CN105203765A - Heavy metal quantitative detecting system and method for quantitatively detecting heavy metal - Google Patents

Heavy metal quantitative detecting system and method for quantitatively detecting heavy metal Download PDF

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Publication number
CN105203765A
CN105203765A CN201510532267.XA CN201510532267A CN105203765A CN 105203765 A CN105203765 A CN 105203765A CN 201510532267 A CN201510532267 A CN 201510532267A CN 105203765 A CN105203765 A CN 105203765A
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heavy metal
colloidal gold
kit
gold
solution
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CN105203765B (en
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刘松杰
米军
吴晓迪
刘文福
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Chengdu Anpro Biotechnology Co Ltd
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Chengdu Anpro Biotechnology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Abstract

The invention discloses a modified colloidal gold preparation method and a gold-labeled antibody preparation method, and further discloses a colloidal gold immunochromatography kit, a heavy metal quantitative detecting system with the kit, and a method for quantitatively detecting heavy metal. A gold-labeled antibody formed by modified colloidal gold and an antibody is stable and is not influenced by the acid environment; when the immunochromatography kit is prepared from the modified colloidal gold, in the acid conditions, heavy metal can still be accurately detected in an oriented mode, traditional atomic absorption spectrometry can be replaced, cost is low, and application prospects are good.

Description

The method of one heavy metal species quantitative detection system and quantitatively detection heavy metal
Technical field
The present invention relates to a heavy metal species quantitative detection system and quantitatively detect the method for heavy metal.
Background technology
Along with fast development and the growth in the living standard of China's economy, people have had higher requirement simultaneously to food security again, due to China in Construction of Market Economy process because the many reasons such as institutional improvement and supervision level cause China's food-safety problem day by day serious.Cause the reason of food-safety problem a lot, heavy metals exceeding standard problem is one of key factor affecting food security.At present, heavy metal is as copper, lead, zinc, iron, cobalt, nickel, manganese, cadmium, mercury, tungsten, molybdenum, gold, silver etc.There is following several element present stage to the heavy metal that our mankind and environment work the mischief: the significant heavy metal elements of bio-toxicity such as mercury, cadmium, lead, chromium, copper and metalloid arsenic.
If heavy metal element is unprocessed just directly enter river, lake or ocean, or enters in soil, because they can not be biodegradable, these rivers, lake, ocean and soil are polluted.Under the biological magnification of food chain, their thousands of hundred times of ground enrichments, finally enter human body.As fish or shellfish accumulation heavy metal eat by the mankind, or heavy metal is eaten by the mankind by after the absorption of the crops such as paddy, wheat, heavy metal will enter human body, makes people produce heavy metal poisoning, light then strange illness (minamata disease, Itai-itai diseases etc.) occurs, heavy then cause death.Secondly, there is strong interaction in heavy metal energy and protein and enzyme etc. in human body, make them lose activity, also can accumulate in some organ of human body, cause slow poisoning, the poisoning that heavy metals exceeding standard causes is too numerous to enumerate.For the heavy metal pollution in water body and soil, also there is no good way now, because the heavy metal be present in water and soil is difficult to degraded, and there is enriching, so the pollution of heavy metal is irreversible.
(1) plumbous: to be the toxic metals can put aside in human body and animal tissue.It enters in body affine with multiple organ by skin, alimentary canal, respiratory tract, and major toxicity effect is anemia, nervous function imbalance and injury of kidney, and vulnerable crowd has children, old man, hypoimmunity crowd.
(2) cadmium: the indispensable element not being human body, healthy by food chain inrichment harm humans, have toxic action to human body multisystem, Cd2+ is classified as the food contaminant of the 3rd priority research by FAO (Food and Agriculture Organization of the United Nation) and the World Health Organization (WHO).The toxicity of cadmium is very large, can put aside, mainly put aside at kidney, cause the changes of function of urinary system in human body; Cadmium can replace calcium in bone, and bone is seriously softened, and bone is broken into pieces, and can cause the dirty functional disturbance of stomach, disturbs the enzyme system of zinc in human body and biosome, causes vascular hypertension to rise.Vulnerable crowd is mining operations person, hypoimmunity crowd.
(3) mercury: mercury and mercuric compounds belongs to extremely toxic substance, can at people's body accumulation.After mercury metal in blood enters brain tissue, accumulate gradually in brain tissue, will cause damage to brain tissue when reaching certain amount, a part of mercury ion transfers to kidney in addition.The inorganic mercury ion entering water body can change the larger organic mercury of toxicity into, enters human body, cause systemic toxicity profiles effect by food chain; Vulnerable crowd has women, especially ready-to-be mother, hobby seafood personage; Seldom mercurous in natural water, be generally no more than 0.1 μ g/L.
(4) arsenic: the non-essential element being human body, the toxicity of element arsenic is extremely low, and the compound of arsenic.All have severe toxicity, trivalent arsenide compound is stronger than other arsenic compound toxicity.Arsenic enters human body by respiratory tract, alimentary canal and skin contact, as intake exceedes excretion, arsenic will in position accumulations such as the liver of human body, kidney, lung, spleen, uterus, placenta, skeleton, muscle, enzyme system in cell is combined, the biological agent of enzyme is suppressed lose activity, particularly accumulates in hair, nail, thus cause arsenicalism, can reach several years latent period, arsenicalism has gastrointestinal symptom, neurological symptom and cutaneous lesions etc. even decades.Arsenic also has carcinogenesis, can cause cutaneum carcinoma.In the day water, arsenic content has very big-difference because water source is different with geographical conditions, fresh water average out to 0.5 μm/L, and seawater is 3.7 μm/L.
(5) chromium: drink as eaten by mistake, can cause the toxicity symptom such as abdominal discomfort and diarrhoea, and cause allergic dermatitis or eczema, breathing enters, and has stimulation and corrosive attack, cause pharyngitis, bronchitis etc. to respiratory tract.The serious Area Inhabitants of water pollutions, often contact or Excess free enthalpy person.Be easy to get rhinitis, tuberculosis, diarrhoea, bronchitis, dermatitis etc.
(5) copper: excess copper is enrichment in human body, there will be the intoxicating phenomenons such as Nausea and vomiting, Upper abdominal pain, Acute hemolytic crisis and renal tubule distortion, severe patient also can cause the harm such as acute renal failure.Copper powder dirt can cause pharyngalgia, cough and cough up phlegm, and gas even uncomfortable in chest is suppressed, fever of feeling cold.
Current conventional heavy metal detection method has Atomic fluorophotometry (AFS), inductive coupling plasma mass (ICP-MS) analytical technology, inductive coupling etc. in vitro atomic emission spectrum (ICP-AES), high performance liquid chromatography etc.Numerously be applied in the analytical technology of detection of heavy metal ion current, atomic absorption spectrography (AAS) is the most frequently used method, the method has higher sensitivity usually, but cost is higher, need professional and technical personnel to operate, obtain the larger sample size of higher sensitivity needs and must to analyze successively and consuming time longer due to sample.Therefore, current batch samples can not be met quick, the easy demand with being suitable for Site Detection.
Immunoassay is a kind of analytical approach with high degree of specificity and sensitivity, and heavy metal analysis has mainly contained Indirect Competitive ELISA method and collaurum fast immune chromatographic method.When adopting immunochromatographyassay assay, need to develop the color with collaurum.
Collaurum is by gold chloride (HAuCl 4) at reductive agent as under the effect such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, can be grouped to a certain size gold grain, and become a kind of stable colloidal state due to electrostatic interaction, form electronegative hydrophobic sol solution, become stable colloidal state due to electrostatic interaction, therefore claim collaurum.
Immunoassay utilizes antigen and antibody specific association reaction, makes the chromogenic reagent of labelled antibody, and detects the analytical approach of various material (medicine, hormone, protein, microorganism etc.).Collaurum is electronegative under mild alkaline conditions, can be combined with the positive charge group of antibody, because this combination is electrostatical binding, so do not affect the biological nature of antibody.When thus utilizing collaurum to carry out immunoassay, there is convenient and swift specific installation and reagent, the result of not needing and judge the advantages such as directly perceived, be used widely.
But, because collaurum only effectively could be combined with antibody under weakly alkaline environment, once the pH of detected object is not weakly alkaline, then cannot accurately detect, and the detection of heavy metal needs to extract with strong acid, although can be detected reluctantly by the mode increasing extension rate, testing result is inaccurate.
Chinese patent application CN201010229009.1 discloses the method for the preparation of a kind of heavy metal Hg antibody and enzyme-linked immunosorbent assay content of beary metal, can be used for detecting huge sum of money mercury, but the method accuracy is looked into, and complicated operation, need professional main equipment.Chinese patent application CN201020521531.2 discloses a kind of huge sum of money test card, and this test card can only detect in particular surroundings again, and can only qualitative detection, cannot quantitatively detect to be.
Therefore need on the market one can fast, accurate quantitative analysis detects heavy metal, and the detection system be convenient for carrying.
Summary of the invention
The object of the present invention is to provide a heavy metal species quantitative detection system.
