CN105974109A

CN105974109A - Fluorescence immunochromatography test paper for detecting ochratoxin A

Info

Publication number: CN105974109A
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: 2016-09-28
Anticipated expiration: 2036-06-14
Also published as: CN105974109B

Abstract

The invention discloses fluorescence immunochromatography test paper for detecting ochratoxin A. The fluorescence immunochromatography test paper comprises a supporting body and an absorbing layer fixed to the supporting body, and the absorbing layer sequentially comprises an absorbing fiber layer, a fluorescent antibody fiber layer, a cellulose membrane layer and a water absorbing material layer from the test end; invisible detection imprinting printed with a coupling OTA carrier protein solution and invisible detection imprinting printed with a goat-anti-mouse IgG, rabbit-anti-mouse IgG and goat-anti-rabbit IgG antibody solution are arranged on the cellulose membrane layer; the fluorescent antibody fiber layer is made of a glass fiber cotton adsorbing fluorescent antibody, and the fluorescent antibody is an OTA monoclonal antibody or polyclonal antibody marked with an oxidized graphene fluorescence nanometer material, NaYF4:Yb, Tm nanometer particles or NaGd(WO4)2:Eu3+ nanometer particles. The test paper strip has the advantages of being high in specificity, sensitivity and stability, good in safety, convenient and fast. On-site quantitative detection can be achieved under a portable fluorescence reading instrument, and requirements of people at different levels can be met.

Description

A kind of fluorescence immune chromatography test paper detecting ochratoxin A

Technical field

The present invention relates to a kind of immune chromatography test paper, particularly relate to a kind of fluorescence immunoassay layer detecting ochratoxin A Analysis reagent paper.

Background technology

Ochratoxin is another mycotoxin causing world's extensive concern after Huang tells mould toxin.It is by aspergillosis One group of important, mycotoxin of contaminated food products that the 7 kinds of aspergillosis belonged to and 6 kinds of penicillium of Penicillium produce, it comprises 7 knots The compound that structure is similar, its toxic is maximum, distribution is the widest, it is the highest, the heaviest to the pollution of agricultural product, with human health to produce poison amount That relation is the closest is ochratoxin A (Ochratoxin A, OTA).OTA mainly poisons kidney and the liver of animal, kidney It is the first target organ, there is stronger teratogenesis, carcinogenic, mutagenic action.In China's GB2761-2011 national food safety standard Regulation, beans and and goods, cereals and to roll OTA limit standard in goods be 5 μ g/kg；On July 5th, 2012, European Union Committee issues (EU) No594/2012 regulation, revises (EC) No. 1881/2006 regulation, and ochratoxin A is at bread basket In MRL be 3.0 μ g/kg；MRL in Fructus Piperis is 15 μ g/kg；Maximum residual in Fructus Capsici Limitation was adjusted to 15 μ g/kg (continuing to use the standard of 30 μ g/kg before this) from 1 day January in 2015；In wheat protein Big residue limits is 8.0 μ g/kg.

The most conventional OTA method for detecting residue is: microbial method, chromatography and immunoassay.Microbial method is simple, Economy, but sensitivity is the highest: chromatography is qualitative and detection by quantitative standard method, have higher accuracy, accuracy and Sensitivity, but its flow process is loaded down with trivial details, apparatus expensive, needs professional and technical personnel, and detection speed is slow；Immunization is simple to operate, cost Low, and have preferable accuracy and susceptiveness, also can carry out qualitative and detection by quantitative, be suitable for the examination to a large amount of samples. Immune test paper method has sxemiquantitative and certain quantitation capabilities, it is provided that the preliminary information of determinand, and this method is highly sensitive, point Analysis process is simple, has unique advantage as examination, is the detection technique needing to first develop.

The graphene oxide of unmodified shows the most weak fluorescence, does not the most substantially observe under ultraviolet light irradiates Fluorescence.This is owing to there are the epoxy bond on a lot of oxy radical, such as nanometer sheet surface and hydroxyl in stannic oxide/graphene nano sheet surface Base, the carboxyl of nanometer sheet side, the non-radiative recombination in these groups generally energy photoinduced electron-hole pair, thus cause aoxidizing stone The fluorescence of ink alkene is the most weak.After n-butylamine is modified, epoxy bond and the carboxyl on stannic oxide/graphene nano sheet surface are all reacted Falling, this will greatly reduce its non-radiative recombination ability.Therefore, after modifying, graphene oxide shows the strongest fluorescence can conduct Novel markings thing is applied in field of biological detection.Although colloid gold test paper is applied relatively wide in this regard, but it can not be accomplished quantitatively Detection, as the research of more sensitive, stable, flexible, safe graphene oxide fluorescent chromatographic reagent paper, is colloid gold immune detection The strong of technology supplements.

Upper conversion fluorescent nano particle is modified and after activation through surface, as novel markings thing in fields such as biological detection Apply.Because of physical arrangement and the optical characteristics of its uniqueness, make up-conversion fluorescence immune test paper method and Physico-chemical tests and micro-life Analyte detection technology is compared, and has highly sensitive, and operating process is simple, low cost and can carry out the feature of mass field detection； Compared with colloid gold test paper, possess the characteristic of detection by quantitative, as the research of more sensitive, stable, flexible, safe UPT-LF, It is that the strong of colloid gold immune detection technique supplements.But the preparation of upconverting fluorescent material at present and the research of reagent paper still suffer from The defects such as luminous efficiency is inadequate, and fluorescent material kind is few, the research of assistant officer's this aspect to be strengthened and discussion.

Down-conversion fluorescent nano-particle has high sensitivity, is a kind of completely inert phosphor, has uniqueness Physical arrangement and optical characteristics, can apply in field of biological detection as novel markings thing.By itself and biology, immunology, material Material is learned and is combined and the down-conversion fluorescent nanoparticle label immune chromatography method quickly detected for OTA developed, sensitive, Stable, flexible aspect shows the characteristic of excellence, is that the favourable of colloid gold immune detection technique is supplemented.

Summary of the invention

The technical problem to be solved is to provide a kind of fluorescence immune chromatography test paper detecting ochratoxin A, This reagent paper has the features such as special, sensitive, quick, easy, the ochratoxin in energy detection by quantitative food, feedstuff, 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 detecting ochratoxin A, including supporter and the suction being fixed on supporter Attached layer, adsorption layer starts to be followed successively by adsorbing fiber layer, fluorescent antibody fibrous layer, cellulose membrane layer and absorbent material from test lead Layer, described cellulose membrane layer is provided with the stealthy detection trace printed with the carrier protein solution of coupling OTA and little with goat-anti The stealthy comparison trace that Mus IgG, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution are printed；Described fluorescent antibody fibrous layer is adopted Making with the glass fibre cotton of absorption fluorescent antibody, fluorescent antibody is graphene oxide fluorescent nano material, NaYF₄: Yb, Tm receive Rice grain or NaGd (WO₄)₂: Eu³⁺The OTA monoclonal antibody of nanoparticle label or polyclonal antibody.

