (111) of adapter sleeve side wall of the described unidirectional air-out apparatus where including being arranged on inlet channel, ventilation lumen (111) it is provided with the ball sealer (113) of spheroidal, the bottom surface of ventilation lumen (111) and is provided with first corresponding with ball sealer Gas outlet (114), the first gas outlet is connected through outlet passageway (115) with inlet channel (132), and ventilation lumen (111) is set sideways The second gas outlet (112) being connected with ambient atmosphere is equipped with, composition can only be outwards vented, the list without normal direction inlet channel air-breathing To air outlet structure.

2. the fluorescence immune chromatography test paper of detection ochratoxin A according to claim 1, it is characterised in that described Supporter includes being arranged on the bottom (15) of adsorption layer bottom surface and is arranged on the surface layer (14) of adsorption layer top surface.

3. the fluorescence immune chromatography test paper of detection ochratoxin A according to claim 1, it is characterised in that described First gas outlet (114) is circle, and its diameter is less than the diameter of ball sealer.

4. the fluorescence immune chromatography test paper of detection ochratoxin A according to claim 1, it is characterised in that described Connector (8) is the hollow structure of upper and lower opening, and the upper end of its lower end and hollow tube (1) is in be tightly connected, connector top Outer wall on be provided with external screw thread, the bottom of adapter sleeve (13) is provided with the blind hole (131) corresponding with connector, blind hole (131) internal thread corresponding with external screw thread is provided with inwall, connector is mounted in adapter sleeve by external screw thread and internal thread rotation In the blind hole (131) of bottom, being provided between the top of connector and blind hole top on adapter sleeve prevents the sealing ring of gas leakage (10)。

5. the fluorescence immune chromatography test paper of detection ochratoxin A according to claim 1, it is characterised in that described Stealth detection trace (5) and stealthy control trace (6) are in the alternate surface for being arranged on cellulose film layer (4), and spacing is 5-8mm；

The inner chamber of described hollow tube (1) is rectangle.

6. the fluorescence immune chromatography test paper of detection ochratoxin A according to claim 1, it is characterised in that described The OTA monoclonal antibodies of graphene oxide fluorescent nano material mark or the preparation method of polyclonal antibody, including following step Suddenly：

(1) graphene oxide fluorescent material is modified：

Take 20mg graphite oxides to grind, be added in 5mL DMF, after ultrasound is mixed, add at 20mL thionyl chlorides, 80 DEG C and heat Centrifuged after backflow 48h, obtain intermediate acid chloride graphene oxide, after being washed twice with tetrahydrofuran, dried；Vacuumizing again Under conditions of nitrogen protection, chloride graphene oxide and 1mL n-butylamines are mixed, 60 DEG C of reaction 72h are cooled to room temperature, obtained To the amine-modified graphene oxide of alkyl, it is scattered in 20mL distilled waters, 8000r/min centrifugations remove unreacted n-butylamine, Remaining reaction solution is evaporated by Rotary Evaporators, the dry after being evaporated is dispersed in distilled water again, is configured to concentration For the graphene oxide fluorescent material aqueous solution of 1mg/mL modification；

(2) preparation of surface markers OTA antibody silver nanoparticle solution：

(a) 100mg silver nitrates are dissolved in 250mL ultra-pure waters, ebuillition of heated, add the citric acid of 10mL mass fractions 1% Sodium solution, 80 DEG C are heated to reflux after 1h stirring under the conditions of 0.5h, 4 DEG C overnight；Take 1mL stay overnight after solution, add 1mM sulfydryl Centrifuged after the μ L of acetic acid 10, stirring 24h, remove unreacted TGA, milli-Q water 3 times is evaporated with Rotary Evaporators, plus It is the silver nanoparticle solution that 1mg/mL TGAs are coated that ultra-pure water, which is configured to concentration,；

(b) 0.1mM EDC 10 μ L and 0.1mM the μ L of NHS 10 are taken, the silver nanoparticle of 1mL TGAs cladding is added step-wise to In grain solution, 1h is reacted, the silver nanoparticle solution of activated carboxylic is obtained；

(c) 0.1mM OTA monoclonal antibodies or the μ L of polyclonal antibody 10 is taken, the silver nanoparticle solution of activated carboxylic is added to In, 4 DEG C of reactions are stayed overnight, and obtain surface markers OTA antibody silver nanoparticle solutions；

(3) mark of graphene oxide fluorescent nano material OTA antibody：

The graphene oxide fluorescent material aqueous solution 5mL of modification is taken, 2mL glutaraldehydes are added, 3h is reacted at room temperature, centrifugation is removed not The glutaraldehyde of reaction, remaining reaction solution is dispersed in 0.01mol/L, pH7.4 PBS cushioning liquid, adds surface markers OTA antibody silver nanoparticle solutions, 4 DEG C of reaction 3h, through centrifugation, the OTA for collecting graphene oxide fluorescent nano material mark is mono- Clonal antibody or polyclonal antibody, 0.01mol/L, pH7.4 PBS washing, are saved backup.

