CN102680695A - Gold magnetic particle labeled chromatography test strip for detecting aflatoxin - Google Patents
Gold magnetic particle labeled chromatography test strip for detecting aflatoxin Download PDFInfo
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Abstract
The invention provides immune magnetic particles for purifying an aflatoxin sample, a chromatography test strip manufactured based on the immune magnetic particles and a detection method, aims to solve the technical problems of complex separation operation, low separation efficiency, and high potential safety hazard in the process of purifying and separating the aflatoxin sample, and discloses a method capable of qualitatively and quantitatively detecting the aflatoxin. By the immune magnetic particles for purifying the aflatoxin sample, gold magnetic particles with the particle size of 20 to 200nm serve as a carrier, an aflatoxin monoclonal antibody serves as a ligand, the gold magnetic particles are connected with the aflatoxin antibody through a covalent reaction or the affinity, and the saturation magnetization of the gold magnetic particles is higher than 30emu/g. The aflatoxin antibody labeled by the gold magnetic particles is used for detection of the chromatography test strip, so that visual qualitative detection can be performed, quantitative detection can be performed by detecting magnetic signals, and the detection sensitivity is greatly improved.
Description
Technical field
The present invention relates to examination of aflatoxin instrument and detection method, be specifically related to a kind of be used to purify the immune magnetic particulate of aflatoxin sample and the chromatograph test strip and the detection method of making based on this immune magnetic particulate.
Background technology
Aflatoxin (AFT) is the secondary metabolite of fungi, mainly by the mould generation in aspergillus flavus, aspergillus parasiticus and Tequ, extensively has soil, and animals and plants are in various nuts, particularly peanut and the walnut.Aflatoxin mainly contains 4 kinds: i.e. B1, B2, G1, G2, wherein B1 is considered to a kind of carcinogen and mutagen the strongest in the present chemical carcinogen.For the food that prevents to be polluted by AFT gets in human consumption's chain, detection in time will be a kind of effective means.
The method of measuring aflatoxin (AFT) at present mainly contains thin-layered chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, mass spectroscopy, radioimmunoassay, fluorophotometric method, chromatograph test strip method, chemiluminescence and electrochemiluminescence method etc.Problem, especially some the sample intractable that all there are sample pre-treatments in now very universal colleges and universities' liquid phase chromatography and enzyme linked immunosorbent assay are like nut fruits, peanut, cereal, Chinese medicine etc.The immune affinity column of selling on the market now costs an arm and a leg, and the unit volume column capacity is relatively low, and sample preparation is limited in one's ability, and quantity of sample handling is relative fixed also, simultaneously the pillar easy blocking.
Not enough below the method for existing detection aflatoxin exists:
1, there is the very big safety problem of safety; Extract the organic solvent that aflatoxin need use multiple poisonous, peculiar smell, during with high performance liquid chromatography and ELISA detection, all need demarcate reference material or do typical curve; This not only poisons operating personnel, and contaminated environment.In addition, radioimmunoassay detects, and possibly also cause radioactive contamination.
2, detect complex steps, detection time is long, as, high performance liquid chromatography and ELISA method.
3, detectable needs refrigeration, like ELISA method, chemiluminescence, electrochemiluminescence.
4, detection needs special instrument and equipment, like HPLC method, ELISA method, electrochemiluminescence, chemiluminescence, radioimmunoassay, fluorophotometric method etc.
5, can only carry out qualitative and can not carry out detection by quantitative, like the colloid gold chromatographic test paper strip method.
Summary of the invention
The object of the present invention is to provide a kind of be used to purify the immune magnetic particulate of aflatoxin sample and the chromatograph test strip and the detection method of making based on this immune magnetic particulate; Solving the technical matters that lock out operation is complicated in the detachment process of aflatoxin sample purification, separation efficiency is low, have bigger potential safety hazard, realization can be qualitative again can the detection by quantitative aflatoxin method.
Technical solution of the present invention is:
A kind of immune magnetic particulate that is used to purify the aflatoxin sample; Its special character is: said immune magnetic particulate is carrier with particle diameter at the gold-magnetic particles of 20nm-200nm; The aflatoxin monoclonal antibody is an aglucon, and gold-magnetic particles is connected with aflatoxin antibody through covalent reaction or affinity interaction; The saturation magnetization of gold-magnetic particles is greater than 30emu/g.
