CN105651992A - Triazophos bio-barcode immunoassay determination kit and application thereof - Google Patents
Triazophos bio-barcode immunoassay determination kit and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of immunoassay for food safety testing, in particular to a triazophos bio-barcode immunoassay determination kit. The kit comprises a kit body, wherein triazophos standards, triazophos magnetic nano-probes, single-standard colloidal gold nano-probes, triazophos double-standard colloidal gold nano-probes, a DNA biochip and a silver staining reagent. The kit can detect the content of triazophos in to-be-detected samples simply, conveniently, rapidly, sensitively, specifically and stably and is good in experimental reproducibility and convenient to operate and produce.
Description
Technical field
The present invention relates to food safety detection technical field of immunoassay, in particular to a kind of triazophos bio-barcode immune analytic reagent kit and application thereof.
Background technology
Along with high-toxic pesticide kind will progressively from market exit, triazophos becomes the principal item substituting methamidophos pesticide, and it makes consumption increase sharply in recent years, is widely used in the preventing and treating of middle and lower reach of Yangtze River basin water chilo suppressalis. At triazophos pesticide while China just comes into the market the busy season, abroad have begun to restriction or forbid use and the sale of triazophos pesticide. On November 28th, 2004, State General Administration for Quality Supervision issues urgent early warning, claims European Union formally to forbid that 320 kinds of pesticide are sold in European Union 31 days in December. It is directed to the Chinese pesticide producing, use and exporting and reaches more than 60 kind, wherein just include triazophos pesticide. The MRL of triazophos be it is also proposed increasingly stricter standard by other countries and area: in Japan's regulation rice, the MRL (MRL) of triazophos must not be and detected; Except European Union is 0.05mg/kg, Semen Gossypii 0.1mg/kg except Folium Camelliae sinensis, all the other samples are 0.02mg/kg; The MRL of China's triazophos is generally defined as 0.05mg/kg. Therefore, the research of triazophos detection method is strengthened significant for ensureing China's food safety and Agricultural Products Trade.
The effect of immune analysis method and status have obtained being widely recognized as of Chinese scholars at present, and the research of immune analysis method is also one of the most popular field of detection method research. In immune analysis method field, China has carried out comparatively deep research in enzyme-linked immune analytic method, prepare including hapten synthesis, antibody, label coupling, in reaction system various physicochemical properties on aspects such as the impact of immune analysis method and hapten structure and the structure activity relationships that obtains prepared by it between antibody. In recent years, Chinese scholar has also carried out more research work in fluorescence immunoassay, chemiluminescence immune assay, biosensor and bio-barcode immune analysis method.
Bio-barcode immune analysis method based on magnetic nano-particle, utilize the separation and concentration effect of bio-barcode signal amplification and magnetic nano-particle, its minimum detectability improves several order of magnitude relative to ELISA method, cause its from be born since, just obtain showing great attention to of scientific circles.Bio-barcode refers to the DNA fragmentation of a large amount of identical sequences that may be incorporated on nanoparticle. The method is for the detection to prostate specific antigen (ProstatespecificAntigen, PSA) the earliest. The method specifically includes that magnetic nano-particle finishing PSA monoclonal antibody, and gold colloidal nano-probe finishing plays the bio-barcode of signal amplification and for the another kind of antibody of PSA. When PSA exists, magnetic nano-particle, PSA, gold colloidal nano-probe form " sandwich " sandwich composite construction by antigen antibody reaction. The complex formed is through magnetic field separation, and the magnetic Nano probe of enrichment passes through high-temperature low salt method degeneration, discharges bio-barcode, just can indirectly obtain the content of PSA by detecting the bio-barcode content disintegrated down.
