CN105699650B - Carbofuran bio-barcode immune analytic reagent kit and its application - Google Patents
Carbofuran bio-barcode immune analytic reagent kit and its application Download PDFInfo
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- CN105699650B CN105699650B CN201610086751.9A CN201610086751A CN105699650B CN 105699650 B CN105699650 B CN 105699650B CN 201610086751 A CN201610086751 A CN 201610086751A CN 105699650 B CN105699650 B CN 105699650B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
The present invention relates to food safety detection technical field of immunoassay, and in particular to a kind of carbofuran bio-barcode immune analytic reagent kit, the kit includes box body, includes in the box body：Carbofuran standard items, carbofuran magnetic Nano probe, single mark colloid gold nano-probe, the double mark colloid gold nano-probes of carbofuran, DNA biochips, silver staining reagent.Kit provided by the present application can easy, quick, sensitive, special, steadily detect the carbofuran content in sample to be tested, test favorable reproducibility, easy to operation and production.
Description
Technical field
The present invention relates to food safety detection technical field of immunoassay, in particular to a kind of carbofuran biology item
Shape code immune analytic reagent kit and its application.
Background technology
Carbofuran is irreversible anticholinesterase, high to toxicity such as people, animal fowl, fish, birds, is sized generally to particle
Agent or seed coat agent carry out soil or seed treatment.The longevity of residure is long in the soil for medicament, and plant can be inhaled by root, stem, leaf or seed
Conduction is received, more in leaf margin accumulation, eating dye medicine plant by mistake may cause to be poisoned.Carbofuran in soil be also possible to by river rising in Ningxia and flowing into central Shaanxi stream and
Osmosis pollution ground and underground water source.Therefore, efficiently quick carbofuran detection technique is established, in environment and agricultural and sideline product
The accurate quick diagnosis and effectively treatment of the monitoring of Determination of carbofuran and carbofuran poisoning, ensure that human health has important meaning
Justice.
The effect of immunoassay method and status have obtained being widely recognized as domestic and foreign scholars at present, immunoassay method
Research is also most popular one of the field of detection method research.In immunoassay method field, China is in ELISA
Carry out in terms of method each in more in-depth study, including the coupling of hapten synthesis, Antibody preparation, marker, reaction system
Kind of physicochemical property influence and haptens structure to immunoassay method and the structure-activity relationship etc. between antibody is obtained prepared by it
Aspect.In recent years, Chinese scholar is exempted from fluoroimmunoassay, chemiluminescence immune assay, biosensor and bio-barcode
More research work is also carried out in terms of epidemic disease analysis method.
Bio-barcode immunoassay method based on magnetic nano-particle, using bio-barcode signal amplification and
The separation and concentration of magnetic nano-particle acts on, and minimum detectability improves several orders of magnitude relative to ELISA method, causes it
Since birth, the highest attention of scientific circles has just been obtained.Bio-barcode refers to may be incorporated in a large amount of on nano-particle
Mutually homotactic DNA fragmentation.This method is for prostate-specific antigen (Prosta tespec if ic earliest
Antigen, PSA) detection.This method includes mainly：Magnetic nano-particle surface modification PSA monoclonal antibodies, colloid Jenner
Rice detecting probe surface has modified the bio-barcode of signal amplification and another antibody for PSA.In the presence of PSA, magnetic
Property nano-particle, PSA, colloid gold nano-probe pass through antigen-antibody reaction formed " sandwich " sandwich composite construction.It is formed
Compound is detached through magnetic field, and the magnetic Nano probe of enrichment is denaturalized by high-temperature low salt method, is discharged bio-barcode, is passed through detection
The bio-barcode content disintegrated down can obtain the content of PSA indirectly.
Bio-barcode analysis method has obtained quick development in trace amount of protein and nucleic acid detection method nearly ten years,
By being combined the bio-barcode immunoassay method of foundation with immune response, the detection limit of immunoassay method is increased to
One new height.However this method is established so far, can only be realized to protein etc. by structure " sandwich " tactic pattern
The detection of macromolecular substances.Since carbofuran is micromolecular compound, only there are one antigen binding sites, therefore biological item at present
" sandwich " tactic pattern that shape code immunoassay method uses is not applied for the detection of carbofuran substantially.