Adopt above-mentioned immune magnetic particulate as gold-magnetic particles mark aflatoxin antibody, make the gold-magnetic particles mark chromatograph test strip that aflatoxin detects that is used for that obtains, comprise sample pad, pad, nitrocellulose membrane and thieving paper; Wherein the material of sample pad and pad is a glass fiber filter;
Sample pad has been passed through sample pad treating fluid immersion treatment, dries subsequent use; Contain 0.1～2g pvp in the sample pad treating fluid, 0.05～2ml TritonX-100 and 0.01～0.2mol/l PB;
Pad has passed through pad treating fluid immersion treatment, dries subsequent use; Contain 0.1%～1% Tween-20 in the pad treating fluid and 0.1%～1% song draws logical;
Sample pad is as sample point sample pond; Be sprayed with gold-magnetic particles mark aflatoxin antibody on the pad; Encapsulate 1～4mg/ml aflatoxin coupled antigen at the detection line place of nitrocellulose membrane, accusing that the line place encapsulates 1～4mg/ml two anti-(antibody of anti-gold-magnetic particles mark aflatoxin antibody).
A kind of method of using above-mentioned chromatograph test strip detection aflatoxin may further comprise the steps:
1] preparation of gold-magnetic particles labelled antibody
A. the aflatoxin monoclonal antibody is in surperficial the fixing of gold-magnetic particles
Get gold-magnetic particles and join in the centrifuge tube, jog is resuspended, places on the magnetic separator, and magnetic resolution is abandoned supernatant; Add ultrapure water again, jog is resuspended, places on the magnetic separator, and magnetic resolution is abandoned supernatant; Aflatoxin antibody is mixed with the antibody-solutions that concentration is 0.1～10mg/ml; Antibody diluent be ultrapure water water or concentration less than the PB of 0.01M, get in an amount of adding gold-magnetic particles, reaction density is 0.5-5mg/mL; In shaking table 25 ℃, 180r/min reacts 1h; Reaction finishes, and magnetic resolution is abandoned supernatant; Add cleaning buffer solution, jog is resuspended; Magnetic resolution is abandoned supernatant, removes the aflatoxin antibody of non-specific adsorption on the gold-magnetic particles surface;
The PB that contains the 0.0005M～0.1M of 0.05% tween in the cleaning buffer solution that this step adopts;
B. the sealing of gold-magnetic particles
In the gold-magnetic particles that step a obtains, add sealer, contain 0.1%～5%BSA in the said sealer, 0.05%～1.5% calf serum, 0.1～2%PEG20000; In shaking table, react, on the sealing magnetic particle not with the site of aflatoxin antibodies; Repeatedly clean with cleaning buffer solution, be suspended from last in the resuspended damping fluid, the concentration of final labelled antibody is at 0.5～2mg/ml; 2～8 ℃ of preservations are subsequent use;
The PB that contains the 0.0005M～0.1M of 0.05% tween in the cleaning buffer solution that adopts in this step;
Contain 0.1%～5%BSA in the resuspended damping fluid that adopts in this step, 1%～10% sucrose, 0.01～1%PEG20000,0.005%～0.5%pvp, 0.1%～1% song draws logical;
2] preparation of test strips
Handle sample pad and pad processing 5～10min respectively with sample pad treating fluid and pad treating fluid, pave then and dry, to increase the water wettability and the release of golden magnetic labelled antibody on pad of sample pad; With step 1] labelled antibody spray on the pad of handling well with 4 μ l/cm～12 μ l/cm; Get the PVC plate; Assemble thieving paper, NC film above; Speed with 1 μ l/cm is drawn 1～4mg/ml, two anti-and 1～4mg/ml coupling aflatoxin antigens respectively in the position of accusing line and detection line; To spray good pad then and be assembled on the test strips with the appearance pad of handling well of going up, it is wide subsequent use at last test strips to be cut into 3～5mm;
Contain 0.1～2g pvp in the sample pad treating fluid, 0.05～2ml Triton X-100,0.01～0.2mol/l PB;
Contain 0.1%～1% Tween-20 in the pad treating fluid and 0.1%～1% song draws logical;
3] examination of aflatoxin
A. the preparation of sample
Sampling originally is dissolved in methanol-water, perhaps in the acetonitrile-water, extracts, and extract dilutes with ultrapure water after filtering;
B. get on sample to be detected 50～150 μ l that prepare appearance to step 2] on the test strips for preparing; Sample liquid is in the chromatography process that makes progress; At first the aflatoxin micromolecule in detection line place sample is competed the incorporation of markings aflatoxin with the coupled antigen that is marked on the detection line; And labelled antibody is trapped in the detectability place, remaining labelled antibody is just in accusing that two resistive connections on the line close;
The pale pink band all occurs if accuse line and detection line, the amount of the aflatoxin of representing to contain in the sample to be detected is less than detectability; If need further to confirm the amount of aflatoxin, then on the magnetic signal detector, further detect;
If accuse the line outlet, detection line does not go out the pale pink band, and the amount of the aflatoxin of representing to contain in the sample to be detected is equal to or greater than detectability.