First, the invention provides a kind of preparation method of modified colloidal gold, it comprises the steps:
(1) cut-off footpath is the colloidal gold solution 100 parts of 10 ~ 60nm, add two (triethanolamine) metatitanic acid diisopropyl ester solution of 0.1 ~ 3% of colloidal gold solution volume, the concentration of two (triethanolamine) metatitanic acid diisopropyl ester solution is 1%, stir 0.5 ~ 2h under 100 ~ 400r/min, obtain mixed solution;
(2) above-mentioned mixed solution pH value to 8 ~ 10 are regulated, add 0.1 ~ 2 part of tetrabutyl titanate, at room temperature with 100 ~ 400r/min stirring reaction 24 ~ 36 hours, centrifugal 5 ~ 60min under 4000 ~ 16000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 ~ 2 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.
In step (1), described colloidal gold solution adopts gold chloride-trisodium citrate reduction method preparation.
In step (2), mixed solution pH value is adjusted to adopt ammoniacal liquor to regulate.
Present invention also offers modified colloidal gold prepared by preceding method.
Present invention also offers a kind of preparation method of golden labeling antibody, step is as follows: get aforesaid modified colloidal gold, add antibody, gold colloid surface activity-OH and antibody Fc section-COOH react to be formed and stablize bonding, form golden labeling antibody.
Preferably, described antibody is Cd monoclonal antibody, Pb monoclonal antibody, Hg monoclonal antibody, As monoclonal antibody, Cr monoclonal anti or Cu monoclonal antibody.
Present invention also offers golden labeling antibody prepared by preceding method.
Present invention also offers aforementioned modified collaurum or the purposes of aforementioned golden labeling antibody in immune colloid gold detects.
Preferably, described immune colloid gold detects is the dyeing of immune colloid gold light microscopic, the dyeing of immune colloid gold Electronic Speculum, colloidal gold immunochromatographimethod.
The present invention detects the colloidal gold immunochromatographykit kit of heavy metal: it comprises Test paper and gets stuck, and test paper is placed in and gets stuck; Described Test paper comprises plastic bottom board 1, upper sample pad 2 is pasted in one end, one end of upper sample pad closely crimps the colloidal gold pad 3 made containing the gold mark that detection heavy metal relative answers antibody to mark to some extent, colloidal gold pad 3 one end closely crimps nitrocellulose filter 4, nitrocellulose filter is coated with detection line T and nature controlling line C5, detection line T wraps by the comlete antigen of heavy metal to be checked, and nature controlling line C is coated with dynamics, and the nitrocellulose filter other end connects inhales sample pad 6; Described position of getting stuck surperficial counter sample pad 2 is provided with well 7, and the position of the surperficial corresponding nitrocellulose filter 4 that gets stuck is provided with observation panel 8; Wherein, the collaurum in colloidal gold pad is modified colloidal gold prepared by preceding method.
Preferably, the golden labeling antibody in described colloidal gold pad is golden labeling antibody prepared by preceding method.
Preferably, the comlete antigen of described heavy metal is after heavy metal and complexing agent complexes, with carrier protein phase coupling shape antigen, wherein, described complexing agent is citric acid, ethylenediamine tetraacetic acid, aminoacetic acid, 1-(the different sulphur cyanobenzyl of 4-) ethylenediamine-N, one or more in N, N', N'-tetraacethyl, diethylenetriamine pentacarboxylic acid salt, glutathione and organic polydentate part; Described carrier protein is one or more in bovine serum albumin(BSA), keyhole limpet hemocyanin, ovalbumin, human albumin.
Preferably, described nitrocellulose filter is the porous spline structure film of aperture 5-12 micron, and described sample pad is glass fibre element film or polyester film, and described suction sample paper is absorbent filter.
Present invention also offers a kind of detection system detecting heavy metal, it comprises aforementioned described colloidal gold immunochromatographykit kit, dry type thermostat and collaurum readout instrument, and wherein, collaurum readout instrument is a kind of hand-held collaurum readout instrument.
Present invention also offers a kind of method detecting heavy metal, it is that aforesaid detection system detects.
Preferably, it comprises the steps:
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens.
2) get measuring samples, add the extraction agent of 2 ~ 8 times of mL/g, earthquake frequency is react 5 ~ 30min under 2 ~ 60cpm condition, and filter, get supernatant, add the dilution of supernatant 2 ~ 40 times of volumes, mixing, obtains mixed liquor; Described extraction agent to be concentration be 1 ~ 15% salpeter solution; Described thinning agent is the buffer solution being added with surfactant, heavy metal complexing agent;
3) by front described colloidal gold immunochromatographykit kit and step 1) mixed liquor prepared puts into dry type thermostat, constant temperature 2 ~ 15min respectively, and temperature is set as 20 ~ 45 DEG C;
4) in dry type thermostat, by step 1) mixed liquor prepared adds in the well of kit, isothermal reaction 5 ~ 30min;
5) by reacted kit, be placed in collaurum readout instrument, read data.
Further preferably, it comprises the steps:
1) get measuring samples, add the extraction agent of 4 times of mL/g, earthquake frequency is react 5min under 2 ~ 60cpm condition, filters, gets supernatant, add the dilution of supernatant 4 times of volumes, and mixing, obtains mixed liquor; Described extraction agent to be concentration be 10% salpeter solution; Described thinning agent for being added with 1%tween20,1 × 10 -7the 0.2MPBS solution of ITCBEg/mL;
2) by aforesaid colloidal gold immunochromatographykit kit and step 1) mixed liquor prepared puts into thermostat, constant temperature 5min respectively, and temperature is set as 37 DEG C;
3) in thermostat, by step 1) mixed liquor prepared adds in the well of kit, isothermal reaction 20min;
4) by reacted kit, be placed in collaurum readout instrument, read data.
Modified colloidal gold of the present invention is the colloid gold particle of the titanate esters modification of surface containing activity-OH, when itself and antibody response, namely there is condensation reaction with-COOH on antibody Fc section in modified colloidal gold surface activity-OH, form stable chemical bonding, obtain the golden labeling antibody that the present invention is stable, instead of the golden labeling antibody that traditional collaurum and antibody are formed by electrostatical binding, the golden labeling antibody that modified colloidal gold of the present invention prepares is stablized, acid and alkali alkali environment and ionic strength affect, little to the requirement of detected object, applied widely, can be applicable to various immune colloid gold detect.
Experimental result explanation, modified colloidal gold of the present invention is stablized with the golden labeling antibody that antibody is formed, the impact of acid and alkali environment, adopt modified colloidal of the present invention gold preparation immune chromatography reagent kit of the present invention, in acid condition, still can accurate oriented detection heavy metal, conventional atom absorption spectroscopy can be substituted, with low cost, application prospect is good.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
The structure of the test paper of Fig. 1 detection kit of the present invention and use schematic diagram.In figure: 1-plastic bottom board, 2-sample pad, 3-colloidal gold pad, 4-nitrocellulose filter, 5-detection line T and nature controlling line C, 6-inhale sample pad.
The structural representation got stuck of Fig. 2 detection kit of the present invention.In figure: 7-well, 8-observation panel.
Embodiment
The preparation of embodiment 1 modified colloidal gold of the present invention
The colloidal gold solution that diameter is 10 ~ 60nm is prepared with gold chloride-trisodium citrate reduction method, the collaurum 100mL prepared adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.1 ~ 3mL concentration is 1%, mix and blend 0.5 ~ 2h under 100 ~ 400r/min.Solution ph to 8 ~ 10 are regulated with ammoniacal liquor, add 0.1 ~ 2mL tetrabutyl titanate, at room temperature continue reaction 24 ~ 36 hours, centrifugal 5 ~ 60min under 4000 ~ 16000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 ~ 2 time, be namely prepared into the colloid gold particle (collaurum of the present invention) of the titanate esters modification of surface containing activity-OH.
The preparation of embodiment 2 the present invention gold labeling antibody
The colloid gold particle of modification prepared by Example 1,0.2 ~ 2mgCd monoclonal antibody is added, reaction 2h, centrifugal 5 ~ 60min under 6000 ~ 12000r/min condition by every 100mL, remove supernatant, be dissolved to 1/20 of colloidal gold solution volume with redissolution liquid and namely obtain golden labeling antibody.
Antibody is heavy metal antibody, such as Cd monoclonal antibody, Pb monoclonal antibody, Hg monoclonal antibody, As monoclonal antibody, Cr monoclonal anti or Cu monoclonal antibody.
Embodiment 3 the present invention detects detection system and the using method thereof of heavy metal
1, detection system
The preparation of 1.1 kits of the present invention
The structure of kit of the present invention is as shown in Fig. 1 ~ 2: it comprises Test paper and gets stuck, and test paper is placed in and gets stuck; Described Test paper comprises plastic bottom board 1, upper sample pad 2 is pasted in one end, one end of upper sample pad closely crimps the colloidal gold pad 3 made containing the gold mark that detection heavy metal relative answers antibody to mark to some extent, colloidal gold pad 3 one end closely crimps nitrocellulose filter 4, nitrocellulose filter is coated with detection line T and nature controlling line C5, detection line T wraps by the comlete antigen of heavy metal to be checked, and nature controlling line C is coated with dynamics, and the nitrocellulose filter other end connects inhales sample pad 6; Described position of getting stuck surperficial counter sample pad 2 is provided with well 7, and the position of the surperficial corresponding nitrocellulose filter 4 that gets stuck is provided with observation panel 8.
