CN107515299A - A kind of bioprobe, the test strips for detecting furazolidone and its application - Google Patents
A kind of bioprobe, the test strips for detecting furazolidone and its application Download PDFInfo
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Abstract
The invention discloses a kind of bioprobe, detect test strips and its application of furazolidone, using the antibody of the anti-furazolidone of nano gold mark as detection probe, the goat anti-mouse immunoglobulin G of nano gold mark is as enhancing probe, two kinds of probes are sprayed on same hydrophobic pad simultaneously, with the flowing of sample solution, successively elute, double probes make gold condense together under the combination of antibody and secondary antibody immediately, form complicated network structure, afterwards with capillarity flow forward, clearly red stripes are formed in T lines, play a part of signal amplification, simultaneously, substantially reduce the dosage of antibody, so as to trigger more keen competition to react, improve sensitivity.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of bioprobe, detect the test strips of furazolidone and its answer
With.
Background technology
Furazolidone, it is that (remaining mainly includes nitrofurazone, furaltadone to one kind the most frequently used in nitrofuran antibiotics
And furantoin), it is metabolized rapidly in vivo, and half-life short, parent drug can not be stabilized.However, the generation of furazolidone
Thanking to product 3- amino -2- oxazolones (AOZ) can combine closely with protein in tissue, be present in the form stable of reference state
In body, and the time remained is longer, and toxicity caused by such medicine is stronger.
There is killing action because furazolidone antibiotic is cheap, and to the pathogen such as bacterium, protozoon and fungi, because
This is widely used in livestock and aquatic products, to treat and prevent the various alimentary infections as caused by bacterium, protozoa, is also made
For domestic animal growth promoter.But because its active compound and its metabolin have potential carcinogenic, teratogenesis side effect, every country to human body
Such medicine has been forbidden to use in livestock non-staple food production process.Therefore strengthen to furans in livestock products, aquatic products and feed
The detection of oxazolone antibiotic residue, particularly speed are surveyed, and are enhancings to understand and grasp the health information of food and feed in time
One important step of foodsafety.
Because furazolidone is metabolized quickly in vivo, therefore the analysis of situation is remained to it using its metabolin AOZ as standard,
And AOZ can not produce immune response as small molecule, then using its derivative as the object detected.Detection furazolidone at present
The conventional method of residual has high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS), enzyme linked immunological examination
Method (ELISA) is tested, and immuno-chromatographic test paper strip overcomes former methodical as novel detection method extremely concerned in recent years
Shortcoming, it is not necessary to expensive Laboratory Instruments and professional operator, have high specificity, high sensitivity, cost it is low, to reality
Test personnel and environment contamination hazard it is small, suitable for live batch detection the advantages that, there is great application value and prospect.Due to
Competitive immunization chromatograph test strip will pass through the antibody of complicated derivatization process and high quality used in Furaxone metabolite detection,
Therefore the research for detecting furazolidone with immuno-chromatographic test paper strip at present is seldom.What is be currently, there are is examined with immuno-chromatographic test paper strip
The research for surveying furazolidone has：
The quantum dot-labeled antibody of Tao Le, Yong Xie, Liqian Zhu, and Lei Zhang groups in 2016
Furaxone metabolite is detected as signal probe, its detection line to detect by an unaided eye is more than 10 μ g/L.2017, the group used
The antibody of colloid gold label detects Furaxone metabolite as signal probe, and its visual detection line is 10 μ g/L.
In recent years, research is directed to replacing label to improve detection sensitivity, but (fluorescence is micro- for new marker material
Ball, quantum dot, magnetic bead, graphene etc.) generally preparation process is complicated, and the bad control of antibody coupling condition, and the material having needs
Signal readout equipment is wanted, is all unsuitable for Site Detection.On the other hand, traditional colloidal gold strip does not reach very high sensitive
Degree, it is impossible to meet the detection demand of current stricter furazolidone limit standard well.But it is related to live inspection
Survey, collaurum still possesses absolute predominance.Therefore, one kind amplified signal on the basis of collaurum is established in research, and is directed to
Furazolidone antibiotic residue has highly sensitive immuno-chromatographic test paper strip, and this is to the furan in Site Detection and monitoring livestock products
Oxazolone of muttering tool has very important significance and application value.
