CN105044346A - Quantum dot fluorescence immunochromatography test strip for double quantification of mycotoxin and preparation method of quantum dot fluorescence immunochromatography test strip - Google Patents
Quantum dot fluorescence immunochromatography test strip for double quantification of mycotoxin and preparation method of quantum dot fluorescence immunochromatography test strip Download PDFInfo
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Abstract
The invention relates to an immuno-chromatographic technology, and aims to provide a quantum dot fluorescence immunochromatography test strip for double quantification of mycotoxin and a preparation method of the quantum dot fluorescence immunochromatography test strip. According to the test strip, a sample pad, a quantum dot antibody marker fixing pad, a nitrocellulose membrane and an absorption pad are sequentially arranged on the surface of a polyvinyl chloride backboard; two detection lines and a quality control line are arranged on the nitrocellulose membrane; a labeled compound of a mycotoxin monoclonal antibody and carboxyl water-soluble quantum dots is fixed on the fixing pad; the quality control line is formed by spraying a goat anti-mouse polyclonal antibody; each detection line is formed by spraying to-be-detected mycotoxin coupling antigen; the two detection lines comprise different mycotoxin coupling antigens. According to the invention, the competition law principle is adopted for qualitative detection and quantitative analysis of mycotoxin, so that two mycotoxins can be detected at the same time, the prepared chromatography test strip has good sensitivity and high stability, is simple in operation and is particularly suitable for rapid diagnosis on site.
Description
Technical field
The present invention relates to immunochromatography technique, specifically, relate to simultaneously to the preparation method of the immuno-chromatographic test paper strip based on quantum dot-labeled mycotoxin monoclonal antibody specific and fluorescence developing that two kinds of mycotoxins in food detect simultaneously.
Background technology
All kinds of poisonings that present stage is often sent out, except some by mineral compound as except potassium cyanide, arsenic causes, the overwhelming majority is all from biotoxin, and microbial toxin is modal, the food poisoning overwhelming majority is that bacteriotoxin or mycotoxin cause, mycotoxin due to pollution range extensively, the feature such as strong toxicity is subject to the great attention in food safety monitoring field day by day.
Mycotoxin is mycetogenetic Small molecular secondary metabolite, is the important pollutant in Fodder and food.The method that the detection of mycotoxin is common is euzymelinked immunosorbent assay (ELISA) (ELISA), although euzymelinked immunosorbent assay (ELISA) is simple to operate, result easily obtains, but need supporting equipment, as constant incubator, microplate reader etc., and often consuming time longer, clinical or on-the-spot quick diagnosis can not be used for.Compare ELISA, immuno-chromatographic test paper strip can be used for on-the-spot quick detection, and does not need other supplementary instruments, can make up the shortcoming that other detection methods are complicated and consuming time.
Summary of the invention
The technical problem to be solved in the present invention is, to overcome in prior art enzyme-labeled immunity technology length consuming time and conventional colloidal gold immuno-chromatography test paper strip sensitivity is not high and the shortcoming such as single channel, provide a kind of the quantum dot fluorescence immuno-chromatographic test paper strip and the preparation method that detect mycotoxin.
For technical solution problem, solution of the present invention is:
A kind of quantum dot fluorescence immuno-chromatographic test paper strip simultaneously detecting two kinds of mycotoxins is provided, comprise the black strip Polyvinylchloride backboard as base material, sample pad, quantum dot antibody labeling thing fixed bolster, nitrocellulose filter and absorption pad is being arranged successively on its surface, adjacent pads or intermembranously to lap one another along carapace length direction; On nitrocellulose filter, be provided with backboard with wide be spaced two detection lines and a nature controlling line, wherein nature controlling line is positioned at adsorptive pads side;
On quantum dot antibody labeling thing fixed bolster, to spray, the mode of spreading or immersion is fixed with the labeled complex of mycotoxin monoclonal antibody and carboxyl water-soluble quantum dot; Described nature controlling line is sprayed on nitrocellulose filter by goat against murine polyclonal antibody to be formed, and described detection line is sprayed on nitrocellulose filter by mycotoxin coupled antigen to be checked to be formed, and contained by two detection lines, mycotoxin coupled antigen is different.