Wherein, the preparation of colloidal gold pad: golden labeling antibody prepared by Example 2, is sprayed on 2cm wide glass fibre element film gold mark pad by 4 μ l/cm, dries 18 ~ 24 hours for 37 DEG C, must make gold mark pad.
Wherein, the holoantigen of described heavy metal is after heavy metal and complexing agent complexes, with carrier protein phase coupling shape antigen, wherein, described complexing agent is citric acid, ethylenediamine tetraacetic acid, aminoacetic acid, 1-(the different sulphur cyanobenzyl of 4-) ethylenediamine-N, one or more in N, N', N'-tetraacethyl, diethylenetriamine pentacarboxylic acid salt, glutathione and organic polydentate part; Described carrier protein is one or more in bovine serum albumin(BSA), keyhole limpet hemocyanin, ovalbumin, human albumin.
Wherein, involved kit NC film is the porous spline structure film of aperture 5-12 micron, and sample pad is glass fibre element film or polyester film, and inhaling sample paper is that absorbent filter is formed.
Wherein, NC film bag by process is: the solution with 0.01MpH7.4 phosphate buffer, metal holoantigen being mixed with 0.1 ~ 3mg/mL, against murine IgG is mixed with the solution of 0.1 ~ 3mg/mL, carrying out line bag respectively by C, T line with drawing film instrument with 1 μ l/cm speed, being placed on 37 degree of oven dryings 5 ~ 16 hours.
The assembling of mentioned reagent box should be 20 ~ 30 DEG C in temperature, humidity is that the indoor of <40% are carried out, get plastic bottom board, the NC film wrapping quilt is placed in the middle part of plastic floor and pastes, gold is marked pad and ride over NC film T line side, gold mark pad rides over 1mm on NC film and pastes, and gold mark pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with test paper lower limb.In NC film side, overlap joint inhales sample paper, rides over 1mm on NC film and pastes.Finally use cutting cutter that the plastic plate posted is cut into the wide test strips of 3 ~ 5mm, reinstall in plastic clip, form kit.
1.2 dry type thermostats, purchased from Hangzhou Rui Cheng Instrument Ltd.;
1.3 hand-held collaurum readout instruments, purchased from Shenzhen Tuo Nengda Science and Technology Ltd., be a kind of portable optical instrument, volume is less than 0.001m 3, easy and simple to handle, multiple project can be detected, there is GPS positioning function, can preserve and print and detect data.For quantitatively judging testing result, being 1 ~ 20 μ g/L to water quality detection scope, is 50 ~ 1000 μ g/kg to grain sensing range, is 100 ~ 2000 μ g/kg to Chinese crude drug sensing range.
2, using method
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens.
2) measuring samples is got, add the extraction agent (choosing different addition according to different sample hygroscopic capacity difference) of 2 ~ 8 times of mL/g, earthquake frequency is react 5 ~ 30min under 2 ~ 60cpm condition, filter, get supernatant, add the dilution (the corresponding respective standard curve of different extension rate ensures to detect Min Lingdu and generally dilutes 4 times) of supernatant 2 ~ 40 times of volumes, mixing, obtains mixed liquor; Described extraction agent to be concentration be 10% salpeter solution; Described thinning agent is the buffer solution being added with surfactant, heavy metal complexing agent;
3) by the colloidal gold immunochromatographykit kit described in claim 4 ~ 7 any one and step 1) mixed liquor prepared puts into dry type thermostat respectively, (after being greater than 2min, metallic ion is basic completely and chelant ties for constant temperature 2 ~ 15min, consider that sample adaptive settings is 5min), temperature is set as 20 ~ 45 DEG C, and (in this temperature range, system all can Accurate Determining heavy metal, too low or too highly all can affect immune response, immune response optimum temperature is 37 DEG C);
4) in dry type thermostat, by step 1) mixed liquor prepared adds in the well of kit, isothermal reaction 5 ~ 30min (reaction detection result keeps stable within the scope of certain hour, stablizes certain hour to be advisable i.e. 20min with lasting maintenance);
5) by reacted kit, be placed in collaurum readout instrument, read data.
Below by the mode of experimental example, beneficial effect of the present invention is described:
The stability test of experimental example 1 the present invention gold labeling antibody
Use titanate esters coupling method and 40nm collaurum heavy metal list cadmium clonal antibody, plumbous monoclonal antibody, mercury monoclonal antibody to carry out mark process respectively, use 40nm collaurum traditionally to carry out mark process to above-mentioned antibody simultaneously.After mark collaurum stable be its color be kermesinus, maximum absorption band is between 530 ~ 532nm, and OD value change fluctuation is little, when destroying its stability due to outside cause, collaurum generation gathering, its color can first purpling, then blackening, is finally colourless; Its maximum absorption band is greater than normal peak gradually; Its OD value diminishes gradually to 0 simultaneously.Mark gold mark is tested as follows:
1) preserve above-mentioned gold and be marked in 4 DEG C of refrigerators, observe and increase in time, use ultraviolet spectrophotometer to measure gold mark absorption peak and the change of OD value.
Result is as following table 1:
From result, new colloidal gold mark mode effectively improves gold mark storage stability.And mark purpling look gathering gradually along with the time increases traditional gold.
2) in above-mentioned gold mark, add different amount 10%NaCl solution, after 30min, use its absorption peak of ultraviolet determination and the change of OD value.
Result is as following table 2:
From result, new colloidal gold mark mode effectively improves the stability of gold mark to salt.
3) in above-mentioned gold mark, add different amount 0.1M hydrochloric acid, after 30min, use its absorption peak of ultraviolet determination and the change of OD value.
Result is as following table 3:
From result, new colloidal gold mark mode effectively improves the stability of gold mark to acid.
4) in above-mentioned gold mark, add different amount 1M NaOH, after 30min, use its absorption peak of ultraviolet determination and the change of OD value.
Result is as following table 3:
From result, new colloidal gold mark mode effectively improves the stability of gold mark to alkali.
As can be seen from table 1 ~ 3, modified colloidal of the present invention gold and antibody in conjunction with highly stable, acid and alkali alkali environment and ionic strength affect, acidproof, alkaline-resisting, salt tolerant.
Heavy metal dust technology process determination of recovery rates in experimental example 2 grain
In current grain, heavy metal analysis detects after needing to carry out thorough clearing up to sample, this method length consuming time, and operator quality requires high, complicated operation, the inadaptable rapid field testing requirement to grain.Therefore the quick simple process of sample is become to the key realizing detecting fast, this patent have studied emphatically the recovery of diluted acid extraction process sample, and realizes the accurate detection to grain samples on this basis.
1 main material
1.1 nitric acid, sulfuric acid, perchloric acid hydrogen peroxide assay are pure: Xi Long chemical industry company limited by shares; Graphite furnace atomic absorption spectrophotometer: Institute of Analysis of Sichuan Province measures; Cadmium standard model, plumbous standard model, mercury standard model, arsenic standard model, chromium standard model, copper standard model: national non-ferrous metal and electronic material Institute of Analysis product;
1.2 each testing agency and collect on the market respectively containing an above-mentioned Heavy Metallic Elements rice, wheat, corn and each 6 increment product of Chinese sorghum;
2. method
2.1 get above-mentioned rice, wheat, corn, each 6 parts of use comminutors of Chinese sorghum sample containing a Heavy Metallic Elements pulverizes, and crosses 20 eye mesh screens;
2.2 respectively compound concentration be the dilute nitric acid solution of 1%, 5%, 10%, 15%, dosage abstraction reaction 30min is extracted by 4 parts, 1 portion of grain, the centrifugal 5min of 4000r/min, with its recovery of first method comparative determination of heavy metal analysis GB in corresponding food (cadmium in foods measures GB/T5009.15-2003, Pb in food measures GB/T5009.12-2010, Mercury In Food measures GB/T5009.17-2003, in Determination of Arsenic in Food GB/T5009.11-2003, food in chromium mark feed GB/T5009.123-2003, food chromium mark feed GB/T5009.13-2003).
2.3 working concentrations are 10% dilute acid soln, respectively by 1 portion of grain and 2 portions of extraction agents, and 1 portion of grain and 4 portions of extraction agents, 1 point of grain and 6 portions of extraction agents, 1 portion of grain and 8 parts of extraction agent abstraction reaction 30min, the centrifugal 5min of 4000r/min, with corresponding its recovery of GB first method comparative determination.
2.4 working concentrations are 10% dilute acid soln, by 1 portion of grain and 4 portions of extraction agents, adopt that the 5min time extracts, the 10min time extracts, the 15min time extracts, the 20min time extracts, the 25min time extracts, the 30min time extracts respectively, the centrifugal 5min of 4000r/min, with corresponding its recovery of GB first method comparative determination.
Result
Dust technology extracts the optimum condition of each heavy metal element in grain as following table:
Under corresponding optimum condition, the recovery is shown as follows:
Heavy metal classification The recovery/% Average recovery rate/%
Cadmium 65.1～73.4 69.8
Plumbous 55.2～65.4 58.6
Mercury 50.7～61.4 56.6
Arsenic 53.7～58.4 55.6
Chromium 64.0～73.5 69
Copper 51.0～55.7 53.5
Prepared by experimental example 3 heavy metal cadmium rapid quantitative detection reagent box
1, cardinal principle
Colloidal gold immunity chromatography detects heavy metal, based on specific recognition and the reaction of antigen and antibody.Because heavy metal belongs to Small molecular, comlete antigen could be formed with protein combination after forming haptens after the functional group that the first coupling of palpus is certain, then just can carry out immunoassay.