The content of the invention
For in the prior art the defects of and deficiency, it is an object of the invention to provide a kind of bioprobe, detection furans azoles
The test strips of ketone and application, the bioprobe can substantially reduce the dosage of antibody, so as to trigger more keen competition to react, improve
Sensitivity, the Visual retrieval line of furazolidone is set to reach 0.3ng/mL.
To reach above-mentioned purpose, the technical scheme that the present invention takes includes：
A kind of bioprobe, including detection probe and enhancing probe, described detection probe are the furans of nano gold mark
Oxazolone monoclonal antibody, described enhancing probe are the goat anti-mouse immunoglobulin G of nano gold mark.
Specifically, detection probe and the volume ratio of enhancing probe are 4~6:5~7, best, detection probe is visited with enhancing
The volume ratio of pin is 5:6.
Again specifically, the particle diameter of described nanogold is 15~20nm；Nanogold and anti-furazolidone monoclonal antibody
Volume ratio is 1000:5~9；Best, the volume ratio of nanogold and anti-furazolidone monoclonal antibody is 1000:7；
Nanogold and goat anti-mouse immunoglobulin G volume ratio are 1000:3~7, best, nanogold is exempted from sheep anti mouse
The volume ratio of epidemic disease Lysozyme is 1000:5.
Further, the furazolidone monoclonal antibody of nano gold mark is prepared using unsaturated labelling method, nanometer
The goat anti-mouse immunoglobulin G of gold mark is prepared using unsaturated labelling method；The particle diameter of nanogold is 15~20nm；Nanometer
The golden and volume ratio of anti-furazolidone monoclonal antibody is 1000:5~9；Nanogold and goat anti-mouse immunoglobulin G volume ratio
For 1000:3~7.
A kind of test strips for detecting furazolidone, described test strips are loaded with described any bioprobe.
Specifically, described test strips include liner plate, nitrocellulose filter, one end of nitrocellulose filter are posted on liner plate
Adsorptive pads are covered, the other end of nitrocellulose filter covers sample pad and pad, the non-covered face of nitrocellulose filter successively
On detection line is transversely set, detection line is coated with CPAOZ-BSA, and pad is loaded with any life described in claim 1-4
Physical prospecting pin.
Further, pad, which is loaded with the preparation method of any bioprobe described in claim 1-4, includes：Will knot
Pad is closed after confining liquid dipping closing, detection probe is sprayed with 5 μ L/cm speed on pad, with 6 μ L/ on pad
Cm speed spraying enhancing probe.
Further, detection line is coated with CPAOZ-BSA preparation method and included：0.5mg/mL CPAOZ-BSA bags
It is coated in by liquid with 0.2 μ L/cm package amount in the detection line of nitrocellulose filter.
Any described bioprobe is used for the application for detecting furazolidone.
The test strips of any described detection furazolidone are used for the application for detecting furazolidone in milk powder.
Compared with prior art, its advantage is with good effect：
(1) high sensitivity.The present invention introduces gold mark secondary antibody and visited as enhancing on the basis of traditional colloidal gold strip
Pin, because the specific recognition of monoclonal antibody and secondary antibody acts on, two kinds of probes can be combined together, so that substantial amounts of nanogold particle
Flock together, form network structure, so as to form apparent red stripes in detection line, play the work of amplified signal
With.
(2) antibody dosage is few.Double probes enhancing signal that the present invention uses, the dosage of antibody, antibody can be substantially reduced
It is fiercer to limited antibody combining site between free analyte and immobilized antigen that the further reduction of dosage will trigger
Competition, so as to bring concentration-response relationship more sensitive between analyte and signal intensity.