In view of this, special propose the present invention.
Invention content
The purpose of the present invention is to provide a kind of carbofuran bio-barcode immune analytic reagent kits, use the reagent
Box specifically can quantitatively detect in water or carbofuran content in food.The kit is detected by competitive reaction system,
It overcomes " sandwich " tactic pattern that current bio-barcode immunoassay method uses and is not applied for carbofuran substantially
The defect of detection.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
A kind of carbofuran bio-barcode immune analytic reagent kit, the kit include box body, in the box body
Include：
Carbofuran standard items, carbofuran magnetic Nano probe, single mark colloid gold nano-probe, the double mark colloid Jenners of carbofuran
Rice probe, DNA biochips, silver staining reagent.
Carbofuran (Carbofuran) molecular formula：C12H15NO3；No. CAS：1563-66-2.
The present invention is established and is competed based on small molecule by the existing reaction pattern of change bio-barcode immunoassay method
The bio-barcode immunoassay method of reaction pattern.By the way that the conjugate of carbofuran and carrier protein is coated in magnetic Nano
Particle surface prepares carbofuran magnetic Nano probe, and combines pesticide antibody signal with to amplify in colloid gold nano-probe surface
The bio-barcode of effect is anti-using the coating on magnetic nano-particle surface to prepare the double mark colloid gold nano-probes of carbofuran
The former antibody with pesticide molecule competitive binding colloid gold nano-probe surface establishes competitive type immuno-chemical reaction system.Utilize magnetic
The antigen antibody complex that piece-rate system separating immune combines promotes colloid gold nano-probe release biology using thermal denaturation technology
Bar code, and bio-barcode is combined using DNA chip probe and single mark colloid gold nano-probe hybridization, and then utilize golden standard silver
Dye method measures bio-barcode content, realizes the quantitative detection to carbofuran pesticide.
The using effect of the kit has many advantages, such as easy, quick, sensitive, special, stable.Also, according to the present invention
Detecting system be open-sky technique, it is easy to be quickly particularly suitable for vast quality inspection organization and promote the use of, be food safety detection
A kind of very valuable detection means is provided.
Preferably, the carbofuran magnetic Nano probe is coupled by magnetic nano-particle and carbofuran comlete antigen；
The carbofuran comlete antigen is formed by carbofuran and carrier protein couplet.
It is further preferred that the carrier protein is chicken ovalbumin；The grain size of the magnetic nano-particle be 18~
22nm。
Chicken ovalbumin (Ovalbumin, OVA) is also referred to as chicken ovalbumin, is made of 386 amino acid, molecular weight is about
43Kd.For OVA as inert protein, it can maintain the colloid-stabilised of colloidal gold, play the skeleton function of dispersion colloid gold particle.
Casein (Casein) or bovine serum albumin(BSA) (BSA) etc. also can be selected in inert protein.
The grain size of magnetic nano-particle has prodigious influence to the coupling quantity of carbofuran comlete antigen, to affect
The sensitivity of end reaction.
Preferably, kit as described above：
Single mark colloid gold nano-probe is obtained by single stranded DNA l coating colloid gold particles；
The double mark colloid gold nano-probes of the carbofuran are obtained by anti-carbofuran antibody and double-stranded DNA coating colloid gold particle
It arrives；
The DNA biochips are made for 2 points by single stranded DNA；
The single stranded DNA and bar code single stranded DNA complementary pairing that the double-stranded DNA is modified by mercaptan form；It is described single-stranded
There are complementary pairing relationships with the bar code single stranded DNA for DNA1 and single stranded DNA 2, and pairing region is not overlapped.
The DNA2 that system is put on DNA biochips is capture dna, can with bar code DNA complementary pairings and captured in core
On piece；The effect of DNA1 on single mark colloid gold nano-probe is that bar code DNA signals are further amplified.