Advantage of the present invention is:
1, but golden magnetic mark chromatograph test strip gets final product the qualitative aflatoxin of detection by quantitative again; Aflatoxin antibody with the gold-magnetic particles mark is used for the chromatograph test strip detection; The optical property that had both had collaurum, but can carry out visualizing qualitative detection (can detect the aflatoxin of 4ng/ml), have the magnetic of magnetic particle again; Can carry out detection by quantitative (can detect the aflatoxin of 0.01ng/ml) through detecting magnetic signal, increase substantially the sensitivity that detects.
2, golden magnetic mark chromatography paper slip stable in properties, resting period are long, and the chromatograph test strip of golden magnetic mark was deposited one month in room temperature, and detection of active is unaffected.
3, it is convenient and swift that golden magnetic mark chromatography paper slip detects aflatoxin, and whole testing process is no more than 10min, both can be visual also can instrument readings.Instrument and equipment is light to be carried easily, and automaticity is high, and is simple to operate, can directly read test result, can be in the small test chamber or on-the-spot the use.
4, detect cost and hang down and can be widely used, do not need large-scale instrument and equipment, the user does not need professional training, and one-time detection only needs several yuans, can be widely used in daily life.
Description of drawings
Fig. 1: the structural drawing of golden magnetic mark immuno-chromatographic test paper strip.
Fig. 2: gold-magnetic particles mark chromatograph test strip detects 4ng/g examination of aflatoxin in the corn, and wherein, left side figure be the experiment photo that detects aflatoxin corn negatives, and right side figure is the experiment photo during the 4ng/g aflatoxin in the detection corn.
Fig. 3: golden magnetic mark aflatoxin chromatograph test strip (detection line is 4ng/ml) detects figure to the detection and the magnetic signal of the aflatoxin sample of 2ng/ml; Wherein, left side figure is the experiment photo of the aflatoxin sample of 2ng/ml, and the magnetic signal of the aflatoxin sample that right figure is 2ng/ml detects figure.
Fig. 4: golden magnetic mark aflatoxin chromatograph test strip (detection line is 4ng/ml) detects figure to the detection and the magnetic signal of the aflatoxin sample of 6ng/ml; Wherein, left side figure is the experiment photo of the aflatoxin sample of 6ng/ml, and the magnetic signal of the aflatoxin sample that right figure is 6ng/ml detects figure.
Embodiment
The present invention is a marker material with the gold-magnetic particles, has prepared the aflatoxin chromatograph test strip.But this chromatograph test strip qualitative and quantitative detection aflatoxin.Gold-magnetic particles is meant that particle diameter at 20nm-200 μ m, has superparamagnetism and the height ratio composite particles than area.Sample refers to that all contain the food of aflatoxin, Chinese medicine equal samples.
The present invention prepares the gold-magnetic particles labelled antibody after reacting a period of time jointly with aflatoxin monoclonal antibody and gold-magnetic particles.The aflatoxin sample that slightly extracts is added on the appearance pad; Under the effect of thieving paper; Aflatoxin layer sample liquid layer is analysed pad and is mixed with golden magnetic mark aflatoxin antibody; And aflatoxin and golden magnetic mark aflatoxin antibodies, the compound that their form continues upwards chromatography along the NC film, T line place not with sample in the gold-magnetic particles labelled antibody that combines of aflatoxin combine with the coupling aflatoxin at T line place and be deposited on T line place; Last remaining golden magnetic labelled antibody chromatography combines to C line anti-with two (antibody of anti-golden magnetic mark aflatoxin antibody); Be deposited on the C line,, can see that the T line and the C line that have golden magnetic hysteresis to stay all are the pink colour band because gold-magnetic particles has the optical property of collaurum.If C line and T line all the amount of the outlet aflatoxin representing to contain in the sample to be detected less than detectability, if think further to confirm that the amount of aflatoxin can further detection on the magnetic signal detector.If the outlet of C line, the amount of the aflatoxin that the not outlet of T line is represented to contain in the sample to be detected is equal to or greater than detectability.
Following examples are to the form example of scheme of the present invention with concrete experimental implementation, and experiment condition wherein and setup parameter should not be regarded as the limitation to basic technical scheme of the present invention.