Described supporter includes being arranged on the bottom of adsorption layer bottom surface and being arranged on the surface layer of adsorption layer end face.

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, fluorescent antibody fibrous layer, cellulose membrane layer and absorbent material layer.

The upper end of described hollow tube is equipped with union joint, and union joint upper end is provided with auxiliary adsorbent equipment, and described is auxiliary Help adsorbent equipment to include with described union joint and in the adapter sleeve being tightly connected and be arranged on the air bag on described adapter sleeve top, air bag Gas outlet be connected with the upper oral part of hollow tube through the inlet channel on adapter sleeve top, the inner chamber of union joint successively, air inlet Being provided with on the adapter sleeve sidewall at passage place can only outwards aerofluxus cannot the unidirectional air-out apparatus of inside air-breathing.

Described unidirectional air-out apparatus includes the ventilation lumen being arranged on the adapter sleeve sidewall at inlet channel place, in ventilation lumen It is provided with the ball sealer of spheroidal, the bottom surface of ventilation lumen has first gas outlet corresponding with ball sealer, the first gas outlet Being connected with inlet channel through outlet passageway, ventilation lumen side is provided with the second gas outlet being connected with ambient atmosphere, constitutes Can only outwards aerofluxus, cannot be to the unidirectional air outlet structure of inlet channel air-breathing.

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

Described union joint 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 Being provided with external screw thread on the outer wall on joint top, the bottom of adapter sleeve is provided with the blind hole corresponding with union joint, blind hole inwall On be provided with the female thread corresponding with external screw thread, union joint is contained in the blind hole of adapter sleeve bottom by external screw thread and internal thread rotation In, on adapter sleeve, it is provided with the sealing ring preventing gas leakage between top and the blind hole top of union joint.

Described stealthy detection trace and stealthy comparison trace are the alternate surface being arranged on cellulose membrane layer, and spacing is 5- 8mm。

The inner chamber of described hollow tube is rectangle.

Described graphene oxide fluorescent nano material be a kind of with graphene oxide for substrate by acyl chloride reaction and After alkylamine is modified, the fluorescent nano material obtained after characterizing with silver nano-grain.

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

(1) graphene oxide fluorescent material is modified:

Take 20mg graphite oxide to grind, join in 5mL DMF, after ultrasonic mixing, add 20mL thionyl chloride, at 80 DEG C It is centrifuged after being heated to reflux 48h, obtains intermediate acid chloride graphene oxide, after washing twice with oxolane, be dried；Taking out again Under conditions of vacuum nitrogen protection, chloride graphene oxide and 1mL n-butylamine are mixed, 60 DEG C of reaction 72h, be cooled to room Temperature, obtain alkylamine modify graphene oxide, be scattered in 20mL distilled water, 8000r/min be centrifuged, remove unreacted just Butylamine, is evaporated remaining reactant liquor 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 the modification of 1mg/mL；

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

A 100mg silver nitrate is dissolved in 250mL ultra-pure water by (), ebuillition of heated, adds the lemon of 10mL mass fraction 1% Lemon acid sodium solution, 80 DEG C are heated to reflux stirring 0.5h after 1h, under the conditions of 4 DEG C overnight；Take 1mL overnight after solution, add 1mM TGA 10 μ L, centrifugal after stirring 24h, remove unreacted TGA, milli-Q water 3 times, steam with Rotary Evaporators Dry, add ultra-pure water and be configured to the silver nanoparticle solution that concentration is 1mg/mL TGA cladding；

B () takes the NHS 10 μ L of EDC 10 μ L and 0.1mM of 0.1mM, be added step-wise to the Yin Na of 1mL TGA cladding In rice grain solution, react 1h, obtain the silver nanoparticle solution of activated carboxylic；

C () takes OTA monoclonal antibody or the polyclonal antibody 10 μ L of 0.1mM, join the silver nano-grain of activated carboxylic In solution, 4 DEG C of reactions overnight, obtain surface markers OTA antibody silver nanoparticle solution；

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

Take the graphene oxide fluorescent material aqueous solution 5mL of modification, add 2mL glutaraldehyde, room temperature reaction 3h, be centrifuged, remove Remove unreacted glutaraldehyde, remaining reactant liquor is dispersed in the PBS buffer solution of 0.01mol/L, pH7.4, add surface Labelling OTA antibody silver nanoparticle solution, 4 DEG C of reaction 3h, by centrifugation, collect graphene oxide fluorescent nano material labelling OTA monoclonal antibody or polyclonal antibody, the PBS washing of 0.01mol/L, pH7.4, save backup.

Described NaYF₄: Yb, Tm nano-particle is with NaYF₄For substrate, Yb³⁺For sensitizer, Yb and Tm codope formed A diameter of 60～80nm send out blue-fluorescence nano-particle.

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

(1) hydrothermal synthesis method is used to prepare NaYF₄: Yb, Tm nano-particle:

Take concentration respectively and be the Y (NO of 0.5mol/L₃)₃Solution 5.5mL, Yb (NO₃)₃Solution 0.5mL and Tm (NO₃)₃Molten Liquid 0.5mL, mix homogeneously, add the sodium citrate solution 2mL of 0.4mol/L, under the conditions of 25 DEG C, lucifuge fully reacts 1h；Add Enter 1.5mol/L NH₄F solution 7mL, under the conditions of 25 DEG C, lucifuge stirring 0.5h, uses HNO₃Regulation pH value, to 7.4, stands 0.5h, adds Distilled water is diluted to 30mL；At 220 DEG C, react 24h again, be cooled to room temperature, through filtration, distilled water washing, be dried, 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 adds in the PBS of 0.01mol/L, pH7.4, is configured to concentration NaYF for 1mg/mL₄: Yb, Tm fluorescent nano particle solution, then 5mL strong aqua ammonia is instilled 5mL NaYF₄: Yb, Tm fluorescence nano In particle solution, it is sufficiently stirred for reacting 20min, adds 2.5mL tetraethyl orthosilicate, under the conditions of 4 DEG C of lucifuges, react 4h； 6000r/min is centrifuged 10min, obtains the fluorescent nano particle of surface siliconization, by washing with alcohol 4 times, is dispersed in methanol, preparation The fluorescent nano particle methanol solution becoming concentration to be 10mg/mL；

(3) surface amination of fluorescent nano particle:

Take 3mL fluorescent nano particle methanol solution, add the acetone of 5mL, magnetic agitation 20min after sealing, then with ultrasonic Ripple is dispersed, adds the APTSA of 3 μ L, reacts 30min at 40 DEG C after sealing, then at 70 DEG C of water-bath 1h, 6000r/min Centrifugal 5min, obtains the fluorescent nano particle of surface amination, and with ethanol cyclic washing 4 times, after vacuum drying, 4 DEG C seal and protect Deposit；

(4) labelling of OTA antibody:

With the PBS buffer solution of 0.01mol/L, pH7.4, the fluorescent nano particle of surface amination is dissolved, be configured to dense Degree is the fluorescent nano particle solution of the surface amination of 10mg/mL；Take the fluorescent nano particle solution of 10mL surface amination Mixing with 5.6mg NHS and 15mg EDC, room temperature lucifuge reaction 3h, 6000r/min are centrifuged 5min, take supernatant；By 1mg/mL OTA monoclonal antibody or polyclonal antibody add in supernatant, OTA monoclonal antibody or polyclonal antibody and supernatant Volume ratio be 1:100,4 DEG C of lucifuges reaction 4h, centrifugal, be dispersed in PBS buffer solution, under the conditions of 4 DEG C after distilled water washing Dialysis 3d, centrifugal, collect precipitation, obtain NaYF₄: the OTA monoclonal antibody of Yb, Tm nanoparticle label or polyclonal antibody, It is placed in PBS buffer solution, 4 DEG C of preservations.

