CN105203765B - 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 PDFInfo
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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
Technical field
The present invention relates to the method for a heavy metal species quantitative detection system and detection by quantitative heavy metal.
Background technology
With the fast-developing and growth in the living standard of China's economy, people are safe to food again higher requirement
Simultaneously as China during Construction of Market Economy because many reasons such as institutional improvement and supervision level cause China's food
Safety problem is increasingly serious.The reason for causing food-safety problem is a lot, and heavy metals exceeding standard problem is the weight for affecting food safety
Want one of factor.At present, heavy metal such as copper, lead, zinc, ferrum, cobalt, nickel, manganese, cadmium, hydrargyrum, tungsten, molybdenum, gold, silver etc..At this stage to us
The heavy metal that the mankind and environment work the mischief has following several elements：The bio-toxicities such as hydrargyrum, cadmium, lead, chromium, copper and metalloid arsenic
Significant heavy metal element.
River, lake or ocean are directly discharged into if heavy metal element is unprocessed, or in soil, due to it
Can not be biodegradable and be contaminated these rivers, lake, ocean and soil.In the biological magnification of food chain
Under, their thousands of Radix Achyranthis Bidentatae ground enrichments finally enter human body.As the heavy metal that Fish or shellfish accumulate is eaten by the mankind, or a huge sum of money
Category will enter human body after being absorbed by crops such as Oryza glutinosa, Semen Tritici aestivis by human consumption, heavy metal, make one to produce heavy metal poisoning,
It is light then strange illness (minamata disease, Itai-itai diseases etc.) occurs, it is heavy then cause death.Secondly, heavy metal can be with protein and enzyme in human body
Deng there is strong interaction, making them lose activity, also can accumulate in some organs of human body, cause chronic poisoning,
The poisoning that heavy metals exceeding standard causes is too numerous to enumerate.For the heavy metal pollution in water body and soil, currently there are no very well
Method because the heavy metal being present in water and soil is difficult degraded, and also with enriching, so the pollution of heavy metal
It is irreversible.
(1) lead：It is the toxic metals that can be put aside in human body and animal tissue.It is by skin, digestive tract, respiratory tract
Into affine with various organs in vivo, major toxicity effect is anemia, nervous function imbalance and injury of kidney, vulnerable crowd
There are child, old man, hypoimmunity crowd.
(2) cadmium：It is not the indispensable element of human body, human health can be endangered by food chain enrichment, to human body polyphyly
System has toxic action, and Cd2+ is classified as the food pollution of the 3rd priority research for FAO (Food and Agriculture Organization of the United Nation) and World Health Organization (WHO)
Thing.The toxicity of cadmium is very big, can put aside in human body, mainly puts aside in kidney, causes the changes of function of urinary system；Cadmium can take
For calcium in bone, skeleton is seriously softened, bone is broken into pieces, the dirty functional disorder of stomach can be caused, disturb the enzyme of human body and biological zinc in vivo
System, causes vascular hypertension to rise.Vulnerable crowd is mining operations person, hypoimmunity crowd.
(3) hydrargyrum：Mercury and mercuric compounds belong to extremely toxic substance, can be in people's body accumulation.Mercury metal in blood enters brain group
After knitting, gradually accumulate in cerebral tissue, cerebral tissue will be caused damage when reaching a certain amount, another part mercury ion turns
Move on to kidney.Inorganic mercury ion into water body can be changed into the bigger organic mercury of toxicity, and by food chain human body is entered, and cause complete
Body intoxication；Vulnerable crowd has women, especially ready-to-be mother, hobby seafood personage；It is mercurous few in natural water, typically
Less than 0.1 μ g/L.
(4) arsenic：It is the non-essential element of human body, the toxicity of element arsenic is extremely low, and the compound of arsenic.There are severe toxicity, trivalent
Arsenic compound is more higher than other arsenic compound toxicity.Arsenic enters human body, such as intake by the contact of respiratory tract, digestive tract and skin
More than excretion, arsenic will be accumulated at positions such as the liver of human body, kidney, lung, spleen, uterus, Placenta Hominiss, skeleton, muscle, in cell
Enzyme system is combined, and the biological agent for making enzyme is suppressed and loses activity, and is accumulated particularly in hair, fingernail, slow so as to cause
Property arseniasiss, have gastrointestinal symptom, neurological symptom and skin up to several years even decades, arseniasiss incubation period
Pathological changes etc..Arsenic also has carcinogenesis, can cause skin carcinoma.Arsenic content has very big because water source is different with geographical conditions in the surface water
Difference, 0.5 μm/L of fresh water average out to, sea water is 3.7 μm/L.
(5) chromium：Such as eat by mistake and drink, the poisoning symptoms such as abdominal discomfort and diarrhoea can be caused, cause allergic dermatitises or eczema, exhale
Inspiration enters, and has stimulation and corrosiveness to respiratory tract, causes pharyngitis, bronchitis etc..The serious Area Inhabitants of water pollution, Jing often connects
Touch or Excess free enthalpy person.Be easy to get rhinitis, tuberculosis, diarrhoea, bronchitis, dermatitis etc.
(5) copper：Excess copper is enriched with human body, occurs that Nausea and vomiting, Upper abdominal pain, Acute hemolytic crisis and renal tubules become
The intoxicating phenomenons such as shape, severe patient can also result in the harm such as acute renal failure.Copper powder dirt can cause pharyngalgia, cough and cough up phlegm, or even breast
Stifling is suppressed, fever of feeling cold.
At present routine heavy metal detection method has Atomic fluorophotometry (AFS), inductive coupling plasma mass (ICP-
MS) in vitro atomic emission spectrum (ICP-AES), high performance liquid chromatography such as analytical technology, inductive coupling etc..Answer currently numerous
For in the analytical technology of detection of heavy metal ion, atomic absorption spectrography (AAS) to be most common method, the method generally has higher
Sensitivity, but it is relatively costly, need professional and technical personnel's operation, obtain higher sensitivity need larger sample size and
Take because sample must be analyzed successively longer.It is easy and be suitable to scene it is thus impossible to it is quick to meet current batch samples
The demand of detection.
Immunoassay is a kind of analysis method with high degree of specificity and sensitivity, between heavy metal analysis mainly have
Connect competitive ELISA method and gold colloidal fast immune chromatographic method.During using immunochromatographyassay assay, need to be developed the color with gold colloidal.
Gold colloidal is by gold chloride (HAuCl4) in the reducing agent such as effect such as white phosphorus, ascorbic acid, sodium citrate, tannic acid
Under, a certain size gold grain is can be grouped to, and because electrostatic interaction becomes a kind of stable colloidal state, formed electronegative
Hydrophobic sol solution, stable colloidal state is become due to electrostatic interaction, therefore claims gold colloidal.
Immunoassay utilizes antigen and antibody specific association reaction, makes the chromogenic reagent of traget antibody, and detects each
Plant the analysis method of material (medicine, hormone, protein, microorganism etc.).Gold colloidal is negatively charged under mild alkaline conditions, can with it is anti-
The positive charge group of body is combined, because this combination is electrostatical binding, so not affecting the biological nature of antibody.Thus utilize glue
When body gold carries out immunoassay, have the advantages that it is convenient and swift do not need special installation and reagent, result to judge directly perceived, obtain extensively
It is general to use.
However, because gold colloidal only could effectively be combined under weakly alkaline environment with antibody, once the pH of detection object
It is not weakly alkaline, then cannot accurately detects, and the detection of heavy metal needs to be extracted with strong acid, although by increasing extension rate
Mode can detect reluctantly, but testing result is inaccurate.
Chinese patent application CN201010229009.1 discloses a kind of heavy metal Hg Antibody preparation and Enzyme-linked Immunosorbent Assay
The method that method determines content of beary metal, can be used to detect huge sum of money hydrargyrum, but the method accuracy is looked into, and complex operation, need
Will professional main equipment.Chinese patent application CN201020521531.2 discloses a kind of huge sum of money detection card, and the detection card can only
Detected in special environment again, and can only qualitative detection, it is impossible to which detection by quantitative is.
Therefore need on the market one kind can quickly, accurate quantitative analysis detect heavy metal, and the detecting system being convenient for carrying.
The content of the invention
It is an object of the invention 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) take 100 parts of the colloidal gold solution of a diameter of 10～60nm, add colloidal gold solution volume 0.1～3% two
(triethanolamine) metatitanic acid diisopropyl ester solution, the concentration of two (triethanolamine) metatitanic acid diisopropyl ester solution is 1%, 100～400r/
0.5～2h is stirred under min, mixed solution is obtained；
(2) adjust above-mentioned mixed solution pH value to 8～10, add 0.1～2 part of tetrabutyl titanate, at room temperature with 100～
400r/min stirring reactions 24～36 hours, are centrifuged 5～60min under the conditions of 4000～16000r/min, suction out supernatant and add
Enter ultra-pure water or buffer solution for cleaning, repeat 1～2 time, that is, be prepared into the ester modified gold colloidal of metatitanic acid of the surface containing active-OH
Grain.
In step (1), the colloidal gold solution is prepared using gold chloride-trisodium citrate reduction method.
In step (2), mixed solution pH value is adjusted to adjust using ammonia.
Present invention also offers modified colloidal gold prepared by preceding method.
Present invention also offers a kind of preparation method of gold labeling antibody, step is as follows：Aforesaid modified colloidal gold is taken, is added
Antibody, gold colloid surface activity-OH react with antibody Fc section-COOH to be formed it is stable be bonded, formed gold labeling antibody.
Preferably, the antibody be Cd monoclonal antibodies, Pb monoclonal antibodies, Hg monoclonal antibodies, As monoclonal antibodies,
Cr monoclonal antis or Cu monoclonal antibodies.
Present invention also offers gold labeling antibody prepared by preceding method.
