CN104007262A - Cadmium ion colloidal gold immunochromatographic assay test paper strip and preparation method and application thereof - Google Patents
Cadmium ion colloidal gold immunochromatographic assay test paper strip and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cadmium ion colloidal gold immunochromatographic assay test paper strip and a preparation method and application thereof, the cadmium ion colloidal gold immunochromatographic assay test paper strip is prepared according to the following preparation method: (1) preparation and purification of a cadmium-ion-resistant monoclonal antibody; (2) preparation of detection antigen cadmium-vinyl diamine-N, N, N ', N '-tetraacetic acid-bovine serum albumin; (3) colloidal gold solution preparation; (4) preparation of a cadmium-ion-resistant monoclonal antibody labeled colloidal gold solution; (5) metal spraying; (6) coating of a cellulose nitrate film with C and T lines; and (7) assembling of the cadmium ion colloidal gold immunochromatographic assay test paper strip. The invention also includes the application of the cadmium ion colloidal gold immunochromatographic assay test paper strip. The cadmium ion colloidal gold immunochromatographic assay test paper strip is low in cost and high in detection precision, and can be used in large-scale, rapid, convenient and accurate detection of residual heavy metal cadmium in grains and products processed by the grains.
Description
Technical field
The present invention relates to a kind of colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof and application, especially relate to a kind of cadmium ion colloidal gold immune chromatography test strip and preparation method thereof and application.
Background technology
Cadmium is present in environment with divalent ion form, is the Heavy Metals in water pollution, can cause toxic reaction after entering human body, and renal function is destroyed, and causes the symptoms such as enterogastritis and anaemia, accumulates the easy cause cancer of too much cadmium in bone.WHO is the foundation of the toxicity based on to kidney to the safety standard of cadmium, and the upper limit is weekly per kilogram of body weight 7 micrograms.The people that this is equivalent to one 60 kilograms, is no more than 60 micrograms every day.The safety standard of China's rice is 0.2 milligram of per kilogram, yet in " cadmium rice " event of China Report in 2013, the content of cadmium reaches as high as 1.005 milligrams of per kilograms, seriously surpasses safety of China production standard.
At present, the detection method of heavy metal cadmium mainly contains:
(1) physics and chemistry method: atomic absorption spectrophotometry (AAS), inductively coupled plasma-atomic emission spectrometry (ICP-AES), inductivity coupled plasma mass spectrometry analysis (ICP-MS), atomic fluorescence spectrometry (AFS) etc.The feature of these methods is sensitive, accurate, but needs complicated instrument and equipment, special analytical technology personnel, and standard items are expensive, and pre-treatment trouble can not detect a large amount of samples simultaneously, is not suitable for scene and applies on a large scale.
(2) immunology detection technology-enzyme-linked immuno-sorbent assay (ELISA): it detects principle is utilize cadmium ion and the competition binding antibody of the cadmium ion in standard items in sample and with this, cadmium in sample carried out to qualitative or quantitative test, the method is easier fast, can process a large amount of samples simultaneously.But expensive, single testing cost is high.
In prior art, also do not use colloidal gold immunity chromatography to detect cadmium ion in food or water.
Summary of the invention
The technical problem to be solved in the present invention is, overcome the deficiencies in the prior art, a kind of cadmium ion colloidal gold immune chromatography test strip and preparation method thereof and application are provided, can on a large scale grain and the residual heavy metal cadmium of converted products thereof are carried out fast, facilitate, be detected accurately.