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

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

(1) hydrothermal synthesis method is used to prepare NaGd (WO₄)₂: Eu³⁺Nano-particle

Weigh 7g Gd₂O₃With 7g Eu₂O₃, it is added separately to the HNO of 50mL₃In, it being heated to 80 DEG C, insulation is concentrated into all Crystallization, prepares Gd (NO₃)₃With Eu (NO₃)₃；By Na₂WO₄·2H₂O is dissolved in deionized water, and being configured to concentration is 0.2mol/L's Na₂WO₄Solution；By prepared Gd (NO₃)₃With Eu (NO₃)₃With deionized water dissolving, prepare the Gd that mass fraction is 80% respectively (NO₃)₃Solution and the Eu (NO that mass fraction is 15%₃)₃Solution, draws the 5mL Gd (NO of preparation₃)₃Solution, 10mL Na₂WO₄ Solution and 5mL Eu (NO₃)₃Solution, joins in reactor, with dust technology, sodium hydroxide regulation pH=5, stirs, closes Reactor, after 200 DEG C of heated at constant temperature 24h, is down to room temperature naturally；Solution in reactor is poured out standing, treats that in solution, powder body sinks Behind shallow lake, remove supernatant, with distilled water wash powder body 3 times, stand 3h every time；Washed powder body is heated to 80 DEG C of dry 12h, Obtain NaGd (WO₄)₂: Eu³⁺Nano-particle；

(2) labelling of OTA antibody

OTA monoclonal antibody or Anti-TNF-α liquid solution 10000r/min are centrifuged 30min, remove impurity；Use deionization Water dissolution 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 is 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 antibody or many Clonal antibody solution 100 μ l, OTA monoclonal antibody or polyclonal antibody solution concentration 1mg/mL, mixing, incubation at room temperature 30min, 4 DEG C, 10000r/min be centrifuged 30min, abandon supernatant, precipitation used 0.02mol/L Na₂B₄O₇Solution is diluted to 10mL, 10000r/ Min is centrifuged 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 membrane layer is made up of nitrocellulose filter, pure cellulose film or carboxylated cellulose film.

The carrier protein of described coupling OTA is bovine serum albumin, chicken egg white or hemocyanin.

Described stealthy detection trace and stealthy comparison trace can also be " 10 " font arrangement trace, " ┬ ┬ " font row Mark, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font of prining arranges trace.

Described surface layer covers on adsorbing fiber layer, fluorescent antibody fibrous layer and absorbent material layer, at adsorbing fiber layer It is printed with sample mark line, this sample mark line deflection adsorbing fiber layer on the surface layer corresponding with fluorescent antibody fibrous layer intersection Side 0.3～0.7cm.

The test strips of the present invention has high specificity, highly sensitive, stability is high, safety is good, easy, quick, result Show feature vivid, directly perceived, applied widely, easy to carry.In situ quantitation inspection can be realized under Portable fluorescence readout instrument Survey.The needs of different levels personnel can be met, add including specialty chemical examination, customs quarantine control, health quarantine, quality-monitoring, livestock products 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.

Accompanying drawing explanation

Fig. 1 is the front view of the reagent paper of embodiment 4, and in figure, 2 is adsorbing fiber layer, and 3 is fluorescent antibody fibrous layer, and 4 is fine Dimension element film layer, 7 is absorbent material layer, and 14 is surface layer, and 15 is bottom.

Fig. 2 is the top view of the reagent paper of embodiment 4, and in figure, 4 is cellulose membrane layer, and 5 is stealthy detection trace, and 6 is stealthy Comparison trace, 14 is surface layer, and 15 is bottom, and 16 is sample mark line.

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

Fig. 4 be the A-A of Fig. 3 in sectional view, figure, 1 is hollow tube, and 4 is cellulose membrane layer, and 6 is stealthy comparison trace.

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

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

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

Detailed description of the invention

Below in conjunction with embodiment, the detailed description of the invention of the present invention is described in further detail.

Embodiment 1

The preparation of fluorescence immune chromatography test paper of detection ochratoxin A, specifically include that OTA artificial antigen preparation, OTA monoclonal antibody or the preparation of polyclonal antibody, the preparation of graphene oxide fluorescent nano material labelling OTA antibody, fiber The steps such as the preparation of element film layer and the assembling of immune chromatography test paper.

1, the preparation of coupling OTA carrier protein

Use active ester method, OTA is carried out coupling with carrier protein, prepares artificial antigen.

It concretely comprises the following steps: accurately weigh 1.3mg OTA, 2mg dicyclohexylcarbodiimide (DCC) and 1.2mg N-hydroxyl Butanimide (NHS) is successively dissolved in 1.0mL methanol solution, room temperature lucifuge stirring 2h, carries out sufficient chemical reaction, name For A liquid.Weigh bovine serum albumin (BSA) 5mg to be dissolved in 2mL PBS (0.01M, pH7.4) buffer, named B liquid.In room Under temperature magnetic agitation, A liquid is slowly added dropwise in B liquid, under the conditions of lucifuge 4 DEG C overnight, with PBS 4 days, changes liquid 6 times/day. After having dialysed, 4000r/min is centrifuged 5min, takes supernatant, is stored in-20 DEG C, standby.

2, the preparation of anti-OTA antibody

(1) preparation of monoclonal antibody

Animal immune: with the OTA carrier protein couplet thing prepared with 30 μ g～50 μ g/ consumption immunity 6～8 week old only Balb/C mice 3～4 times, each immunization interval time 3～5 weeks, determine that antibody titer carries out superpower immunity after meeting the requirements, and Its suppression valency was detected before merging.