Present invention also offers the purposes of aforementioned modified gold colloidal or aforementioned gold labeling antibody in immune colloid gold detection.
Preferably, the immune colloid gold detection is the dyeing of immune colloid gold light microscopic, immune colloid gold electronic speculum dyeing, colloid
Golden immunochromatography.
The colloidal gold immunochromatographykit kit of present invention detection heavy metal：It includes Test paper and gets stuck that reagent paper is placed in
In getting stuck；The Test paper includes plastic bottom board 1, and upper sample pad 2 is pasted in one end, and one end of upper sample pad closely crimps and contains
The colloidal gold pad 3 that heavy metal relative answers the gold mark of antibody institute labelling to make is detected, the one end of colloidal gold pad 3 closely crimps nitre
Acid cellulose film 4, is coated with detection line T and nature controlling line C5 on nitrocellulose filter, detection line T is coated with the complete of heavy metal to be checked
Holoantigen, is coated with dynamics, nitrocellulose filter other end connection sample suction pad 6 on nature controlling line C；The surface of getting stuck
The position of counter sample pad 2 is provided with well 7, and the position of the surface correspondence nitrocellulose filter 4 that gets stuck is provided with observation panel 8；Wherein,
Gold colloidal in colloidal gold pad is modified colloidal gold prepared by preceding method.
Preferably, the gold labeling antibody in the colloidal gold pad is gold labeling antibody prepared by preceding method.
Preferably, the complete antigen of the heavy metal is after heavy metal and chelating agent complexation, with carrier protein shape to be mutually coupled
Antigen, wherein, the chelating agent is citric acid, ethylenediaminetetraacetic acid, glycine, 1- (the different sulfur cyanobenzyls of 4-) vinyl two
One or more in amine-N, N, N', N'- tetraacethyl, diethylenetriamine pentacarboxylic acid salt, glutathion and organic polydentate part；
The carrier protein is one or more in bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, human albumin.
Preferably, the nitrocellulose filter is the porous spline structure film of aperture 5-12 microns, and the sample pad is glass
Cellulose membrane or polyester film, the suction sample paper is absorbent filter.
Present invention also offers a kind of detecting system of detection heavy metal, it includes aforementioned described colloidal gold immunochromatographimethod
Test kit, dry type thermostat and gold colloidal readout instrument, wherein, gold colloidal readout instrument is a kind of hand-held gold colloidal readout instrument.
Present invention also offers a kind of method of detection heavy metal, it is that aforesaid detecting system is detected.
Preferably, it comprises the steps：
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens.
2) take measuring samples, add the extractant of 2～8 times of mL/g, earthquake frequency be reaction 5 under the conditions of 2～60cpm～
30min, filters, and takes supernatant, adds the diluent of 2～40 times of volumes of supernatant, mixes, and obtains mixed liquor；The extractant is concentration
For 1～15% salpeter solution；The diluent is the buffer solution for being added with surfactant, heavy metal complexing agent；
3) by front described colloidal gold immunochromatographykit kit and step 1) prepare mixed liquor be respectively put into dry type constant temperature
In instrument, 2～15min of constant temperature, temperature is set as 20～45 DEG C；
4) in dry type thermostat, by step 1) prepare mixed liquor add test kit well in, isothermal reaction 5～
30min；
5) by reacted test kit, in being placed in gold colloidal readout instrument, data are read.
It is further preferred that it comprises the steps：
1) measuring samples are taken, the extractant of 4 times of mL/g is added, earthquake frequency are to react 5min under the conditions of 2～60cpm,
Filter, take supernatant, add the diluent of 4 times of volumes of supernatant, mix, obtain mixed liquor；The extractant is the nitre that concentration is 10%
Acid solution；The diluent is to be added with 1%tween20,1 × 10-7The 0.2M PBS solutions of ITCBE g/mL；
2) by aforesaid colloidal gold immunochromatographykit kit and step 1) prepare mixed liquor be respectively put in thermostat,
Constant temperature 5min, temperature is set as 37 DEG C；
3) in thermostat, by step 1) prepare mixed liquor add test kit well in, isothermal reaction
20min；
4) by reacted test kit, in being placed in gold colloidal readout instrument, data are read.
Modified colloidal gold of the present invention is the ester modified colloid gold particle of metatitanic acid of the surface containing active-OH, and it is anti-with antibody
At once, with-COOH on antibody Fc section there is condensation reaction in modified colloidal gold surface activity-OH, form stable chemical bonding,
Stable gold labeling antibody of the invention, rather than the gold labeling antibody that traditional gold colloidal is formed with antibody by electrostatical binding are obtained, this
The gold labeling antibody that bright modified colloidal gold is prepared is stablized, and not by acid or alkali environment and ionic strength affect, detection object is wanted
Ask little, it is applied widely, can be applicable to various immune colloid gold detections.
Experimental result illustrates that modified colloidal gold of the present invention is stablized with the gold labeling antibody that antibody is formed, not by sour environment
Affect, immune chromatography reagent kit of the present invention is prepared using modified colloidal of the present invention gold, in acid condition, still can accurately determine
To detection heavy metal, conventional atom absorption spectrometry can be substituted, with low cost, application prospect is good.
Obviously, the above of the invention, according to the ordinary technical knowledge and customary means of this area, without departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other various ways can also be made, is replaced or is changed.
By the following examples the specific embodiment of form, remakes further specifically to the above of the present invention
It is bright.But this scope for being interpreted as above-mentioned theme of the invention should not be only limitted to Examples below.It is all based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Description of the drawings
The structure of the reagent paper of Fig. 1 detection kit of the present invention and use schematic diagram.In figure：1- plastic bottom boards, 2- sample pad,
3- colloidal gold pads, 4- nitrocellulose filters, 5- detection lines T and nature controlling line C, 6- sample suction pad.
The structural representation for getting stuck of Fig. 2 detection kit of the present invention.In figure：7- wells, 8- observation panels.
Specific embodiment
The preparation of the modified colloidal of the present invention of embodiment 1 gold
With the colloidal gold solution that gold chloride-trisodium citrate reduction method prepares a diameter of 10～60nm, the glue for completing is prepared
Body gold 100mL adds two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution that 0.1～3mL concentration is 1%, 100～
0.5～2h is mixed under 400r/min.Solution ph is adjusted to 8～10, add 0.1～2mL tetrabutyl titanates with ammonia,
Continue to react 24～36 hours under room temperature, 5～60min is centrifuged under the conditions of 4000～16000r/min, suction out supernatant and add
Ultra-pure water or buffer solution for cleaning, repeat 1～2 time, that is, be prepared into the ester modified colloid gold particle of metatitanic acid of the surface containing active-OH
(gold colloidal of the present invention).
The preparation of the gold labeling antibody of the present invention of embodiment 2
Modified colloid gold particle prepared by Example 1, adds 0.2～2mg Cd monoclonal antibodies, instead by every 100mL
2h is answered, 5～60min is centrifuged under the conditions of 6000～12000r/min, remove supernatant, with redissolution liquid colloidal gold solution body is dissolved to
Long-pending 1/20 obtains final product gold labeling antibody.
Antibody be heavy metal antibody, such as Cd monoclonal antibodies, Pb monoclonal antibodies, Hg monoclonal antibodies, As monoclonals
Antibody, Cr monoclonal antis or Cu monoclonal antibodies.
The detecting system and its using method of the present invention detection heavy metal of embodiment 3
1st, detecting system
The preparation of 1.1 test kits of the present invention
The structure of test kit of the present invention is as shown in Fig. 1～2：It includes Test paper and gets stuck that reagent paper is placed in getting stuck；Institute
Test paper is stated including plastic bottom board 1, upper sample pad 2 is pasted in one end, one end of upper sample pad closely crimps to contain and detected
Heavy metal relative answers the colloidal gold pad 3 that the gold mark of antibody institute labelling makes, and the one end of colloidal gold pad 3 closely crimps celluloid
Film 4, is coated with detection line T and nature controlling line C5 on nitrocellulose filter, detection line T is coated with the complete antigen of heavy metal to be checked,
Dynamics, nitrocellulose filter other end connection sample suction pad 6 are coated with nature controlling line C；The surface counter sample that gets stuck
The position of pad 2 is provided with well 7, and the position of the surface correspondence nitrocellulose filter 4 that gets stuck is provided with observation panel 8.
Wherein, the preparation of colloidal gold pad：Gold labeling antibody prepared by Example 2, by 4 μ l/cm 2cm width glass fibre is sprayed on
In plain film gold standard pad, 37 DEG C dry 18～24 hours, obtain final product and make gold standard pad.
Wherein, the holoantigen of the heavy metal is after heavy metal and chelating agent complexation, with carrier protein shape antigen to be mutually coupled,
Wherein, the chelating agent is citric acid, ethylenediaminetetraacetic acid, glycine, 1- (the different sulfur cyanobenzyls of 4-) ethylenediamine-N, N,
One or more in N', N'- tetraacethyl, diethylenetriamine pentacarboxylic acid salt, glutathion and organic polydentate part；The carrier
Albumen is one or more in bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, human albumin.
Wherein, involved test kit NC films are the porous spline structure film of aperture 5-12 microns, and sample pad is glass fibre element
Film or polyester film, inhale sample paper for absorbent filter composition.
Wherein, NC films coating process is：Metal holoantigen is configured to into 0.1 with 0.01M pH7.4 phosphate buffers～
The solution of 3mg/mL, by anti-Mus IgG the solution of 0.1～3mg/mL is configured to, and carries out respectively drawing with 1 μ l/cm speed with film instrument is drawn
Line is coated with C, T line, is placed on 37 degree of oven dryings 5～16 hours.