Bio-barcode is analyzed method and is obtained quick development at trace amount of protein and nucleic acid detection method nearly ten years, by the bio-barcode immune analysis method set up that combines with immunoreation, the detection limit of immune analysis method is brought up to a new height. But the method is set up so far, realize the detection to macromolecular substances such as protein only by building " sandwich " structural models. Owing to triazophos is micromolecular compound, under normal circumstances only one of which antigen binding site, " sandwich " structural models that therefore bio-barcode immune analysis method adopts at present is not applied for the detection of triazophos substantially.
In view of this, the special proposition present invention.
Summary of the invention
It is an object of the invention to provide a kind of triazophos bio-barcode immune analytic reagent kit, use this test kit can triazophos content in detection by quantitative water or in food specifically. This test kit is detected by competitive reaction system, overcomes " sandwich " structural models that current bio-barcode immune analysis method adopts and is substantially not applied for the defect of detection of triazophos.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
A kind of triazophos bio-barcode immune analytic reagent kit, described test kit includes box body, includes in described box body:
Triazophos standard substance, triazophos magnetic Nano probe, single mark gold colloidal nano-probe, triazophos double; two mark gold colloidal nano-probe, DNA biochip, silver transfection reagent.
Triazophos (Triazophos) molecular formula: C12H16N3O3PS; No. CAS: 24017-47-8.
The present invention, by changing the existing reaction pattern of bio-barcode immune analysis method, sets up the bio-barcode immune analysis method based on little molecule competitive mechanism. Triazophos magnetic Nano probe is prepared by the conjugate of triazophos Yu carrier protein being coated in magnetic nano particle sub-surface, and in gold colloidal nano-probe surface combination pesticide antibody and the bio-barcode of signal amplification in order to prepare triazophos double; two mark gold colloidal nano-probe, utilize the envelope antigen of magnetic nano particle sub-surface and the antibody on pesticide molecule competition binding gold colloidal nano-probe surface to set up competitive type immuno-chemical reaction system. Utilize the antigen antibody complex that magnetic separation system separating immune combines, thermal denaturation technology is adopted to promote gold colloidal nano-probe release bio-barcode, and utilize DNA chip probe and single mark gold colloidal nano-probe to hybridize in conjunction with bio-barcode, and then utilize gold nanoparticle to measure bio-barcode content, it is achieved the detection by quantitative to triazophos pesticide.
The result of use of this test kit has simplicity, the advantage such as quick, sensitive, special, stable. Further, it is open-sky technique according to the detection system of the present invention, easy to be quick, it is particularly suitable for vast quality inspection organization and promotes the use of, provide the very valuable detection means of one for food safety detection.
Preferably, described triazophos magnetic Nano probe is formed by magnetic nano-particle and triazophos complete antigen coupling; Described triazophos complete antigen is formed by triazophos and carrier protein couplet.
It is further preferred that described carrier protein is chicken ovalbumin; The particle diameter of described magnetic nano-particle is 18��22nm.
Chicken ovalbumin (Ovalbumin, OVA), also referred to as chicken ovalbumin, is made up of 386 aminoacid, and molecular weight is about 43Kd. OVA is as inert protein, and it can maintain the colloid-stabilised of gold colloidal, plays the skeleton function of dispersion colloid gold grain.
Inert protein also can be selected for casein (Casein) or bovine serum albumin (BSA) etc.
The coupling quantity of triazophos complete antigen is had very big impact by the particle diameter of magnetic nano-particle, thus affecting the sensitivity of end reaction.
Preferably, test kit as above:
Described single mark gold colloidal nano-probe is coated colloid gold particle by single stranded DNA 1 and obtains;
Described triazophos double; two mark gold colloidal nano-probe is coated colloid gold particle by anti-triazophos antibody and double-stranded DNA and obtains;
Described DNA biochip is prepared by single stranded DNA 2;
Single stranded DNA and bar code single stranded DNA complementary pairing that described double-stranded DNA is modified by mercaptan form; There is complementary pairing relation with described bar code single stranded DNA in described single stranded DNA 1 and single stranded DNA 2, and pairing region is not overlapping.