Cell merges: after superpower immunity 3～4 days, take a blood sample by hole under immune mouse socket of the eye, separation positive serum；De-neck is lethal, Alcohol-pickled mice 5～10min with 75% is sterilized body surface, aseptic takes its spleen, is shredded by spleen and grinds, through 120 mesh Buddhist nuns Dragon filtered through gauze, 1000r/min is centrifuged 10min, collects splenocyte.By 1 × 10⁸Splenocyte and NS₀Myeloma cell presses 10:1 Ratio mixing, 1000r/min is centrifuged 10min and abandons supernatant, cell precipitate is slowly added in 37 DEG C of water-baths 0.7～ The 50%PEG4000 of 1.0mL, adds in 1min, and front 30s adds 0.1～0.3mL, and middle 15s adds 0.2～0.4mL, and last 15s adds Complete；Then serum-free 1640 culture medium 15mL it is slowly added into, to terminate the effect of PEG, 37 DEG C of water-baths 5～10min, 1000r/ Min is centrifuged 10min and abandons supernatant, is resuspended in HAT Selective agar medium by cell precipitate, and adds in 96 porocyte culture plates (8 ～10 pieces), 100 μ L～200 μ L/ holes, it is placed in 37 DEG C, the CO of 5%₂Incubator is cultivated.

The screening of monoclonal antibody: cultivate 10～14 days, carries out positive hole sizer with indirect elisa method and selects, selection strong positive, Suppression ratio is high, the eugonic hole of cell carries out 3～6 limited dilution clonings and (until cell clone is monoclonal, detects each Individual clone hole titer, suppression valency are basically identical), then amplification culture, set up hybridoma cell strain.Prepared hybridoma The monoclonal antibody of secretion can be reacted with OTA specifically, 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 epitope, it is used for preparing graphite oxide The glass fibre cotton of alkene fluorescent nano material traget antibody.

(2) preparation of polyclonal antibody

By OTA carrier protein couplet thing immunity New Zealand white rabbit, immunizing dose is 200 μ g～500 μ g/ time, dorsal sc Points 4～6 injections.Head exempts from, and the OTA carrier protein couplet thing prepared with aseptic PBS dissolving, with equivalent Freund's complete adjuvant (FCA) mixing, fully emulsified；Booster immunization, dissolves OTA carrier protein couplet thing with aseptic PBS, not exclusively helps with equivalent Freund Agent (FIA) mixes, fully emulsified, and head is carried out after exempting from for 2～3 weeks, continuous immunity 4～5 times, every minor tick 2～3 weeks, for the last time After immunity 10～15 days, survey it with ELISA method and determine titer and reach 10⁵Time above, take a blood sample and separate and collect hyper-immune serum.

Press salting out method with saturated sulphuric acid and extract IgG antibody, i.e. take 1 portion of hyper-immune serum and add 2 parts of PBS (pH7.2) mixings, add The saturated sulphuric acid of volume presses solution mixing, under the conditions of putting 4 DEG C after static 12h, is centrifuged 15min with 2500r/min, abandons supernatant, adds PBS (pH7.2) dissolution precipitation, puts the 4 DEG C of interior PBS of refrigerator (pH7.2) dialysis 72h, and liquid is changed for several times in centre, and 12000r/min is centrifuged 15min, collects supernatant, obtains the anti-OTA polyclonal antibody of purification, and-20 DEG C frozen, is used for preparing graphene oxide fluorescence nano material The glass fibre cotton of material traget antibody.

3, the OTA monoclonal antibody of graphene oxide fluorescent nano material labelling or the preparation of polyclonal antibody

(1) graphene oxide fluorescent material is modified:

Take 20mg graphite oxide to grind, join in 5mL dimethylformamide (DMF), after ultrasonic mixing, add 20mL bis- Chlorine sulfoxide, centrifugal after being heated to reflux 48h at 80 DEG C, obtain intermediate acid chloride graphene oxide, wash twice with oxolane After, it is dried；Again under conditions of evacuation nitrogen is protected, chloride graphene oxide and 1mL n-butylamine are mixed, 60 DEG C of reactions 72h, is cooled to room temperature, obtains the graphene oxide that alkylamine is modified, is scattered in 20mL distilled water, and 8000r/min is centrifuged, and removes Removing unreacted n-butylamine, be evaporated by Rotary Evaporators by remaining reactant liquor, the dry after being evaporated is dispersed in double again Steam in water, be configured to the graphene oxide fluorescent material aqueous solution of the modification that concentration is 1mg/mL；

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

B () takes the NHS10 μ of 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) 10 μ L and 0.1mM of 0.1mM L, is added step-wise in the silver nanoparticle solution of 1mL TGA cladding, reacts 1h, obtain the silver nano-grain of activated carboxylic Solution；

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

4, the fibrolaminar preparation of fluorescent antibody

The OTA antibody of the graphene oxide fluorescent nano material labelling diluted by 1:100～1:500 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 traget antibody.

5, the preparation of adsorptive cellulose film layer:

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

6, the assembling of immune chromatography test paper

Start to be attached to successively from test lead by adsorbing fiber layer, fluorescent antibody fibrous layer, cellulose membrane layer, absorbent material layer On bottom with binding agent, then cover upper layer, and be cut into the wide reagent paper of 3-4cm.

7, detection reaction principle

After reagent paper test lead inserts testing sample solution, under siphonage drives, in solution to be measured, OTA and fluorescence resist Fluorescent antibody in body fibrous layer spreads to cellulose membrane layer together, until to absorbent material layer.

In diffusion process, OTA to be measured can combine with absorption OTA antibody-silver nano-grain on graphene oxide, Because of Ag-Ab effect and electrostatic attraction effect, cause OTA antibody-silver nano-grain to be stripped out from graphene oxide, enter And discharge the fluorescence that graphene oxide is distributed, and and the carrier protein combination of coupling OTA.Sheep anti-mouse antibody then can be with OTA Antigen combines, and excites lower stealthy detection trace line (T line) and stealthy comparison trace line (C line) all can occur at 350nm ultraviolet Absworption peak, reads, by fluorescence, the content that bar instrument reads in T line peak value detection by quantitative testing sample in OTA.Otherwise, nothing in sample During OTA, then T line and C line do not have ultraviolet absorption peak.

Embodiment 2

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

(2) surface siliconization of fluorescent nano particle:

(3) surface amination of fluorescent nano particle:

Take 3mL fluorescent nano particle methanol solution, add the acetone of 5mL, magnetic agitation 20min after sealing, then with ultrasonic Ripple is dispersed, adds N-(2-the aminoethyl)-3-aminopropyl trimethoxysilane (APTSA) of 3 μ L, 40 DEG C of reactions after sealing 30min, then at 70 DEG C of water-bath 1h, 6000r/min is centrifuged 5min, obtains the fluorescent nano particle of surface amination, uses second Alcohol cyclic washing 4 times, after vacuum drying, 4 DEG C seal preservation；

(4) labelling of OTA antibody:

Detection reaction principle

In diffusion process, OTA to be measured can combine with fluorescent antibody, and then closes the antigen knot of OTA in fluorescent antibody Chalaza, stops the detection trace of fluorescent antibody OTA artificial antigen on cellulose membrane to be combined, it is impossible to display detection trace, and Sheep or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody then can be combined with fluorescent antibody, read bar by fluorescence under infrared excitation Absworption peak would not occur at instrument T line, at C line, there will be absworption peak.Otherwise time in sample solution without OTA, then can not stop glimmering Photoactivated antibody detection trace of coupling OTA carrier protein on cellulose membrane is combined, and reads to arise that suction at bar instrument T line by fluorescence Receiving peak, same goat-anti or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody also can be combined with fluorescent-labeled antibody, read by fluorescence Also absworption peak is there will be at bar instrument C line.If there is no T line and C line absorption peak on cellulose membrane, then show that test strips lost efficacy.