The assembling of mentioned reagent box should be 20～30 DEG C in temperature, and humidity is<40% interior is carried out, and takes plastic bottom board,
Coated NC films are placed on into plastic floor middle part to paste, gold standard pad is ridden over into NC film T lines side, gold standard pad is ridden on NC films
1mm pastes, and gold standard pad opposite side overlap joint pastes upper sample pad, and sample pad is alignd with reagent paper lower limb.In NC films side, overlap joint is inhaled
Sample paper, rides over 1mm on NC films and pastes.Finally the plastic plate for posting is cut into into 3～5mm width test strips using cutting machine, is reinstalled
In plastic clip, test kit is formed.
1.2 dry type thermostats, purchased from Hangzhou Rui Cheng Instrument Ltd.；
1.3 hand-held gold colloidal readout instruments, it can be a kind of portable optical instrument up to Science and Technology Ltd. to open up purchased from Shenzhen
Device, volume is less than 0.001m3, it is easy to operate, multiple projects can be detected, with GPS location function, can preserve and print detection
Data.It is 1～20 μ g/L to water quality detection scope for quantitatively judging to testing result, is 50 to grain detection range
～1000 μ g/kg, are 100～2000 μ g/kg to Chinese crude drug detection range.
2nd, using method
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens.
2) measuring samples are taken, the extractant for adding 2～8 times of mL/g (adds according to the different differences of choosing of different sample hygroscopic capacities
Enter amount), earthquake frequency is that 5～30min is reacted under the conditions of 2～60cpm, is filtered, and takes supernatant, adds 2～40 times of volumes of supernatant
Diluent (different extension rates correspondence respective standard curves, it is ensured that detection sensitivity degree typically 4 times of dilution), mix, must mix
Liquid；The extractant is the salpeter solution that concentration is 10%；The diluent is to be added with surfactant, heavy metal complexing agent
Buffer solution；
3) by the colloidal gold immunochromatographykit kit and step 1 described in claim 4～7 any one) prepare mixing
Liquid is respectively put in dry type thermostat, 2～15min of constant temperature (more than metal ion after 2min substantially completely and chelant ties,
Consider that sample adaptive settings are 5min), temperature is set as 20～45 DEG C, and (system can Accurate Determining weight in this temperature range
Metal, it is too low or it is too high can all affect immunoreation, immunoreation optimum temperature is 37 DEG C)；
4) in dry type thermostat, by step 1) prepare mixed liquor add test kit well in, isothermal reaction 5～
(reaction detection result keeps stable to 30min in the range of certain hour, persistently to keep stable certain hour to be advisable i.e.
20min)；
5) by reacted test kit, in being placed in gold colloidal readout instrument, data are read.
Illustrate beneficial effect of the present invention with the mode of experimental example below：
The stability test of the gold labeling antibody of the present invention of experimental example 1
Respectively using titanate esters coupling method and 40nm gold colloidal heavy metal list cadmium clonal antibodies, lead monoclonal antibody, hydrargyrum
Monoclonal antibody is marked process, while being traditionally marked process to above-mentioned antibody using 40nm gold colloidals.Mark
Gold colloidal is being stably its color in kermesinus after note, and maximum absorption band is between 530～532nm, and the fluctuation of OD value changes is little, when
When destroying its stability due to outside cause, gold colloidal produces aggregation, its color can first purpling, then blackening, finally for colourless；Its
Maximum absorption band is increasingly greater than normal peak；Simultaneously its OD value is tapered into 0.Labelling gold mark is tested as follows：
1) preserve above-mentioned gold and be marked in 4 DEG C of refrigerators, observe increases with the time, determine gold mark using ultraviolet spectrophotometer and absorb
Peak and OD value changes.
As a result such as table 1 below：
From result, new colloidal gold mark mode effectively increases gold mark storage stability.And increase over time
Traditional gold mark gradually becomes purple aggregation.
2) not commensurability 10%NaCl solution is added in above-mentioned gold mark, using its absworption peak of ultraviolet determination and OD after 30min
Value changes.
As a result such as table 2 below：
From result, new colloidal gold mark mode effectively increases stability of the gold mark to salt.
3) not commensurability 0.1M hydrochloric acid is added in above-mentioned gold mark, is become using its absworption peak of ultraviolet determination and OD values after 30min
Change.
As a result such as table 3 below：
From result, new colloidal gold mark mode effectively increases stability of the gold mark to acid.
4) not commensurability 1M sodium hydroxide is added in above-mentioned gold mark, using its absworption peak of ultraviolet determination and OD values after 30min
Change.
As a result such as table 3 below：
From result, new colloidal gold mark mode effectively increases stability of the gold mark to alkali.
By table 1～3 as can be seen that the combination of modified colloidal of the present invention gold and antibody is highly stable, not by acid or alkali environment with
Ionic strength affect, acidproof, alkaline-resisting, salt tolerant.
Heavy metal dust technology processes determination of recovery rates in the grain of experimental example 2
At present heavy metal analysis need that sample is carried out to detect after thoroughly clearing up this method takes in grain
Long, operator quality has high demands, complex operation, is not suitable with the rapid field detection to grain and requires.Therefore to the fast of sample
Fast simple process becomes the key for realizing quick detection, and this patent have studied emphatically the response rate of diluted acid extraction process sample, and
The accurate detection to grain samples is realized on this basis.
1 main material
1.1 nitric acid, sulphuric acid, perchloric acid hydrogen peroxide assay are pure：Xi Long chemical industry company limited by shares；Sampling Graphite Furnace Atomic
Absorption spectrometer：Institute of Analysis of Sichuan Province determines；Cadmium standard sample, lead standard sample, hydrargyrum standard sample, arsenic standard sample
Product, chromium standard sample, copper standard sample：National non-ferrous metal and electronic material Institute of Analysis product；
1.2 each testing agency and collect on the market respectively contain above-mentioned Heavy Metallic Elements rice, Semen Tritici aestivi, Semen Maydiss
6 parts of samples each with Sorghum vulgare Pers.；
2. method
2.1 take each 6 parts of the above-mentioned rice containing a Heavy Metallic Elements, Semen Tritici aestivi, Semen Maydiss, Sorghum vulgare Pers. sample uses pulverizer powder
It is broken, cross 20 eye mesh screens；
2.2 respectively compound concentration be 1%, 5%, 10%, 15% dilute nitric acid solution, extract dosage by 4 parts of 1 portion of grain and carry
Negate and answer 30min, (cadmium in foods determines GB/ to heavy metal analysis GB in 4000r/min centrifugation 5min, with corresponding food
T5009.15-2003, Pb in food determine GB/T5009.12-2010, Mercury In Food and determine GB/T5009.17-2003, food
Chromium mark feed GB/ in chromium mark feed GB/T5009.123-2003, food in middle arsenic measure GB/T5009.11-2003, food
T5009.13-2003 first method comparative determination its response rate).
2.3 concentrations are 10% dilute acid soln, and respectively by 1 portion of grain and 2 portions of extractants, 1 portion of grain and 4 parts are extracted
Agent, 1 point of grain and 6 portions of extractants, 1 portion of grain and 8 portions of extractants extract reaction 30min, 4000r/min centrifugation 5min, with phase
Answer GB the first method comparative determination its response rate.
2.4 concentrations be 10% dilute acid soln, by 1 portion of grain and 4 portions of extractants, be respectively adopted the 5min times extract,
The 10min times extract, the 15min times extract, the 20min times extract, the 25min times extract, the 30min times extract, 4000r/
Min is centrifuged 5min, with its response rate of corresponding GB the first method comparative determination.
As a result
Dust technology extracts the optimal condition such as following table of each heavy metal element in grain：
The following table of the response rate under corresponding optimal condition：
| Heavy metal classification | The response rate/% | Average recovery rate/% |
| Cadmium | 65.1～73.4 | 69.8 |
| Lead | 55.2～65.4 | 58.6 |
| Hydrargyrum | 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 |
It is prepared by the heavy metal cadmium rapid quantitative detection reagent box of experimental example 3
1st, cardinal principle
Colloidal gold immunity chromatography detects heavy metal, the specific recognition and reaction based on antigen and antibody.Due to a huge sum of money
Belong to small molecule, must first be coupled after certain functional group after formation hapten could form complete antigen with protein binding,
Then immunoassay can just be carried out.
Cleaning Principle is, with nitrocellulose filter as carrier, to be coated with heavy metal holoantigen (T lines), add measuring samples it
Afterwards, heavy metal ion sequestrant is combined with colloid gold label monoclonal antibody, unconjugated colloid gold label monoclonal antibody with
Detect that antigen (T lines) occurs specific binding and is trapped, the red stripes for manifesting completely.And pass through Instrument measuring T line colors
The depth, and contrasted with the standard curve being preset in instrument, so as to be accurately judged to metal ion content.
2nd, main material and instrument
2.1 cadmiums (Cd) standard sample：National non-ferrous metal and electronic material Institute of Analysis product；Cd specificity Dan Ke
Grand antibody, Cd holoantigens, sheep anti-mouse igg：Chengdu An Punuo bio tech ltd product；Gold chloride：Sigma companies produce
Product；Nitrocellulose filter：MILLIPORE (U.S.), M135；Bovine serum albumin (BSA), Macrogol 2000：Sigma is produced
Product；Glass fibre element film, adsorptive pads, base plate：Shanghai one biological product of outstanding person；Other common agents are analytical reagent.
2.2 contain heavy metal Cd grain samples commercially or testing agency's acquisition, totally 7 parts, using GB GB/
First method of T5009.15-2003 determines its content, and wherein Cd content distribution is interval for 2 parts of 0～200 μ g/kg, Cd contents point
It is 2 parts of 400～1000 μ g/kg for 3 parts of 200～400 μ g/kg, Cd content distribution interval that cloth is interval.