Cleaning Principle take nitrocellulose filter as carrier, be coated with heavy metal holoantigen (T line), after adding measuring samples, heavy metal ion sequestrant is combined with colloid gold label monoclonal antibody, there is specific binding and be trapped, the red stripes manifested in unconjugated colloid gold label monoclonal antibody and complete detectable antigens (T line).And by the Instrument measuring T line color depth, and contrast with the typical curve be preset in instrument, thus accurately judge metal ion content.
2, main material and instrument
2.1 cadmiums (Cd) standard model: national non-ferrous metal and electronic material Institute of Analysis product; Cd monoclonal antibody specific, Cd holoantigen, sheep anti-mouse igg: An Punuo bio tech ltd, Chengdu product; Gold chloride: Sigma Products; Nitrocellulose filter: MILLIPORE (U.S.), M135; Bovine serum albumin(BSA) (BSA), Macrogol 2000: Sigma product; Glass fibre element film, adsorptive pads, base plate: the outstanding biologics in Shanghai; Other common agents is analytical reagent.
2.2 containing heavy metal Cd grain samples commercially or testing agency obtain, totally 7 parts, use the first method of GB GB/T5009.15-2003 to measure its content, wherein Cd content distribution interval is 0 ~ 200 μ g/kg2 part, Cd content distribution interval is 200 ~ 400 μ g/kg3 parts, Cd content distribution interval is 400 ~ 1000 μ g/kg2 parts.
2.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.; Hand-held collaurum readout instrument, purchased from Shenzhen Tuo Nengda Science and Technology Ltd..
3, method
The preparation of 3.1 kits
3.1.1Cd monoclonal antibody colloid gold label
The colloidal gold solution that diameter is 40nm is prepared with gold chloride-trisodium citrate reduction method, the collaurum prepared 100 parts adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.5 part of concentration is 1%, mix and blend 2h under 200r/min.Solution ph to 8.5 is regulated with ammoniacal liquor, add 0.5 part of tetrabutyl titanate, at room temperature continue reaction 24 hours, centrifugal 15min under 12000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.Get the collaurum 3 parts that the titanate esters modification of activity-OH is contained on above-mentioned surface, add 0.2 by every 100mL, 0.5,1.0,1.5,2mgCd monoclonal antibody, reaction 2h.Under 12000r/min condition, centrifugal 15min, removes supernatant, is dissolved to 1/20 of colloidal gold solution volume with redissolution liquid.Be sprayed on 2cm wide glass fibre element film gold mark pad by 4 μ l/cm, dry 18 ~ 24 hours for 37 DEG C, gold mark pad must be made.
3.1.2NC film bag by process is: Cd holoantigen is mixed with 0.1 with 0.01MpH7.4 phosphate buffer, 0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, against murine IgG is mixed with 0.1,0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, carrying out line bag respectively by C, T line with drawing film instrument with 1 μ l/cm speed, being placed on 37 degree of oven dryings 12 hours.
3.1.3 the assembling of kit: it is 20 ~ 30 DEG C that kit is assemblied in temperature, humidity is that the indoor of <40% are carried out, get plastic bottom board, the NC film wrapping quilt is placed in the middle part of plastic floor and pastes, gold is marked pad and ride over NC film T line side, gold mark pad rides over 1mm on NC film and pastes, and gold mark pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with test paper lower limb.In NC film side, overlap joint inhales sample paper, rides over 1mm on NC film and pastes.Finally use cutting cutter that the plastic plate posted is cut into the wide test strips of 5mm, reinstall in plastic clip, form kit.
3.1.4 test strips technological parameter debugging: the reagent of marks different for concentration, bag quilt is carried out combination pairing, be prepared into sample, uses Cd standard model to test reagent, finds out best of breed.
3.2 extraction agents: be the dilute nitric acid solution of 10%.
3.3 dilutions: use 0.01M, 0.02M, 0.04MPBS solution respectively, add the tween-20 that final concentration is respectively 0.5%, 1%, 1.5,2%, and adding final concentration is 1 × 10 -9, 1 × 10 -8, 1 × 10 -7, 1 × 10 -6, 1 × 10 -5, 1 × 10 -4, 2 × 10 -the complexing agent ITCBE of 3g/mL is configured to dilution.
3.4 typical curve preparations, conversion and typing: after determining test paper technological parameter, with the Cd standard model that concentration is 0,50,100,200,400,600,800,1000 μ g/kg, kit is measured respectively, the standard model of variable concentrations demonstrates different intensity T lines, uses collaurum readout instrument to measure its intensity.Standard is added the recovery 69.8% of sample concentration divided by Cd as Cd concentration value in working sample; Make production standard curve, and relevant parameter is imported in readout instrument, complete instrument parameter of curve and arrange.
3.5 detection method
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens;
2) use small-sized balance precise 1g to detect measuring samples in extraction flask, add 4mL extraction agent, under room temperature, earthquake frequency is react 5min under 2 ~ 60cpm condition;
3) use hydro-extractor or filtration devices, get supernatant, accurately pipette in 40 μ l clear liquid to 160 μ l dilutions with pipettor device and mix;
4) take out kit and kit and above-mentioned mixed liquor are put into dry type thermostat 5min, temperature is set as 37 DEG C;
5) get the above-mentioned mixed liquor of 120 μ l with pipettor to add in kit well, continue to react 20min under 37 DEG C of isoperibols;
6) kit reacted is put into the concentration that hand-held collaurum readout instrument can demonstrate sample Cd.
3.6 pairs commercially or testing agency obtain 7 increment product use heavy metal cadmium rapid quantitative detection reagent boxes carry out detections also and atomic absorption spectrography (AAS) result detect its accuracy of comparative analysis; Compound concentration is respectively 200ppb, 400ppb, 800ppb standard model solution and uses heavy metal cadmium rapid quantitative detection reagent box and analyze detection, and each Concentration Testing analyzes its precision 6 times.
4 results
4.1 redissolution liquid are that 20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20; The suitableeest labelled amount of modified colloidal gold is 1mg/100mL; Best Cd holoantigen package amount 1.5mg/mL; Dilution top condition is 0.2MPBS, and final concentration is 1%tween-20,1 × 10 -7g/mLEDTA.
4.2 accuracy testing results
Paired sample T test method is done to this group data, t=0.707, t two tail critical (0.05,6)=2.447, t<t two tail critical (0.05,6), illustrate that heavy metal cadmium rapid quantitative detection reagent box testing result and atomic absorption spectrography (AAS) testing result are without remarkable result difference.
4.3 precision testing results
From result and analyze, the heavy metal cadmium rapid quantitative detection reagent box duplicate detection result coefficient of variation is that between 10.6 ~ 12.2, average coefficient of variation is less than 15%.
Testing result show the detection kit of heavy metal cadmium of the present invention and detection system functional, cadmium content in measuring samples can be detected fast and accurately, be applicable to the Quantitative detection at laboratory and sampling scene, meet client to heavy metal cadmium Quantitative detection requirement in food.
Prepared by experimental example 4 heavy metal lead rapid quantitative detection reagent box
1, main material
1.1 lead (Pb) standard model: national non-ferrous metal and electronic material Institute of Analysis product; Pb monoclonal antibody specific, Pb holoantigen, sheep anti-mouse igg: An Punuo bio tech ltd, Chengdu product; Gold chloride: Sigma Products; Nitrocellulose filter: MILLIPORE (U.S.), M135; Bovine serum albumin(BSA) (BSA), Macrogol 2000: Sigma product; Glass fibre element film, adsorptive pads, base plate: the outstanding biologics in Shanghai; Other common agents is analytical reagent.
1.2 containing heavy metal Pb grain samples commercially or testing agency obtain, totally 6 parts, use the first method of GB GB/T5009.12-2010 to measure its content, wherein Pb content distribution interval is that 0 ~ 200 μ g/kg2 part Pb content distribution interval is 200 ~ 400 μ g/kg2 parts, Pb content distribution interval is 400 ~ 1000 μ g/kg2 parts.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.; Hand-held collaurum readout instrument, purchased from Shenzhen Tuo Nengda Science and Technology Ltd..
2, method
The preparation of 2.1 kits
2.1.1Pb monoclonal antibody colloid gold label:
The colloidal gold solution that diameter is 40nm is prepared with gold chloride-trisodium citrate reduction method, the collaurum prepared 100 parts adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.5 part of concentration is 1%, mix and blend 2h under 200r/min.Solution ph to 8.5 is regulated with ammoniacal liquor, add 0.5 part of tetrabutyl titanate, at room temperature continue reaction 24 hours, centrifugal 15min under 12000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.Get the collaurum 3 parts that the titanate esters modification of activity-OH is contained on above-mentioned surface, add 0.2 by every 100mL, 0.5,1.0,1.5,2mgPb monoclonal antibody, reaction 2h.Under 12000r/min condition, centrifugal 15min, removes supernatant, is dissolved to 1/20 of colloidal gold solution volume with redissolution liquid.Be sprayed on 2cm wide glass fibre element film gold mark pad by 4 μ l/cm, dry 18 ~ 24 hours for 37 DEG C, gold mark pad must be made.