Embodiment 3

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

(2) labelling of OTA antibody

Detection reaction principle

After reagent 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⁺Traget antibody together to Cellulose membrane 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 combines, and then closes down-conversion fluorescent nanometer The antigen-combining site of OTA on particle marker antibody, stops down-conversion fluorescent nanoparticle label antibody and coupling on cellulose membrane The detection trace of OTA carrier protein combines, and reagent paper cannot show detection trace, and sheep or rabbit anti-mouse IgG (or goat-anti rabbit IgG) antibody then can read bar instrument T line by fluorescence with down-conversion fluorescent nanoparticle label antibodies under infrared excitation Would not there is absworption peak in place, there will be absworption peak at C line.If otherwise without OTA in sample solution, then lower conversion can not be stoped glimmering Light nanoparticle label antibody detection trace of coupling OTA carrier protein on cellulose membrane is combined, and reads bar instrument T line by fluorescence Place arises that absworption peak, same goat-anti or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody also can be with down-conversion fluorescent nanometers Grain traget antibody combines, and reads also to there will be absworption peak at bar instrument C line by fluorescence.If not having T line and C line to inhale on cellulose membrane Receive peak, then show that test strips lost efficacy.

Structure and the detection method of immune chromatography test paper is illustrated below in conjunction with other embodiments

Embodiment 4

The fluorescence immune chromatography test paper of the detection ochratoxin A of the present embodiment, with reference to Fig. 1-2, including supporter with solid The adsorption layer being scheduled on supporter, adsorption layer starts to be followed successively by adsorbing fiber layer 2, fluorescent antibody fibrous layer 3, fiber from test lead Element film layer 4 and absorbent material layer 7, described cellulose membrane layer is provided with the stealth printed with the carrier protein solution of coupling OTA Detection trace 5 and the stealthy comparison trace 6 printed with goat anti-mouse igg, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution；Institute The fluorescent antibody fibrous layer stated uses the glass fibre cotton of absorption fluorescent antibody to make, and fluorescent antibody is that graphene oxide fluorescence is received The OTA monoclonal antibody of rice material marking or polyclonal antibody.

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

Described surface layer covers on adsorbing fiber layer, fluorescent antibody fibrous layer and absorbent material layer, at adsorbing fiber layer It is printed with sample mark line 16, this sample mark line deflection adsorbing fiber on the surface layer corresponding with fluorescent antibody fibrous layer intersection Layer side 0.3～0.7cm.

For superior technique effect, described stealthy detection trace 5 and stealthy comparison trace 6 are arranged on fiber in alternate The surface of element film layer 4, i.e. two trace band one-tenth arranged in parallel | | ", spacing is 5～8mm.

Covering the surface layer on adsorbing fiber layer and fluorescent antibody fibrous layer is white, covers on absorbent material layer Surface layer be other color (such as yellow), test sample mark line is positioned at adsorbing fiber layer and fluorescent antibody fibrous layer intersection pair At the white surface layer deflection adsorbing fiber layer side about 0.5cm answered, on the right side of mark line, on surface layer, it is printed on arrow and max printed words.

(1) preparation of testing sample and detecting step:

Detection corn-like: by sample comminution, make the sample suspension of 1:10 (m/v) with dehydrated alcohol dilution.

Operational approach: being inserted by OTA reagent paper test lead in testing sample, insertion depth is less than mark line, about 10～20 Second take out reagent paper, put into after 5min fluorescence read the direct readings of bar instrument.

Result judges:

A () is positive judges: read all to occur at bar instrument T line and C line absworption peak by fluorescence, represents that testing result is positive, Illustrate in testing sample containing OTA；

B () is negative judges: read all to occur without at bar instrument T line and C line absworption peak by fluorescence, represents that testing result is cloudy Property, illustrate in testing sample without OTA；

C () fail-ure criterion: read bar instrument T line by fluorescence and occur without absworption peak, occurs occurring at absworption peak, or T line at C line Absworption peak, occurs without absworption peak at C line, then show that reagent paper lost efficacy.

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

The detection of susceptiveness: with dehydrated alcohol be respectively configured concentration be 8,4,2,1,0.5,0.25,0.125,0ng/mL OTA standard substance, after the present embodiment reagent paper loading 100 μ L, reaction 5min, read bar instrument by fluorescence and directly read peak figure.With peak Value or peak area are vertical coordinate, with the logarithm value of different OTA concentration as abscissa, draw standard suppression curve, are correlated with back Return analysis, calculate this reagent paper IC to OTA₅₀And lowest detectable limit.After measured, the Regression Equations of OTA is by this reagent paper: y =-0.2356x+0.7659, correlation coefficient is R²=0.9889, go out this reagent paper IC to OTA according to regression equation calculation₅₀For 13.44ng/mL, the lowest detection of this reagent paper is limited to 0.27ng/mL, shows that OTA is had by immune chromatography test paper higher sensitive Degree.

Specific detection: with aflatoxin B₁, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, vomitoxin, patulin, ergotoxin make For competitor, configure above-mentioned mark product concentration and be 1mg/mL, detect its suppression ratio with graphene oxide fluorescence immune chromatography test paper, Cross reacting rate is calculated according to formula.

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

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

The test strips of the present embodiment has the advantage that

(1) high specificity, highly sensitive.The test strips of the present embodiment is with graphene oxide fluorescent nano material labelling Make based on OTA antibody, owing to the graphene oxide fluorescent material of modified mistake has outstanding optical characteristics and good life The thing compatibility, simultaneous oxidation Graphene electron transmission efficiency is high so that this Novel immune method is highly sensitive, detectable limit Low, minimum only 0.27ng/mL.

(2) stability is high, and safety is good.The OTA of the test strips graphene oxide fluorescent nano material labelling of the present embodiment Make based on antibody, nano-Ag particles surface with protein with contrary charge groups, the strong bonded because of Electrostatic Absorption； Graphene specific surface area is big, can be combined by chemical reaction with multiple proteins biomolecule, and affects its biological activity very Little, stability is high, and motility is good.

(3) Semen Maydis, DDGS, feedstuff etc. can be detected by the graphene oxide fluorescence immune chromatography test paper of the present embodiment. Jointly being acted on by silver nanoparticle and graphene oxide, detection signal strengthens, and reduces antibody consumption, saves antibody cost.