The technical scheme that the present invention solves its technical matters employing is:
The present invention's cadmium ion colloidal gold immune chromatography test strip, is prepared from accordance with the following methods:
(1) preparation of Cadmium resistance ion monoclonal antibody and purifying: with difunctional vinyl diamines-N, N, N ', N '-tetraacethyl sequestrant (iEDTA) is coupled to cadmium ion on bovine serum albumin, mix incomplete Freund's adjuvant immunity BALB/c mouse, get immune mouse spleen cell and SP2/0 myeloma cell and merge, filter out the positive cell strain of stably excreting Cadmium resistance ion monoclonal antibody and expand and cultivate, injection cell enters in Mice Body, to induce ascites; Get 2-4 ml ascites, add and be equivalent to ascites volume 1.8-2.2 sodium-acetate buffer doubly, regulate pH to 4.0-5.0, obtain mixed liquor; It is sad under stirring at room, dropwise to add, and described volume ratio sad and described mixed liquor is 30-40 μ l/ml; Standing at 2-8 ℃, centrifugal, obtain supernatant; Regulate pH to 7.0-7.8, under 2-8 ℃ of cooling bath, add ammonium sulfate, controlling ammonium sulfate final concentration is preferably 0.277 g/ml of 0.25-0.30g/ml(), standing 1-2h; Then 2-8 ℃, the centrifugal 10-20min of 8000-12000r/min, abandon supernatant, precipitation is dissolved in the PBS solution (phosphate buffered solution) of 0.008-0.012mol/L and dialyses, 2-8 ℃ of standing over night, the centrifugal 10-20min of 8000-12000r/min, abandons precipitation; Obtain Cadmium resistance ion monoclonal antibody;
The volumetric molar concentration of described sodium-acetate buffer is 0.05-0.08mol/L, and pH value is 4.5-5.5, and the volumetric molar concentration of described phosphate buffered solution is 0.008-0.012mol/L, and pH value is 7.0-7.8;
(2) detectable antigens cadmium-vinyl diamines-N, N, N ', the preparation of N '-tetraacethyl-bovine serum albumin: get vinyl diamines-N, N, N ', N '-tetraacethyl be take mass volume ratio and as 15-25mg/ml is dissolved in, in dimethyl sulfoxide (DMSO), is formed A liquid; The anti-bovine serum albumin of rabbit be take to mass volume ratio as 15-16mg/ml is dissolved in Tri(Hydroxymethyl) Amino Methane Hydrochloride, add respectively the triethylamine and the Macrogol 600 that account for Pehanorm hydrochloride volume 1-2%, form B liquid; A liquid is dropwise added in B liquid, and described A liquid and B liquid volume ratio are 1:0.8-1.2, concussion 12-24h; Centrifugal with super filter tube, obtain vinyl diamines-N, N, N ', N '-tetraacethyl-bovine serum albumin; Getting cadmium nitrate take mass volume ratio and as 10-12mg/ml is dissolved in, in dimethyl sulfoxide (DMSO), forms C liquid; C liquid is dropwise joined to vinyl diamines-N, N, N ', in N '-tetraacethyl-bovine serum albumin, vinyl diamines-N, N, N ', the volume ratio of N '-tetraacethyl-bovine serum albumin and C liquid is 1:0.8-1.2, concussion 2-5h; Centrifugal with super filter tube, obtain detectable antigens cadmium-vinyl diamines-N, N, N ', N '-tetraacethyl-bovine serum albumin;
(3) preparation of colloidal gold solution: getting mass concentration is the aqueous solution of chloraurate 80-120ml of 0.008-0.012%, be heated with stirring to boiling, the disposable mass concentration that adds is fast the trisodium citrate aqueous solution 2-3ml of 0.8-1.2%, continue agitating heating 0.5-24h, until solution is claret, room temperature is cooling, obtains containing the colloidal gold solution that particle diameter is the colloid gold particle of 20-40nm;
(4) preparation of the colloidal gold solution of Cadmium resistance ion monoclonal antibody mark: get step (3) gained colloidal gold solution 80-120ml, the solution of potassium carbonate that is 0.2-0.3mol/L by volumetric molar concentration is adjusted to 8.3-8.8 by its pH value; Step (1) gained Cadmium resistance ion monoclonal antibody 1.2-2.0mg is joined in colloidal gold solution, stir, standing 0.5-24 hour; Dropwise adding mass concentration is the aseptic bovine serum albumin 8-14ml of 8-12%, stirs 0.5-24h, 4 ℃ of-30 ℃ of standing over night; By the colloidal gold solution after mark, in the centrifugal 20-60min of 12000-14000r/min, abandoning supernatant, adds the phosphate buffer 2-6ml of volumetric molar concentration 0.01-0.02mol/L resuspended, obtains the colloidal gold solution of Cadmium resistance ion monoclonal antibody mark;