2.1.2NC film bag by process is: Pb holoantigen is mixed with 0.1 with 0.01MpH7.4 phosphate buffer, 0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, against murine IgG is mixed with 0.1,0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, carrying out line bag respectively by C, T line with drawing film instrument with 1 μ l/cm speed, being placed on 37 degree of oven dryings 12 hours.
2.1.3 the assembling of kit: it is 20 ~ 30 DEG C that kit is assemblied in temperature, humidity is that the indoor of <40% are carried out, get plastic bottom board, the NC film wrapping quilt is placed in the middle part of plastic floor and pastes, gold is marked pad and ride over NC film T line side, gold mark pad rides over 1mm on NC film and pastes, and gold mark pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with test paper lower limb.In NC film side, overlap joint inhales sample paper, rides over 1mm on NC film and pastes.Finally use cutting cutter that the plastic plate posted is cut into the wide test strips of 5mm, reinstall in plastic clip, form kit.
2.1.4 test strips technological parameter debugging: the reagent of concentration not isolabeling, bag quilt is carried out combination pairing, be prepared into sample, use Pb standard model is tested reagent, finds out best of breed.
2.2 extraction agents: be the dilute nitric acid solution of 10%.
2.3 dilutions: use 0.01M, 0.02M, 0.04MPBS solution respectively, add the tween-20 that final concentration is respectively 0.5%, 1%, 1.5,2%, and adding final concentration is 1 × 10 -9, 1 × 10 -8, 1 × 10 -7, 1 × 10 -6, 1 × 10 -5, 1 × 10 -4, 2 × 10 -3the complexing agent ITCBE of g/mL is configured to dilution.
2.4 typical curve preparations, conversion and typing: after determining test paper technological parameter, with the Pb standard model that concentration is 0,50,100,200,400,600,800,1000 μ g/kg, kit is measured respectively, the standard model of variable concentrations demonstrates different intensity T lines, uses collaurum readout instrument to measure its intensity.Standard is added the recovery 58.6% of sample concentration divided by Pb as Pb concentration value in working sample; Make to be used as special software production standard curve, and relevant parameter is imported in readout instrument, complete instrument parameter of curve and arrange.
2.5 detection method
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens;
2) use small-sized balance precise 1g to detect measuring samples in extraction flask, add 4mL extraction agent, under room temperature, earthquake frequency is react 10min under 2 ~ 60cpm condition;
3) use hydro-extractor or filtration devices to get clear liquid, accurately pipette in 40 μ l clear liquid to 160 μ l dilutions with pipettor device and mix;
4) take out kit and kit and above-mentioned mixed liquor are put into dry type thermostat 5min, temperature is set as 37 DEG C;
5) get the above-mentioned mixed liquor of 120 μ l with pipettor to add in kit well, continue to react 20min under 37 DEG C of isoperibols;
6) kit reacted is put into the concentration that hand-held collaurum readout instrument can demonstrate sample Pb.
2.6 pairs commercially or testing agency obtain 6 increment product use heavy metal lead rapid quantitative detection reagent boxes carry out detections also and atomic absorption spectrography (AAS) result detect its accuracy of comparative analysis; Compound concentration is respectively 200ppb, 400ppb, 800ppb standard model solution and uses heavy metal lead rapid quantitative detection reagent box and analyze detection, and each Concentration Testing analyzes its precision 6 times.
3, result
3.1 the suitableeest labelled amounts are 1.5mg/100mL; The liquid that redissolves is that 20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20; The suitableeest labelled amount of modified colloidal gold is 1.5mg/100mL; Best Pb holoantigen package amount 1.0mg/mL; Dilution top condition is 0.2MPBS, and final concentration is 1%tween-20,1 × 10 -6g/mLITCBE; Above technique different batches may need suitable adjusting process.
3.2 accuracy testing results
Paired sample T test method is done to this group data, t=0.018, t two tail critical (0.05,5)=2.571, t<t two tail critical (0.05,5), illustrate that heavy metal lead rapid quantitative detection reagent box testing result and atomic absorption spectrography (AAS) testing result are without remarkable result difference.
3.3 precision testing results
From result and analyze, the heavy metal lead rapid quantitative detection reagent box duplicate detection result coefficient of variation is that between 12.3 ~ 12.8, average coefficient of variation is less than 15%.
Testing result show the detection kit of heavy metal lead of the present invention and detection system functional, lead content in measuring samples can be detected fast and accurately, be applicable to the Quantitative detection at laboratory and sampling scene, meet client to heavy metal lead Quantitative detection requirement in food.
Prepared by experimental example 5 heavy metal Hg rapid quantitative detection reagent box
1, main material
1.1 mercury (Hg) standard model: national non-ferrous metal and electronic material Institute of Analysis product; Hg monoclonal antibody specific, Hg holoantigen, sheep anti-mouse igg: An Punuo bio tech ltd, Chengdu product; Gold chloride: Sigma Products; Nitrocellulose filter: MILLIPORE (U.S.), M135; Bovine serum albumin(BSA) (BSA), Macrogol 2000: Sigma product; Glass fibre element film, adsorptive pads, base plate: the outstanding biologics in Shanghai; Other common agents is analytical reagent.
1.2 containing heavy metal Hg grain samples commercially or testing agency obtain, totally 5 parts, use the first method of GB GB/T5009.17-2003 to measure its content, wherein Hg content distribution interval is that 0 ~ 200 μ g/kg2 part Hg content distribution interval is 200 ~ 400 μ g/kg2 parts, Hg content distribution interval is 400 ~ 1000 μ g/kg1 parts.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.; Hand-held collaurum readout instrument, purchased from Shenzhen Tuo Nengda Science and Technology Ltd..
2, method
The preparation of 2.1 kits
2.1.1Hg monoclonal antibody colloid gold label
The colloidal gold solution that diameter is 40nm is prepared with gold chloride-trisodium citrate reduction method, the collaurum prepared 100 parts adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.5 part of concentration is 1%, mix and blend 2h under 200r/min.Solution ph to 8.5 is regulated with ammoniacal liquor, add 0.5 part of tetrabutyl titanate, at room temperature continue reaction 24 hours, centrifugal 15min under 12000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.Get the collaurum 3 parts that the titanate esters modification of activity-OH is contained on above-mentioned surface, add 0.2 by every 100mL, 0.5,1.0,1.5,2mgHg monoclonal antibody, reaction 2h.Under 12000r/min condition, centrifugal 15min, removes supernatant, is dissolved to 1/20 of colloidal gold solution volume with redissolution liquid.Be sprayed on 2cm wide glass fibre element film gold mark pad by 4 μ l/cm, dry 18 ~ 24 hours for 37 DEG C, gold mark pad must be made.
2.1.2NC film bag by process is: Hg holoantigen is mixed with 0.1 with 0.01MpH7.4 phosphate buffer, 0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, against murine IgG is mixed with 0.1,0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, carrying out line bag respectively by C, T line with drawing film instrument with 1 μ l/cm speed, being placed on 37 degree of oven dryings 12 hours.
2.1.3 the assembling of kit: it is 20 ~ 30 DEG C that kit is assemblied in temperature, humidity is that the indoor of <40% are carried out, get plastic bottom board, the NC film wrapping quilt is placed in the middle part of plastic floor and pastes, gold is marked pad and ride over NC film T line side, gold mark pad rides over 1mm on NC film and pastes, and gold mark pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with test paper lower limb.In NC film side, overlap joint inhales sample paper, rides over 1mm on NC film and pastes.Finally use cutting cutter that the plastic plate posted is cut into the wide test strips of 5mm, reinstall in plastic clip, form kit.
2.1.4 test strips technological parameter debugging: the reagent of concentration not isolabeling, bag quilt is carried out combination pairing, be prepared into sample, use Hg standard model is tested reagent, finds out best of breed.
2.2 extraction agents: be the dilute nitric acid solution of 10%.
2.3 use 0.01M, 0.02M, 0.04MPBS solution respectively, add the tween-20 that final concentration is respectively 0.5%, 1%, 1.5,2%, and adding final concentration is 1 × 10 -9, 1 × 10 -8, 1 × 10 -7, 1 × 10 -6, 1 × 10 -5, 1 × 10 -4, 2 × 10 -3the complexing agent ITCBE of g/mL is configured to dilution.
2.4 typical curve preparations, conversion and typing: after determining test paper technological parameter, with the Hg standard model that concentration is 0,50,100,200,400,600,800,1000 μ g/kg, kit is measured respectively, the standard model of variable concentrations demonstrates different intensity T lines, uses collaurum readout instrument to measure its intensity.Standard is added the recovery 56.6% of sample concentration divided by Hg as Hg concentration value in working sample; Make to be used as special software production standard curve, and relevant parameter is imported in readout instrument, complete instrument parameter of curve and arrange.
2.5 detection method
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens;
2) use small-sized balance precise 1g to detect measuring samples in extraction flask, add 4mL extraction agent, under room temperature, earthquake frequency is react 5min under 2 ~ 60cpm condition;
3) use hydro-extractor or filtration devices to get clear liquid, accurately pipette in 40 μ l clear liquid to 160 μ l dilutions with pipettor device and mix;
4) take out kit and kit and above-mentioned mixed liquor are put into dry type thermostat 5min, temperature is set as 37 DEG C;
5) get the above-mentioned mixed liquor of 120 μ l with pipettor to add in kit well, continue to react 20min under 37 DEG C of isoperibols;
6) kit reacted is put into the concentration that hand-held collaurum readout instrument can demonstrate sample Hg.