The DNA2 putting system on DNA biochip is capture dna, can with bar code DNA complementary pairing caught on chip; The effect of the DNA1 on single mark gold colloidal nano-probe is in that amplification bar code DNA signal further.
Double; two mark gold colloidal nano-probes are the anti-checking matter antibody that double-stranded DNA is coated colloid gold particle and anti-checking matter, and 1 in double-stranded DNA is connected by Au S key with colloid gold particle, and another 1 DNA is used to the bar code DNA of instruction checking matter. Being marked with the bar code DNA of a lot of bar on each colloid gold particle, thus play the effect of method signal, sensitivity is higher.
In practical operation, bar code DNA is chosen as prior art, as long as specificity and sensitivity are enough good.
Preferably, the particle diameter of described colloid gold particle is 13��15nm.
The combination that key technical point is that by forming triazophos magnetic Nano probe-triazophos double; two mark gold colloidal nano-probe of the application, the combination with triazophos-triazophos double; two mark gold colloidal nano-probe is at war with, and the separation that the former combination should be homogeneous. The effective of bar code DNA that the efficient recovery that it is critical only that checking matter of sensitivity raising is corresponding with as each checking matter identification binding site discharges. The amount (the triazophos magnetic Nano probe suppression ratio to the competitive binding of tested triazophos) of the corresponding checking matter of amount of bar code DNA, therefore can pass through the checking matter of quantitative bar code DNA indirect quantification trace. Triazophos magnetic Nano probe-triazophos double; two mark gold colloidal nano-probe is the triazophos complete antigen in magnetic Nano probe and the combination of the anti-triazophos antibody in double; two mark gold colloidal nano-probes in conjunction with essence, and the bond strength of the two is closely related with the amount of antibody/antigen, the amount of antibody/antigen is then closely related with the particle diameter of colloid gold particle/magnetic nano-particle.
Preferably, test kit as above, described triazophos double; two mark gold colloidal nano-probe is specifically prepared by following methods:
1) it is added thereto to anti-triazophos antibody, stirring mixing, standing 25��35min after, taking colloidal gold solution tune pH to 8.8��9.2; Being subsequently adding the single stranded DNA chain that mercaptan is modified, 8��12 DEG C stand 36��44h; Adjust pH to 7.3��7.5 again, add NaCl to the centrifugal 8��12min of concentration 0.08��0.12mol/L, 8000��11000r/min, abandon supernatant, obtain the gold colloidal containing oligonucleotide;
2), with the resuspended described gold colloidal containing oligonucleotide of the phosphate buffer that bovine serum albumin concentration is 0.8��1.2%, 1��3h is hatched; Adding the bio-barcode DNA that the single stranded DNA chain modified with described mercaptan is complementary, hybridization 3��5h, 8000��11000r/min are centrifugal for room temperature, abandon supernatant, obtain described triazophos double; two mark gold colloidal nano-probe.
It is further preferred that when adding NaCl to set concentration, in 120��180min, point 2��4 equivalent add, every minor tick 40��60min.
Add the process of NaCl and the ageing process of DNA. Nanometer gold and DNA are electronegative, so can be mutually exclusive, add NaCl and can mask some electric charges on nanometer gold surface, reduce the repulsion between nanoparticle. But the NaCl of excessive concentrations can cause that ionic intension is excessive, easily causes DNA to reunite, so being gradually added, it is prevented that moment ionic strength excessive.
Preferably, test kit as above, described anti-triazophos antibody is monoclonal antibody.
Monoclonal antibody owing to can be produced by cell strain, stable in properties between each batch, and high specificity, thus be more suitable for reagent kit product.
The triazophos bio-barcode immune analytic reagent kit as above application in triazophos qualitative and quantitative analysis.