2.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.；Hand-held gold colloidal reading
Instrument, opening up purchased from Shenzhen can reach Science and Technology Ltd..
3rd, method
The preparation of 3.1 test kits
3.1.1 Cd monoclonal antibody colloid gold labels
With the colloidal gold solution that gold chloride-trisodium citrate reduction method prepares a diameter of 40nm, the gold colloidal for completing is prepared
100 parts of additions, 0.5 part of concentration is 1% two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution, is mixed under 200r/min
Close stirring 2h.Solution ph is adjusted to 8.5, adds 0.5 part of tetrabutyl titanate, continue to react 24 hours at room temperature with ammonia,
15min is centrifuged under the conditions of 12000r/min, supernatant is suctioned out and is added ultra-pure water or buffer solution for cleaning, be repeated 1 times, that is, be prepared into
The ester modified colloid gold particle of metatitanic acid of the surface containing active-OH.Take the ester modified glue of metatitanic acid of the above-mentioned surface containing active-OH
3 parts of body gold, by every 100mL additions 0.2,0.5,1.0,1.5,2mg Cd monoclonal antibodies, reacts 2h.In 12000r/min conditions
Lower centrifugation 15min, removes supernatant, and with redissolution liquid the 1/20 of colloidal gold solution volume is dissolved to.2cm width glass is sprayed on by 4 μ l/cm
In cellulose membrane gold standard pad, 37 DEG C dry 18～24 hours, obtain final product and make gold standard pad.
3.1.2 NC films coating process is：Cd holoantigens are configured to into 0.1 with 0.01M pH7.4 phosphate buffers,
0.5th, 1.0,1.5,2.0, the 2.5, solution of 3.0mg/mL, anti-Mus IgG is configured to into 0.1,0.5,1.0,1.5,2.0,2.5,
The solution of 3.0mg/mL, line coating C, T line is carried out respectively with film instrument is drawn with 1 μ l/cm speed, is placed on 37 degree of oven dryings 12
Hour.
3.1.3 the assembling of test kit：It is 20～30 DEG C that test kit is assemblied in temperature, and humidity is<40% interior is carried out, and is taken
Plastic bottom board, is placed on coated NC films plastic floor middle part and pastes, and gold standard pad is ridden over into NC film T lines side, gold standard pad
Ride over 1mm on NC films to paste, gold standard pad opposite side overlap joint pastes upper sample pad, sample pad is alignd with reagent paper lower limb.In NC films
Side overlap joint inhales sample paper, rides over 1mm on NC films and pastes.Finally the plastic plate for posting is cut into into 5mm width test strips using cutting machine,
Reinstall in plastic clip, form test kit.
3.1.4 test strips technological parameter is debugged：The different labelling of concentration, coated reagent are combined into pairing, are prepared
Into sample, reagent is tested using Cd standard sample, search out best of breed.
3.2 extractant：For 10% dilute nitric acid solution.
3.3 diluent：0.01M, 0.02M, 0.04M PBS solution is used respectively, add final concentration to be respectively 0.5%,
1%th, 1.5,2% tween-20, adds final concentration of 1 × 10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、2×10-The chelating agent ITCBE of 3g/mL is configured to diluent.
3.4 standard curves prepare, convert and typing：After determining reagent paper technological parameter, respectively with concentration be 0,50,100,
200th, the Cd standard sample of 400,600,800,1000 μ g/kg is measured to test kit, and the standard sample of variable concentrations shows
Go out different intensity T lines, using gold colloidal readout instrument its intensity is determined.Standard is added into the response rate of the sample concentration divided by Cd
69.8% used as Cd concentration values in determination sample；Making standard curve is made, and relevant parameter is imported in readout instrument, complete instrument
Parameter of curve is arranged.
3.5 detection method
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens；
2) using small-sized balance precise 1g detect measuring samples in extraction flask, add 4mL extractants, under room temperature
Concussion frequency is to react 5min under the conditions of 2～60cpm；
3) filtered using centrifuge or defecator, take supernatant, 40 μ l clear liquids are accurately pipetted to 160 μ l with pipettor device
Mix homogeneously in diluent；
4) take out test kit and test kit and above-mentioned mixed liquor are put into into 5min in dry type thermostat, temperature is set as 37 DEG C；
5) the above-mentioned mixed liquors of 120 μ l are taken with pipettor to add in test kit well, continues anti-under 37 DEG C of isoperibols
Answer 20min；
6) test kit for having reacted is put in hand-held gold colloidal readout instrument the concentration that can be shown that Cd in sample.
3.6 pairs commercially or testing agency obtain 7 parts of samples carried out using heavy metal cadmium rapid quantitative detection reagent box
Detection and with atomic absorption spectrography (AAS) result detection relative analyses its accuracy；Compound concentration be respectively 200ppb, 400ppb,
800ppb standard sample solution is using heavy metal cadmium rapid quantitative detection reagent box and analyzes detection, 6 times points of each Concentration Testing
Analyse its precision.
4 results
4.1 redissolution liquid contain 1%BSA, 5% sucrose, 0.1%tween-20 for 20mM PBS；The most suitable mark of modified colloidal gold
Note amount is 1mg/100mL；Optimal Cd holoantigen package amount 1.5mg/mL；Diluent optimum condition is 0.2M PBS, final concentration of
1%tween-20,1 × 10-7g/mL EDTA。
4.2 accuracy testing results
Paired sample T test method, t=0.707, t are done to this group of dataDouble tails it is critical (0.05,6)=2.447, t<tDouble tails it is critical (0.05,6),
Illustrate heavy metal cadmium rapid quantitative detection reagent box testing result with atomic absorption spectrography (AAS) testing result without notable result difference.
4.3 precision testing results
From result and analysis, the heavy metal cadmium rapid quantitative detection reagent box duplicate detection result coefficient of variation is 10.6
Between～12.2, average coefficient of variation is less than 15%.
Testing result shows that the detection kit and detecting system of heavy metal cadmium of the present invention is functional, can quickly,
Accurately detect cadmium content in measuring samples, it is adaptable to the Quantitative detection at laboratory and sampling scene, meet client to food
Heavy metal cadmium Quantitative detection is required in product.
It is prepared by the heavy metal lead rapid quantitative detection reagent box of experimental example 4
1st, main material
1.1 lead (Pb) standard sample：National non-ferrous metal and electronic material Institute of Analysis product；Pb specificity Dan Ke
Grand antibody, Pb holoantigens, sheep anti-mouse igg：Chengdu An Punuo bio tech ltd product；Gold chloride：Sigma companies produce
Product；Nitrocellulose filter：MILLIPORE (U.S.), M135；Bovine serum albumin (BSA), Macrogol 2000：Sigma is produced
Product；Glass fibre element film, adsorptive pads, base plate：Shanghai one biological product of outstanding person；Other common agents are analytical reagent.
1.2 contain heavy metal Pb grain samples commercially or testing agency's acquisition, totally 6 parts, using GB GB/
First method of T5009.12-2010 determines its content, and it is 0～200 μ g/kg, 2 parts of Pb content distribution that wherein Pb content distribution is interval
It is 2 parts of 400～1000 μ g/kg that interval is 2 parts of 200～400 μ g/kg, Pb content distribution is interval.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.；Hand-held gold colloidal reading
Instrument, opening up purchased from Shenzhen can reach Science and Technology Ltd..
2nd, method
The preparation of 2.1 test kits
2.1.1 Pb monoclonal antibody colloid gold labels：
With the colloidal gold solution that gold chloride-trisodium citrate reduction method prepares a diameter of 40nm, the gold colloidal for completing is prepared
100 parts of additions, 0.5 part of concentration is 1% two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution, is mixed under 200r/min
Close stirring 2h.Solution ph is adjusted to 8.5, adds 0.5 part of tetrabutyl titanate, continue to react 24 hours at room temperature with ammonia,
15min is centrifuged under the conditions of 12000r/min, supernatant is suctioned out and is added ultra-pure water or buffer solution for cleaning, be repeated 1 times, that is, be prepared into
The ester modified colloid gold particle of metatitanic acid of the surface containing active-OH.Take the ester modified glue of metatitanic acid of the above-mentioned surface containing active-OH
3 parts of body gold, by every 100mL additions 0.2,0.5,1.0,1.5,2mg Pb monoclonal antibodies, reacts 2h.In 12000r/min conditions
Lower centrifugation 15min, removes supernatant, and with redissolution liquid the 1/20 of colloidal gold solution volume is dissolved to.2cm width glass is sprayed on by 4 μ l/cm
In cellulose membrane gold standard pad, 37 DEG C dry 18～24 hours, obtain final product and make gold standard pad.
2.1.2 NC films coating process is：Pb holoantigens are configured to into 0.1 with 0.01M pH7.4 phosphate buffers,
0.5th, 1.0,1.5,2.0, the 2.5, solution of 3.0mg/mL, anti-Mus IgG is configured to into 0.1,0.5,1.0,1.5,2.0,2.5,
The solution of 3.0mg/mL, line coating C, T line is carried out respectively with film instrument is drawn with 1 μ l/cm speed, is placed on 37 degree of oven dryings 12
Hour.
2.1.3 the assembling of test kit：It is 20～30 DEG C that test kit is assemblied in temperature, and humidity is<40% interior is carried out, and is taken
Plastic bottom board, is placed on coated NC films plastic floor middle part and pastes, and gold standard pad is ridden over into NC film T lines side, gold standard pad
Ride over 1mm on NC films to paste, gold standard pad opposite side overlap joint pastes upper sample pad, sample pad is alignd with reagent paper lower limb.In NC films
Side overlap joint inhales sample paper, rides over 1mm on NC films and pastes.Finally the plastic plate for posting is cut into into 5mm width test strips using cutting machine,
Reinstall in plastic clip, form test kit.