2.6 pairs commercially or testing agency obtain 5 increment product use heavy metal Hg rapid quantitative detection reagent boxes carry out detections also and atomic absorption spectrography (AAS) result detect its accuracy of comparative analysis; Compound concentration is respectively 200ppb, 400ppb, 800ppb standard model solution and uses heavy metal Hg rapid quantitative detection reagent box and analyze detection, and each Concentration Testing analyzes its precision 6 times.
3, result
3.1 the suitableeest labelled amounts are 1.5mg/100mL; The liquid that redissolves is that 20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20; The suitableeest labelled amount of modified colloidal gold is 1.5mg/100mL; Best Hg holoantigen package amount 1.0mg/mL; Dilution top condition is 0.2MPBS, and final concentration is 1.5%tween-20,1 × 10 -7g/mLITCBE; Above technique different batches may need suitable adjusting process.
3.2 accuracy testing results
Paired sample T test method is done to this group data, t=0.018, t two tail critical (0.05,4)=2.776, t<t two tail critical (0.05,4), illustrate that heavy metal Hg rapid quantitative detection reagent box testing result and atomic absorption spectrography (AAS) testing result are without remarkable result difference.
3.3 precision testing results
From result and analyze, the heavy metal Hg rapid quantitative detection reagent box duplicate detection result coefficient of variation is that between 7.7 ~ 14.1, average coefficient of variation is less than 15%.
Testing result show the detection kit of heavy metal Hg of the present invention and detection system functional, mercury content in measuring samples can be detected fast and accurately, be applicable to the Quantitative detection at laboratory and sampling scene, meet client to heavy metal Hg Quantitative detection requirement in food.
Prepared by embodiment 6 heavy metal arsenic rapid quantitative detection reagent box
1, main material
1.1 arsenic (As) standard model: national non-ferrous metal and electronic material Institute of Analysis product; As monoclonal antibody specific, As holoantigen, sheep anti-mouse igg: An Punuo bio tech ltd, Chengdu product; Gold chloride: Sigma Products; Nitrocellulose filter: MILLIPORE (U.S.), M135; Bovine serum albumin(BSA) (BSA), Macrogol 2000: Sigma product; Glass fibre element film, adsorptive pads, base plate: the outstanding biologics in Shanghai; Other common agents is analytical reagent.
1.2 containing heavy metal As grain samples commercially or testing agency obtain, totally 6 parts, use the first method of GB GB/T5009.11-2003 to measure its content, wherein As content distribution interval is that 0 ~ 200 μ g/kg2 part As content distribution interval is 200 ~ 400 μ g/kg3 parts, As content distribution interval is 400 ~ 1000 μ g/kg1 parts.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.; Hand-held collaurum readout instrument, purchased from Shenzhen Tuo Nengda Science and Technology Ltd..
2, method
The preparation of 2.1 kits
2.1.1As monoclonal antibody colloid gold label
The colloidal gold solution that diameter is 40nm is prepared with gold chloride-trisodium citrate reduction method, the collaurum prepared 100 parts adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.5 part of concentration is 1%, mix and blend 2h under 200r/min.Solution ph to 8.5 is regulated with ammoniacal liquor, add 0.5 part of tetrabutyl titanate, at room temperature continue reaction 24 hours, centrifugal 15min under 12000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.Get the collaurum 3 parts that the titanate esters modification of activity-OH is contained on above-mentioned surface, add 0.2 by every 100mL, 0.5,1.0,1.5,2mgAs monoclonal antibody, reaction 2h.Under 12000r/min condition, centrifugal 15min, removes supernatant, is dissolved to 1/20 of colloidal gold solution volume with redissolution liquid.Be sprayed on 2cm wide glass fibre element film gold mark pad by 4 μ l/cm, dry 18 ~ 24 hours for 37 DEG C, gold mark pad must be made.
2.1.2NC film bag by process is: As holoantigen is mixed with 0.1 with 0.01MpH7.4 phosphate buffer, 0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, against murine IgG is mixed with 0.1,0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, carrying out line bag respectively by C, T line with drawing film instrument with 1 μ l/cm speed, being placed on 37 degree of oven dryings 12 hours.
2.1.3 the assembling of kit: it is 20 ~ 30 DEG C that kit is assemblied in temperature, humidity is that the indoor of <40% are carried out, get plastic bottom board, the NC film wrapping quilt is placed in the middle part of plastic floor and pastes, gold is marked pad and ride over NC film T line side, gold mark pad rides over 1mm on NC film and pastes, and gold mark pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with test paper lower limb.In NC film side, overlap joint inhales sample paper, rides over 1mm on NC film and pastes.Finally use cutting cutter that the plastic plate posted is cut into the wide test strips of 5mm, reinstall in plastic clip, form kit.
2.1.4 test strips technological parameter debugging: the reagent of concentration not isolabeling, bag quilt is carried out combination pairing, be prepared into sample, use As standard model is tested reagent, finds out best of breed.
2.2 extraction agents: be the dilute nitric acid solution of 10%.
2.3 dilutions: use 0.01M, 0.02M, 0.04MPBS solution respectively, add the tween-20 that final concentration is respectively 0.5%, 1%, 1.5,2%, and adding final concentration is 1 × 10 -9, 1 × 10 -8, 1 × 10 -7, 1 × 10 -6, 1 × 10 -5, 1 × 10 -4, 2 × 10 -3the complexing agent ITCBE of g/mL is configured to dilution.
2.4 typical curve preparations, conversion and typing: after determining test paper technological parameter, with the As standard model that concentration is 0,50,100,200,400,600,800,1000 μ g/kg, kit is measured respectively, the standard model of variable concentrations demonstrates different intensity T lines, uses collaurum readout instrument to measure its intensity.Standard is added the recovery 55.6% of sample concentration divided by As As concentration value in working sample; Make to be used as special software production standard curve, and relevant parameter is imported in readout instrument, complete instrument parameter of curve and arrange.
2.5 detection method
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens;
2) use small-sized balance precise 1g to detect measuring samples in extraction flask, add 4mL extraction agent, under room temperature, earthquake frequency is react 10min under 2 ~ 60cpm condition;
3) use hydro-extractor or filtration devices to get clear liquid, accurately pipette in 40 μ l clear liquid to 160 μ l dilutions with pipettor device and mix;
4) take out kit and kit and above-mentioned mixed liquor are put into dry type thermostat 5min, stablize and be set as 37 DEG C;
5) get the above-mentioned mixed liquor of 120 μ l with pipettor to add in kit well, continue to react 20min under 37 DEG C of isoperibols;
6) kit reacted is put into the concentration that hand-held collaurum readout instrument can demonstrate sample As.
2.6 pairs commercially or testing agency obtain 6 increment product use heavy metal arsenic rapid quantitative detection reagent boxes carry out detections also and atomic absorption spectrography (AAS) result detect its accuracy of comparative analysis; Compound concentration is respectively 200ppb, 400ppb, 800ppb standard model solution and uses heavy metal arsenic rapid quantitative detection reagent box and analyze detection, and each Concentration Testing analyzes its precision 6 times.
3, result
3.1 traditional method colloid gold label pH value are 8.5; The suitableeest labelled amount is 1.0mg/100mL; The liquid that redissolves is that 20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20; The suitableeest labelled amount of modified colloidal gold is 1.0mg/100mL; Best As holoantigen package amount 1.0mg/mL; Dilution top condition is 0.2MPBS, and final concentration is 1.5%tween-20,1 × 10 -5g/mLITCBE; Above technique different batches may need suitable adjusting process.
3.2 accuracy testing results
Paired sample T test method is done to this group data, t=0.255, t two tail critical (0.05,5)=2.571, t<t two tail critical (0.05,5), illustrate that heavy metal arsenic rapid quantitative detection reagent box testing result and atomic absorption spectrography (AAS) testing result are without remarkable result difference.
3.3 precision testing results
From result and analyze, the heavy metal arsenic rapid quantitative detection reagent box duplicate detection result coefficient of variation is that between 13.8 ~ 14.4, average coefficient of variation is less than 15%
Testing result show the detection kit of heavy metal arsenic of the present invention and detection system functional, arsenic content in measuring samples can be detected fast and accurately, be applicable to the Quantitative detection at laboratory and sampling scene, meet client to heavy metal arsenic Quantitative detection requirement in food.
Prepared by embodiment 7 heavy metal chromium rapid quantitative detection reagent box
1, main material
1.1 chromium (Cr) standard model: national non-ferrous metal and electronic material Institute of Analysis product; Cr monoclonal antibody specific, Cr holoantigen, sheep anti-mouse igg: An Punuo bio tech ltd, Chengdu product; Gold chloride: Sigma Products; Nitrocellulose filter: MILLIPORE (U.S.), M135; Bovine serum albumin(BSA) (BSA), Macrogol 2000: Sigma product; Glass fibre element film, adsorptive pads, base plate: the outstanding biologics in Shanghai; Other common agents is analytical reagent.