Double mark colloid gold nano-probes are the anti-checking matter antibody of double-stranded DNA coating colloid gold particle and anti-checking matter, double
1 in chain DNA is connected with colloid gold particle by Au--S keys, and another 1 DNA chain is used to refer to show the bar code of checking matter
DNA.Label has the bar code DNA of item on each colloid gold particle, thus plays the role of method signal, sensitivity compared with
It is high.
In practical operation, bar code DNA's is selected as the prior art, as long as specificity and sensitivity are enough good.
Preferably, the grain size of the colloid gold particle is 13~15nm.
The technical essential of the application is by forming the double mark colloid gold nano-probes of carbofuran magnetic Nano probe-carbofuran
Combination, and the combinations of the double mark colloid gold nano-probes of carbofuran-carbofuran are at war with, and the former combination should can be with
Uniform separation.The key that sensitivity improves is effective recycling of checking matter and identifies binding site pair as each checking matter
The bar code DNA chain answered is released effectively.The amount of bar code DNA corresponds to the amount of checking matter, and (carbofuran magnetic Nano probe is to quilt
Examine the inhibiting rate of the competitive binding of carbofuran), therefore the checking matter of quantitative bar code DNA indirect quantification traces can be passed through.
The combination of the double mark colloid gold nano-probes of carbofuran magnetic Nano probe-carbofuran is substantially the carbofuran in magnetic Nano probe
The combination of comlete antigen and the anti-carbofuran antibody in double mark colloid gold nano-probe, and the bond strength of the two and antibody/anti-
Former amount is closely related, and the amount of antibody/antigen is then closely related with the grain size of colloid gold particle/magnetic nano-particle.
Preferably, kit as described above, the double mark colloid gold nano-probes of the carbofuran are specifically by following methods system
It is standby to obtain：
1) anti-carbofuran antibody is added thereto after, taking colloidal gold solution tune pH to 8.8~9.2, is stirred, stands 25
~35min；Then the single stranded DNA chain of mercaptan modification, 8~12 DEG C of 36~44h of standing are added；PH to 7.3~7.5 is adjusted again, is added
Enter NaCl to 0.08~0.12mol/L of concentration, 8000~11000r/min centrifuges 8~12min, abandons supernatant, obtains containing few nucleosides
The colloidal gold of acid；
2) glue containing oligonucleotides, is resuspended with the phosphate buffer of bovine serum albumin(BSA) a concentration of 0.8~1.2%
Body gold, is incubated 1~3h；1~3h；Add the bio-barcode DNA chain for the single stranded DNA chain complementation modified with the mercaptan, room
Temperature hybridization 3~5h, 8000~11000r/min centrifugation, abandons supernatant, obtains the double mark colloid gold nano-probes of the carbofuran.
It is further preferred that when NaC1 is added to set concentration, point 2~4 equivalent are added in 120~180min,
Per 40~60min of minor tick.
The ageing process of process, that is, DNA of NaC1 is added.Nanogold and DNA are negatively charged, so can be mutually exclusive, it is added
NaCl can mask some charges of nanometer gold surface, reduce the repulsion between nano-particle.But the NaCl of excessive concentrations can be led
It causes ionic intension excessive, easily DNA is caused to reunite, so being gradually added, prevent the excessive of moment ionic strength.
Preferably, kit as described above, the anti-carbofuran antibody are monoclonal antibody.
Monoclonal antibody by cell strain due to can be produced, and property is stablized between each batch, and high specificity, because
And it is more suitable for reagent kit product.
Carbofuran bio-barcode immune analytic reagent kit as described above is in carbofuran qualitative and quantitative analysis
Using.