2.1.4 test strips technological parameter is debugged：By concentration, isolabeling, coated reagent are not combined pairing, are prepared into
Sample, is tested reagent using Pb standard sample, searches out best of breed.
2.2 extractant：For 10% dilute nitric acid solution.
2.3 diluent：0.01M, 0.02M, 0.04M PBS solution is used respectively, add final concentration to be respectively 0.5%,
1%th, 1.5,2% tween-20, adds final concentration of 1 × 10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、2×10-3The chelating agent ITCBE of g/mL is configured to diluent.
2.4 standard curves prepare, convert and typing：After determining reagent paper technological parameter, respectively with concentration be 0,50,100,
200th, the Pb standard sample of 400,600,800,1000 μ g/kg is measured to test kit, and the standard sample of variable concentrations shows
Go out different intensity T lines, using gold colloidal readout instrument its intensity is determined.Standard is added into the response rate of the sample concentration divided by Pb
58.6% used as Pb concentration values in determination sample；Standard curve is made using special software is done, and relevant parameter is imported into reading
In instrument, the setting of instrument parameter of curve is completed.
2.5 detection method
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens；
2) using small-sized balance precise 1g detect measuring samples in extraction flask, add 4mL extractants, under room temperature
Concussion frequency is to react 10min under the conditions of 2～60cpm；
3) clear liquid is filtered to take using centrifuge or defecator, accurately pipettes 40 μ l clear liquids with pipettor device dilute to 160 μ l
Release mix homogeneously in liquid；
4) take out test kit and test kit and above-mentioned mixed liquor are put into into 5min in dry type thermostat, temperature is set as 37 DEG C；
5) the above-mentioned mixed liquors of 120 μ l are taken with pipettor to add in test kit well, continues anti-under 37 DEG C of isoperibols
Answer 20min；
6) test kit for having reacted is put in hand-held gold colloidal readout instrument the concentration that can be shown that Pb in sample.
2.6 pairs commercially or testing agency obtain 6 parts of samples carried out using heavy metal lead rapid quantitative detection reagent box
Detection and with atomic absorption spectrography (AAS) result detection relative analyses its accuracy；Compound concentration be respectively 200ppb, 400ppb,
800ppb standard sample solution is using heavy metal lead rapid quantitative detection reagent box and analyzes detection, 6 times points of each Concentration Testing
Analyse its precision.
3rd, result
3.1 most suitable labelled amounts are 1.5mg/100mL；Redissolve liquid for 20mM PBS contain 1%BSA, 5% sucrose, 0.1%
tween-20；The most suitable labelled amount of modified colloidal gold is 1.5mg/100mL；Optimal Pb holoantigen package amount 1.0mg/mL；Diluent
Optimum condition is 0.2M PBS, final concentration of 1%tween-20,1 × 10-6g/mL ITCBE；Process above different batches may
Need appropriate adjusting process.
3.2 accuracy testing results
Paired sample T test method, t=0.018, t are done to this group of dataDouble tails it is critical (0.05,5)=2.571, t<tDouble tails it is critical (0.05,5),
Illustrate heavy metal lead rapid quantitative detection reagent box testing result with atomic absorption spectrography (AAS) testing result without notable result difference.
3.3 precision testing results
From result and analysis, the heavy metal lead rapid quantitative detection reagent box duplicate detection result coefficient of variation is 12.3
Between～12.8, average coefficient of variation is less than 15%.
Testing result shows that the detection kit and detecting system of heavy metal lead of the present invention is functional, can quickly,
Accurately detect lead content in measuring samples, it is adaptable to the Quantitative detection at laboratory and sampling scene, meet client to food
Heavy metal lead Quantitative detection is required in product.
It is prepared by the heavy metal Hg rapid quantitative detection reagent box of experimental example 5
1st, main material
1.1 hydrargyrum (Hg) standard sample：National non-ferrous metal and electronic material Institute of Analysis product；Hg specificity Dan Ke
Grand antibody, Hg holoantigens, sheep anti-mouse igg：Chengdu An Punuo bio tech ltd product；Gold chloride：Sigma companies produce
Product；Nitrocellulose filter：MILLIPORE (U.S.), M135；Bovine serum albumin (BSA), Macrogol 2000：Sigma is produced
Product；Glass fibre element film, adsorptive pads, base plate：Shanghai one biological product of outstanding person；Other common agents are analytical reagent.
1.2 contain heavy metal Hg grain samples commercially or testing agency's acquisition, totally 5 parts, using GB GB/
First method of T5009.17-2003 determines its content, and it is 0～200 μ g/kg, 2 parts of Hg content distribution that wherein Hg content distribution is interval
It is 1 part of 400～1000 μ g/kg that interval is 2 parts of 200～400 μ g/kg, Hg content distribution is interval.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.；Hand-held gold colloidal reading
Instrument, opening up purchased from Shenzhen can reach Science and Technology Ltd..
2nd, method
The preparation of 2.1 test kits
2.1.1 Hg monoclonal antibody colloid gold labels
With the colloidal gold solution that gold chloride-trisodium citrate reduction method prepares a diameter of 40nm, the gold colloidal for completing is prepared
100 parts of additions, 0.5 part of concentration is 1% two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution, is mixed under 200r/min
Close stirring 2h.Solution ph is adjusted to 8.5, adds 0.5 part of tetrabutyl titanate, continue to react 24 hours at room temperature with ammonia,
15min is centrifuged under the conditions of 12000r/min, supernatant is suctioned out and is added ultra-pure water or buffer solution for cleaning, be repeated 1 times, that is, be prepared into
The ester modified colloid gold particle of metatitanic acid of the surface containing active-OH.Take the ester modified glue of metatitanic acid of the above-mentioned surface containing active-OH
3 parts of body gold, by every 100mL additions 0.2,0.5,1.0,1.5,2mg Hg monoclonal antibodies, reacts 2h.In 12000r/min conditions
Lower centrifugation 15min, removes supernatant, and with redissolution liquid the 1/20 of colloidal gold solution volume is dissolved to.2cm width glass is sprayed on by 4 μ l/cm
In cellulose membrane gold standard pad, 37 DEG C dry 18～24 hours, obtain final product and make gold standard pad.
2.1.2 NC films coating process is：Hg holoantigens are configured to into 0.1 with 0.01M pH7.4 phosphate buffers,
0.5th, 1.0,1.5,2.0, the 2.5, solution of 3.0mg/mL, anti-Mus IgG is configured to into 0.1,0.5,1.0,1.5,2.0,2.5,
The solution of 3.0mg/mL, line coating C, T line is carried out respectively with film instrument is drawn with 1 μ l/cm speed, is placed on 37 degree of oven dryings 12
Hour.
2.1.3 the assembling of test kit：It is 20～30 DEG C that test kit is assemblied in temperature, and humidity is<40% interior is carried out, and is taken
Plastic bottom board, is placed on coated NC films plastic floor middle part and pastes, and gold standard pad is ridden over into NC film T lines side, gold standard pad
Ride over 1mm on NC films to paste, gold standard pad opposite side overlap joint pastes upper sample pad, sample pad is alignd with reagent paper lower limb.In NC films
Side overlap joint inhales sample paper, rides over 1mm on NC films and pastes.Finally the plastic plate for posting is cut into into 5mm width test strips using cutting machine,
Reinstall in plastic clip, form test kit.
2.1.4 test strips technological parameter is debugged：By concentration, isolabeling, coated reagent are not combined pairing, are prepared into
Sample, is tested reagent using Hg standard sample, searches out best of breed.
2.2 extractant：For 10% dilute nitric acid solution.
2.3 use respectively 0.01M, 0.02M, 0.04M PBS solution, add final concentration to be respectively 0.5%, 1%, 1.5,
2% tween-20, adds final concentration of 1 × 10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、2×10- 3The chelating agent ITCBE of g/mL is configured to diluent.
2.4 standard curves prepare, convert and typing：After determining reagent paper technological parameter, respectively with concentration be 0,50,100,
200th, the Hg standard sample of 400,600,800,1000 μ g/kg is measured to test kit, and the standard sample of variable concentrations shows
Go out different intensity T lines, using gold colloidal readout instrument its intensity is determined.Standard is added into the response rate of the sample concentration divided by Hg
56.6% used as Hg concentration values in determination sample；Standard curve is made using special software is done, and relevant parameter is imported into reading
In instrument, the setting of instrument parameter of curve is completed.
2.5 detection method
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens；
2) using small-sized balance precise 1g detect measuring samples in extraction flask, add 4mL extractants, under room temperature
Concussion frequency is to react 5min under the conditions of 2～60cpm；
3) clear liquid is filtered to take using centrifuge or defecator, accurately pipettes 40 μ l clear liquids with pipettor device dilute to 160 μ l
Release mix homogeneously in liquid；
4) take out test kit and test kit and above-mentioned mixed liquor are put into into 5min in dry type thermostat, temperature is set as 37 DEG C；
5) the above-mentioned mixed liquors of 120 μ l are taken with pipettor to add in test kit well, continues anti-under 37 DEG C of isoperibols
Answer 20min；
6) test kit for having reacted is put in hand-held gold colloidal readout instrument the concentration that can be shown that Hg in sample.
2.6 pairs commercially or testing agency obtain 5 parts of samples carried out using heavy metal Hg rapid quantitative detection reagent box
Detection and with atomic absorption spectrography (AAS) result detection relative analyses its accuracy；Compound concentration be respectively 200ppb, 400ppb,
800ppb standard sample solution is using heavy metal Hg rapid quantitative detection reagent box and analyzes detection, 6 times points of each Concentration Testing
Analyse its precision.