In the present invention, the mycotoxin monoclonal antibody on described quantum dot antibody labeling thing fixed bolster and the labeled complex of carboxyl water-soluble quantum dot, its preparation method is:
Be that the borate buffer solution of 0.01M, pH=7.4 configures coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solution respectively with concentration, concentration is 10mg/ml; The water-soluble quantum dot getting 50 μ l carboxyl modified is managed to 2mlEP, add 2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid that 200 μ l concentration are 0.05M, pH=6, and add EDC and NHS solution according to the carboxyl number on water-soluble quantum dot; The MES damping fluid of 1ml containing pH=6,0.05M of 5 μ g specificity mycotoxin monoclonal antibodies is added after the centrifugal 30min of 15000rpm abandons supernatant after room temperature shaker activation 30min; After room temperature shaker reaction 2h, the centrifugal 30min of 15000rpm abandons supernatant, and resuspended, centrifugal with the phosphate buffer (PBS) of 1ml0.01M, pH=7.4, washes away unreacted antibody; Repeat above-mentioned resuspended, centrifugally operated three times; The borate buffer solution finally adding 0.05M, pH=8.0 of 100 μ l, as conserving liquid, obtains the labeled complex of mycotoxin monoclonal antibody and carboxyl water-soluble quantum dot, 4 DEG C of placements, for subsequent use.
In the present invention, the mycotoxin corresponding to described mycotoxin monoclonal antibody is any one in ochratoxin A, aflatoxin B1, zearalenone, vomitoxin etc.
In the present invention, the width of this test strips is 0.4cm, and the length of sample pad, quantum dot antibody labeling thing fixed bolster, nitrocellulose filter and absorption pad is respectively 1.7cm, 0.8cm, 2.5cm, 1.7cm; Wherein, the sample pad length that overlaps with the overlap joint of quantum dot antibody labeling thing fixed bolster is 0.2cm, and the quantum dot antibody labeling thing fixed bolster length that overlaps with the overlap joint of nitrocellulose filter is 0.2cm, and the nitrocellulose filter length that overlaps with the overlap joint of absorption pad is 0.3cm; On nitrocellulose filter, two detection lines and the spacing between detection line-2 and nature controlling line are respectively 0.4cm, 0.4cm.
Invention further provides the method for the front described quantum dot fluorescence immuno-chromatographic test paper strip of preparation, comprise the steps:
(1) quantum dot antibody labeling thing fixed bolster is prepared
Be that the borate buffer solution of 0.01M, pH=7.4 configures coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solution respectively with concentration, concentration is 10mg/ml; The water-soluble quantum dot getting 50 μ l carboxyl modified is managed to 2mlEP, add 2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid that 200 μ l concentration are 0.05M, pH=6, and add EDC and NHS solution according to the carboxyl number on water-soluble quantum dot; The MES damping fluid of 1ml containing 0.05M, pH=6 of 5 μ g specificity mycotoxin monoclonal antibodies is added after the centrifugal 30min of 15000rpm abandons supernatant after room temperature shaker activation 30min; After room temperature shaker reaction 2h, the centrifugal 30min of 15000rpm abandons supernatant, and resuspended, centrifugal with the phosphate buffer (PBS) of 1ml0.01M, pH=7.4, washes away unreacted antibody; Repeat above-mentioned resuspended, centrifugally operated three times; The borate buffer solution finally adding 0.05M, pH=8.0 of 100 μ l is as conserving liquid, and the labeled complex 4 DEG C obtaining mycotoxin monoclonal antibody specific and carboxyl modified water-soluble quantum dot is placed, for subsequent use;
In the mode of spraying, spreading or immersion, the labeled complex of mycotoxin monoclonal antibody specific and carboxyl modified water-soluble quantum dot is fixed on quantum dot antibody labeling thing fixed bolster, for subsequent use 37 DEG C of dryings 2 hours;
(2) get on strip Polyvinylchloride backboard that nitrocellulose filter is affixed on as base material, some film instrument sprays two detection lines and a nature controlling line on nitrocellulose filter simultaneously, and wherein nature controlling line is positioned at side; The spray coating liquor of nature controlling line is goat against murine polyclonal antibody, and the spray coating liquor of two detection lines is different types of mycotoxin coupled antigens to be checked; 37 DEG C of dryings after 2 hours, be placed in polyglycol solution and soak 30 minutes, for subsequent use 37 DEG C of dryings 2 hours after drying;
(3) sample pad, quantum dot antibody labeling thing fixed bolster and absorption pad are arranged on Polyvinylchloride backboard, and sample pad, quantum dot antibody labeling thing fixed bolster, nitrocellulose filter and absorption pad are arranged in order, the nature controlling line of nitrocellulose filter is positioned at adsorptive pads side, and adjacent pads or intermembranously to lap one another;
(4) material of Polyvinylchloride backboard and attaching thereof is cut into the wide chromatography strip of 4mm, chromatography strip is put into plastics board, use card press machine compacting.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention replaces conventional collaurum with quantum dot fluorescence material and marks mycotoxin monoclonal antibody specific, competition law principle is adopted to carry out qualitative detection and quantitative test to mycotoxin, nitrocellulose filter wrap simultaneously by two detection lines, be respectively mycotoxin coupled antigen to be checked, can detect two kinds of mycotoxins simultaneously, prepared chromatograph test strip has good sensitivity and stability, and simple and efficient to handle, is specially adapted to on-the-spot quick diagnosis.