(3) it is easy to operate.Double probes that the present invention uses are sprayed on same pad, without adding extra reagent
And operation, so that analysis process is quick, easy, and cost is saved, can be achieved to show the high sensitivity of Furaxone metabolite
Field detection.Therefore miniaturization, portable field quick detection device can be used as, particularly in clinical diagnosis, food hygiene, ring
There is good application prospect in the field such as border monitoring and biochemistry.
Brief description of the drawings
Fig. 1 is immuno-chromatographic test paper strip configuration picture of the present invention；
Fig. 2 is immuno-chromatographic test paper strip enhancing schematic diagram of the present invention；
Fig. 3 is immuno-chromatographic test paper strip detection positive procedure chart prepared by the present invention；
Fig. 4 is immuno-chromatographic test paper strip detection negative sample procedure chart prepared by the present invention；
Fig. 5 is immuno-chromatographic test paper strip detection powdered milk sample sensitivity prepared by the present invention；
Fig. 6 is immuno-chromatographic test paper strip and traditional colloidal gold strip Sensitivity comparison prepared by the present invention；
Fig. 7 is that the immuno-chromatographic test paper strip prepared to the present invention is verified；
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Embodiment
The bioprobe of the present invention is used as detection including the use of the furazolidone monoclonal antibody (monoclonal antibody) of nano gold mark
Probe, the goat anti-mouse immunoglobulin G (secondary antibody) of nano gold mark are sprayed on same combination as enhancing probe, two kinds of probes
On pad, detection line is only coated with nitrocellulose membrane；Due to the specific binding of monoclonal antibody and secondary antibody, two kinds of probes are combined together,
So as to which nanogold thereon flocks together, a gold mark monoclonal antibody can combine several gold medal mark secondary antibodies, and a gold mark secondary antibody is again
Several gold medal mark monoclonal antibodies can be combined, so as to form huge network structure, assemble more nanogold, can be at detection line (T)
It is upper to form apparent red stripes.
Furazolidone monoclonal antibody can be existing monoclonal antibody in the prior art, and preferably applicant seminar oneself grinds
The high sensitivity furazolidone monoclonal antibody of hair, with the main nitrofuran antibiotics no cross reaction of its excess-three kind.Furans azoles
Ketone monoclonal antibody is in publication according to Zhang Daohong et al.《Analytical Chimica Acta》Volume 635
63-69 page titles are " Production of ultrasensitive generic monoclonal antibodies
In the texts of against major aflatoxins using a modified two-step screening procedure " one
What the method for record was prepared：By anti-furazolidone cell infusion and mouse web portion, it is induced to produce Furaxone metabolite
Antiserum, after its ascites is acquired, purified with sad ammonium sulfate method, with polyacrylate hydrogel acid amides electrophoresis to purifying
As a result identified, to obtain the monoclonal antibody of high specific.
Specifically, detection probe and the volume ratio of enhancing probe are 4~6:5~7, best, detection probe is visited with enhancing
The volume ratio of pin is 5:6.
Again specifically, the particle diameter of described nanogold is 15~20nm；Nanogold and anti-furazolidone monoclonal antibody
Volume ratio is 1000:5~9；Best, the volume ratio of nanogold and anti-furazolidone monoclonal antibody is 1000:7；
Nanogold and goat anti-mouse immunoglobulin G volume ratio are 1000:3~7, best, nanogold is exempted from sheep anti mouse
The volume ratio of epidemic disease Lysozyme is 1000:5.
See Fig. 2, its operation principle is：Two kinds of gold mark probes of gold mark monoclonal antibody and gold mark secondary antibody are sprayed on same pad
On, after sample solution is added in the sample pad of test strips lower end, sample solution is by capillarity along test strips to suction
Water cushion direction is moved, and when being moved to pad, two kinds of gold mark probes are successively dissolved, and in the specific binding of antibody and secondary antibody
Lower formation network structure, as sample solution moves to adsorptive pads direction together.When containing object in sample, it will and be tied
Close the gold mark monoclonal antibody combination eluted on pad and together upward swimming, when reaching the detection line of fixed antigen, immobilized antigen
Antigen binding site limited on monoclonal antibody being marked with object competition binding gold, Furaxone metabolite content is higher in sample,
The gold mark probe that immobilized antigen in detection line can combine will be fewer, and the developed band color of formation is more shallow.When immobilized antigen institute
With reference to gold mark probe be less than certain quantity when, will not have red lines appearance at detection line.No matter whether contain in sample
There is furazolidone, the network structure or its conjugate with Furaxone metabolite that the gold mark probe that not tested survey line is intercepted and captured is formed
Will move on to adsorptive pads, thus in sample not Furanzolidon-containing (feminine gender) when be a red stripes, contain a certain amount of furan
For than compareing shallow red stripes or no red stripes during oxazolone (positive) of muttering.