The method that carbofuran bio-barcode immune analytic reagent kit as described above measures carbofuran, including：
A) carbofuran standard items, extraction, are marked into colloids with isometric carbofuran pair respectively with purified sample to be tested
Au probe and the mixing of carbofuran magnetic Nano probe are incubated；
B), washing is incubated product and hydridization is gone to obtain and each standard items bio-barcode corresponding with sample to be tested；
C), by the bio-barcode and DNA biochip hybridizations, and single mark Nano-Au probe amplified signal is added；It is miscellaneous
It hands over and silver staining reagent is added after reaction and measures each hole gray value with chip scanning analyzer；
D), with a concentration of abscissa of carbofuran standard items, the gray scale inhibiting rate corresponding to each concentration is ordinate, is established
Standard curve, and the carbofuran concentration in each sample is calculated according to standard curve.
Compared with prior art, beneficial effects of the present invention are：
(1) present invention specifically can quantitatively detect carbofuran content.With easy, quick, sensitive, special, stable etc.
Advantage.Also, detecting system according to the present invention is open-sky technique, easy to be quick, is particularly suitable for vast quality inspection organization and pushes away
It is wide to use, provide a kind of very valuable detection means for food safety detection.
(2) kit according to the present invention, carbofuran haptens in magnetic Nano probe and grams hundred in detection sample
The carbofuran monoclonal antibody that prestige competes on the double mark colloidal gold probes of carbofuran forms direct competitive system, therefore the competition that this law uses is anti-
System is answered, that is, ensures the sensitivity of detection, also can effectively ensure that the good reproduction of testing result.In addition, this pattern is also just
In operation and production.
(3) kit of the invention uses silver staining method, and by detecting the gray value on biochip, sensitivity is big
It is big to improve, more sensitive, quick, reliable foundation can be provided for the detection of carbofuran in vegetable and fruit.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 be hybridization reaction after DNA biochips scanning figure；
Fig. 2 is in embodiment 2 with a concentration of abscissa of carbofuran standard items, and the gray scale inhibiting rate corresponding to each concentration is
Ordinate, the standard curve of foundation.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The preparation method of carbofuran bio-barcode immune analytic reagent kit
The preparation of s11, single mark colloidal gold probe
1), gold nano grain synthetic method
98mL deionized waters are separately added into the beaker good to silication, then enter 2mL50mM gold chloride stostes respectively, keep chlorine golden
A concentration of 1mM of acid, 1000rpm/min stirrings, 250 DEG C of heating, continue to boil 2min after liquid boiling on magnetic stirring apparatus；
10mL 38.8mM trisodium citrates are drawn, is added at one time in beaker rapidly, is kept stirring speed and heating temperature is constant, after
It is continuous to boil 6min, it is possible to find solution colour gradually becomes purple by colourless, then becomes claret, also according to addition trisodium citrate
The difference of former dosage ultimately becomes different colors, original volume is refilled with deionized water after liquid cooling, in 4 DEG C of closed guarantors
It deposits, the size controlling of the gold nano grain of synthesis is in 13~15nm.
2) sulfhydrylation DNA1 solution, is taken, three (2- carboxyethyls) phosphine solution (TCEP) of 100mmol/L are added, at room temperature
It is placed 2 hours on shaking table.For ratio between reducing agent TCEP and DNA1 close to 200: 1, a high proportion of gap accelerates reaction speed,
Improve reduction effect.At room temperature, newly synthesized nano gold spherical 20 centrifugation minute under 6000rcf rotating speeds, nano gold spherical is taken out
It is dissolved in ultra-pure water.It takes the nano gold spherical 10000rcf of certain volume to centrifuge 20 minutes, removes supernatant, 0.5 × Tris- boron is added
Sour (TBE) buffer solution, is then added the amount of taken sulfhydrylation DNA, and sodium chloride solution to sodium chloride concentration is added and is
50mmol/L under room temperature, reacts 24 hours.It gradually adds sodium chloride and (is added three times, per septum secundum 1 to a concentration of 500mmol/L
It is added within a hour primary), 8 hours are stood under room temperature.10000rcf is centrifuged 20 minutes, is removed supernatant, is dissolved in ultra-pure water, is repeated
3 times, obtain the nano gold spherical GNP solution of sulfhydrylation DNA1 modifications.