Triazophos bio-barcode immune analytic reagent kit as above measures the method for triazophos, including:
A), by triazophos standard substance, extract to mix with isopyknic triazophos double; two mark colloidal gold probe and triazophos magnetic Nano probe respectively with the testing sample after purification and hatch;
B), washing hatches product and roguing obtains the bio-barcode corresponding with testing sample with each standard substance;
C), by described bio-barcode and DNA biochip hybridization, and single mark Nano-Au probe amplification signal is added; Hybridization adds silver transfection reagent and measures each hole gray value with chip scanner analyzer after terminating;
D), with the concentration of triazophos standard substance for abscissa, the gray scale suppression ratio corresponding to each concentration is vertical coordinate, Criterion curve, and calculates the triazophos concentration in each sample according to standard curve.
Compared with prior art, the invention have the benefit that
(1) present invention can detection by quantitative triazophos content specifically. There is simplicity, the advantage such as quick, sensitive, special, stable. Further, it is open-sky technique according to the detection system of the present invention, easy to be quick, it is particularly suitable for vast quality inspection organization and promotes the use of, provide the very valuable detection means of one for food safety detection.
(2) test kit according to the present invention, triazophos hapten in magnetic Nano probe forms direct competitive system with the triazophos monoclonal antibody on the triazophos competition triazophos double; two mark colloidal gold probe in detection sample, therefore the competitive reaction system that this law adopts, namely the sensitivity of detection is guaranteed, it is possible to be effectively ensured the good reproduction of testing result.It addition, this pattern is also convenient for operation and produces.
(3) test kit of the present invention uses silver staining method, and by detecting the gray value on biochip, sensitivity is greatly improved, and can provide foundation more sensitive, quick, reliable for the detection of triazophos in vegetable and fruit.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below.
Fig. 1 is the scanning figure that hybridization terminates rear DNA biochip;
Fig. 2 is with the concentration of triazophos standard substance for abscissa in embodiment 2, and the gray scale suppression ratio corresponding to each concentration is vertical coordinate, the standard curve of foundation.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, it will be appreciated by those skilled in the art that the following example is merely to illustrate the present invention, and are not construed as restriction the scope of the present invention. Unreceipted actual conditions person in embodiment, conventionally the condition of condition or manufacturer's suggestion carries out. Agents useful for same or the unreceipted production firm person of instrument, be and can pass through the commercially available conventional products bought and obtain.
Embodiment 1
The preparation method of triazophos bio-barcode immune analytic reagent kit
S11, single preparation marking colloidal gold probe
1), gold nano grain synthetic method
Being separately added into 98mL deionized water in the beaker that silication is good, then enter 2mL50mM gold chloride stock solution respectively, the concentration making gold chloride is 1mM, and on magnetic stirring apparatus, 1000rpm/min stirring, 250 DEG C of heating, continue after liquid boiling to boil 2min; Draw 10mL38.8mM trisodium citrate, in rapid disposable addition beaker, it is kept stirring for speed and heating-up temperature is constant, continue to boil 6min, it is possible to find solution colour is by the colourless purple that gradually becomes, then becomes claret, different colors is ultimately become according to the difference adding trisodium citrate reduction dosage, refilling original volume with deionized water after liquid cools down, in 4 DEG C of airtight preservations, the size controlling of the gold nano grain of synthesis is at 13��15nm.
2), take sulfhydrylation DNA1 solution, add three (2-carboxyethyl) phosphine solution (TCEP) of 100mmol/L, at room temperature shaking table is placed 2 hours. Ratio between reducing agent TCEP and DNA1 is close to 200:1, and a high proportion of gap accelerates response speed, improves reduction effect. Under room temperature, newly synthesized nano gold spherical under 6000rcf rotating speed 20 centrifugal minutes, takes out nano gold spherical and is dissolved in ultra-pure water. The nano gold spherical 10000rcf taking certain volume is centrifuged 20 minutes, removes supernatant, adds 0.5 �� Tris-boric acid (TBE) buffer solution, it is subsequently adding the amount of the sulfhydrylation DNA taken, adding sodium chloride solution is 50mmol/L to sodium chloride concentration, under room temperature, reacts 24 hours. Gradually adding sodium chloride to concentration is 500mmol/L (point 3 additions, every septum secundum adds once for 1 hour), stands 8 hours under room temperature. Centrifugal 20 minutes of 10000rcf, removes supernatant, is dissolved in ultra-pure water, repeats 3 times, obtains the sulfhydrylation DNA1 nano gold spherical GNP solution modified.