Embodiment 1：
The preparation method of furazolidone monoclonal antibody
The preparation method of furazolidone monoclonal antibody, comprises the following steps：
1. ascites gathers：
Furazolidone cell is cultivated in advance, and when cell length to kilter, cell line injection is used into atoleine in advance
Treated BALB/c mouse, when mouse web portion obvious swell, collect the ascites of the mouse.
2. antibody purification：
Using caprylic acid-ammonium antibody purification, concrete operations are：Ascites is filtered with double-layer filter paper, 4 DEG C, 12000r/
Min centrifuges 15min, draws supernatant.Gained ascites supernatant mixes with the acetate buffer of 4 times of volumes, is slowly added under stirring
The μ L/mL ascites of caprylic acid 33, mixed at room temperature 30min, 4 DEG C of standing 2h.4 DEG C afterwards, 12000r/min centrifugation 30min, abandon precipitation.
After supernatant is filtered with double-layer filter paper, the phosphate buffer of the 0.1mol/LpH values 7.4 of 1/10 filtrate volume is added, uses 2mol/L
Sodium hydroxide solution adjusts mixed liquor pH value to 7.4.4 DEG C of precoolings of supernatant, it is final concentration of to ammonium sulfate to be slowly added to ammonium sulfate
0.277g/mL, 4 DEG C of standing 2h.4 DEG C again, 12000r/min centrifugation 30min, abandon supernatant.Gained 1/10 former ascites volume of precipitation
0.01mol/L phosphate buffers be resuspended, load bag filter, dialysed with pure water.The protein solution fully dialysed is put -80
DEG C refrigerator freezing, is freezed with freeze drier afterwards, is collected freeze-dried powder, is produced purified anti-furazolidone monoclonal antibody,
Antibody is put standby in -20 DEG C of refrigerators.
Acetate buffer is 0.29g sodium acetates, and 0.141mL acetic acid adds water to be settled to obtained by 100mL.
0.1mol/L phosphate buffer is 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride,
Potassium dihydrogen phosphate 0.02g adds water to be settled to obtained by 100mL.Described is that 80g sodium hydroxides add with 2mol/L sodium hydroxide solutions
Water is settled to obtained by 1000mL.
3. the Property Identification of monoclonal antibody：
Its sensitivity and cross reacting rate to Furaxone metabolite is identified with indirect competitive ELISA method.Use polypropylene
Acrylamide gel electrophoresis is identified purification effect.
The anti-furazolidone monoclonal antibody that following embodiment 2-6 are used is what embodiment 1 was prepared.
Embodiment 2：
Nano-Au solution is prepared using reduction of sodium citrate method.Its specific method is：96mL distilled water is taken in taper
In bottle, after heating is boiled, in the state of heating and magnetic stirring, 1mLHAuCl is successively added4It is molten with 4mL trisodium citrates
Liquid, color change is observed, from indigo plant to purple again to red, after solution is changed into red, continue to heat 10min under stirring.It is molten
After liquid cools down at room temperature, 100mL is settled to distilled water in volumetric flask, it is standby to put 4 DEG C of refrigerators.The particle diameter of nanogold is 15
~20nm.