The preparation of the double mark colloidal gold probes of S12, carbofuran
Certain volume colloidal gold solution is taken, pH=9.0 is adjusted to 0.1mol/L K2C03；15min is stood, is added certain
The carbofuran monoclonal antibody of amount under stiring mixes colloidal gold and antibody, stands 30min.Then mercaptan modification is added
DNA chain, 10 DEG C of standing 40h, then adjusted to pH=7.4 with phosphate buffer (PBS), NaCl concentration is improved, NaCl divides 3 times and adds
Enter, be added once every 1 hour, until final concentration 0.1mol/L, 10000r/min centrifuge 10min to get to containing oligonucleotides
Colloidal gold.The colloidal gold containing oligonucleotides is resuspended with 1mL PBS, stands 10~15 minutes, it is pure then to add 5% ox blood
Albumen (BSA) makes BSA final concentration of 1%, closes 1h.Add the biological item for the single stranded DNA chain complementation modified with mercaptan
Shape code DNA chain, room temperature hybridize 4h, and 10000r/min centrifuges to obtain the double mark colloid gold nano-probes of carbofuran.
The preparation of S13, carbofuran magnetic Nano probe
1), the preparation of chicken ovalbumin (OVA) label carbofuran haptens
The preparation of horseradish peroxidase-labeled carbofuran haptens, the specific method is as follows：
Haptens 0.25mmol is taken, 1mL n,N-Dimethylformamide (DMF) is dissolved in, is added with stirring 60 μ, 1 positive tri-n-butylamines
With 30 μ l ethyl chloroformates, reaction 1h is stirred at room temperature.Extract reaction solution the carbonic acid that 300 μ l are slowly dropped to 6mL dissolved with 120mg OVA
In salt buffer (CBS, 0.0lmol/L), after reaction 2h is stirred at room temperature, it is packed into bag filter, is first dialysed 3 times with distilled water, then
With the PBS dialysis 3d of 0.01mol/L, dialyzate is changed daily 3-4 times.Packing is taken out to be stored in -20 DEG C of refrigerator.
2), magnetic nano-particle synthetic method
The forming process of magnetic microsphere：Take 8.1g FeCl3·6H2O and 3.3g FeCl2·4H2150mL deionizations are added in O
Water, wiring solution-forming then move into three-necked flask.In N2In environmental protection and it is vigorously stirred under state to Fe2+And Fe3+Mixing
The concentrated ammonia liquor that 18mL mass fractions are 25% in solution starts the Fe for generating sepia in solution3O4Particle adjusts the pH of solution
For value between 9~11, reaction temperature is 70 DEG C this moment, to ensure certain mixing speed during reaction, reaction from
Sub- equation is：
Fe2++2Fe3++ 8OH=Fe3O4+4H2O
FeaO4The synthesis of/oleic acid：Fe is added in 5.0g oleic acid (C18H3402) under the state that is vigorously stirred3O4Liquid
In, then in N2Under environmental protection, temperature is that a hour is reacted in 70 DEG C of water-baths, and oleic acid is made to be uniformly coated on particle table
Face.After reaction, black sol shape substance is obtained, is isolated the precipitation of gained from reaction system using externally-applied magnetic field
Come, washing 3 times with ethyl alcohol removes extra oleic acid, then is washed with deionized to pH=7, obtains what oleic acid uniformly coated at this time
Fe3O4Particle.
The Fe of single layer carboxyl-functional3O4Particle synthesizes：Using potassium permanganate oxidation oleic acid method synthesis azelaic acid (HOOC
(CH2) 7C0OH) form carboxylated magnetic nano-particle.The KMnO of a concentration of 10mg/mL of 160mL is added4Solution is being vigorously stirred
State, temperature react 8h under the conditions of being 50 DEG C, utilize externally-applied magnetic field to precipitate gained after reaction and are detached from reaction medium
Out, it and is successively washed with deionized 3 times, products therefrom is dried in vacuo at 40 DEG C, the particle after drying is dissolved in water-soluble
Liquid just obtains stable carboxylated magnetic nano-particle.The size controlling of magnetic nano-particle is in 18~22nm.