Accompanying drawing explanation
Fig. 1 is the structural representation of quantum dot fluorescence immuno-chromatographic test paper strip in the present invention.
Reference numeral in figure: 1, sample pad; 2, quantum dot antibody labeling thing fixed bolster; 3, nitrocellulose filter; 4, absorption pad; 5, detection line-1; 6, detection line-2; 7, nature controlling line.
Embodiment
Inventive principle describes:
Quantum dot fluorescence immuno-chromatographic test paper strip in the present invention is based on quantum dot fluorescence material and immunochromatography technique.(CdSe is for core with the water-soluble cadmium selenide/ZnS quantum dots (CdSe/ZnS) of carboxyl modified for this kind of immunochromatography bar, ZnS is shell, quantum dot surface can carry out covalent coupling with specific biological molecules after carboxyl functional group is modified) replace conventional collaurum, covalent coupling is carried out with mycotoxin monoclonal antibody under the effect of coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS), competition law principle is adopted to carry out qualitative detection and quantitative test to mycotoxin levels in food.
This can detect the quantum dot fluorescence immuno-chromatographic test paper strip of two kinds of mycotoxins simultaneously, comprise the black strip Polyvinylchloride backboard as base material, sample pad (1), quantum dot antibody labeling thing fixed bolster (2), nitrocellulose filter (3) and absorption pad (4) is being arranged successively on its surface, adjacent pads or intermembranously to lap one another along carapace length direction; On nitrocellulose filter (3), be provided with backboard with the wide detection line (5) be spaced, detection line (6) and nature controlling line (7), wherein nature controlling line (7) is positioned at the side of adsorptive pads (4); On the fixed bolster (3) of quantum dot antibody labeling thing, to spray, the mode of spreading or immersion is fixed with mycotoxin monoclonal antibody specific and carboxyl modified water-soluble quantum dot labeled complex; Nature controlling line (7) goat against murine polyclonal antibody is sprayed at the upper formation of nitrocellulose filter (3), detection line-1 (5) and detection line-2 (6) mycotoxin coupled antigen to be checked are sprayed at the upper formation of nitrocellulose filter (3), and detection line-1 (5) and the contained mycotoxin coupled antigen of detection line-2 (6) are different.
The width of this test strips is 0.4cm, and the length of sample pad (1), quantum dot antibody labeling thing fixed bolster (2), nitrocellulose filter (3) and absorption pad (4) is respectively 1.7cm, 0.8cm, 2.5cm, 1.7cm; Wherein, sample pad (1) length that overlaps with the overlap joint of quantum dot antibody labeling thing fixed bolster 2 is 0.2cm, quantum dot antibody labeling thing fixed bolster (2) length that overlaps with the overlap joint of nitrocellulose filter (3) is 0.2cm, and nitrocellulose filter (3) length that overlaps with the overlap joint of absorption pad (4) is 0.3cm; On nitrocellulose filter (3), the spacing of detection line-1 (5), detection line-2 (6) and nature controlling line (7) is respectively 0.4cm, 0.4cm.