The preparation method of detection probe includes：
50.0mL nano-Au solution is measured, is 5.5 with 0.1mol/L solution of potassium carbonate regulation solution ph；In stirring
0.35mL 1mg/mL furazolidone monoclonal antibody solution is slowly added under state, continues to stir 30min；Add 10% N
Serum albumin solution to its final concentration of 1%, continue stir 30min；After 4 DEG C are placed 2h, 1500r/min centrifugation 15min,
Supernatant is taken, abandons precipitation；By supernatant 12,000r/min centrifugation 30min, abandoning supernatant, add 50.0mL mark washings and protect
Liquid storage；30min is centrifuged with 12,000r/min again, abandoning supernatant, is washed with mark and preserves liquid resuspension, it is dense to respectively obtain 5.0mL
Contracting thing, it is standby to put 4 DEG C of refrigerators；
The preparation method of enhancing probe includes：
50.0mL nano-Au solution is measured, is 5.5 with 0.1mol/L solution of potassium carbonate regulation solution ph；In stirring
0.25mL 1mg/mL goat anti-mouse immunoglobulin G solution is slowly added under state, continues to stir 30min；Add 10% ox blood
Pure protein solution to its final concentration of 1%, continue stir 30min；After 4 DEG C are placed 2h, 1500r/min centrifugation 15min, take
Supernatant, abandon precipitation；By supernatant 12,000r/min centrifugation 30min, abandoning supernatant, add 50.0mL mark washings and preserve
Liquid；30min is centrifuged with 12,000r/min again, abandoning supernatant, is washed with mark and preserves liquid resuspension, respectively obtains 5.0mL concentrations
Thing, it is standby to put 4 DEG C of refrigerators；
0.1mol/L solution of potassium carbonate is：13.8g potassium carbonate is dissolved in pure water and is settled to 1000mL, 0.22 μm of membrane filtration institute
；1mg/mL Furaxone metabolites monoclonal antibody solution is that to be dissolved in 1mL pure for 1mg Furaxone metabolite monoclonal antibodies
It is made in water；10% bovine serum albumin solution is that 10g bovine serum albumin(BSA)s are dissolved in 100mL pure water, 0.22 μm of filter membrane mistake
Filter gained；Mark washing preserves liquid：2.0g PEG-400s, 0.2g Sodium azides, 0.1235g boric acid, pure water are settled to
1000mL, 0.22 μm of membrane filtration gained；
Embodiment 3：
The preparation of quick detection Furaxone metabolite immuno-chromatographic test paper strip
The high-sensitivity immunochromatographitest test strip of quick detection Furaxone metabolite, is shown in Fig. 1, and it is provided with liner plate, lining
It is pasted with adsorptive pads, nitrocellulose filter on plate successively from top to bottom, containing two kinds of gold labeling antibodies (gold mark monoclonal antibody and gold mark two
It is anti-) pad, sample pad, wherein, detection line is laterally set on nitrocellulose filter, and detection line is coated with furazolidone metabolite
Thing-bovine serum albumin(BSA) conjugate (CPAOZ-BSA).
The preparation method of quick detection Furaxone metabolite high-sensitivity immunochromatographitest test strip, comprises the following steps：
1) preparation of adsorptive pads
Blotting paper is cut out to the wide 3mm of growth 18mm specification, produces adsorptive pads
2) preparation of nitrocellulose filter
The coating of detection line：
The conjugate (CPAOZ-BSA) of furazolidone metabolite derivative CPAOZ- bovine serum albumin(BSA)s is configured to
0.5mg/mL solution；With hatched manner by its with 2 μ L/cm package amount be laterally coated in away from nitrocellulose filter along 30mm
Position, then under the conditions of 37 DEG C dry 15min.
3) preparation of sample pad：Glass fibre membrane is cut out to the wide 3mm of growth 8mm specification, is put into confining liquid A and soaks,
10-16h is dried under the conditions of 37 DEG C, sample pad is obtained, then puts room temperature preservation in drier；
Described confining liquid A is 1g bovine serum albumin(BSA)s, 2g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g ten
Phosphate dihydrate disodium hydrogen, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphates add water to be settled to obtained by 100mL.