1.2 containing heavy metal Cr grain samples commercially or testing agency obtain, totally 5 parts, use the first method of GB GB/T5009.123-2003 to measure its content, wherein Cr content distribution interval is that 0 ~ 200 μ g/kg3 part Cr content distribution interval is 200 ~ 400 μ g/kg1 parts, Cr content distribution interval is 400 ~ 1000 μ g/kg1 parts.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.; Hand-held collaurum readout instrument, purchased from Shenzhen Tuo Nengda Science and Technology Ltd..
2, method
The preparation of 2.1 kits
2.1.1Cr monoclonal antibody colloid gold label
The colloidal gold solution that diameter is 40nm is prepared with gold chloride-trisodium citrate reduction method, the collaurum prepared 100 parts adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.5 part of concentration is 1%, mix and blend 2h under 200r/min.Solution ph to 8.5 is regulated with ammoniacal liquor, add 0.5 part of tetrabutyl titanate, at room temperature continue reaction 24 hours, centrifugal 15min under 12000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.Get the collaurum 3 parts that the titanate esters modification of activity-OH is contained on above-mentioned surface, add 0.2 by every 100mL, 0.5,1.0,1.5,2mgCr monoclonal antibody, reaction 2h.Under 12000r/min condition, centrifugal 15min, removes supernatant, is dissolved to 1/20 of colloidal gold solution volume with redissolution liquid.Be sprayed on 2cm wide glass fibre element film gold mark pad by 4 μ L/cm, dry 18 ~ 24 hours for 37 DEG C, gold mark pad must be made.
2.1.2NC film bag by process is: Cr holoantigen is mixed with 0.1 with 0.01MpH7.4 phosphate buffer, 0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, against murine IgG is mixed with 0.1,0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, carrying out line bag respectively by C, T line with drawing film instrument with 1 μ L/cm speed, being placed on 37 degree of oven dryings 12 hours.
2.1.3 the assembling of kit: it is 20 ~ 30 DEG C that kit is assemblied in temperature, humidity is that the indoor of <40% are carried out, get plastic bottom board, the NC film wrapping quilt is placed in the middle part of plastic floor and pastes, gold is marked pad and ride over NC film T line side, gold mark pad rides over 1mm on NC film and pastes, and gold mark pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with test paper lower limb.In NC film side, overlap joint inhales sample paper, rides over 1mm on NC film and pastes.Finally use cutting cutter that the plastic plate posted is cut into the wide test strips of 5mm, reinstall in plastic clip, form kit.
2.1.4 test strips technological parameter debugging: the reagent of concentration not isolabeling, bag quilt is carried out combination pairing, be prepared into sample, use Cr standard model is tested reagent, finds out best of breed.
2.2 extraction agents: be the dilute nitric acid solution of 10%.
2.3 use 0.01M, 0.02M, 0.04MPBS solution respectively, add the tween-20 that final concentration is respectively 0.5%, 1%, 1.5,2%, and adding final concentration is 1 × 10 -9, 1 × 10 -8, 1 × 10 -7, 1 × 10 -6, 1 × 10 -5, 1 × 10 -4, 2 × 10 -3the complexing agent ITCBE of g/mL is configured to dilution.
2.4 typical curve preparations, conversion and typing: after determining test paper technological parameter, with the Cr standard model that concentration is 0,50,100,200,400,600,800,1000 μ g/kg, kit is measured respectively, the standard model of variable concentrations demonstrates different intensity T lines, uses collaurum readout instrument to measure its intensity.Standard is added the recovery 69% of sample concentration divided by Cr as Cr concentration value in working sample; Make to be used as special software production standard curve, and relevant parameter is imported in readout instrument, complete instrument parameter of curve and arrange.
2.5 detection method
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens;
2) use small-sized balance precise 1g to detect measuring samples in extraction flask, add 4mL extraction agent, under room temperature, earthquake frequency is react 5min under 2 ~ 60cpm condition;
3) use hydro-extractor or filtration devices to get clear liquid, accurately pipette in 40 μ l clear liquid to 160 μ l dilutions with pipettor device and mix;
4) take out kit and kit and above-mentioned mixed liquor are put into dry type thermostat 5min, stablize and be set as 37 DEG C;
5) get the above-mentioned mixed liquor of 120 μ l with pipettor to add in kit well, continue to react 20min under 37 DEG C of isoperibols;
6) kit reacted is put into the concentration that hand-held collaurum readout instrument can demonstrate sample Cr.
2.6 pairs commercially or testing agency obtain 6 increment product use heavy metal chromium rapid quantitative detection reagent boxes carry out detections also and atomic absorption spectrography (AAS) result detect its accuracy of comparative analysis; Compound concentration is respectively 200ppb, 400ppb, 800ppb standard model solution and uses heavy metal chromium rapid quantitative detection reagent box and analyze detection, and each Concentration Testing analyzes its precision 6 times.
3, result
3.1 traditional method colloid gold label pH value are 8.5; The suitableeest labelled amount is 1.0mg/100mL; The liquid that redissolves is that 20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20; The suitableeest labelled amount of modified colloidal gold is 1.0mg/100mL; Best Cr holoantigen package amount 0.5mg/mL; Dilution top condition is 0.2MPBS, and final concentration is 1%tween-20,1 × 10 -6g/mLITCBE; Above technique different batches may need suitable adjusting process.
3.2 accuracy testing results
Paired sample T test method is done to this group data, t=0.649, t two tail critical (0.05,4)=2.776, t<t two tail critical (0.05,4), illustrate that heavy metal chromium rapid quantitative detection reagent box testing result and atomic absorption spectrography (AAS) testing result are without remarkable result difference.
3.3 precision testing results
From result and analyze, the heavy metal chromium rapid quantitative detection reagent box duplicate detection result coefficient of variation is that between 10.9 ~ 14.1, average coefficient of variation is less than 15%
Testing result show the detection kit of heavy metal chromium of the present invention and detection system functional, chromium content in measuring samples can be detected fast and accurately, be applicable to the Quantitative detection at laboratory and sampling scene, meet client to huge sum of money chromium cadmium Quantitative detection requirement in food.
Prepared by embodiment 8 heavy metal copper rapid quantitative detection reagent box
1, main material
1.1 bronze medals (Cu) standard model: national non-ferrous metal and electronic material Institute of Analysis product; Cu monoclonal antibody specific, Cu holoantigen, sheep anti-mouse igg: An Punuo bio tech ltd, Chengdu product; Gold chloride: Sigma Products; Nitrocellulose filter: MILLIPORE (U.S.), M135; Bovine serum albumin(BSA) (BSA), Macrogol 2000: Sigma product; Glass fibre element film, adsorptive pads, base plate: the outstanding biologics in Shanghai; Other common agents is analytical reagent.
1.2 containing heavy metal Cu grain samples commercially or testing agency obtain, totally 7 parts, use the first method of GB GB/T5009.13-2003 to measure its content, wherein Cu content distribution interval is that 0 ~ 200 μ g/kg3 part Cu content distribution interval is 200 ~ 400 μ g/kg3 parts, Cu content distribution interval is 400 ~ 1000 μ g/kg1 parts.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.; Hand-held collaurum readout instrument, purchased from Shenzhen Tuo Nengda Science and Technology Ltd..
2, method
The preparation of 2.1 kits
2.1.1Cu monoclonal antibody colloid gold label
The colloidal gold solution that diameter is 40nm is prepared with gold chloride-trisodium citrate reduction method, the collaurum prepared 100 parts adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.5 part of concentration is 1%, mix and blend 2h under 200r/min.Solution ph to 8.5 is regulated with ammoniacal liquor, add 0.5 part of tetrabutyl titanate, at room temperature continue reaction 24 hours, centrifugal 15min under 12000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.Get the collaurum 3 parts that the titanate esters modification of activity-OH is contained on above-mentioned surface, add 0.2 by every 100mL, 0.5,1.0,1.5,2mgCu monoclonal antibody, reaction 2h.Under 12000r/min condition, centrifugal 15min, removes supernatant, is dissolved to 1/20 of colloidal gold solution volume with redissolution liquid.Be sprayed on 2cm wide glass fibre element film gold mark pad by 4 μ l/cm, dry 18 ~ 24 hours for 37 DEG C, gold mark pad must be made.
2.1.2NC film bag by process is: Cu holoantigen is mixed with 0.1 with 0.01MpH7.4 phosphate buffer, 0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, against murine IgG is mixed with 0.1,0.5,1.0,1.5,2.0,2.5, the solution of 3.0mg/mL, carrying out line bag respectively by C, T line with drawing film instrument with 1 μ l/cm speed, being placed on 37 degree of oven dryings 12 hours.
2.1.3 the assembling of kit: it is 20 ~ 30 DEG C that kit is assemblied in temperature, humidity is that the indoor of <40% are carried out, get plastic bottom board, the NC film wrapping quilt is placed in the middle part of plastic floor and pastes, gold is marked pad and ride over NC film T line side, gold mark pad rides over 1mm on NC film and pastes, and gold mark pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with test paper lower limb.In NC film side, overlap joint inhales sample paper, rides over 1mm on NC film and pastes.Finally use cutting cutter that the plastic plate posted is cut into the wide test strips of 5mm, reinstall in plastic clip, form kit.
2.1.4 test strips technological parameter debugging: the reagent of concentration not isolabeling, bag quilt is carried out combination pairing, be prepared into sample, use Cu standard model is tested reagent, finds out best of breed.