Mycotoxin monoclonal antibody specific on described quantum dot antibody labeling thing fixed bolster (2) and carboxyl modified water-soluble quantum dot labeled complex, its preparation method is: be that the borate buffer solution of 0.01M, pH=7.4 configures coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solution respectively with concentration, concentration is 10mg/ml; The water-soluble quantum dot getting 50 μ l carboxyl modified is managed to 2mlEP, add 2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid that 200 μ l concentration are 0.05M, pH=6, and add EDC and NHS solution according to the carboxyl quantity on carboxyl water-soluble quantum dot; After room temperature shaker activation 30min, the centrifugal 30min of 15000rpm abandons supernatant, adds the MES damping fluid of 1ml containing 0.05M, pH=6 of 5 μ g specificity mycotoxin monoclonal antibodies; After room temperature shaker reaction 2h, the centrifugal 30min of 15000rpm is also resuspended, centrifugal with the phosphate buffer of 1ml0.01M, pH=7.4 after abandoning supernatant, washes away unreacted antibody; Repeat above-mentioned resuspended, centrifugally operated three times; Finally add the conserving liquid of borate buffer solution as the quantum dot-labeled monoclonal antibody obtained of 0.05M, pH=8.0 of 100 μ l, 4 DEG C of placements, for subsequent use.
Mycotoxin corresponding to mycotoxin monoclonal antibody of the present invention, in the present invention, the mycotoxin corresponding to described mycotoxin monoclonal antibody is any one in ochratoxin A, aflatoxin B1, zearalenone, vomitoxin.
The preparation method of quantum dot fluorescence immuno-chromatographic test paper strip, comprises the steps:
1, quantum dot antibody labeling thing fixed bolster is prepared
Be that the borate buffer solution of 0.01M, pH=7.4 configures coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solution respectively with concentration, concentration is 10mg/ml; The water-soluble quantum dot getting 50 μ l carboxyl modified is managed to 2mlEP, add 2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid that 200 μ l concentration are 0.05M, pH=6, and add EDC and NHS solution according to the carboxyl quantity on water-soluble quantum dot; The MES damping fluid of 1ml containing the pH=6 of 5 μ g specificity mycotoxin monoclonal antibodies is added after the centrifugal 30min of 15000rpm abandons supernatant after room temperature shaker activation 30min; After room temperature shaker reaction 2h, the centrifugal 30min of 15000rpm abandons supernatant, and resuspended, centrifugal with the phosphate buffer (PBS) of 1ml0.01M, pH=7.4, washes away unreacted antibody; Repeat above-mentioned resuspended, centrifugally operated three times; The borate buffer solution finally adding 0.05M, pH=8.0 of 100 μ l, as conserving liquid, obtains mycotoxin monoclonal antibody specific and carboxyl modified water-soluble quantum dot labeled complex 4 DEG C is placed, for subsequent use;
In the mode of spraying, spreading or immersion, mycotoxin monoclonal antibody specific and carboxyl modified water-soluble quantum dot labeled complex are fixed on quantum dot antibody labeling thing fixed bolster 2, for subsequent use 37 DEG C of dryings 2 hours;
2, the spraying of detection line and nature controlling line: get on strip Polyvinylchloride backboard that nitrocellulose filter (3) is affixed on as base material, point film instrument sprays detection line-1 (5), detection line-2 (6), nature controlling line (7) at nitrocellulose filter (3) simultaneously, wherein nature controlling line (7) is positioned at side; The spray coating liquor of nature controlling line (7) be goat anti mouse polyclonal antibody, the spray coating liquor of detection line-1 (5) and detection line-2 (6) is different types of mycotoxin coupled antigen to be checked; 37 DEG C of dryings after 2 hours, be placed in polyglycol solution and soak 30 minutes, for subsequent use 37 DEG C of dryings 2 hours after drying;
3, the assembling of immuno-chromatographic test paper strip: sample pad (1), quantum dot antibody labeling thing fixed bolster (2) and absorption pad (4) are arranged on Polyvinylchloride backboard, and sample pad (1), quantum dot antibody labeling thing fixed bolster (2), nitrocellulose filter (3) and absorption pad (4) are arranged in order, nature controlling line (7) is positioned at adsorptive pads (4) side, and adjacent pads or intermembranously to lap one another; The material of Polyvinylchloride backboard and attaching thereof is cut into the wide chromatography strip of 4mm, chromatography strip is put into plastics board, use card press machine compacting.