(5) metal spraying: the colloidal gold solution of step (4) gained Cadmium resistance ion monoclonal antibody mark is sprayed on pad by the discharge rate of 2-10 μ l/cm, and 37 ℃ of-45 ℃ of exhausting are dried, and the time is 5-24h, obtains the pad after metal spraying;
(6) coated C on nitrocellulose filter, T line: coated C line and T line on nitrocellulose filter, wherein C line is nature controlling line, coated sheep anti mouse immunoglobulin G, concentration is 0.2-1.5mg/ml; T line is detection line, detectable antigens cadmium-vinyl diamines-N of coated cadmium, and N, N ', N '-tetraacethyl-bovine serum albumin, concentration is 0.1-0.9mg/ml; 37 ℃ of-45 ℃ of exhausting are dried, and the time is 2-24h; Nitrocellulose filter after being coated with;
On nitrocellulose filter after described being coated with, arrange and detect T line and Quality Control C line, wherein detection line T line is near sample pad one end;
(7) assembling of cadmium ion colloidal gold immune chromatography rapid detecting test paper strip: by Polyvinylchloride base plate, sample pad, step (nitrocellulose filter after the pad after 5) Suo Tinkling metal sprayings, step (6) gained are coated with and thieving paper assembling, cutting slivering, obtains cadmium ion colloidal gold immune chromatography rapid detecting test paper strip;
Assembling mode is as follows: take Polyvinylchloride base plate as bottom support, the pad after sample pad, metal spraying, nitrocellulose filter after coated are sticked on Polyvinylchloride base plate and made in the mode being connected successively with thieving paper; Wherein sample pad and thieving paper are positioned at two ends, the two ends of the nitrocellulose filter after coated lay respectively at pad and thieving paper below, one end of pad be arranged at sample pad below, the other end be arranged at nitrocellulose filter after coated above;
Described pad and sample pad are by polyester film process mixed liquid dipping 5-24h, after taking-up, in 37-45 ℃ of exhausting, dry and obtain; Described mixed liquor is that volumetric molar concentration is that phosphate buffer, the mass volume ratio of 0.01-0.02mol/L is 3g-6g/(100ml phosphate buffer) non-ionic surfactant, volume ratio be 1ml-4ml/(100ml phosphate buffer) polysorbas20 and mass volume ratio be 5g-20g/(100ml phosphate buffer) Macrogol 6000 form, the pH value of described phosphate buffer is 7.0-7.5;
Described non-ionic surfactant preferred fat alcohol polyoxyethylene ether, APES, fatty acid polyethylene glycol ester etc.
The application of the present invention's cadmium ion colloidal gold immune chromatography test strip, comprises following operation steps:
(1) in cadmium ion titer or sample digestion solution, according to the volume ratio of 1:1, adding volumetric molar concentration is vinyl diamines-N of 1mmol/L, N, N ', N '-tetraacethyl sequestrant, mixes, and makes cadmium ion and vinyl diamines-N, N, N ', the abundant chelating of N '-tetraacethyl, obtains detected sample solution;
Described sample digestion solution is that acid adding after testing sample (rice accurately weighs 100mg) ashing is carried out to microwave or heating and decompose, then uses nano titanium oxide enrichment, then adjusts pH to neutrality and be concentrated to the solution that 0.5ml obtains;
Described high temperature ashing is that sample is placed in crucible and burns and incinerate;
(2) with dropper, draw step (1) gained sample solution 80-100 to be checked μ l, drip in the sample pad of test strips, after dropping sample, start timing;
(3) 3-5min reading result after dripping sample, while reading, is vertically placed in observer front by test strips in the downward mode in sample pad one end;
(4) result judgement: the shown in red lines of C line, when the colour developing of T line, result is negative, and the concentration of cadmium ions in testing sample is less than 50ng/ml; When T line does not develop the color, result is positive, and the concentration of cadmium ions in testing sample is greater than 50ng/ml.