The preparation of embodiment 1 gold-magnetic particles mark chromatograph test strip and the detailed process that detects aflatoxin in the food:
1] preparation of gold-magnetic particles labelled antibody
A. aflatoxin monoclonal antibody fixing on gold-magnetic particles surface: get in the centrifuge tube of 1mg gold-magnetic particles to a 2ml capacity, jog is resuspended.Place on the magnetic separator, magnetic resolution is abandoned supernatant.Add the 1ml ultrapure water, jog is resuspended.Place on the magnetic separator, magnetic resolution is abandoned supernatant.Repetitive operation once.Aflatoxin monoclonal antibody 100ug is configured to the solution that concentration is 0.1～10mg/ml, gets in an amount of adding gold-magnetic particles, in shaking table 25 ℃, 180r/min reacts 1h.Reaction finishes, and magnetic resolution is abandoned supernatant.Add 1ml cleaning buffer solution (PB that contains the 0.0025M of 0.05% polysorbas20), the resuspended magnetic grain of jog.Magnetic resolution is abandoned supernatant.
B. the sealing of gold-magnetic particles: in the magnetic grain in a last step, added the sealer of 1ml, in shaking table 25 ℃, 180r/min reacts 2h.(PB that contains 0.05% polysorbas20 0.0025M) cleans 3 times with the cleaning buffer solution of 2ml, is suspended from last in the resuspended damping fluid of 1ml, and 2～8 ℃ of preservations are subsequent use.
2] preparation of test strips: use appearance pad treating fluid and pad treating fluid and handle appearance pad and pad processing 10min respectively, pave then and dry.With 1] step golden magnetic labelled antibody spray on the pad of handling well with 1ul/cm.Get the PVC plate of 30cm; Assemble thieving paper, NC film above; Speed with 1ul/cm is drawn 2mg/ml quality-control product and 2mg/ml coupling aflatoxin antigen in the position of accusing line and detection line respectively; To spray good pad then and be assembled on the test strips with the appearance pad of handling well of going up, it is wide subsequent use at last test strips to be cut into 0.38cm.
3] examination of aflatoxin
A. the preparation of sample: this 5g that takes a sample, be dissolved in the 25mL methanol-water, perhaps acetonitrile-water extracts, and extract dilutes with ultrapure water after filtering.
B. get on the sample to be detected 100 μ l that prepare appearance to 2] on the test strips for preparing of step; If accuse line and detection line all the amount of the outlet aflatoxin representing to contain in the sample to be detected less than detectability, if think further to confirm that the amount of aflatoxin can further detection on the magnetic signal detector.If accuse the line outlet, the amount of the aflatoxin that not outlet of detection line is represented to contain in the sample to be detected is equal to or greater than detectability.
Can find out from accompanying drawing; Capital magnetic mark chromatograph test strip has good detection sensitivity; But the aflatoxin that can visualizing detects 4ng/g; The amount of aflatoxin can see significantly that less than 4ng/ml the pink colour band is arranged in sample, and detecting through the magnetic signal to 2ng/ml aflatoxin in the sample to prove (like Fig. 3).Detection line did not have the pink colour band when amount of aflatoxin was greater than 4ng/ml in the sample, and the magnetic signal of 6ng/ml aflatoxin in the sample is detected can prove (like Fig. 4).
Claims (3)
1. immune magnetic particulate that is used to purify the aflatoxin sample; It is characterized in that: said immune magnetic particulate is carrier with particle diameter at the gold-magnetic particles of 20nm-200nm; The aflatoxin monoclonal antibody is an aglucon, and gold-magnetic particles is connected with aflatoxin antibody through covalent reaction or affinity interaction; The saturation magnetization of gold-magnetic particles is greater than 30emu/g.
2. adopt immune magnetic particulate as claimed in claim 1 as gold-magnetic particles mark aflatoxin antibody; The gold-magnetic particles mark chromatograph test strip that is used for the aflatoxin detection that making obtains is characterized in that: comprise sample pad, pad, nitrocellulose membrane and thieving paper; Wherein the material of sample pad and pad is a glass fiber filter;
Sample pad has been passed through sample pad treating fluid immersion treatment, dries subsequent use; Contain 0.1～2g pvp in the sample pad treating fluid, 0.05～2ml TritonX-100 and 0.01～0.2mol/l PB;
Pad has passed through pad treating fluid immersion treatment, dries subsequent use; Contain 0.1%～1% Tween-20 in the pad treating fluid and 0.1%～1% song draws logical;
Sample pad is sprayed with gold-magnetic particles mark aflatoxin antibody as sample point sample pond on the pad, encapsulate 1～4mg/ml aflatoxin coupled antigen at the detection line place of nitrocellulose membrane, is accusing that the line place encapsulates 1～4mg/ml two and resists.