Embodiment 1: the preparation of ochratoxin A and aflatoxin B1 quantum dot fluorescence immuno-chromatographic test paper strip in dual quantitative detection food
Concrete preparation process is as follows:
1, quantum dot-ochratoxin A monoclonal antibody label and quantum dot-aflatoxin B1 labeling of monoclonal antibody thing fixed bolster is prepared
The conventional method preparation also anti-ochratoxin A of purifying and aflatoxin B1 mouse monoclonal antibody, concentration is respectively 5mg/ml and 3mg/ml, for subsequent use;
Prepare the labeled complex of mycotoxin monoclonal antibody and carboxyl water-soluble quantum dot: use borate buffer solution (0.01M respectively, pH=7.4) coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solution is configured, concentration is 10mg/ml, get the water-soluble quantum dot of carboxyl modified (purchased from Haiwang Yingtelong Biological Technology Co., Ltd., Shenzhen City, article No. QD618-C-S001) 50 μ l are in 2mlEP pipe and add the MES damping fluid of 200 μ l0.05M, after adding coupling agent EDC and NHS of 3 μ l respectively according to mol ratio (the carboxyl number on water-soluble quantum dot: EDC:NHS=1:5000:5000), room temperature shaker activation 30min, supernatant is abandoned after the centrifugal 30min of 15000rpm, add the 0.05M of 1ml containing the ochratoxin A monoclonal antibody specific of 5 μ g, the MES damping fluid of pH=7.4, room temperature shaker reaction 2h, supernatant is abandoned after the centrifugal 30min of 15000rpm, and with 1ml0.01M phosphate buffer (0.01M, pH=7.4) resuspended, centrifugal, wash away unreacted antibody, repeat above-mentioned resuspended, centrifugally operated three times, finally add conserving liquid (the 0.05M borate buffer solution of 100 μ l, pH=8.0) after resuspended, 4 DEG C of placements, for subsequent use.According to identical method coupling aflatoxin B1 monoclonal antibody and carboxyl water-soluble quantum dot, obtain labelled antibody compound;
The spray parameters of set point film instrument is 30.0 μ l/cm, will be connected to the C pipe joint of a film instrument after sub-for two amounts point-monoclonal antibody complex equal-volume mixing.Getting specification is that the quantum dot antibody labeling thing fixed bolster 2 of 30cm × 1cm is positioned on a motion platform of film instrument.Opening point film instrument, quantum dot antibody labeling thing fixed bolster 2 sprays antibody quantum dot compound, 37 DEG C of dryings 2 hours.
2, the spraying of quantum dot immune chromatograph test strip detection line and nature controlling line: get nitrocellulose filter 3 and be affixed on low background PVC backboard, the spray parameters of set point film instrument is 2 μ l/cm.Get the aflatoxin B1 coupled antigen AFB1-OVA1.5ml of ochratoxin A coupled antigen OTA-OVA1.5ml and 2mg/ml of 5mg/ml, receive A, B pipe joint place of a film instrument respectively, and the goat against murine polyclonal antibody getting 4mg/ml receives the C pipe joint of a film instrument simultaneously, wherein A, B, the pipeline opening spacing of C is 0.4cm, is placed in by the PVC backboard posting nitrocellulose filter 3 on the to-and-fro movement platform of a film instrument.Opening point film instrument, nitrocellulose filter 3 sprays mycotoxin coupled antigen and Goat anti-mouse antibodies respectively, 37 DEG C of dryings 2 hours.Backboard is placed in polyglycol solution immersion and dries rear 37 DEG C of dryings 2 hours after 30 minutes.
3, the assembling of immuno-chromatographic test paper strip: stick absorption pad (4) in the one end being fixed on the close Goat anti-mouse antibodies of the nitrocellulose filter in PVC board (3), nitrocellulose filter (3) and absorption pad (4) overlap 0.3cm in length, quantum dot antibody labeling thing fixed bolster (2) and sample pad (1) is being sticked successively near mycotoxin coupled antigen one end, quantum dot antibody labeling thing fixed bolster (2) and nitrocellulose filter (3) overlap 0.2cm in length, sample pad (1) and 0.2cm that quantum dot antibody labeling thing fixed bolster (2) length direction overlaps, finally the material of PVC backboard and attaching thereof is cut into the wide chromatography strip of 4mm, chromatography strip is put into plastics board, use card press machine compacting.