3), the connection of magnetic microsphere and OVA- haptens
A), the activation of microballoon.With pH=6.0,2- (N- morpholines) ethanesulfonic acid buffers (MES) of 15mmol/L are as work
Change buffer solution, take 1mL magnetic beads in 2.0mL centrifuge tubes, after activation buffer solution is added, supernatant is sopped up；Again with activation
Buffer solution relaunders magnetic bead 2 times, then is separately added into carbodiimide and n-hydroxysuccinimide, is mixed in vortex oscillator
Uniformly, room temperature activates 30min.
B), microballoon and OVA- hapten conjugations.With pH=7.4, and the phosphate tween of 0.01mol/L (PBST, 1%
Tween-20) solution makees coupling buffer, and activated magnetic bead is relaundered 3 times with activation buffer, then is buffered with coupling
Liquid washs magnetic bead 2 times；Then magnetic bead is resuspended with coupling buffer, adds OVA- haptens so that the carboxylic of magnetic bead surfaces activation
Base reacts 1h with the amino of OVA under 37C, by OVA- hapten conjugations in magnetic bead surfaces, obtains immunomagnetic beads.
C) it, closes.The coupling buffer closing containing 2%BSA is added in magnetic bead 2 times after washing coupling with coupling buffer
Magnetic bead 60min.Finally obtain immune magnetic probe.
The preparation of S14, carbofuran standard items
It is prepared with carbofuran sterling, concentration is respectively 0ng/mL, 0.05ng/mL, 0.01ng/mL, 0.5ng/mL, 1ng/
ML, 5ng/mL, 10ng/mL, totally 7 bottles；
The carbofuran sterling purity is not less than 90%.
S15, reaction system working concentration are selected
Optimized selected magnetic Nano probe dilution multiple is 100 times, and colloid gold nano-probe extension rate is 10 times.
It is prepared by S16, DNA chip
The capturing probe DNA2 that can be combined with its partial complementarity according to bar code detecting probe sequence design；By amination modification
Capturing probe by chip point sample instrument point to surface aldehydes on slide, 100 μm of spot diameter, put 500 μm of spacing, point sample amount
For 0.7nL, DNA2 concentration and probe concentrations are 75 μm of ol/L, 37 DEG C of fixations.Unbonded probe is rinsed with deionized water, is soaked in sulfydryl
30min is to be passivated remaining binding site in succinic acid solution, and after repeatedly being rinsed with deionized water, 4 DEG C spare.
S17, semi-finished product and finished product composition
Aliquots are semi-finished product obtained by above-mentioned steps, extract three parts out by specificity, accuracy, sensitivity and stabilization
Property assay approval can just be assembled into carbofuran bio-barcode immune analytic reagent kit, and being assembled into after kit also needs to inspect by random samples
It could dispatch from the factory after qualification.
To sum up, in the research process of the present invention, the present inventor first screens raw material used
Experiment and Quality Identification, include the activity of antibody and carbofuran haptens, the grain size of colloid gold particle, the grain of magnetic nanoparticle
Diameter and magnetism etc..Then by the reaction pattern of kit, the reaction time, coupling efficiency, DNA biochip point samples size,
The silver staining time waits a series of condition to be optimized, and establishes highly sensitive and good reproduction carbofuran biology bar shaped
Code immune analysis determination method.
Embodiment 2
The kit application method of the present invention
The concrete operations of carbofuran bio-barcode immune analytic reagent kit prepared by the above case study on implementation 1 are as follows：
S21, kit, equilibrium at room temperature 10 minutes are taken out from 4 DEG C of refrigerators；
S22, sample extraction and purification
Weigh 10.0g (being accurate to 0.01g) sample；10mL acetonitriles are added, it is anhydrous that 4.0g is added in high-speed homogenization 0.5min
Be vortexed at a high speed 1min after MgSO4 and 1.0g NaC1, at 4 DEG C with 10000r/min high speed centrifugations 5min.Accurately pipette 2mL acetonitriles
50mg N- propyl ethylenediamines (PSA) and 50mg C18 solid phase dispersion extracting and purifyings is added in 10mL plastic centrifuge tubes in extracting solution
Agent, after high speed vortex 0.5min, at 4 DEG C with 10000r/min high speed centrifugations 5min.1mL supernatants are taken, nitrogen is blown at 30 DEG C
Dry, residue PBS buffer solution of the 1mL containing 10% methanol dissolves.