3. the said chromatograph test strip of application such as claim 2 detects the method for aflatoxin, may further comprise the steps:
1] preparation of gold-magnetic particles labelled antibody
A. the aflatoxin monoclonal antibody is in surperficial the fixing of gold-magnetic particles
Get gold-magnetic particles and join in the centrifuge tube, jog is resuspended, places on the magnetic separator, and magnetic resolution is abandoned supernatant; Add ultrapure water again, jog is resuspended, places on the magnetic separator, and magnetic resolution is abandoned supernatant; Aflatoxin antibody is mixed with the antibody-solutions that concentration is 0.1～10mg/ml; Antibody diluent be ultrapure water water or concentration less than the PB of 0.01M, get in an amount of adding gold-magnetic particles, reaction density is 0.5-5mg/mL; In shaking table 25 ℃, 180r/min reacts 1h; Reaction finishes, and magnetic resolution is abandoned supernatant; Add cleaning buffer solution, jog is resuspended; Magnetic resolution is abandoned supernatant, removes the aflatoxin antibody of non-specific adsorption on the gold-magnetic particles surface;
The PB that contains the 0.0005M～0.1M of 0.05% tween in the cleaning buffer solution that this step adopts;
B. the sealing of gold-magnetic particles
In the gold-magnetic particles that step a obtains, add sealer, contain 0.1%～5%BSA in the said sealer, 0.05%～1.5% calf serum, 0.1～2%PEG20000; In shaking table, react, on the sealing magnetic particle not with the site of aflatoxin antibodies; Repeatedly clean with cleaning buffer solution, be suspended from last in the resuspended damping fluid, the concentration of final labelled antibody is at 0.5～2mg/ml; 2～8 ℃ of preservations are subsequent use;
The PB that contains the 0.0005M～0.1M of 0.05% tween in the cleaning buffer solution that adopts in this step;
Contain 0.1%～5%BSA in the resuspended damping fluid that adopts in this step, 1%～10% sucrose, 0.01～1%PEG20000,0.005%～0.5%pvp, 0.1%～1% song draws logical;
2] preparation of test strips
Handle sample pad and pad processing 5～10min respectively with sample pad treating fluid and pad treating fluid, pave then and dry, to increase the water wettability and the release of golden magnetic labelled antibody on pad of sample pad; With step 1] labelled antibody spray on the pad of handling well with 4 μ l/cm～12 μ l/cm; Get the PVC plate; Assemble thieving paper, NC film above; Speed with 1 μ l/cm is drawn 1～4mg/ml, two anti-and 1～4mg/ml coupling aflatoxin antigens respectively in the position of accusing line and detection line; To spray good pad then and be assembled on the test strips with the appearance pad of handling well of going up, it is wide subsequent use at last test strips to be cut into 3～5mm;
Contain 0.1～2g pvp in the sample pad treating fluid, 0.05～2ml TritonX-100,0.01～0.2mol/l PB;
Contain 0.1%～1% Tween-20 in the pad treating fluid and 0.1%～1% song draws logical;
3] examination of aflatoxin
A. the preparation of sample
Sampling originally is dissolved in methanol-water, perhaps in the acetonitrile-water, extracts, and extract dilutes with ultrapure water after filtering;
B. get on sample to be detected 50～150 μ l that prepare appearance to step 2] on the test strips for preparing; Sample liquid is in the chromatography process that makes progress; At first the aflatoxin micromolecule in detection line place sample is competed the incorporation of markings aflatoxin with the coupled antigen that is marked on the detection line; And labelled antibody is trapped in the detectability place, remaining labelled antibody is just in accusing that two resistive connections on the line close;
If the pale pink band all appears in nature controlling line and detection line, the amount of the aflatoxin of representing to contain in the sample to be detected is less than detectability; If need further to confirm the amount of aflatoxin, then on the magnetic signal detector, further detect;
If accuse the line outlet, detection line does not go out the pale pink band, and the amount of the aflatoxin of representing to contain in the sample to be detected is equal to or greater than detectability.
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| CN110441538A (en) * | 2019-08-23 | 2019-11-12 | 北京丹大生物技术有限公司 | A kind of immuno-chromatographic test paper strip and its application for detecting digoxin |
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Application publication date: 20120919 |