7. the fluorescence immune chromatography test paper of detection ochratoxin A according to claim 1, it is characterised in that described NaYF₄:The OTA monoclonal antibodies or the preparation method of polyclonal antibody of Yb, Tm nanoparticle label, comprise the following steps：

Y (the NO that concentration is 0.5mol/L are taken respectively₃)₃Solution 5.5mL, Yb (NO₃)₃Solution 0.5mL and Tm (NO₃)₃Solution 0.5mL, is well mixed, and lucifuge fully reacts 1h under the conditions of adding 0.4mol/L sodium citrate solution 2mL, 25 DEG C；Add 1.5mol/L NH₄F solution 7mL, lucifuge stirring 0.5h under the conditions of 25 DEG C, uses HNO₃PH value is adjusted to 7.4,0.5h is stood, plus it is double Steam water and be diluted to 30mL；24h is reacted at 220 DEG C again, room temperature is cooled to, through filtering, distilled water washing, dries, is turned blue The NaYF of coloured light₄:Yb, Tm fluorescent nano particle；

(2) surface siliconization of fluorescent nano particle：

By NaYF₄:Yb, Tm fluorescent nano particle are added in 0.01mol/L, pH7.4 PBS, are configured to concentration for 1mg/ ML NaYF₄:Yb, Tm fluorescent nano particle solution, then 5mL concentrated ammonia liquors are instilled into 5mL NaYF₄:Yb, Tm fluorescent nano particle are molten In liquid, it is sufficiently stirred for reacting 20min, adds under the conditions of 2.5mL tetraethyl orthosilicates, 4 DEG C of lucifuges and react 4h；6000r/min 10min is centrifuged, the fluorescent nano particle of surface siliconization is obtained, is washed with ethanol 4 times, is disperseed in methyl alcohol, being configured to concentration is 10mg/mL fluorescent nano particle methanol solution；

(3) surface amination of fluorescent nano particle：

Take 3mL fluorescent nano particle methanol solutions, add 5mL acetone, magnetic agitation 20min after sealing, then it is equal with ultrasonic wave It is even scattered, 3 μ L APTSA is added, 30min is reacted after sealing at 40 DEG C, then in 70 DEG C of water-bath 1h, 6000r/min centrifugations 5min, obtains the fluorescent nano particle of surface amination, with ethanol cyclic washing 4 times, after vacuum drying, 4 DEG C of sealing preserves；

(4) mark of OTA antibody：

The fluorescent nano particle of surface amination is dissolved with 0.01mol/L, pH7.4 PBS cushioning liquid, being configured to concentration is The fluorescent nano particle solution of 10mg/mL surface amination；Take the fluorescent nano particle solution of 10mL surface aminations with 5.6mg NHS and 15mg EDC are mixed, room temperature lucifuge reaction 3h, 6000r/min centrifugation 5min, take supernatant；By 1mg/mL's OTA monoclonal antibodies or polyclonal antibody are added in supernatant, OTA monoclonal antibodies or polyclonal antibody and supernatant Volume ratio is 1:100,4 DEG C of lucifuges react 4h, and centrifugation is dispersed in PBS cushioning liquid, under the conditions of 4 DEG C thoroughly after distilled water washing 3d is analysed, centrifugation collects precipitation, obtains NaYF₄:The OTA monoclonal antibodies or polyclonal antibody of Yb, Tm nanoparticle label, put In PBS cushioning liquid, 4 DEG C of preservations.

8. the fluorescence immune chromatography test paper of detection ochratoxin A according to claim 1, it is characterised in that described NaGd(WO₄)₂：Eu³⁺The OTA monoclonal antibodies or the preparation method of polyclonal antibody of nanoparticle label, including following step Suddenly：

Weigh 7g Gd₂O₃With 7g Eu₂O₃, it is added separately to 50mL HNO₃In, 80 DEG C are heated to, insulation is concentrated into whole knots Crystalline substance, is made Gd (NO₃)₃With Eu (NO₃)₃；By Na₂WO₄·2H₂O is dissolved in deionized water, and it is 0.2mol/L's to be configured to concentration Na₂WO₄Solution；By obtained Gd (NO₃)₃With Eu (NO₃)₃With deionized water dissolving, the Gd that mass fraction is 80% is prepared respectively (NO₃)₃Solution and the Eu (NO that mass fraction is 15%₃)₃Solution, draws the 5mL Gd (NO prepared₃)₃Solution, 10mLNa₂WO₄It is molten Liquid and 5mL Eu (NO₃)₃Solution, is added in reactor, adjusts pH=5 with dust technology, sodium hydroxide, stirs, closing is anti- Answer after kettle, 200 DEG C of heated at constant temperature 24h, room temperature is down to naturally；Solution in reactor is poured out into standing, treats that powder is precipitated in solution Afterwards, supernatant is removed, powder is washed with distilled water 3 times, 3h is stood every time；Washed powder is heated to 80 DEG C of dry 12h, obtained To NaGd (WO₄)₂：Eu³⁺Nano particle；

(2) mark of OTA antibody

By OTA monoclonal antibodies or Anti-TNF-α liquid solution 10000r/min centrifugation 30min, impurity is removed；It is molten with deionized water Solve NaGd (WO₄)₂：Eu³⁺Nano particle, is configured to the NaGd (WO that concentration is 0.1mg/mL₄)₂：Eu³⁺Nanoparticles solution, then Use 0.1mol/L K₂CO₃Solution regulation NaGd (WO₄)₂：Eu³⁺Nanoparticles solution is to pH 8.2；

Take 10mL NaGd (WO₄)₂：Eu³⁺Nanoparticles solution, under stirring, adds OTA monoclonal antibodies or Anti-TNF-α μ l, the OTA monoclonal antibodies of liquid solution 100 or polyclonal antibody solution concentration 1mg/mL, are mixed, incubation at room temperature 30min, 4 DEG C, 10000r/min centrifuges 30min, abandons supernatant, and precipitation is used into 0.02mol/L Na₂B₄O₇Solution is diluted to 10mL, 10000r/min 30min is centrifuged, precipitation uses 0.02mol/L Na₂B₄O₇Solution is diluted to 1mL, and 4 DEG C save backup.