Embodiment 5

The fluorescence immune chromatography test paper of the detection ochratoxin A of the present embodiment, with reference to Fig. 3-7, including supporter with solid The adsorption layer being scheduled on supporter, described supporter includes transparent hollow tube 1, and adsorption layer is filled in hollow tube Portion, adsorption layer starts to be followed successively by adsorbing fiber layer 2, fluorescent antibody fibrous layer 3, cellulose membrane layer 4 and absorbent material from test lead Layer 7, described cellulose membrane layer is provided with the stealthy detection trace 5 printed with the carrier protein solution of coupling OTA and uses goat-anti The stealthy comparison trace 6 that mouse IgG, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution are printed；Described fluorescent antibody fiber Layer uses the glass fibre cotton of absorption fluorescent antibody to make, and fluorescent 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 union joint 8, and union joint 8 upper end is provided with auxiliary adsorbent equipment, described Auxiliary adsorbent equipment includes with described union joint in the adapter sleeve 13 being tightly connected with the air bag that is arranged on described adapter sleeve top 12, the gas outlet of air bag 12 is successively through the inlet channel 132 on adapter sleeve 13 top, the inner chamber 9 of union joint 8 and hollow tube 1 Upper oral part is connected, the adapter sleeve sidewall at inlet channel place is provided with can only outwards aerofluxus cannot inwardly air-breathing unidirectional go out Device of air 11.

Described unidirectional air-out apparatus includes the ventilation lumen 111 being arranged on the adapter sleeve sidewall at inlet channel place, ventilation It is provided with the ball sealer 113 of spheroidal in chamber 111, the bottom surface of ventilation lumen 111 has first corresponding with ball sealer and gives vent to anger Mouth 114, the first gas outlet is connected with inlet channel 132 through outlet passageway 115, and ventilation lumen 111 side is provided with big with the external world Gas phase connection the second gas outlet 112, constitute can only outwards aerofluxus, cannot be to the unidirectional air outlet structure of inlet channel air-breathing.

For superior technique effect, the first described gas outlet 114 is circular, and its diameter is less than the diameter of ball sealer. Such setting can make the first gas outlet be combined more closely with the ball sealer of spheroidal, it is ensured that the sealing in space.

Described union joint 8 is the hollow structure of upper and lower opening, the upper end of its lower end and hollow tube 1 in being tightly connected, Being provided with external screw thread on the outer wall on union joint top, the bottom of adapter sleeve 13 is provided with obtain blind hole 131 corresponding with union joint, blind Being provided with the female thread corresponding with external screw thread on the inwall of hole 131, union joint is contained in adapter sleeve by external screw thread and internal thread rotation In the blind hole 131 of bottom, on adapter sleeve, between top and the blind hole top of union joint, it is provided with the sealing ring 10 preventing gas leakage.

Described stealthy detection trace 5 and stealthy comparison trace 6 are in the alternate surface being arranged on cellulose membrane layer 4, spacing It is 5～8mm.

The inner chamber of described hollow tube 1 is rectangle.

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

During use, adapter sleeve and union joint 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 union joint and adapter sleeve sidewall, now the spheroidal in ventilation lumen The gas that ball sealer is squeezed out from air bag lifts, and the gas in air bag is discharged smoothly by the first gas outlet；Will examination Paper inserts in testing sample solution, then unclamps air bag, now, the inner chamber of hollow tube, the inner chamber of union joint and inlet channel Interior generation negative pressure, under the attraction of negative pressure, ball sealer tightly adsorbs on the first gas outlet on the bottom surface of ventilation lumen, by its envelope Close, thus help sample solution more quickly to infiltrate reagent paper, thus reduce the time of sample solution infiltration test strips, improve inspection Survey efficiency.

By above-mentioned situation it should be apparent that the present embodiment novel structure is unique, advantages of simple, by whole by supporter Body is revised as closed structure, increases air bag at the top of supporter simultaneously, makes absorption more rapid, thus is effectively improved detection Speed and accuracy, cell parts and supporter part connect for sealing reassembling type simultaneously, only need to change support after detection Body portion, easy accessibility, easily operate, using effect is good, is the innovation on Test paper.

(1) preparation of testing sample and detecting step:

Detection DDGS sample: by sample comminution, make the sample suspension of 1:10 (m/v) with dehydrated alcohol dilution.

Operational approach: extruding gasbag, discharges the gas in air bag, is inserted by OTA reagent paper test lead in testing sample, inserts Enter the degree of depth and be less than mark line, unclamp air bag, within about 3-5 second, take out reagent paper, put into fluorescence after 4min and read the direct readings of bar instrument.

Result judges:

A () is positive judges: reads to occur without absworption peak at bar instrument T line by fluorescence, absworption peak occurs, represent detection at C line Result is positive, illustrates in testing sample containing ochratoxin A；

B () is negative judges: read all to occur at bar instrument T line and C line absworption peak by fluorescence, represents that testing result is negative, Illustrate in testing sample without ochratoxin A；

(c) fail-ure criterion: read all to occur without absworption peak at bar instrument T line and C line by fluorescence, then show that reagent paper lost efficacy.

The detection of susceptiveness: with dehydrated alcohol be respectively configured concentration be 8,4,2,1,0.5,0.25,0.125,0ng/mL OTA standard substance, after the present embodiment reagent paper loading 100 μ L, reaction 4min, read bar instrument by fluorescence and directly read peak figure.With peak Value or peak area are vertical coordinate, with the logarithm value of different OTA concentration as abscissa, draw standard suppression curve, are correlated with back Return analysis, calculate this reagent paper IC to OTA₅₀And lowest detectable limit.After measured, the Regression Equations of OTA is by this reagent paper: y =-0.4319x+0.9367, correlation coefficient is R²=0.9689, go out this reagent paper IC to OTA according to regression equation calculation₅₀For 10.26ng/mL, the lowest detection of this reagent paper is limited to 0.21ng/mL, shows that OTA is had by immune chromatography test paper higher sensitive Degree.

Specific detection: with aflatoxin B₁, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, vomitoxin, patulin, ergotoxin make For competitor, configure above-mentioned mark product concentration and be 1mg/mL, use NaYF₄: Yb, Tm nano fluorescent immune chromatography test paper detects it to be pressed down Rate processed, calculates cross reacting rate according to formula.

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

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

Compound	Half-inhibition concentration (ng/mL)	Cross reacting rate (%)
			OTA	10.26	100
6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone	＞ 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 has the advantage that

(1) high specificity, highly sensitive.The present embodiment up-conversion fluorescence immune chromatography test paper is to turn blue spectrochrome upper turn Change fluorescent nano material NaYF₄: make, due to NaYF based on the OTA antibody of Yb, Tm labelling₄: Yb, Tm nano-particle luminescence is imitated Rate is high, can effectively eliminate sample detection bias light, and sensitivity is greatly improved, and lowest detection is limited to 0.21ng/mL.