4) preparation of pad：Glass fibre membrane is cut out to the wide 3mm of growth 15mm specification, is put into confining liquid B and soaks,
Take out, 10-16h is dried under the conditions of 37 DEG C, be laterally sprayed at two kinds of Nano-Au probe solution with a spray mode dried
On pad, the speed of detection probe solution is 5 μ L/cm, and the speed for strengthening probe solution is 6 μ L/cm, and then vacuum refrigeration is done
Dry 3h, put room temperature preservation in drier；
Described confining liquid B is 1g bovine serum albumin(BSA)s, 0.1mL triton x-100s, 0.3g polyvinylpyrrolidones, 2g
Sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride, potassium dihydrogen phosphate
0.02g adds water to be settled to obtained by 100mL.
5) assembling of test strips：Nitrocellulose filter is attached on liner plate first, then sample pad pressure pad 1-
3mm, pad pressure nitrocellulose filter 1-3mm, adsorptive pads pressure nitrocellulose filter 1-3mm are attached on liner plate, produced successively
The immuno-chromatographic test paper strip of quick detection Furaxone metabolite, is shown in Fig. 1.
Embodiment 4：
Immuno-chromatographic test paper strip made from embodiment 3 is used for the application for detecting Furaxone metabolite in powdered milk sample,
Comprise the following steps：
1g skimmed milk powers sample is weighed in 10mL centrifuge tubes, is dissolved with 8mL ultra-pure waters.Then add in the sample different
The Furaxone metabolite standard liquid of concentration, by mixture vortex mixed at room temperature.Then, by 200 μ L dissolved with to carboxyl
The dimethyl sulfoxide (DMSO) of benzaldehyde (0.05M) is added in sample, and each sample is incubated into 16h at 37 DEG C.Then will be derivative
Sample with 10,000rpm centrifuge 10min, collect supernatant, take 100 μ L samples solution as detection liquid be placed in centrifuge tube,
Sample pad one end of test strips is inserted in centrifuge tube, while takes 100 μ L to be not added with the powdered milk sample of Furaxone metabolite and makees
For negative controls, result is read after 10min.
Testing result：The detection line of test strip does not develop the color or color is more shallow than control ELISA test strip line color, sentences
When for the positive, seeing Fig. 3, and detecting line color with control stripes bar solid colour, feminine gender is judged to, sees Fig. 4.As a result show, the examination
Paper slip sensitivity can reach 0.3ng/mL, see Fig. 5.
Embodiment 5：
In order to prove that the immuno-chromatographic test paper strip that the present invention uses is sensitiveer than traditional colloidal gold strip, the present inventor
Also it has been following contrast experiment：
Gold mark list is only sprayed with unlike preparation process in immuno-chromatographic test paper strip prepared by embodiment 3 at pad
It is anti-, nature controlling line (C) is also laterally set on nitrocellulose membrane, coating thing is goat anti-mouse immunoglobulin G, concentration 1mg/mL,
Speed is 1 μ L/cm)；
Test strips prepared by embodiment 3 and embodiment 5 are all used for detecting furazolidone standard liquid, prepared by embodiment 3
The concentration of Furaxone metabolite of ELISA test strip be arranged to 0,0.1,0.3,0.5,0.8,1,3ng/mL, embodiment 5 is made
The concentration of the Furaxone metabolite of standby ELISA test strip is arranged to 0,0.5,1,2,3,5,7ng/mL, after each reaction 10min,
Detection line and nature controlling line colored intensity are visually observed, and records result；
See Fig. 6, its detection of traditional colloidal gold strip is limited to 1ng/mL, and the line concentration that disappears is 5ng/mL, and the present invention makes
Immuno-chromatographic test paper strip detection is limited to 0.3ng/mL, and the line concentration that disappears is 1ng/mL.The above results explanation, what the present invention used
The sensitivity of immuno-chromatographic test paper strip improves 5 times compared to traditional test strips.