2.2 extraction agents: be the dilute nitric acid solution of 10%.
2.3 use 0.01M, 0.02M, 0.04MPBS solution respectively, add the tween-20 that final concentration is respectively 0.5%, 1%, 1.5,2%, and adding final concentration is 1 × 10 -9, 1 × 10 -8, 1 × 10 -7, 1 × 10 -6, 1 × 10 -5, 1 × 10 -4, 2 × 10 -3the complexing agent ITCBE of g/mL is configured to dilution.
2.4 typical curve preparations, conversion and typing: after determining test paper technological parameter, with the Cu standard model that concentration is 0,50,100,200,400,600,800,1000 μ g/kg, kit is measured respectively, the standard model of variable concentrations demonstrates different intensity T lines, uses collaurum readout instrument to measure its intensity.Standard is added the recovery 53.5% of sample concentration divided by Cu as Cu concentration value in working sample; Make to be used as special software production standard curve, and relevant parameter is imported in readout instrument, complete instrument parameter of curve and arrange.
2.5 detection method
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens;
2) use small-sized balance precise 1g to detect measuring samples in extraction flask, add 4mL extraction agent, under room temperature, earthquake frequency is react 5min under 2 ~ 60cpm condition;
3) use hydro-extractor or filtration devices to get clear liquid, accurately pipette in 40 μ l clear liquid to 160 μ l dilutions with pipettor device and mix;
4) take out kit and kit and above-mentioned mixed liquor are put into dry type thermostat 5min, stablize and be set as 37 DEG C;
5) get the above-mentioned mixed liquor of 120 μ l with pipettor to add in kit well, continue to react 20min under 37 DEG C of isoperibols;
6) kit reacted is put into the concentration that hand-held collaurum readout instrument can demonstrate sample Cu.
2.6 pairs commercially or testing agency obtain 6 increment product use heavy metal copper rapid quantitative detection reagent boxes carry out detections also and atomic absorption spectrography (AAS) result detect its accuracy of comparative analysis; Compound concentration is respectively 200ppb, 400ppb, 800ppb standard model solution and uses heavy metal copper rapid quantitative detection reagent box and analyze detection, and each Concentration Testing analyzes its precision 6 times.
3, result
3.1 traditional method colloid gold label pH value are 8.5; The suitableeest labelled amount is 1.0mg/100mL; The liquid that redissolves is that 20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20; The suitableeest labelled amount of modified colloidal gold is 1.0mg/100mL; Best Cu holoantigen package amount 0.5mg/mL; Dilution top condition is 0.2MPBS, and final concentration is 1%tween-20,1 × 10 -5g/mLITCBE; Above technique different batches may need suitable adjusting process.
3.2 accuracy testing results
Paired sample T test method is done to this group data, t=0.514, t two tail critical (0.05,6)=2.447, t<t two tail critical (0.05,6), illustrate that heavy metal copper rapid quantitative detection reagent box testing result and atomic absorption spectrography (AAS) testing result are without remarkable result difference.
3.3 precision testing results
From result and analyze, the heavy metal copper rapid quantitative detection reagent box duplicate detection result coefficient of variation is that between 11.2 ~ 14.2, average coefficient of variation is less than 15%
Testing result show the detection kit of heavy metal copper of the present invention and detection system functional, copper content in measuring samples can be detected fast and accurately, be applicable to the Quantitative detection at laboratory and sampling scene, meet client to heavy metal copper Quantitative detection requirement in food.

Claims (10)

1. a preparation method for modified colloidal gold, is characterized in that: it comprises the steps:
(1) cut-off footpath is the colloidal gold solution 100 parts of 10 ~ 60nm, add two (triethanolamine) metatitanic acid diisopropyl ester solution of 0.1 ~ 3% of colloidal gold solution volume, the concentration of two (triethanolamine) metatitanic acid diisopropyl ester solution is 1%, stir 0.5 ~ 2h under 100 ~ 400r/min, obtain mixed solution;
(2) above-mentioned mixed solution pH value to 8 ~ 10 are regulated, add 0.1 ~ 2 part of tetrabutyl titanate, at room temperature with 100 ~ 400r/min stirring reaction 24 ~ 36 hours, centrifugal 5 ~ 60min under 4000 ~ 16000r/min condition, sucking-off supernatant adds ultrapure water or buffer solution for cleaning, repeat 1 ~ 2 time, be namely prepared into the colloid gold particle of the titanate esters modification of surface containing activity-OH.
2. the preparation method of a golden labeling antibody, it is characterized in that: prepare modified colloidal gold according to the method for claim 1,0.2 ~ 2mg antibody is added by every 100mL, reaction 2h, centrifugal 5 ~ 60min under 6000 ~ 12000r/min condition, remove supernatant, be dissolved to 1/20 of colloidal gold solution volume with redissolution liquid, obtain golden labeling antibody.
3. preparation method according to claim 2, is characterized in that: described antibody is Cd monoclonal antibody, Pb monoclonal antibody, Hg monoclonal antibody, As monoclonal antibody, Cr monoclonal anti or Cu monoclonal antibody.
4. detect a colloidal gold immunochromatographykit kit for heavy metal, it is characterized in that: it comprises Test paper and gets stuck, and test paper is placed in and gets stuck; Described Test paper comprises plastic bottom board (1), upper sample pad (2) is pasted in one end, one end of upper sample pad closely crimps the colloidal gold pad (3) made containing the gold mark that detection heavy metal relative answers antibody to mark to some extent, colloidal gold pad (3) one end closely crimps nitrocellulose filter (4), nitrocellulose filter is coated with detection line T and nature controlling line C (5), detection line T wraps by the comlete antigen of heavy metal to be checked, nature controlling line C is coated with dynamics, and the nitrocellulose filter other end connects inhales sample pad (6); Described position of getting stuck surperficial counter sample pad (2) is provided with well (7), and the position of surperficial corresponding nitrocellulose filter (4) of getting stuck is provided with observation panel (8);
Wherein, the collaurum in colloidal gold pad is modified colloidal gold prepared by the method for claim 1.
5. kit according to claim 4, is characterized in that: the golden labeling antibody in described colloidal gold pad is golden labeling antibody prepared by the method for Claims 2 or 3.
6. kit according to claim 5, it is characterized in that: the comlete antigen of described heavy metal is after heavy metal and complexing agent complexes, with carrier protein phase coupling shape antigen, wherein, described complexing agent is citric acid, ethylenediamine tetraacetic acid, aminoacetic acid, 1-(the different sulphur cyanobenzyl of 4-) ethylenediamine-N, one or more in N, N', N'-tetraacethyl, diethylenetriamine pentacarboxylic acid salt, glutathione and organic polydentate part; Described carrier protein is one or more in bovine serum albumin(BSA), keyhole limpet hemocyanin, ovalbumin, human albumin.
7. kit according to claim 5, is characterized in that: described nitrocellulose filter is the porous spline structure film of aperture 5-12 micron, and described sample pad is glass fibre element film or polyester film, and described suction sample paper is absorbent filter.
8. a heavy metal species quantitative detection system, is characterized in that: it comprises colloidal gold immunochromatographykit kit, dry type thermostat and collaurum readout instrument described in claim 4 ~ 7 any one, and wherein, collaurum readout instrument is a kind of hand-held collaurum readout instrument.
9. quantitatively detect a method for heavy metal, it is characterized in that: it is that detection system according to claim 8 detects.
10. method according to claim 9, is characterized in that: it comprises the steps:
1) use comminutor to pulverize required detection sample, and cross 20 eye mesh screens.
2) get measuring samples, add the extraction agent of 2 ~ 8 times of mL/g, earthquake frequency is react 5 ~ 30min under 2 ~ 60cpm condition, and filter, get supernatant, add the dilution of supernatant 2 ~ 40 times of volumes, mixing, obtains mixed liquor; Described extraction agent to be concentration be 1 ~ 15% salpeter solution; Described thinning agent is the buffer solution being added with surfactant, heavy metal complexing agent;
3) by the colloidal gold immunochromatographykit kit described in claim 4 ~ 7 any one and step 1) mixed liquor prepared puts into dry type thermostat, constant temperature 2 ~ 15min respectively, and temperature is set as 20 ~ 45 DEG C;
4) in dry type thermostat, by step 1) mixed liquor prepared adds in the well of kit, isothermal reaction 5 ~ 30min;
5) by reacted kit, be placed in collaurum readout instrument, read data.
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CN105911048A (en) * 2016-04-08 2016-08-31 合肥工业大学 Carbon nanotube labeled test paper, production method thereof, and rapid Hg<2+> detection method
CN105954506A (en) * 2016-04-28 2016-09-21 苏州市天灵中药饮片有限公司 Biochemical detection method for detecting heavy metal in traditional Chinese medicine decoction pieces
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CN108717122A (en) * 2018-06-02 2018-10-30 暨南大学 It is a kind of detection copper ion immunofluorescence chromatograph test strip and its application
CN111442960A (en) * 2020-04-20 2020-07-24 北京仪达仪器有限责任公司 Kit and method for rapidly extracting heavy metals in soil
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CN112881376A (en) * 2021-01-15 2021-06-01 山东省海洋资源与环境研究院(山东省海洋环境监测中心、山东省水产品质量检验中心) Method for rapidly detecting residual quantity of heavy metal cadmium in aquatic products
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