When adding detected sample, be positioned at quantum dot antibody complex on quantum dot antibody labeling thing fixed bolster (2) by moving to absorption pad one end after chromatography effect and sample blending, when kind of the mycotoxin to be checked of two in sample exceeds standard (>=5 μ g/kg), antibody quantum dot compound can be combined completely, make not have the coupled antigen on unnecessary quantum dot-mycotoxin monoclonal antibody complex and detection line-1 (5) and detection line-2 (6) to combine, now under uviol lamp shines, detection line-1 (5) and detection line-2 (6) do not have fluorescence, be positive, show that material to be checked exceeds standard.If there is fluorescence, is judged to be that detected sample is negative, shows content of material to be checked in allowed limits.Whether two kinds of content of material to be checked no matter in measuring samples exceed standard, and antibody quantum dot compound all can move to nature controlling line (7) place, is combined with Goat anti-mouse antibodies, shows fluorescent bands under ultraviolet irradiation, points out this detection system effective; If this place does not show fluorescent bands, show that this detection system lost efficacy, testing result is invalid.If the positive of being judged to be, two kinds of mycotoxin standard items of configurable variable concentrations, read detection line and nature controlling line fluorescent value, calculate the fluorescence ratio of detection line/nature controlling line, with mycotoxin standard concentration logarithm for horizontal ordinate, with corresponding fluorescence ratio for ordinate Criterion curve, quantitative test is carried out to the concentration of mycotoxin in sample.
Apply dual quantitative quantum dot fluorescence immuno-chromatographic test paper strip to detect the 60 parts of food samples gathered, and carried out contrasting (ochratoxin A: article No. R1311, aflatoxin B1: article No. R1211 with ELISA import reagent box testing result; All be purchased from Germany visit send out).(ochratoxin A content >=5 μ g/kg during qualitative detection, aflatoxin B1 content >=5 μ g/kg), ochratoxin A positive sample is 5, aflatoxin B1 positive sample is 7, 100% is reached with the coincidence rate of kit, and through quantitative test, the content of ochratoxin A is between 1.7 μ g/kg-4.3 μ g/kg, the content of aflatoxin B1 is between 1.2 μ g/kg-4.8 μ g/kg, the sensitivity of quantitative detection is up to state standards (5 μ g/kg), the fast qualitative that can be used for ochratoxin A and aflatoxin B1 in food detects and quantitative test.
Special instruction:
Although only list competition principle in embodiments of the invention to carry out qualitative detection and quantitative test to two kinds of mycotoxins simultaneously.The invention provides described based on quantum dot fluorescence material and immunochromatography technique while detect the method for two kinds of mycotoxins after, the technical ability that those skilled in the art can grasp according to it be drawn inferences about other cases from one instance, and realizes the object detecting other target substances.
Be more than in conjunction with specific embodiments son the present invention is done further describe.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; under the premise without departing from the principles of the invention; the present invention and application thereof also have various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (5)
1. dual quantitative mycotoxin quantum dot fluorescence immuno-chromatographic test paper strip, comprise the black strip Polyvinylchloride backboard as base material, it is characterized in that, sample pad, quantum dot antibody labeling thing fixed bolster, nitrocellulose filter and absorption pad is being arranged successively on its surface, adjacent pads or intermembranously to lap one another along carapace length direction; On nitrocellulose filter, be provided with backboard with wide be spaced two detection lines and a nature controlling line, wherein nature controlling line is positioned at adsorptive pads side;
On quantum dot antibody labeling thing fixed bolster, to spray, the mode of spreading or immersion is fixed with the labeled complex of mycotoxin monoclonal antibody and carboxyl water-soluble quantum dot; Described nature controlling line is sprayed on nitrocellulose filter by goat against murine polyclonal antibody to be formed, and described detection line is sprayed on nitrocellulose filter by mycotoxin coupled antigen to be checked to be formed, and contained by two detection lines, mycotoxin coupled antigen is different.
2. quantum dot fluorescence immuno-chromatographic test paper strip according to claim 1, is characterized in that, the mycotoxin monoclonal antibody on described quantum dot antibody labeling thing fixed bolster and the labeled complex of carboxyl water-soluble quantum dot, and its preparation method is:
Be that the borate buffer solution of 0.01M, pH=7.4 configures coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide solution respectively with concentration, concentration is 10mg/ml; The water-soluble quantum dot getting 50 μ l carboxyl modified is managed to 2mlEP, adds 2-(N-morpholino) ethanesulfonic acid buffer that 200 μ l concentration are 0.05M, pH=6, and adds EDC and NHS solution according to the carboxyl quantity on water-soluble quantum dot; The MES damping fluid of 1ml containing 0.05M, pH=6 of 5 μ g specificity mycotoxin monoclonal antibodies is added after the centrifugal 30min of 15000rpm abandons supernatant after room temperature shaker activation 30min; After room temperature shaker reaction 2h, the centrifugal 30min of 15000rpm abandons supernatant, and resuspended with the phosphate buffer of 1ml0.01M, pH=7.4, centrifugal, washes away unreacted antibody; Repeat above-mentioned resuspended, centrifugally operated three times; The borate buffer solution finally adding 0.05M, pH=8.0 of 100 μ l, as conserving liquid, obtains the labeled complex of mycotoxin monoclonal antibody and carboxyl water-soluble quantum dot, 4 DEG C of placements, for subsequent use.