The preparation of S12, triazophos double; two mark colloidal gold probe
Take certain volume colloidal gold solution, use 0.1mol/LK2CO3Regulate to pH=9.0; Stand 15min, add a certain amount of triazophos monoclonal antibody, under agitation gold colloidal and antibody are mixed, stand 30min. It is subsequently adding the DNA that mercaptan is modified, 10 DEG C stand 40h, adjust to pH=7.4 with phosphate buffer (PBS) again, improve NaCl concentration, NaCl divides 3 additions, adds once every 1 hour, to final concentration 0.1mol/L, 10000r/min is centrifuged 10min, namely obtains the gold colloidal containing oligonucleotide.With the resuspended gold colloidal containing oligonucleotide of 1mLPBS buffer, stand 10��15 minutes, then add 5% bovine serum albumin (BSA), make BSA final concentration of 1%, close 1h. Adding the bio-barcode DNA that the described single stranded DNA chain modified with mercaptan is complementary, room temperature hybridization 4h, 10000r/min is centrifugal obtains double; two mark gold colloidal nano-probe.
S13, triazophos magnetic Nano probe preparation
1), the haptenic preparation of chicken ovalbumin (OVA) labelling triazophos
The haptenic preparation of horseradish peroxidase-labeled triazophos, concrete grammar is as follows:
Taking hapten 0.25mmol, be dissolved in 1mLN, dinethylformamide (DMF), stirring is lower adds the 60 positive tri-n-butylamines of �� l and 30 �� l ethyl chloroformates, and reaction 1h is stirred at room temperature. Extract reaction solution 300 �� l and be slowly added dropwise 6mL dissolved with, in the carbonate buffer solution (CBS, 0.01mol/L) of 120mgOVA, being stirred at room temperature after reacting 2h, load bag filter, first dialysing 3 times with distilled water, then with the PBS 3d of 0.01mol/L, every day changes dialysis solution 3-4 time. Take out subpackage to be stored in the refrigerator of-20 DEG C.
2), magnetic nano-particle synthetic method
The forming process of magnetic microsphere: take 8.1gFeCl3��6H2O and 3.3gFeCl2��4H2O adds 150mL deionized water, wiring solution-forming, then moves in there-necked flask. At N2Protect in environment and be stirred vigorously under state to Fe2+And Fe3+Mixed solution in 18mL mass fraction be the strong aqua ammonia of 25%, solution starts generate tan Fe3O4Particle, regulates the pH value of solution between 9��11, and reaction temperature is 70 DEG C this moment, to ensure certain mixing speed in the process of reaction, and the ionic equation of reaction is:
Fe2++2Fe3++8OH-=Fe3O4+4H2O
Fe3O4The synthesis of/oleic acid: by 5.0g oleic acid (C under being stirred vigorously state18H34O2) add Fe3O4Liquid in, then at N2Under protection environment, temperature is react one hour in 70 DEG C of water-baths, makes oleic acid be uniformly coated on particle surface. Reaction obtains black sol shape material, utilizes externally-applied magnetic field the precipitation of gained to be separated from reaction system after terminating, and removes unnecessary oleic acid 3 times by washing with alcohol, then with deionized water wash to pH=7, now obtains the Fe that oleic acid is uniformly coated with3O4Particle.