3rd, result
3.1 most suitable labelled amounts are 1.5mg/100mL；Redissolve liquid for 20mM PBS contain 1%BSA, 5% sucrose, 0.1%
tween-20；The most suitable labelled amount of modified colloidal gold is 1.5mg/100mL；Optimal Hg holoantigen package amount 1.0mg/mL；Diluent
Optimum condition is 0.2M PBS, final concentration of 1.5%tween-20,1 × 10-7g/mL ITCBE；Process above different batches can
Appropriate adjusting process can be needed.
3.2 accuracy testing results
Paired sample T test method, t=0.018, t are done to this group of dataDouble tails it is critical (0.05,4)=2.776, t<tDouble tails it is critical (0.05,4),
Illustrate heavy metal Hg rapid quantitative detection reagent box testing result with atomic absorption spectrography (AAS) testing result without notable result difference.
3.3 precision testing results
From result and analysis, the heavy metal Hg rapid quantitative detection reagent box duplicate detection result coefficient of variation is 7.7
Between～14.1, average coefficient of variation is less than 15%.
Testing result shows that the detection kit and detecting system of heavy metal Hg of the present invention is functional, can quickly,
Accurately detect mercury content in measuring samples, it is adaptable to the Quantitative detection at laboratory and sampling scene, meet client to food
Heavy metal Hg Quantitative detection is required in product.
It is prepared by the heavy metal arsenic rapid quantitative detection reagent box of embodiment 6
1st, main material
1.1 arsenic (As) standard sample：National non-ferrous metal and electronic material Institute of Analysis product；As specificity Dan Ke
Grand antibody, As holoantigens, sheep anti-mouse igg：Chengdu An Punuo bio tech ltd product；Gold chloride：Sigma companies produce
Product；Nitrocellulose filter：MILLIPORE (U.S.), M135；Bovine serum albumin (BSA), Macrogol 2000：Sigma is produced
Product；Glass fibre element film, adsorptive pads, base plate：Shanghai one biological product of outstanding person；Other common agents are analytical reagent.
1.2 contain heavy metal As grain samples commercially or testing agency's acquisition, totally 6 parts, using GB GB/
First method of T5009.11-2003 determines its content, and it is 0～200 μ g/kg, 2 parts of As content distribution that wherein As content distribution is interval
It is 1 part of 400～1000 μ g/kg that interval is 3 parts of 200～400 μ g/kg, As content distribution is interval.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.；Hand-held gold colloidal reading
Instrument, opening up purchased from Shenzhen can reach Science and Technology Ltd..
2nd, method
The preparation of 2.1 test kits
2.1.1 As monoclonal antibody colloid gold labels
With the colloidal gold solution that gold chloride-trisodium citrate reduction method prepares a diameter of 40nm, the gold colloidal for completing is prepared
100 parts of additions, 0.5 part of concentration is 1% two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution, is mixed under 200r/min
Close stirring 2h.Solution ph is adjusted to 8.5, adds 0.5 part of tetrabutyl titanate, continue to react 24 hours at room temperature with ammonia,
15min is centrifuged under the conditions of 12000r/min, supernatant is suctioned out and is added ultra-pure water or buffer solution for cleaning, be repeated 1 times, that is, be prepared into
The ester modified colloid gold particle of metatitanic acid of the surface containing active-OH.Take the ester modified glue of metatitanic acid of the above-mentioned surface containing active-OH
3 parts of body gold, by every 100mL additions 0.2,0.5,1.0,1.5,2mg As monoclonal antibodies, reacts 2h.In 12000r/min conditions
Lower centrifugation 15min, removes supernatant, and with redissolution liquid the 1/20 of colloidal gold solution volume is dissolved to.2cm width glass is sprayed on by 4 μ l/cm
In cellulose membrane gold standard pad, 37 DEG C dry 18～24 hours, obtain final product and make gold standard pad.
2.1.2 NC films coating process is：As holoantigens are configured to into 0.1 with 0.01M pH7.4 phosphate buffers,
0.5th, 1.0,1.5,2.0, the 2.5, solution of 3.0mg/mL, anti-Mus IgG is configured to into 0.1,0.5,1.0,1.5,2.0,2.5,
The solution of 3.0mg/mL, line coating C, T line is carried out respectively with film instrument is drawn with 1 μ l/cm speed, is placed on 37 degree of oven dryings 12
Hour.
2.1.3 the assembling of test kit：It is 20～30 DEG C that test kit is assemblied in temperature, and humidity is<40% interior is carried out, and is taken
Plastic bottom board, is placed on coated NC films plastic floor middle part and pastes, and gold standard pad is ridden over into NC film T lines side, gold standard pad
Ride over 1mm on NC films to paste, gold standard pad opposite side overlap joint pastes upper sample pad, sample pad is alignd with reagent paper lower limb.In NC films
Side overlap joint inhales sample paper, rides over 1mm on NC films and pastes.Finally the plastic plate for posting is cut into into 5mm width test strips using cutting machine,
Reinstall in plastic clip, form test kit.
2.1.4 test strips technological parameter is debugged：By concentration, isolabeling, coated reagent are not combined pairing, are prepared into
Sample, is tested reagent using As standard sample, searches out best of breed.
2.2 extractant：For 10% dilute nitric acid solution.
2.3 diluent：0.01M, 0.02M, 0.04M PBS solution is used respectively, add final concentration to be respectively 0.5%,
1%th, 1.5,2% tween-20, adds final concentration of 1 × 10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、2×10-3The chelating agent ITCBE of g/mL is configured to diluent.
2.4 standard curves prepare, convert and typing：After determining reagent paper technological parameter, respectively with concentration be 0,50,100,
200th, the As standard sample of 400,600,800,1000 μ g/kg is measured to test kit, and the standard sample of variable concentrations shows
Go out different intensity T lines, using gold colloidal readout instrument its intensity is determined.Standard is added into the response rate of the sample concentration divided by As
55.6% used as As concentration values in determination sample；Standard curve is made using special software is done, and relevant parameter is imported into reading
In instrument, the setting of instrument parameter of curve is completed.
2.5 detection method
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens；
2) using small-sized balance precise 1g detect measuring samples in extraction flask, add 4mL extractants, under room temperature
Concussion frequency is to react 10min under the conditions of 2～60cpm；
3) clear liquid is filtered to take using centrifuge or defecator, accurately pipettes 40 μ l clear liquids with pipettor device dilute to 160 μ l
Release mix homogeneously in liquid；
4) take out test kit and test kit and above-mentioned mixed liquor are put into into 5min in dry type thermostat, be stably set as 37 DEG C；
5) the above-mentioned mixed liquors of 120 μ l are taken with pipettor to add in test kit well, continues anti-under 37 DEG C of isoperibols
Answer 20min；
6) test kit for having reacted is put in hand-held gold colloidal readout instrument the concentration that can be shown that As in sample.
2.6 pairs commercially or testing agency obtain 6 parts of samples carried out using heavy metal arsenic rapid quantitative detection reagent box
Detection and with atomic absorption spectrography (AAS) result detection relative analyses its accuracy；Compound concentration be respectively 200ppb, 400ppb,
800ppb standard sample solution is using heavy metal arsenic rapid quantitative detection reagent box and analyzes detection, 6 times points of each Concentration Testing
Analyse its precision.
3rd, result
3.1 traditional method colloid gold label pH value are 8.5；Most suitable labelled amount is 1.0mg/100mL；Redissolving liquid is
20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20；The most suitable labelled amount of modified colloidal gold is 1.0mg/100mL；Most
Good As holoantigens package amount 1.0mg/mL；Diluent optimum condition be 0.2M PBS, final concentration of 1.5%tween-20,1 ×
10-5g/mL ITCBE；Process above different batches may need appropriate adjusting process.
3.2 accuracy testing results
Paired sample T test method, t=0.255, t are done to this group of dataDouble tails it is critical (0.05,5)=2.571, t<tDouble tails it is critical (0.05,5),
Illustrate heavy metal arsenic rapid quantitative detection reagent box testing result with atomic absorption spectrography (AAS) testing result without notable result difference.
3.3 precision testing results
From result and analysis, the heavy metal arsenic rapid quantitative detection reagent box duplicate detection result coefficient of variation is 13.8
Between～14.4, average coefficient of variation is less than 15%
Testing result shows that the detection kit and detecting system of heavy metal arsenic of the present invention is functional, can quickly,
Accurately detect arsenic content in measuring samples, it is adaptable to the Quantitative detection at laboratory and sampling scene, meet client to food
Heavy metal arsenic Quantitative detection is required in product.
It is prepared by the heavy metal chromium rapid quantitative detection reagent box of embodiment 7
1st, main material
1.1 chromium (Cr) standard sample：National non-ferrous metal and electronic material Institute of Analysis product；Cr specificity Dan Ke
Grand antibody, Cr holoantigens, sheep anti-mouse igg：Chengdu An Punuo bio tech ltd product；Gold chloride：Sigma companies produce
Product；Nitrocellulose filter：MILLIPORE (U.S.), M135；Bovine serum albumin (BSA), Macrogol 2000：Sigma is produced
Product；Glass fibre element film, adsorptive pads, base plate：Shanghai one biological product of outstanding person；Other common agents are analytical reagent.
1.2 contain heavy metal Cr grain samples commercially or testing agency's acquisition, totally 5 parts, using GB GB/
First method of T5009.123-2003 determines its content, and wherein Cr content distribution is interval for 0～200 μ g/kg, 3 parts of Cr contents point
It is 1 part of 400～1000 μ g/kg for 1 part of 200～400 μ g/kg, Cr content distribution interval that cloth is interval.
1.3 dry type thermostat purchases dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.；Hand-held gold colloidal reading
Instrument, opening up purchased from Shenzhen can reach Science and Technology Ltd..