This ELISA test strip precision meets the GB standard of country to cadmium content in rice.
The present invention's cadmium ion colloidal gold immune chromatography test strip, by chelating cadmium ion, make antigen, and produce antibody by mixing the method for incomplete Freund's adjuvant immune mouse, effectively reduce production costs, improve the high sensitivity of antibody, improve accuracy of detection.It is standard that the content of the cadmium in rice in national standard is take in the present invention, the sensing range of association colloid gold test paper strip, change the amount of raw material to be measured, and by raw material being carried out to the ashing of pre-service-high temperature, clear up, overcome first the deficiency that colloidal gold method in prior art cannot detect cadmium ion in rice.
The present invention's test strips can carry out fast, facilitate, detect accurately grain and the residual heavy metal cadmium of converted products thereof on a large scale, and detection sensitivity is high, and result is stable, reliable.
The present invention's test strips, except the cadmium metal that can detect in grain and converted products thereof, can also detect concentration of cadmium ions in other foods and tap water and whether surpass national standard (city tap-water national standard is that cadmium is no more than 10 μ g/ml).The whether super object detection method of concentration of cadmium ions in tap water, concrete operations are: get testing sample and be concentrated into original volume 1/5, draw the sample after 80-100 μ l concentrates, drip in the sample pad of test strips, other methods of operating are with the detection of cadmium in rice.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
The cadmium ion colloidal gold immune chromatography test strip of the present embodiment, its preparation method, comprises following operation steps:
(1) preparation of Cadmium resistance ion monoclonal antibody and purifying: with difunctional vinyl diamines-N, N, N ', N '-tetraacethyl sequestrant (iEDTA) is coupled to cadmium ion on bovine serum albumin, mix incomplete Freund's adjuvant immunity BALB/c mouse, get immune mouse spleen cell and SP2/0 myeloma cell and merge, filter out the positive cell strain of stably excreting Cadmium resistance ion monoclonal antibody and expand and cultivate, injection cell enters in Mice Body, to induce ascites; Get ascites, add the sodium-acetate buffer that is equivalent to 2 times of ascites volumes, regulate pH to 4.5, obtain mixed liquor; It is sad under stirring at room, dropwise to add, and described volume ratio sad and described mixed liquor is 33 μ l/ml; Standing at 4 ℃, centrifugal, obtain supernatant; Regulate pH to 7.4, under 4 ℃ of cooling baths, add ammonium sulfate, controlling ammonium sulfate final concentration is 0.277 g/ml, standing 1h, then 4 ℃, the centrifugal 10min of 10000r/min, abandon supernatant, precipitation is dissolved in the PBS solution of 0.01mol/L and dialyses, 4 ℃ of standing over night, the centrifugal 10min of 10000r/min, abandons precipitation; Obtain Cadmium resistance ion monoclonal antibody;
The volumetric molar concentration of described sodium-acetate buffer is 0.06mol/L, and pH value is 5.0, and the volumetric molar concentration of described phosphate buffered solution is 0.01mol/L, and pH value is 7.4;
(2) detectable antigens cadmium-vinyl diamines-N, N, N ', the preparation of N '-tetraacethyl-bovine serum albumin: get vinyl diamines-N, N, N ', N '-tetraacethyl be take mass volume ratio and as 20mg/ml is dissolved in, in dimethyl sulfoxide (DMSO), is formed A liquid; The anti-bovine serum albumin of rabbit be take to mass volume ratio as 15.4mg/ml is dissolved in Tri(Hydroxymethyl) Amino Methane Hydrochloride, add respectively the triethylamine and the Macrogol 600 that account for Pehanorm hydrochloride volume 1.3%, form B liquid; A liquid is dropwise added to (the volume ratio 1:1 of A, B liquid) in B liquid, concussion 24h; Centrifugal with super filter tube, obtain vinyl diamines-N, N, N ', N '-tetraacethyl-bovine serum albumin; Getting cadmium nitrate take mass volume ratio and as 11mg/ml is dissolved in, in dimethyl sulfoxide (DMSO), forms C liquid; C liquid is dropwise joined to vinyl diamines-N, N, N ', in N '-tetraacethyl-bovine serum albumin (vinyl diamines-N, N, N ', the volume ratio of N '-tetraacethyl-bovine serum albumin and C liquid is 1:1), concussion 3h; Centrifugal with super filter tube, obtain detectable antigens cadmium-vinyl diamines-N, N, N ', N '-tetraacethyl-bovine serum albumin;