The Fe of monolayer carboxyl-functional3O4Particle synthesizes: adopt potassium permanganate oxidation oleic acid method synthesis Azelaic Acid (HOOC (CH2)7COOH) carboxylated magnetic nano-particle is formed. Adding 160mL concentration is the KMnO of 10mg/mL4Solution is being stirred vigorously state, temperature is reaction 8h under 50 DEG C of conditions, reaction utilizes externally-applied magnetic field gained precipitation to be separated from reaction medium after terminating, and successively use deionized water wash 3 times, products therefrom is vacuum drying at 40 DEG C, dried particle is dissolved in aqueous solution and just obtains stable carboxylated magnetic nano-particle. The size controlling of magnetic nano-particle is at 18��22nm.
3), magnetic microsphere and the haptenic connection of OVA-
A), the activation of microsphere. With the 2-(N-morpholine) ethanesulfonic acid buffer (MES) of pH=6.0,15mmol/L as activation buffer solution, take 1mL magnetic bead in 2.0mL centrifuge tube, after adding activation buffer solution, supernatant is sopped up; Relaunder magnetic bead 2 times with activation buffer again, then be separately added into carbodiimide and N-hydroxy-succinamide, mix homogeneously in vortex oscillator, room temperature activation 30min.
B), microsphere and OVA-hapten conjugation. Make coupling buffer with phosphate tween (PBST, the 1%Tween-20) solution of pH=7.4,0.01mol/L, relaunder activated magnetic bead 3 times with activation buffer, then wash magnetic bead 2 times with coupling buffer;Then with the resuspended magnetic bead of coupling buffer, OVA-hapten is added so that the carboxyl of magnetic bead surfaces activation reacts 1h with the amino of OVA under 37C, by OVA-hapten conjugation in magnetic bead surfaces, obtains immunomagnetic beads.
C), close. With magnetic bead 2 times after coupling buffer washing coupling, add the coupling buffer containing 2%BSA and close magnetic bead 60min. Finally give immune magnetic probe.
S14, triazophos standard substance preparation
Preparing by triazophos sterling, concentration is 0ng/mL, 0.05ng/mL, 0.01ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL respectively, totally 7 bottles;
Described triazophos 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 gold colloidal nano-probe extension rate is 10 times.
Prepared by S16, DNA chip
According to the capturing probe DNA2 that bar code detecting probe sequential design can be combined with its partial complementarity; By amination modify capturing probe by chip point sample instrument point to surface aldehydes on slide, spot diameter 100 ��m, dot spacing 500 ��m, point sample amount is 0.7nL, DNA2 concentration and probe concentration is 75 ��m of ol/L, 37 DEG C fix. Unconjugated probe, with deionized water rinsing, is soaked in mercaptosuccinic acid solution 30min to be passivated all the other binding sites, and after repeatedly rinsing with deionized water, 4 DEG C standby.
S17, semi-finished product and finished product composition
Above-mentioned steps gained aliquots is semi-finished product, extract three parts out and just can be assembled into triazophos bio-barcode immune analytic reagent kit through specificity, elaboration, sensitivity and stability assay approval, just can dispatch from the factory after also needing sampling observation qualified after being assembled into test kit.
To sum up, in the research process of the present invention, first raw material used has been carried out screening test and Quality Identification by the present inventor, including the haptenic activity of antibody and triazophos, the particle diameter of colloid gold particle, the particle diameter of magnetic nanoparticle and magnetic etc. Then pass through the reaction pattern to test kit, response time, coupling efficiency, DNA biochip point sample size, silver dye time, it is optimized etc. a series of condition, establishes the triazophos bio-barcode immune analysis determination method of high sensitivity and good reproduction.