2nd, method
The preparation of 2.1 test kits
2.1.1 Cr monoclonal antibody colloid gold labels
With the colloidal gold solution that gold chloride-trisodium citrate reduction method prepares a diameter of 40nm, the gold colloidal for completing is prepared
100 parts of additions, 0.5 part of concentration is 1% two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution, is mixed under 200r/min
Close stirring 2h.Solution ph is adjusted to 8.5, adds 0.5 part of tetrabutyl titanate, continue to react 24 hours at room temperature with ammonia,
15min is centrifuged under the conditions of 12000r/min, supernatant is suctioned out and is added ultra-pure water or buffer solution for cleaning, be repeated 1 times, that is, be prepared into
The ester modified colloid gold particle of metatitanic acid of the surface containing active-OH.Take the ester modified glue of metatitanic acid of the above-mentioned surface containing active-OH
3 parts of body gold, by every 100mL additions 0.2,0.5,1.0,1.5,2mg Cr monoclonal antibodies, reacts 2h.In 12000r/min conditions
Lower centrifugation 15min, removes supernatant, and with redissolution liquid the 1/20 of colloidal gold solution volume is dissolved to.2cm width glass is sprayed on by 4 μ L/cm
In cellulose membrane gold standard pad, 37 DEG C dry 18～24 hours, obtain final product and make gold standard pad.
2.1.2 NC films coating process is：Cr holoantigens are configured to into 0.1 with 0.01M pH7.4 phosphate buffers,
0.5th, 1.0,1.5,2.0, the 2.5, solution of 3.0mg/mL, anti-Mus IgG is configured to into 0.1,0.5,1.0,1.5,2.0,2.5,
The solution of 3.0mg/mL, line coating C, T line is carried out respectively with film instrument is drawn with 1 μ L/cm speed, is placed on 37 degree of oven dryings 12
Hour.
2.1.3 the assembling of test kit：It is 20～30 DEG C that test kit is assemblied in temperature, and humidity is<40% interior is carried out, and is taken
Plastic bottom board, is placed on coated NC films plastic floor middle part and pastes, and gold standard pad is ridden over into NC film T lines side, gold standard pad
Ride over 1mm on NC films to paste, gold standard pad opposite side overlap joint pastes upper sample pad, sample pad is alignd with reagent paper lower limb.In NC films
Side overlap joint inhales sample paper, rides over 1mm on NC films and pastes.Finally the plastic plate for posting is cut into into 5mm width test strips using cutting machine,
Reinstall in plastic clip, form test kit.
2.1.4 test strips technological parameter is debugged：By concentration, isolabeling, coated reagent are not combined pairing, are prepared into
Sample, is tested reagent using Cr standard sample, searches out best of breed.
2.2 extractant：For 10% dilute nitric acid solution.
2.3 use respectively 0.01M, 0.02M, 0.04M PBS solution, add final concentration to be respectively 0.5%, 1%, 1.5,
2% tween-20, adds final concentration of 1 × 10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、2×10- 3The chelating agent ITCBE of g/mL is configured to diluent.
2.4 standard curves prepare, convert and typing：After determining reagent paper technological parameter, respectively with concentration be 0,50,100,
200th, the Cr standard sample of 400,600,800,1000 μ g/kg is measured to test kit, and the standard sample of variable concentrations shows
Go out different intensity T lines, using gold colloidal readout instrument its intensity is determined.Standard is added into the response rate of the sample concentration divided by Cr
69% used as Cr concentration values in determination sample；Standard curve is made using special software is done, and relevant parameter is imported into readout instrument
In, complete the setting of instrument parameter of curve.
2.5 detection method
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens；
2) using small-sized balance precise 1g detect measuring samples in extraction flask, add 4mL extractants, under room temperature
Concussion frequency is to react 5min under the conditions of 2～60cpm；
3) clear liquid is filtered to take using centrifuge or defecator, accurately pipettes 40 μ l clear liquids with pipettor device dilute to 160 μ l
Release mix homogeneously in liquid；
4) take out test kit and test kit and above-mentioned mixed liquor are put into into 5min in dry type thermostat, be stably set as 37 DEG C；
5) the above-mentioned mixed liquors of 120 μ l are taken with pipettor to add in test kit well, continues anti-under 37 DEG C of isoperibols
Answer 20min；
6) test kit for having reacted is put in hand-held gold colloidal readout instrument the concentration that can be shown that Cr in sample.
2.6 pairs commercially or testing agency obtain 6 parts of samples carried out using heavy metal chromium rapid quantitative detection reagent box
Detection and with atomic absorption spectrography (AAS) result detection relative analyses its accuracy；Compound concentration be respectively 200ppb, 400ppb,
800ppb standard sample solution is using heavy metal chromium rapid quantitative detection reagent box and analyzes detection, 6 times points of each Concentration Testing
Analyse its precision.
3rd, result
3.1 traditional method colloid gold label pH value are 8.5；Most suitable labelled amount is 1.0mg/100mL；Redissolving liquid is
20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20；The most suitable labelled amount of modified colloidal gold is 1.0mg/100mL；Most
Good Cr holoantigens package amount 0.5mg/mL；Diluent optimum condition is 0.2M PBS, final concentration of 1%tween-20,1 × 10- 6g/mL ITCBE；Process above different batches may need appropriate adjusting process.
3.2 accuracy testing results
Paired sample T test method, t=0.649, t are done to this group of dataDouble tails it is critical (0.05,4)=2.776, t<tDouble tails it is critical (0.05,4),
Illustrate heavy metal chromium rapid quantitative detection reagent box testing result with atomic absorption spectrography (AAS) testing result without notable result difference.
3.3 precision testing results
From result and analysis, the heavy metal chromium rapid quantitative detection reagent box duplicate detection result coefficient of variation is 10.9
Between～14.1, average coefficient of variation is less than 15%
Testing result shows that the detection kit and detecting system of heavy metal chromium of the present invention is functional, can quickly,
Accurately detect chromium content in measuring samples, it is adaptable to the Quantitative detection at laboratory and sampling scene, meet client to food
Huge sum of money chromium cadmium Quantitative detection is required in product.
It is prepared by the heavy metal copper rapid quantitative detection reagent box of embodiment 8
1st, main material
1.1 bronze medals (Cu) standard sample：National non-ferrous metal and electronic material Institute of Analysis product；Cu specificity Dan Ke
Grand antibody, Cu holoantigens, sheep anti-mouse igg：Chengdu An Punuo bio tech ltd product；Gold chloride：Sigma companies produce
Product；Nitrocellulose filter：MILLIPORE (U.S.), M135；Bovine serum albumin (BSA), Macrogol 2000：Sigma is produced
Product；Glass fibre element film, adsorptive pads, base plate：Shanghai one biological product of outstanding person；Other common agents are analytical reagent.
1.2 contain heavy metal Cu grain samples commercially or testing agency's acquisition, totally 7 parts, using GB GB/
First method of T5009.13-2003 determines its content, and it is 0～200 μ g/kg, 3 parts of Cu content distribution that wherein Cu content distribution is interval
It is 1 part of 400～1000 μ g/kg that interval is 3 parts of 200～400 μ g/kg, Cu content distribution is interval.
1.3 dry type thermostats purchase dry type thermostat, purchased from Hangzhou Rui Cheng Instrument Ltd.；Hand-held gold colloidal reading
Instrument, opening up purchased from Shenzhen can reach Science and Technology Ltd..
2nd, method
The preparation of 2.1 test kits
2.1.1 Cu monoclonal antibody colloid gold labels
With the colloidal gold solution that gold chloride-trisodium citrate reduction method prepares a diameter of 40nm, the gold colloidal for completing is prepared
100 parts of additions, 0.5 part of concentration is 1% two (triethanolamine) metatitanic acid diisopropyl ester (TMC-TE) solution, is mixed under 200r/min
Close stirring 2h.Solution ph is adjusted to 8.5, adds 0.5 part of tetrabutyl titanate, continue to react 24 hours at room temperature with ammonia,
15min is centrifuged under the conditions of 12000r/min, supernatant is suctioned out and is added ultra-pure water or buffer solution for cleaning, be repeated 1 times, that is, be prepared into
The ester modified colloid gold particle of metatitanic acid of the surface containing active-OH.Take the ester modified glue of metatitanic acid of the above-mentioned surface containing active-OH
3 parts of body gold, by every 100mL additions 0.2,0.5,1.0,1.5,2mg Cu monoclonal antibodies, reacts 2h.In 12000r/min conditions
Lower centrifugation 15min, removes supernatant, and with redissolution liquid the 1/20 of colloidal gold solution volume is dissolved to.2cm width glass is sprayed on by 4 μ l/cm
In cellulose membrane gold standard pad, 37 DEG C dry 18～24 hours, obtain final product and make gold standard pad.
2.1.2 NC films coating process is：Cu holoantigens are configured to into 0.1 with 0.01M pH7.4 phosphate buffers,
0.5th, 1.0,1.5,2.0, the 2.5, solution of 3.0mg/mL, anti-Mus IgG is configured to into 0.1,0.5,1.0,1.5,2.0,2.5,
The solution of 3.0mg/mL, line coating C, T line is carried out respectively with film instrument is drawn with 1 μ l/cm speed, is placed on 37 degree of oven dryings 12
Hour.
2.1.3 the assembling of test kit：It is 20～30 DEG C that test kit is assemblied in temperature, and humidity is<40% interior is carried out, and is taken
Plastic bottom board, is placed on coated NC films plastic floor middle part and pastes, and gold standard pad is ridden over into NC film T lines side, gold standard pad
Ride over 1mm on NC films to paste, gold standard pad opposite side overlap joint pastes upper sample pad, sample pad is alignd with reagent paper lower limb.In NC films
Side overlap joint inhales sample paper, rides over 1mm on NC films and pastes.Finally the plastic plate for posting is cut into into 5mm width test strips using cutting machine,
Reinstall in plastic clip, form test kit.