(3) preparation of colloidal gold solution: get mass concentration and be 0.01% aqueous solution of chloraurate 100ml, be heated with stirring to boiling, the disposable mass concentration that adds is fast 1% trisodium citrate aqueous solution 2.2ml, continue agitating heating 0.5-24h, until solution is claret, room temperature is cooling, obtains containing the colloidal gold solution that particle diameter is the colloid gold particle of 20-40nm;
(4) preparation of the colloidal gold solution of Cadmium resistance ion monoclonal antibody mark: get step (3) gained colloidal gold solution 100ml, the solution of potassium carbonate that is 0.25mol/L by volumetric molar concentration is adjusted to 8.5 by its pH value; Step (1) gained Cadmium resistance ion monoclonal antibody 1.6mg is joined in colloidal gold solution, stir, standing 12 hours; Dropwise adding mass concentration is 10% aseptic bovine serum albumin 11ml, stirs 5h, 20 ℃ of standing over night; By the colloidal gold solution after mark, in the centrifugal 40min of 12000r/min, abandoning supernatant, adds the phosphate buffer 4ml of volumetric molar concentration 0.015mol/L resuspended, obtains the colloidal gold solution of Cadmium resistance ion monoclonal antibody mark;
(5) metal spraying: the colloidal gold solution of step (4) gained Cadmium resistance ion monoclonal antibody mark is sprayed on pad by the discharge rate of 5 μ l/cm, and 40 ℃ of exhausting are dried, and the time is 12h, obtains the pad after metal spraying;
(6) coated C on nitrocellulose filter, T line: coated C line and T line on nitrocellulose filter, wherein C line is nature controlling line, coated sheep anti mouse immunoglobulin G, concentration is 1.0 mg/ml; T line is detection line, detectable antigens cadmium-vinyl diamines-N of coated cadmium, and N, N ', N '-tetraacethyl-bovine serum albumin, concentration is 0.5mg/ml; 40 ℃ of exhausting are dried, and the time is 12h; Nitrocellulose filter after being coated with;
On nitrocellulose filter after described being coated with, arrange and detect T line and Quality Control C line, wherein detection line T line is near sample pad one end;
(7) assembling of cadmium ion colloidal gold immune chromatography rapid detecting test paper strip: by Polyvinylchloride base plate, sample pad, step (nitrocellulose filter after the pad after 5) Suo Tinkling metal sprayings, step (6) gained are coated with and thieving paper assembling, cutting slivering, obtains cadmium ion colloidal gold immune chromatography rapid detecting test paper strip;
Assembling mode is as follows: take Polyvinylchloride base plate as bottom support, the pad after sample pad, metal spraying, nitrocellulose filter after coated are sticked on Polyvinylchloride base plate and made in the mode being connected successively with thieving paper; Wherein sample pad and thieving paper are positioned at two ends, the two ends of the nitrocellulose filter after coated lay respectively at pad and thieving paper below, one end of pad be arranged at sample pad below, the other end be arranged at nitrocellulose filter after coated above;
Described pad and sample pad are by polyester film process mixed liquid dipping 12h, after taking-up, in 40 ℃ of exhausting, dry and obtain; Described mixed liquor is that volumetric molar concentration is that phosphate buffer, the mass volume ratio of 0.015mol/L is 6g/(100ml phosphate buffer) AEO, volume ratio be 2ml/(100ml phosphate buffer) polysorbas20 and mass volume ratio be 10g/(100ml phosphate buffer) Macrogol 6000 form, the pH value of described phosphate buffer is 7.4.