Embodiment 2
The test kit using method of the present invention
The concrete operations of the triazophos bio-barcode immune analytic reagent kit of above case study on implementation 1 preparation are as follows:
S21, in 4 DEG C of refrigerators take out test kit, equilibrium at room temperature 10 minutes;
S22, sample extraction and purification
Weigh 10.0g (being accurate to 0.01g) sample; Add 10mL acetonitrile, high-speed homogenization 0.5min, add the anhydrous MgSO of 4.0g4With high speed vortex 1min after 1.0gNaCl, at 4 DEG C with 10000r/min high speed centrifugation 5min. Accurately pipette 2mL acetonitrile extraction liquid in 10mL plastic centrifuge tube, add 50mgN-propyl group ethylenediamine (PSA) and 50mgC18 solid phase dispersion extracting and purifying agent, after high speed vortex 0.5min, at 4 DEG C with 10000r/min high speed centrifugation 5min. Taking 1mL supernatant, at 30 DEG C, nitrogen is blown to do, and the residue 1mL PBS containing 10% methanol dissolves.
S23, reacting hole add 20 �� L triazophos double; two mark colloidal gold probe, then reacting hole adds each concentration triazophos standard substance (0ng/mL, 0.05ng/mL, 0.01ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL) and each 20 �� L of sample obtained after pre-treatment respectively, if a repetition, then every hole adds triazophos magnetic Nano probe 20 �� L, fully vibrate with micro oscillator mixing, being subsequently placed in temperature is 37 DEG C, relative humidity be 70% constant temperature and humidity incubator in incubation 1h;
S24, use PBST liquid wash three times, and roguing obtains bio-barcode;
S25, chip hybridization
4 �� L bio-barcode DNA and the mixing of 15 �� L hybridization solutions, uniformly drip on chip, and 48 DEG C of hybridization 1h, 0.2 �� SSC washing liquids clean chip 1min, dry stand-by. By mono-for 4 �� L mark Nano-Au probe solution and 15 �� L hybridization solutions (0,01MPBS buffer of the sodium lauryl sulphate containing 0.1%) mixing, room temperature closes 30min. List mark nano-Au solution after closing uniformly is dripped in the dot matrix area drying chip, 48 DEG C of hybridization 1h. 0.2 �� SSC washing liquid, cleans chip 1min, dries. Every dot matrix is uniformly added into brand-new silver dye liquor 20 �� L (lucifuge operation), room temperature reaction 10min and is placed on deionized water to terminate reaction, Microscopic observation.
S26, use chip scanner analyzer measure each hole gray value;
S27, with the concentration of triazophos standard substance for abscissa, the gray scale suppression ratio corresponding to each concentration is vertical coordinate, Criterion curve, and calculates the triazophos concentration in each sample according to standard curve; Canonical plotting is shown in Fig. 2.
Experimental example 1
The methodology verification result of test kit of the present invention
According to vertification regulation common in this area, the test kit of preparation in case study on implementation 1 being identified, result is in Table 1.
The methodology verification result of table 1 test kit of the present invention
Result above shows that the accuracy of " triazophos bio-barcode immune analytic reagent kit ", specificity, elaboration, sensitivity and stability comply fully with condition.
Experimental example 2
Triazophos TIANZHU XINGNAO Capsul is tested
In order to check the practical application performance of triazophos bio-barcode immune analytic reagent kit, triazophos standard solution is joined tap water is simulated the water sample polluted by triazophos, add recovery experiment by standard and measure the concentration of triazophos in tap water, interpolation concentration is the triazophos of 1ng/mL, 5ng/mL and 10ng/mL, and testing result is in Table 2.
The TIANZHU XINGNAO Capsul of triazophos in table 2 sample
Although illustrate and describing the present invention with specific embodiment, however it will be appreciated that may be made that when without departing substantially from the spirit and scope of the present invention many other change and amendment. It is, therefore, intended that include all such changes and modifications belonging in the scope of the invention in the following claims.