The double mark colloidal gold probes of 20 μ L carbofurans are added in S23, reacting hole, then add each concentration gram hundred in reacting hole respectively
Prestige standard items (0ng/mL, 0.05ng/mL, 0.01ng/mL, 0.5ng/mL, lng/mL, 5ng/mL, 10ng/mL) and through pre-treatment
Then 20 μ L of carbofuran magnetic Nano probe are added per hole, use micro oscillator if a repetition by each 20 μ L of sample obtained afterwards
Fully oscillation mixing, it is 37 DEG C to be subsequently placed in temperature, and 1h is incubated in the constant temperature and humidity incubator that relative humidity is 70%；
S24, it is washed three times with PBST liquid, hydridization is gone to obtain bio-barcode；
S25, chip hybridization
4 μ L bio-barcodes DNA and 15 μ L hybridization solution mixings, uniformly drop is on chip, and 48 DEG C hybridize 1h, and 0.2 × SSC is washed
Liquid cleans chip 1min, dries for use.The mono- mark Nano-Au probe solution of 4 μ L and 15 μ L hybridization solutions (are contained into 0.1% dodecane
0, the 01M PBS buffer solution of base sodium sulphate) mixing, room temperature closing 30min.By single mark nano-Au solution after closing uniformly drop in
Dry the dot matrix area of chip, 48 DEG C of hybridization 1h.0.2 × SSC washing lotions are cleaned chip 1min, are dried.It is uniformly added into brand-new per dot matrix
20 μ L of silver staining liquid (being protected from light operation), room temperature reaction 10min are placed on deionized water to terminate reaction, under the microscope.
S26, each hole gray value is measured with chip scanning analyzer；
S27, a concentration of abscissa with carbofuran standard items, the gray scale inhibiting rate corresponding to each concentration is ordinate, is built
Day-mark directrix curve, and the carbofuran concentration in each sample is calculated according to standard curve；Canonical plotting is shown in Fig. 2.
Experimental example 1
The methodology verification result of kit of the present invention
The kit prepared in case study on implementation 1 is identified according to vertification regulation common in this field, the results are shown in Table
1。
The methodology verification result of 1 kit of the present invention of table
The above result shows that the accuracy of " carbofuran bio-barcode immune analytic reagent kit ", specificity, precision
Property, sensitivity and stability comply fully with condition.
Experimental example 2
Carbofuran TIANZHU XINGNAO Capsul is tested
In order to examine the practical application performance of carbofuran bio-barcode immune analytic reagent kit, by carbofuran standard
Solution is added to the water sample simulated in tap water and polluted by carbofuran, adds recovery experiment by standard to measure in tap water
The concentration of carbofuran, adds the carbofuran of a concentration of 1ng/mL, 5ng/mL and 10ng/mL, and testing result is shown in Table 2.