(2) stability is high, and safety is good.Upconversion fluorescence nano material NaYF in the reagent paper of the present embodiment₄: Yb, Tm energy Effectively launch blue color spectrum, it is entirely avoided the luminous cancellation that other conditions cause.NaYF₄: Yb, Tm material has inorganic lazy Property, infrared ray excited, the feature of VISIBLE LIGHT EMISSION, use this reagent paper to carry out detecting for surrounding people with environment without any danger Evil.

(3) easy, quickly.Utilizing fluorescence to read bar instrument, this reagent paper can direct readings, it is achieved scene Quantitative detection.Only Reagent paper is inserted test sample 3～5 seconds, i.e. can read result after 4 minutes, at T line, represent test sample without absworption peak In containing OTA；Otherwise, without OTA.Visual result, accurately, simple and clear, avoid artificial erroneous judgement to greatest extent.

(4), when prepared by the reagent paper of the present embodiment, fluorescent antibody uses NaYF₄: the OTA Dan Ke of Yb, Tm nanoparticle label Grand antibody or polyclonal antibody, will amidized NaYF₄: Yb, Tm nano-particle is directly connected with antibody, labeling process letter Single, Conjugate ratio is high, thus is effectively improved based on NaYF₄: the sensitivity of the immune test paper of Yb, Tm material.

Embodiment 6

The structure of the fluorescence immune chromatography test paper of the detection ochratoxin A of the present embodiment is with embodiment 5, difference , the fluorescent antibody of the present embodiment is NaGd (WO₄)₂: Eu³⁺The OTA monoclonal antibody of nanoparticle label or Anti-TNF-α Body.

(1) preparation of testing sample and detecting step:

Result judges:

The detection of susceptiveness: with dehydrated alcohol be respectively configured concentration be 8,4,2,1,0.5,0.25,0.125,0ng/mL OTA standard substance, after the present embodiment reagent paper loading 100 μ L, reaction 4min, read bar instrument by fluorescence and directly read peak figure.With peak Value or peak area are vertical coordinate, with the logarithm value of different OTA concentration as abscissa, draw standard suppression curve, are correlated with back Return analysis, calculate this reagent paper IC to OTA₅₀And lowest detectable limit.After measured, the Regression Equations of OTA is by this reagent paper: y =-0.4154x+1.1123, correlation coefficient is R²=0.9779, go out this reagent paper IC to OTA according to regression equation calculation₅₀For 9.83ng/mL, the lowest detection of this reagent paper is limited to 1.07ng/mL, shows that OTA is had by immune chromatography test paper higher sensitive Degree.

Specific detection: with aflatoxin B₁, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, vomitoxin, patulin, ergotoxin make For competitor, configure above-mentioned mark product concentration and be 1mg/mL, with NaGd (WO₄)₂: Eu³⁺Nano fluorescent immune chromatography test paper detects Its suppression ratio, calculates cross reacting rate according to formula.

Measurement result see table 3, this 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.

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

Compound	Half-inhibition concentration (ng/mL)	Cross reacting rate (%)
			OTA	9.83	100
6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone	＞ 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 has the advantage that

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

(2) stability is high.NaGd(WO₄)₂: Eu³⁺Nano-particle belongs to completely inert phosphor, these character Make it have extraordinary light stability, photobleaching does not occur under the irradiation of uviol lamp so that readings is stable.

(3) easy, quickly.Use the present embodiment reagent paper can read bar instrument with fluorescence to be used in combination, direct readings, it is achieved on-the-spot Detection by quantitative, only need to insert test strips in test sample 3-5 second, i.e. can determine that testing result in taking out latter 4 minutes.

(4) result display is vivid, directly perceived, accurately.The present embodiment fluorescent nano particle labeled immunochromatographyassay assay test is with T line Whether the standard that absworption peak is positive and negative as detection occurs.If without absworption peak at T line, representing in test sample and contain OTA, on the contrary then represent in test sample without OTA.Visual result, accurately, is difficult to occur that false positive and false negative etc. are the most by mistake Sentence.

Claims

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

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

The fluorescence immune chromatography test paper of detection ochratoxin A the most according to claim 1, it is characterised in that described Supporter includes transparent hollow tube (1), and adsorption layer is filled in inside hollow tube, and adsorption layer starts to be followed successively by from test lead Adsorbing fiber layer, fluorescent antibody fibrous layer, cellulose membrane layer and absorbent material layer.

The fluorescence immune chromatography test paper of detection ochratoxin A the most according to claim 3, it is characterised in that described The upper end of hollow tube (1) is equipped with union joint (8), and union joint (8) upper end is provided with auxiliary adsorbent equipment, described auxiliary absorption Device includes with described union joint in the adapter sleeve (13) being tightly connected with the air bag (12) being arranged on described adapter sleeve top, gas The gas outlet of capsule (12) is successively through the inlet channel (132) on adapter sleeve (13) top, the inner chamber (9) of union joint (8) and hollow pipe The upper oral part of body (1) is connected, and the adapter sleeve sidewall at inlet channel place is provided with can only outwards aerofluxus cannot inside air-breathing Unidirectional air-out apparatus (11)；

Described unidirectional air-out apparatus includes the ventilation lumen (111) being arranged on the adapter sleeve sidewall at inlet channel place, ventilation lumen (111) it is provided with the ball sealer (113) of spheroidal in, the bottom surface of ventilation lumen (111) has first corresponding with ball sealer Gas outlet (114), the first gas outlet is connected with inlet channel (132) through outlet passageway (115), and ventilation lumen (111) side sets Be equipped with the second gas outlet (112) being connected with ambient atmosphere, constitute can only outwards aerofluxus, cannot be to the list of inlet channel air-breathing To air outlet structure.

The fluorescence immune chromatography test paper of detection ochratoxin A the most according to claim 4, it is characterised in that described First gas outlet (114) is circular, and its diameter is less than the diameter of ball sealer.

The fluorescence immune chromatography test paper of detection ochratoxin A the most according to claim 4, it is characterised in that described Union joint (8) is the hollow structure of upper and lower opening, the upper end of its lower end and hollow tube (1) in being tightly connected, union joint 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 union joint, blind hole (131) being provided with the female thread corresponding with external screw thread on inwall, union joint is contained in adapter sleeve by external screw thread and internal thread rotation In the blind hole (131) of bottom, on adapter sleeve, between top and the blind hole top of union joint, it is provided with the sealing ring preventing gas leakage (10)。

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

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

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

(1) graphene oxide fluorescent material is modified:

Take 20mg graphite oxide to grind, join in 5mL DMF, after ultrasonic mixing, add 20mL thionyl chloride, heat at 80 DEG C It is centrifuged after backflow 48h, obtains intermediate acid chloride graphene oxide, after washing twice with oxolane, be dried；Again at evacuation Under conditions of nitrogen protection, chloride graphene oxide and 1mL n-butylamine are mixed, 60 DEG C of reaction 72h, be cooled to room temperature, The graphene oxide modified to alkylamine, is scattered in 20mL distilled water, and 8000r/min is centrifuged, and removes unreacted n-butylamine, Being evaporated by Rotary Evaporators by remaining reactant liquor, the dry after being evaporated is dispersed in distilled water again, is configured to concentration Graphene oxide fluorescent material aqueous solution for the modification of 1mg/mL；