Claims (10)
1. a triazophos bio-barcode immune analytic reagent kit, it is characterised in that described test kit includes box body, includes in described box body:
Triazophos standard substance, triazophos magnetic Nano probe, single mark gold colloidal nano-probe, triazophos double; two mark gold colloidal nano-probe, DNA biochip, silver transfection reagent.
2. test kit according to claim 1, it is characterised in that:
Described triazophos magnetic Nano probe is formed by magnetic nano-particle and triazophos complete antigen coupling; Described triazophos complete antigen is formed by triazophos and carrier protein couplet.
3. test kit according to claim 2, it is characterised in that:
Described carrier protein is chicken ovalbumin; The particle diameter of described magnetic nano-particle is 18��22nm.
4. test kit according to claim 1, it is characterised in that:
Described single mark gold colloidal nano-probe is coated colloid gold particle by single stranded DNA 1 and obtains;
Described triazophos double; two mark gold colloidal nano-probe is coated colloid gold particle by anti-triazophos antibody and double-stranded DNA and obtains;
Described DNA biochip is prepared by single stranded DNA 2;
Single stranded DNA and bar code single stranded DNA complementary pairing that described double-stranded DNA is modified by mercaptan form; There is complementary pairing relation with described bar code single stranded DNA in described single stranded DNA 1 and single stranded DNA 2, and pairing region is not overlapping.
5. test kit according to claim 4, it is characterised in that:
The particle diameter of described colloid gold particle is 13��15nm.
6. test kit according to claim 5, it is characterised in that described triazophos double; two mark gold colloidal nano-probe is specifically prepared by following methods:
1) it is added thereto to anti-triazophos antibody, stirring mixing, standing 25��35min after, taking colloidal gold solution tune pH to 8.8��9.2; Being subsequently adding the single stranded DNA chain that mercaptan is modified, 8��12 DEG C stand 36��44h; Adjust pH to 7.3��7.5 again, add NaCl to the centrifugal 8��12min of concentration 0.08��0.12mol/L, 8000��11000r/min, abandon supernatant, obtain the gold colloidal containing oligonucleotide;
2), with the resuspended described gold colloidal containing oligonucleotide of the phosphate buffered solution that bovine serum albumin concentration is 0.8��1.2%, 1��3h is hatched; Adding the bio-barcode DNA that the single stranded DNA chain modified with described mercaptan is complementary, hybridization 3��5h, 8000��11000r/min are centrifugal for room temperature, abandon supernatant, obtain described triazophos double; two mark gold colloidal nano-probe.
7. test kit according to claim 6, it is characterised in that:
When adding NaCl to set concentration, in 120��180min, point 2��4 equivalent add, every minor tick 40��60min.
8. the test kit according to claim 4 or 6, it is characterised in that described anti-triazophos antibody is monoclonal antibody.
9. the application in triazophos qualitative and quantitative analysis of the triazophos bio-barcode immune analytic reagent kit described in any one of claim 1��8.
10. the method that the triazophos bio-barcode immune analytic reagent kit described in any one of claim 1��8 measures triazophos, it is characterised in that including:
A), by triazophos standard substance, extract to mix with isopyknic triazophos double; two mark colloidal gold probe and triazophos magnetic Nano probe respectively with the testing sample after purification and hatch;
B), washing hatches product and roguing obtains the bio-barcode corresponding with testing sample with each standard substance;
C), by described bio-barcode and DNA biochip hybridization, and single mark Nano-Au probe amplification signal is added; Hybridization adds silver transfection reagent and measures each hole gray value with chip scanner analyzer after terminating;
D), with the concentration of triazophos standard substance for abscissa, the gray scale suppression ratio corresponding to each concentration is vertical coordinate, Criterion curve, and calculates the triazophos concentration in each sample according to standard curve.
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| CN113567658B (en) * | 2021-07-27 | 2023-07-21 | 中国农业科学院农业质量标准与检测技术研究所 | Organophosphorus pesticide multi-residue biological bar code immunodetection kit based on hairpin self-assembly and application thereof |
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