2.1.4 test strips technological parameter is debugged：By concentration, isolabeling, coated reagent are not combined pairing, are prepared into
Sample, is tested reagent using Cu standard sample, searches out best of breed.
2.2 extractant：For 10% dilute nitric acid solution.
2.3 use respectively 0.01M, 0.02M, 0.04M PBS solution, add final concentration to be respectively 0.5%, 1%, 1.5,
2% tween-20, adds final concentration of 1 × 10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、2×10- 3The chelating agent ITCBE of g/mL is configured to diluent.
2.4 standard curves prepare, convert and typing：After determining reagent paper technological parameter, respectively with concentration be 0,50,100,
200th, the Cu standard sample of 400,600,800,1000 μ g/kg is measured to test kit, and the standard sample of variable concentrations shows
Go out different intensity T lines, using gold colloidal readout instrument its intensity is determined.Standard is added into the response rate of the sample concentration divided by Cu
53.5% used as Cu concentration values in determination sample；Standard curve is made using special software is done, and relevant parameter is imported into reading
In instrument, the setting of instrument parameter of curve is completed.
2.5 detection method
1) detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens；
2) using small-sized balance precise 1g detect measuring samples in extraction flask, add 4mL extractants, under room temperature
Concussion frequency is to react 5min under the conditions of 2～60cpm；
3) clear liquid is filtered to take using centrifuge or defecator, accurately pipettes 40 μ l clear liquids with pipettor device dilute to 160 μ l
Release mix homogeneously in liquid；
4) take out test kit and test kit and above-mentioned mixed liquor are put into into 5min in dry type thermostat, be stably set as 37 DEG C；
5) the above-mentioned mixed liquors of 120 μ l are taken with pipettor to add in test kit well, continues anti-under 37 DEG C of isoperibols
Answer 20min；
6) test kit for having reacted is put in hand-held gold colloidal readout instrument the concentration that can be shown that Cu in sample.
2.6 pairs commercially or testing agency obtain 6 parts of samples carried out using heavy metal copper rapid quantitative detection reagent box
Detection and with atomic absorption spectrography (AAS) result detection relative analyses its accuracy；Compound concentration be respectively 200ppb, 400ppb,
800ppb standard sample solution is using heavy metal copper rapid quantitative detection reagent box and analyzes detection, 6 times points of each Concentration Testing
Analyse its precision.
3rd, result
3.1 traditional method colloid gold label pH value are 8.5；Most suitable labelled amount is 1.0mg/100mL；Redissolving liquid is
20mMPBS contains 1%BSA, 5% sucrose, 0.1%tween-20；The most suitable labelled amount of modified colloidal gold is 1.0mg/100mL；Most
Good Cu holoantigens package amount 0.5mg/mL；Diluent optimum condition is 0.2M PBS, final concentration of 1%tween-20,1 × 10- 5g/mL ITCBE；Process above different batches may need appropriate adjusting process.
3.2 accuracy testing results
Paired sample T test method, t=0.514, t are done to this group of dataDouble tails it is critical (0.05,6)=2.447, t<tDouble tails it is critical (0.05,6),
Illustrate heavy metal copper rapid quantitative detection reagent box testing result with atomic absorption spectrography (AAS) testing result without notable result difference.
3.3 precision testing results
From result and analysis, the heavy metal copper rapid quantitative detection reagent box duplicate detection result coefficient of variation is 11.2
Between～14.2, average coefficient of variation is less than 15%
Testing result shows that the detection kit and detecting system of heavy metal copper of the present invention is functional, can quickly,
Accurately detect copper content in measuring samples, it is adaptable to the Quantitative detection at laboratory and sampling scene, meet client to food
Heavy metal copper Quantitative detection is required in product.
Claims (10)
1. the preparation method of a kind of modified colloidal gold, it is characterised in that：It comprises the steps：
（1）100 parts of the colloidal gold solution of a diameter of 10～60nm is taken, the 0.1 ~ 3% of colloidal gold solution volume two (three second are added
Hydramine) metatitanic acid diisopropyl ester solution, the concentration of two (triethanolamine) metatitanic acid diisopropyl ester solution is 1%, under 100～400r/min
0.5～2h of stirring, obtains mixed solution；
（2）Above-mentioned mixed solution pH value is adjusted to 8～10,0.1～2 part of tetrabutyl titanate is added, at room temperature with 100～
400r/min stirring reactions 24～36 hours, are centrifuged 5～60min under the conditions of 4000～16000r/min, suction out supernatant and add
Enter ultra-pure water or buffer solution for cleaning, repeat 1～2 time, that is, be prepared into the ester modified gold colloidal of metatitanic acid of the surface containing active-OH
Grain.
2. a kind of preparation method of gold labeling antibody, it is characterised in that：Method according to claim 1 prepares modified colloidal gold, presses
0.2～2mg antibody is added per 100mL, 2h is reacted, 5～60min is centrifuged under the conditions of 6000～12000r/min, remove supernatant,
The 1/20 of colloidal gold solution volume is dissolved to redissolution liquid, gold labeling antibody is obtained final product.
3. preparation method according to claim 2, it is characterised in that：The antibody is Cd monoclonal antibodies, Pb monoclonals
Antibody, Hg monoclonal antibodies, As monoclonal antibodies, Cr monoclonal antibodies or Cu monoclonal antibodies.
4. it is a kind of detection heavy metal colloidal gold immunochromatographykit kit, it is characterised in that：It includes Test paper and gets stuck, tries
Paper is placed in getting stuck；The Test paper includes plastic bottom board（1）, sample pad is pasted in one end（2）, one end of sample pad is closely pressed
Then detecting containing colloid gold label heavy metal relative answers the colloidal gold pad of antibody（3）, colloidal gold pad（3）Closely press one end
Then nitrocellulose filter（4）, detection line T and nature controlling line C are coated with nitrocellulose filter（5）, detection line T is coated with to be checked
The complete antigen of heavy metal, is coated with dynamics, nitrocellulose filter other end connection sample suction pad on nature controlling line C（6）；
The surface counter sample pad that gets stuck（2）Position be provided with well（7）, get stuck surface correspondence nitrocellulose filter（4）Position
Observation panel is installed（8）；
Wherein, the modified colloidal gold that the gold colloidal in colloidal gold pad is prepared for the method for claim 1.
5. test kit according to claim 4, it is characterised in that：Gold labeling antibody in the colloidal gold pad is claim
Gold labeling antibody prepared by 2 or 3 method.
6. test kit according to claim 5, it is characterised in that：The complete antigen of the heavy metal is heavy metal and complexation
After agent complexation, mutually it is coupled to form antigen with carrier protein, wherein, the chelating agent is citric acid, ethylenediaminetetraacetic acid, amino second
Acid, 1- (the different sulfur cyanobenzyls of 4-) ethylenediamine-N, N, N', N'- tetraacethyl, diethylenetriamine pentacarboxylic acid salt, glutathion and
One or more in organic polydentate part；The carrier protein be bovine serum albumin, keyhole limpet hemocyanin, ovalbumin,
One or more in human albumin.
7. test kit according to claim 5, it is characterised in that：The nitrocellulose filter is many of aperture 5-12 microns
Hole spline structure film, the sample pad is glass fibre element film or polyester film, and the sample suction pad is absorbent filter.
8. a heavy metal species quantitative detection system, it is characterised in that：It includes the gold colloidal described in claim 4 ~ 7 any one
Immune chromatography reagent kit, dry type thermostat and gold colloidal readout instrument, wherein, gold colloidal readout instrument is that a kind of hand-held gold colloidal is read
Number instrument.
9. a kind of method of detection by quantitative heavy metal, it is characterised in that：It is that the detecting system described in claim 8 is examined
Survey.
10. method according to claim 9, it is characterised in that：It comprises the steps：
1）Detection sample needed for being crushed using pulverizer, and cross 20 eye mesh screens；
2）Measuring samples are taken, extractant is added, 2～8 times of mL extractants are added per g samples, earthquake frequency is 2～60cpm bars
5～30min is reacted under part, is filtered, take supernatant, add the diluent of 2～40 times of volumes of supernatant, mixed, obtain mixed liquor；It is described to carry
Take the salpeter solution that agent is that concentration is 1～15%；The diluent is the buffering for being added with surfactant, heavy metal complexing agent
Solution；
3）By colloidal gold immunochromatographykit kit and step 2 described in claim 4 ~ 7 any one）The mixed liquor difference of preparation
In being put into dry type thermostat, 2～15min of constant temperature, temperature is set as 20～45 DEG C；
4）In dry type thermostat, by step 2）The mixed liquor of preparation is added in the well of test kit, and isothermal reaction 5～
30min；
5）By reacted test kit, in being placed in gold colloidal readout instrument, data are read.
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| CN105954506B (en) * | 2016-04-28 | 2018-02-09 | 苏州市天灵中药饮片有限公司 | Heavy metal biochemical detection methods in a kind of prepared slices of Chinese crude drugs |
| WO2019000353A1 (en) * | 2017-06-28 | 2019-01-03 | 南通发宝贸易有限公司 | Use of fiber-optic immunosensor in environment detection |
| 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 |
| CN111442960B (en) * | 2020-04-20 | 2023-12-29 | 北京仪达仪器有限责任公司 | Kit and method for rapidly extracting heavy metals in soil |
| 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 |
| CN114034868A (en) * | 2021-11-05 | 2022-02-11 | 上海市农业科学院 | Quantitative detection method for heavy metal cadmium ions |
| CN114563572B (en) * | 2022-04-27 | 2022-08-02 | 成都安普诺生物科技有限公司 | Tobacco leaf all-in-one heavy metal rapid quantitative detection card and detection method |
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