Embodiment 2
The present embodiment is used the cadmium ion colloidal gold immune chromatography test strip of embodiment 1 to detect the cadmium ion in rice, comprises following operation steps:
(1) detect rice: accurately weigh 100mg rice, high temperature ashing, adds nitric acid (0.5mol/L) to dissolve, and regulates pH, and constant volume is solution to be measured, or to use titania enrichment, regulator solution amount be 0.5ml; In cadmium ion titer or sample digestion solution or liquid to be detected, according to the volume ratio of 1:1, adding volumetric molar concentration is vinyl diamines-N of 1mmol/L, N, N ', N '-tetraacethyl sequestrant, mixes, make cadmium ion and vinyl diamines-N, N, N ', the abundant chelating of N '-tetraacethyl, obtain detected sample solution, constant volume is 1ml;
Described sample digestion solution is that acid adding after testing sample (rice accurately weighs 100mg) ashing is carried out to microwave or heating and decompose, then uses nano titanium oxide enrichment, then adjusts pH to neutrality and be concentrated to the solution that 0.5ml obtains;
(2) with dropper, draw step (1) gained sample solution 80-100 to be checked μ l, drip in the sample pad of test strips, after dropping sample, start timing;
(3) 3-5min reading result after dripping sample, while reading, is vertically placed in observer front by test strips in the downward mode in sample pad one end;
(4) result judgement: the shown in red lines of C line, when the colour developing of T line, result is negative, and the concentration of cadmium ions in testing sample is less than 50ng/ml; When T line does not develop the color, result is positive, and the concentration of cadmium ions in solution to be measured is greater than 50ng/ml, and the concentration of cadmium ions in testing sample is greater than 0.2mg/kg.
Embodiment 3
The present embodiment is used the cadmium ion colloidal gold immune chromatography test strip of embodiment 1 to detect the cadmium ion in city tap-water, comprises following operation steps:
(1) detect city tap-water: get testing sample and be concentrated into original volume 1/5, obtain liquid to be detected, in cadmium ion titer or sample digestion solution or liquid to be detected, according to the volume ratio of 1:1, adding volumetric molar concentration is vinyl diamines-N of 1mmol/L, N, N ', N '-tetraacethyl sequestrant, mixes, make cadmium ion and vinyl diamines-N, N, N ', the abundant chelating of N '-tetraacethyl, obtain detected sample solution, constant volume is 1ml;
(2) with dropper, draw step (1) gained sample solution 80-100 to be checked μ l, drip in the sample pad of test strips, after dropping sample, start timing;
(3) 3-5min reading result after dripping sample, while reading, is vertically placed in observer front by test strips in the downward mode in sample pad one end;
(4) result judgement: the shown in red lines of C line, when the colour developing of T line, result is negative, and the concentration of cadmium ions in testing sample is less than 50ng/ml; When T line does not develop the color, result is positive, and the concentration of cadmium ions in solution to be measured is greater than 50ng/ml, and the concentration of cadmium ions in testing sample is greater than 0.2mg/kg.