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

A 100mg silver nitrate is dissolved in 250mL ultra-pure water by (), ebuillition of heated, adds the citric acid of 10mL mass fraction 1% Sodium solution, 80 DEG C are heated to reflux stirring 0.5h after 1h, under the conditions of 4 DEG C overnight；Take 1mL overnight after solution, add 1mM sulfydryl Acetic acid 10 μ L, centrifugal after stirring 24h, remove unreacted TGA, milli-Q water 3 times, be evaporated with Rotary Evaporators, add Ultra-pure water is configured to the silver nanoparticle solution that concentration is 1mg/mL TGA cladding；

B () takes the NHS 10 μ L of EDC 10 μ L and 0.1mM of 0.1mM, be added step-wise to the silver nanoparticle of 1mL TGA cladding In grain solution, react 1h, obtain the silver nanoparticle solution of activated carboxylic；

C () takes OTA monoclonal antibody or the polyclonal antibody 10 μ L of 0.1mM, join the silver nanoparticle solution of activated carboxylic In, 4 DEG C of reactions overnight, obtain surface markers OTA antibody silver nanoparticle solution；

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

Taking the graphene oxide fluorescent material aqueous solution 5mL of modification, add 2mL glutaraldehyde, room temperature reaction 3h, centrifugal, removing is not The glutaraldehyde of reaction, is dispersed in remaining reactant liquor in the PBS buffer solution of 0.01mol/L, pH7.4, adds surface markers OTA antibody silver nanoparticle solution, 4 DEG C of reaction 3h, by centrifugation, the OTA collecting graphene oxide fluorescent nano material labelling is mono- Clonal antibody or polyclonal antibody, the PBS washing of 0.01mol/L, pH7.4, save backup.

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

Take concentration respectively and be the Y (NO of 0.5mol/L₃)₃Solution 5.5mL, Yb (NO₃)₃Solution 0.5mL and Tm (NO₃)₃Solution 0.5mL, mix homogeneously, add the sodium citrate solution 2mL of 0.4mol/L, under the conditions of 25 DEG C, lucifuge fully reacts 1h；Add 1.5mol/L NH₄F solution 7mL, under the conditions of 25 DEG C, lucifuge stirring 0.5h, uses HNO₃Regulation pH value, to 7.4, stands 0.5h, adds double Steam water and be diluted to 30mL；At 220 DEG C, react 24h again, be cooled to room temperature, through filtration, distilled water washing, be dried, 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 adds in the PBS of 0.01mol/L, pH7.4, and being configured to concentration is 1mg/ The NaYF of mL₄: Yb, Tm fluorescent nano particle solution, then 5mL strong aqua ammonia is instilled 5mL NaYF₄: Yb, Tm fluorescent nano particle is molten In liquid, it is sufficiently stirred for reacting 20min, adds 2.5mL tetraethyl orthosilicate, under the conditions of 4 DEG C of lucifuges, react 4h；6000r/min Centrifugal 10min, obtains the fluorescent nano particle of surface siliconization, by washing with alcohol 4 times, is dispersed in methanol, and being configured to concentration is The fluorescent nano particle methanol solution of 10mg/mL；

(3) surface amination of fluorescent nano particle:

Take 3mL fluorescent nano particle methanol solution, add the acetone of 5mL, magnetic agitation 20min after sealing, more equal with ultrasound wave Even dispersion, adds the APTSA of 3 μ L, reacts 30min at 40 DEG C after sealing, more centrifugal at 70 DEG C of water-bath 1h, 6000r/min 5min, obtains the fluorescent nano particle of surface amination, and with ethanol cyclic washing 4 times, after vacuum drying, 4 DEG C seal and preserve；

(4) labelling of OTA antibody:

Being dissolved by the fluorescent nano particle of surface amination with the PBS buffer solution of 0.01mol/L, pH7.4, being configured to concentration is The fluorescent nano particle solution of the surface amination of 10mg/mL；Take the fluorescent nano particle solution of 10mL surface amination with 5.6mg NHS and 15mg EDC mixing, room temperature lucifuge reaction 3h, 6000r/min are centrifuged 5min, take supernatant；By 1mg/mL's OTA monoclonal antibody or polyclonal antibody add in supernatant, OTA monoclonal antibody or polyclonal antibody and supernatant Volume ratio is 1:100,4 DEG C of lucifuge reaction 4h, centrifugal, is dispersed in PBS buffer solution, under the conditions of 4 DEG C thoroughly after distilled water washing Analysis 3d, centrifugal, collect precipitation, obtain NaYF₄: the OTA monoclonal antibody of Yb, Tm nanoparticle label or polyclonal antibody, put In PBS buffer solution, 4 DEG C of preservations.

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

Weigh 7g Gd₂O₃With 7g Eu₂O₃, it is added separately to the HNO of 50mL₃In, it being heated to 80 DEG C, insulation is concentrated into all knots Crystalline substance, prepares Gd (NO₃)₃With Eu (NO₃)₃；By Na₂WO₄·2H₂O is dissolved in deionized water, and being configured to concentration is 0.2mol/L's Na₂WO₄Solution；By prepared Gd (NO₃)₃With Eu (NO₃)₃With deionized water dissolving, prepare the Gd that mass fraction is 80% respectively (NO₃)₃Solution and the Eu (NO that mass fraction is 15%₃)₃Solution, draws the 5mL Gd (NO of preparation₃)₃Solution, 10mLNa₂WO₄Molten Liquid and 5mL Eu (NO₃)₃Solution, joins in reactor, with dust technology, sodium hydroxide regulation pH=5, stirs, closes anti- Answer still, after 200 DEG C of heated at constant temperature 24h, be naturally down to room temperature；Solution in reactor is poured out standing, treats powder body precipitation in solution After, remove supernatant, with distilled water wash powder body 3 times, stand 3h every time；Washed powder body is heated to 80 DEG C of dry 12h, To NaGd (WO₄)₂: Eu³⁺Nano-particle；

(2) labelling of OTA antibody

OTA monoclonal antibody or Anti-TNF-α liquid solution 10000r/min are centrifuged 30min, remove impurity；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 antibody or Anti-TNF-α Liquid solution 100 μ l, OTA monoclonal antibody or polyclonal antibody solution concentration 1mg/mL, mixing, incubation at room temperature 30min, 4 DEG C, 10000r/min is centrifuged 30min, abandons supernatant, and precipitation is used 0.02mol/L Na₂B₄O₇Solution is diluted to 10mL, 10000r/min Centrifugal 30min, precipitation uses 0.02mol/L Na₂B₄O₇Solution is diluted to 1mL, and 4 DEG C save backup.