3. quantum dot fluorescence immuno-chromatographic test paper strip according to claim 1, it is characterized in that, mycotoxin corresponding to described mycotoxin monoclonal antibody is any one in ochratoxin A, aflatoxin B1, zearalenone, vomitoxin.
4. according to the quantum dot fluorescence immuno-chromatographic test paper strip described in claims 1 to 3 any one, it is characterized in that, the width of this test strips is 0.4cm, and the length of sample pad, quantum dot antibody labeling thing fixed bolster, nitrocellulose filter and absorption pad is respectively 1.7cm, 0.8cm, 2.5cm, 1.7cm; Wherein, the sample pad length that overlaps with the overlap joint of quantum dot antibody labeling thing fixed bolster is 0.2cm, and the quantum dot antibody labeling thing fixed bolster length that overlaps with the overlap joint of nitrocellulose filter is 0.2cm, and the nitrocellulose filter length that overlaps with the overlap joint of absorption pad is 0.3cm; On nitrocellulose filter, two detection lines and the spacing between detection line-2 and nature controlling line are respectively 0.4cm, 0.4cm.
5. prepare the method for quantum dot fluorescence immuno-chromatographic test paper strip described in claim 1, it is characterized in that, comprise the steps:
(1) quantum dot antibody labeling thing fixed bolster is prepared
Be that the borate buffer solution of 0.01M, pH=7.4 configures coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide solution respectively with concentration, concentration is 10mg/ml; The water-soluble quantum dot getting 50 μ l carboxyl modified is managed to 2mlEP, adds 2-(N-morpholino) ethanesulfonic acid buffer that 200 μ l concentration are 0.05M, pH=6, and adds EDC and NHS solution according to the carboxyl quantity on water-soluble quantum dot; After room temperature shaker activation 30min, the centrifugal 30min of 15000rpm abandons supernatant, adds the MES damping fluid of 1ml containing 0.05M, pH=6 of 5 μ g specificity mycotoxin monoclonal antibodies; After room temperature shaker reaction 2h, the centrifugal 30min of 15000rpm abandons supernatant, and resuspended with the phosphate buffer of 1ml0.01M, pH=7.4, centrifugal, washes away unreacted antibody; Repeat above-mentioned resuspended, centrifugally operated three times; The borate buffer solution finally adding 0.05M, pH=8.0 of 100 μ l, as conserving liquid, obtains the labeled complex of mycotoxin monoclonal antibody specific and carboxyl modified water-soluble quantum dot, 4 DEG C of placements, for subsequent use;
In the mode of spraying, spreading or immersion, the labeled complex of mycotoxin monoclonal antibody specific and carboxyl modified water-soluble quantum dot is fixed on quantum dot antibody labeling thing fixed bolster, for subsequent use 37 DEG C of dryings 2 hours;
(2) spraying of detection line and nature controlling line: get on strip Polyvinylchloride backboard that nitrocellulose filter is affixed on as base material, some film instrument sprays two detection lines and a nature controlling line on nitrocellulose filter simultaneously, and wherein nature controlling line is positioned at side; The spray coating liquor of nature controlling line is goat against murine polyclonal antibody, and the spray coating liquor of two detection lines is different types of mycotoxin coupled antigens to be checked; 37 DEG C of dryings after 2 hours, be placed in polyglycol solution and soak 30 minutes, for subsequent use 37 DEG C of dryings 2 hours after drying;
(3) assembling of immuno-chromatographic test paper strip: sample pad, quantum dot antibody labeling thing fixed bolster and absorption pad are arranged on Polyvinylchloride backboard, and sample pad, quantum dot antibody labeling thing fixed bolster, nitrocellulose filter and absorption pad are arranged in order, the nature controlling line of nitrocellulose filter is positioned at adsorptive pads side, and adjacent pads or intermembranously to lap one another; The material of Polyvinylchloride backboard and attaching thereof is cut into the wide chromatography strip of 4mm, chromatography strip is put into plastics board, use card press machine compacting.
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