Claims (4)
1. cadmium ion colloidal gold immune chromatography test strip, is characterized in that, is prepared from accordance with the following methods:
(1) preparation of Cadmium resistance ion monoclonal antibody and purifying: with difunctional vinyl diamines-N, N, N ', N '-tetraacethyl sequestrant is coupled to cadmium ion on bovine serum albumin, mix incomplete Freund's adjuvant immunity BALB/c mouse, get immune mouse spleen cell and SP2/0 myeloma cell and merge, filter out the positive cell strain of stably excreting Cadmium resistance ion monoclonal antibody and expand and cultivate, injection cell enters in Mice Body, to induce ascites; Get 2-4 ml ascites, add and be equivalent to ascites volume 1.8-2.2 sodium-acetate buffer doubly, regulate pH to 4.0-5.0, obtain mixed liquor; It is sad under stirring at room, dropwise to add, and described volume ratio sad and described mixed liquor is 30-40 μ l/ml; Standing at 2-8 ℃, centrifugal, obtain supernatant; Regulate pH to 7.0-7.8, under 2-8 ℃ of cooling bath, add ammonium sulfate, controlling ammonium sulfate final concentration is 0.25-0.30g/ml, standing 1-2h; Then 2-8 ℃, the centrifugal 10-20min of 8000-12000r/min, abandon supernatant, and precipitation is dissolved in the phosphate buffered solution of 0.008-0.012mol/L and dialyses, 2-8 ℃ of standing over night, the centrifugal 10-20min of 8000-12000r/min, abandons precipitation; Obtain Cadmium resistance ion monoclonal antibody;
(2) detectable antigens cadmium-vinyl diamines-N, N, N ', the preparation of N '-tetraacethyl-bovine serum albumin: get vinyl diamines-N, N, N ', N '-tetraacethyl be take mass volume ratio and as 15-25mg/ml is dissolved in, in dimethyl sulfoxide (DMSO), is formed A liquid; The anti-bovine serum albumin of rabbit be take to mass volume ratio as 15-16mg/ml is dissolved in Tri(Hydroxymethyl) Amino Methane Hydrochloride, add respectively the triethylamine and the Macrogol 600 that account for Pehanorm hydrochloride volume 1-2%, form B liquid; A liquid is dropwise added in B liquid, and described A liquid and B liquid volume ratio are 1:0.8-1.2, concussion 12-24h; Centrifugal with super filter tube, obtain vinyl diamines-N, N, N ', N '-tetraacethyl-bovine serum albumin; Getting cadmium nitrate take mass volume ratio and as 10-12mg/ml is dissolved in, in dimethyl sulfoxide (DMSO), forms C liquid; C liquid is dropwise joined to vinyl diamines-N, N, N ', in N '-tetraacethyl-bovine serum albumin, vinyl diamines-N, N, N ', the volume ratio of N '-tetraacethyl-bovine serum albumin and C liquid is 1:0.8-1.2, concussion 2-5h; Centrifugal with super filter tube, obtain detectable antigens cadmium-vinyl diamines-N, N, N ', N '-tetraacethyl-bovine serum albumin;
(3) preparation of colloidal gold solution: getting mass concentration is the aqueous solution of chloraurate 80-120ml of 0.008-0.012%, be heated with stirring to boiling, the disposable mass concentration that adds is fast the trisodium citrate aqueous solution 2-3ml of 0.8-1.2%, continue agitating heating 0.5-24h, until solution is claret, room temperature is cooling, obtains containing the colloidal gold solution that particle diameter is the colloid gold particle of 20-40nm;
(4) preparation of the colloidal gold solution of Cadmium resistance ion monoclonal antibody mark: get step (3) gained colloidal gold solution 80-120ml, the solution of potassium carbonate that is 0.2-0.3mol/L by volumetric molar concentration is adjusted to 8.3-8.8 by its pH value; Step (1) gained Cadmium resistance ion monoclonal antibody 1.2-2.0mg is joined in colloidal gold solution, stir, standing 0.5-24 hour; Dropwise adding mass concentration is the aseptic bovine serum albumin 8-14ml of 8-12%, stirs 0.5-24h, 4 ℃ of-30 ℃ of standing over night; By the colloidal gold solution after mark, in the centrifugal 20-60min of 12000-14000r/min, abandoning supernatant, adds the phosphate buffer 2-6ml of volumetric molar concentration 0.01-0.02mol/L resuspended, obtains the colloidal gold solution of Cadmium resistance ion monoclonal antibody mark;
(5) metal spraying: the colloidal gold solution of step (4) gained Cadmium resistance ion monoclonal antibody mark is sprayed on pad by the discharge rate of 2-10 μ l/cm, and 37 ℃ of-45 ℃ of exhausting are dried, and the time is 5-24h, obtains the pad after metal spraying;