Embodiment 6：
In order to prove that the immuno-chromatographic test paper strip that the present invention uses is effective, the present inventor has also done following experiment：
On the basis of immuno-chromatographic test paper strip prepared by embodiment 3, nature controlling line, matter are laterally set on nitrocellulose membrane
The coating thing controlled on line is goat anti-mouse immunoglobulin G, and concentration 1mg/mL, discharge rate is 1 μ L/cm；
Furaxone metabolite standard liquid is configured to 1ng/mL with PBS, takes 100 μ L to be placed in centrifuge tube, will implement
In sample pad one end insertion centrifuge tube of test strips prepared by example 6, result is observed after 10min；
Negative control is used as using PBS simultaneously；
Experimental result is shown in Fig. 7, and during negative control, nature controlling line and detection line have obvious red stripes, work as furazolidone
Metabolite concentration is 1ng/mL, and nature controlling line has clearly red stripes, and detection line does not have, the results showed that, what the present invention developed exempts from
Epidemic disease chromatograph test strip is effective.
Claims (10)
1. a kind of bioprobe, it is characterised in that including detection probe and enhancing probe, described detection probe is nanogold mark
The furazolidone monoclonal antibody of note, described enhancing probe are the goat anti-mouse immunoglobulin G of nano gold mark.
2. bioprobe as claimed in claim 1, it is characterised in that detection probe is 4~6 with the volume ratio for strengthening probe:5
~7.
3. bioprobe as claimed in claim 1 or 2, it is characterised in that the particle diameter of described nanogold is 15~20nm；
The volume ratio of nanogold and anti-furazolidone monoclonal antibody is 1000:5~9；
Nanogold and goat anti-mouse immunoglobulin G volume ratio are 1000:3~7.
4. bioprobe as claimed in claim 1 or 2, it is characterised in that the furazolidone monoclonal antibody of nano gold mark
It is prepared using unsaturated labelling method, the goat anti-mouse immunoglobulin G of nano gold mark is prepared into using unsaturated labelling method
Arrive；
The particle diameter of nanogold is 15~20nm；
The volume ratio of nanogold and anti-furazolidone monoclonal antibody is 1000:5~9；
Nanogold and goat anti-mouse immunoglobulin G volume ratio are 1000:3~7.
5. a kind of test strips for detecting furazolidone, it is characterised in that described test strips are loaded with described in claim 1-4
Any bioprobe.
6. the test strips of detection furazolidone as claimed in claim 5, it is characterised in that described test strips include liner plate,
Nitrocellulose filter is posted on liner plate, one end covering adsorptive pads of nitrocellulose filter, the other end of nitrocellulose filter is successively
Sample pad and pad are covered, detection line is transversely set on the non-covered face of nitrocellulose filter, detection line is coated with
CPAOZ-BSA, pad are loaded with any bioprobe described in claim 1-4.
7. the test strips of detection furazolidone as claimed in claim 6, it is characterised in that pad is loaded with claim 1-
The preparation method of any bioprobe described in 4 includes：By pad after confining liquid dipping closing, with 5 μ L/ on pad
Cm speed spraying detection probe, with 6 μ L/cm speed spraying enhancing probe on pad.
8. the test strips of detection furazolidone as claimed in claim 6, it is characterised in that detection line is coated with CPAOZ-BSA
Preparation method include：0.5mg/mL CPAOZ-BSA coating buffers are coated in nitrocellulose filter with 0.2 μ L/cm package amount
Detection line on.
9. any described bioprobes of claim 1-4 are used for the application for detecting furazolidone.
10. the test strips of any described detection furazolidones of claim 5-8 are used for the application for detecting furazolidone in milk powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710552301.9A CN107515299A (en) | 2017-07-07 | 2017-07-07 | A kind of bioprobe, the test strips for detecting furazolidone and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710552301.9A CN107515299A (en) | 2017-07-07 | 2017-07-07 | A kind of bioprobe, the test strips for detecting furazolidone and its application |
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| CN107515299A true CN107515299A (en) | 2017-12-26 |
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| CN201710552301.9A Pending CN107515299A (en) | 2017-07-07 | 2017-07-07 | A kind of bioprobe, the test strips for detecting furazolidone and its application |
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