The TIANZHU XINGNAO Capsul of carbofuran in 2 sample of table
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (3)
1. a kind of carbofuran bio-barcode immune analytic reagent kit, which is characterized in that the kit includes box body, institute
It states in box body and includes：
Carbofuran standard items, carbofuran magnetic Nano probe, single mark colloid gold nano-probe, the double mark colloid gold nanos of carbofuran are visited
Needle, DNA biochips, silver staining reagent；
Single mark colloid gold nano-probe is coated with colloid gold particle by single stranded DNA 1 and obtains；The grain size of the colloid gold particle is
13~15nm；
The double mark colloid gold nano-probes of the carbofuran are obtained by anti-carbofuran antibody and double-stranded DNA coating colloid gold particle；Institute
It is monoclonal antibody to state anti-carbofuran antibody；The double mark colloid gold nano-probes of the carbofuran are specifically prepared by following methods
It arrives：
1) anti-carbofuran antibody is added thereto after, taking colloidal gold solution tune pH to 8.8~9.2, is stirred, standing 25~
35min；Then the single stranded DNA chain of mercaptan modification, 8~12 DEG C of 36~44h of standing are added；PH to 7.3~7.5 is adjusted again, 120
NaCl to 0.08~0.12mol/L of concentration is added in point 2~4 equivalent in~180min, per 40~60min of minor tick, 8000~
11000r/min centrifuges 8~12min, abandons supernatant, obtains the colloidal gold containing oligonucleotides；
2) colloid containing oligonucleotides, is resuspended with the phosphate buffer of bovine serum albumin(BSA) a concentration of 0.8~1.2%
Gold is incubated 1~3h；Add the bio-barcode DNA chain for the single stranded DNA chain complementation modified with the mercaptan, room temperature hybridization 3~
5h, 8000~11000r/min are centrifuged, and abandon supernatant, obtain the double mark colloid gold nano-probes of the carbofuran；
The DNA biochips are made for 2 points by single stranded DNA；
The single stranded DNA and bar code single stranded DNA complementary pairing that the double-stranded DNA is modified by mercaptan form；1 He of the single stranded DNA
There are complementary pairing relationships with the bar code single stranded DNA for single stranded DNA 2, and pairing region is not overlapped；
The carbofuran magnetic Nano probe is coupled by magnetic nano-particle and carbofuran comlete antigen；The carbofuran is complete
Holoantigen is formed by carbofuran and carrier protein couplet；The carrier protein is chicken ovalbumin；The magnetic nano-particle
Grain size is 18~22nm.
2. carbofuran bio-barcode immune analytic reagent kit described in claim 1 is in carbofuran qualitative and quantitative analysis
Application.
3. the method that carbofuran bio-barcode immune analytic reagent kit described in claim 1 measures carbofuran, special
Sign is, including：
A), carbofuran standard items, extraction are visited with the double mark colloidal golds of isometric carbofuran respectively with purified sample to be tested
Needle and the mixing of carbofuran magnetic Nano probe are incubated；
B), washing is incubated product and hydridization is gone to obtain and each standard items bio-barcode corresponding with sample to be tested；
C), by the bio-barcode and DNA biochip hybridizations, and single mark Nano-Au probe amplified signal is added；Hybridization is anti-
Silver staining reagent is added after answering and measures each hole gray value with chip scanning analyzer；
D), with a concentration of abscissa of carbofuran standard items, the gray scale inhibiting rate corresponding to each concentration is ordinate, establishes standard
Curve, and the carbofuran concentration in each sample is calculated according to standard curve.
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| CN106771221A (en) * | 2016-12-16 | 2017-05-31 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of chlopyrifos analysis determines kit and its application |
| CN106596915A (en) * | 2016-12-16 | 2017-04-26 | 中国农业科学院农业质量标准与检测技术研究所 | Method for qualitatively and quantitatively analyzing carbofuran, and kit used by method |
| CN107402297A (en) * | 2017-06-04 | 2017-11-28 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Enterotoxin B detection kit and detection method based on immune bio-barcode |
| CN107271417A (en) * | 2017-07-11 | 2017-10-20 | 黄柯影 | A kind of biochip and its detector and system and its annotation mark and discrimination method |
| CN113238041B (en) * | 2021-01-13 | 2024-01-16 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Beta-cyclodextrin modification-based immune biological bar code sensitization probe and method for rapidly detecting atrazine |
| CN113933500A (en) * | 2021-09-30 | 2022-01-14 | 山东省农业科学院原子能农业应用研究所(山东省辐照中心、山东省农业科学院农产品研究所) | Portable high-sensitivity immunoassay kit for ractopamine and application thereof |
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