(6) coated C on nitrocellulose filter, T line: coated C line and T line on nitrocellulose filter, wherein C line is nature controlling line, coated sheep anti mouse immunoglobulin G, concentration is 0.2-1.5mg/ml; T line is detection line, detectable antigens cadmium-vinyl diamines-N of coated cadmium, and N, N ', N '-tetraacethyl-bovine serum albumin, concentration is 0.1-0.9mg/ml; 37 ℃ of-45 ℃ of exhausting are dried, and the time is 2-24h; Nitrocellulose filter after being coated with;
On nitrocellulose filter after described being coated with, arrange and detect T line and Quality Control C line, wherein detection line T line is near sample pad one end;
(7) assembling of cadmium ion colloidal gold immune chromatography rapid detecting test paper strip: nitrocellulose filter and thieving paper assembling after the pad after Polyvinylchloride base plate, sample pad, step (5) gained metal spraying, step (6) gained are coated with, cutting slivering, obtains cadmium ion colloidal gold immune chromatography rapid detecting test paper strip;
Assembling mode is as follows: take Polyvinylchloride base plate as bottom support, the pad after sample pad, metal spraying, nitrocellulose filter after coated are sticked on Polyvinylchloride base plate and made in the mode being connected successively with thieving paper; Wherein sample pad and thieving paper are positioned at two ends, the two ends of the nitrocellulose filter after coated lay respectively at pad and thieving paper below, one end of pad be arranged at sample pad below, the other end be arranged at nitrocellulose filter after coated above;
Described pad and sample pad are by polyester film process mixed liquid dipping 5-24h, after taking-up, in 37-45 ℃ of exhausting, dry and obtain; Described mixed liquor is that volumetric molar concentration is that phosphate buffer, the mass volume ratio of 0.01-0.02mol/L is 3g-6g/(100ml phosphate buffer) non-ionic surfactant, volume ratio be 1ml-4ml/(100ml phosphate buffer) polysorbas20 and mass volume ratio be 5g-20g/(100ml phosphate buffer) Macrogol 6000 form, the pH value of described phosphate buffer is 7.0-7.5.
2. cadmium ion colloidal gold immune chromatography test strip according to claim 1, it is characterized in that, in step (1), the volumetric molar concentration of described sodium-acetate buffer is 0.05-0.08mol/L, pH value is 4.5-5.5, the volumetric molar concentration of described phosphate buffered solution is 0.008-0.012mol/L, and pH value is 7.0-7.8.
3. cadmium ion colloidal gold immune chromatography test strip according to claim 1 and 2, is characterized in that, in step (7), described non-ionic surfactant is AEO, APES or fatty acid polyethylene glycol ester.
4. the application of cadmium ion colloidal gold immune chromatography test strip as described in one of claim 1-3, comprises following operation steps:
(1) in cadmium ion titer or sample digestion solution, according to the volume ratio of 1:1, adding volumetric molar concentration is vinyl diamines-N of 1mmol/L, N, N ', N '-tetraacethyl sequestrant, mixes, and makes cadmium ion and vinyl diamines-N, N, N ', the abundant chelating of N '-tetraacethyl, obtains detected sample solution;
Described sample digestion solution is that acid adding after testing sample ashing is carried out to microwave or heating and decompose, then uses nano titanium oxide enrichment, then adjusts pH to neutrality and be concentrated to the solution that 0.5ml obtains;
(2) with dropper, draw step (1) gained sample solution 80-100 to be checked μ l, drip in the sample pad of test strips, after dropping sample, start timing;
(3) 3-5min reading result after dripping sample, while reading, is vertically placed in observer front by test strips in the downward mode in sample pad one end;
(4) result judgement: the shown in red lines of C line, when the colour developing of T line, result is negative, and the concentration of cadmium ions in testing sample is less than 50ng/ml; When T line does not develop the color, result is positive, and the concentration of cadmium ions in testing sample is greater than 50ng/ml.
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