CN108508213A - The detection kit of heavy metal lead ion and its application - Google Patents
The detection kit of heavy metal lead ion and its application Download PDFInfo
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- CN108508213A CN108508213A CN201810517009.8A CN201810517009A CN108508213A CN 108508213 A CN108508213 A CN 108508213A CN 201810517009 A CN201810517009 A CN 201810517009A CN 108508213 A CN108508213 A CN 108508213A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of detection kit of heavy metal lead ion and its applications.Kit provided by the present invention contains test strips and the first antibody marked through superparamagnetism fluorescence compound particle (can specific bond heavy metal lead ion)；The test strips by be sequentially connected and be fixed on basal layer sample pad, equipped with the detection line being separated from each other (close to sample pad, it is coated with carrier protein couplet heavy metal lead ion antigen) and nature controlling line (close water absorption pad, it is coated with secondary antibody, resist the first antibody antibody) coated film and water absorption pad composition.Heavy metal lead ion is detected using the kit, high sensitivity, high specificity, detection time is short, and testing cost is low, easily operated and popularization.
Description
Technical field
The present invention relates to the detection of heavy metal ion in biotechnology, and in particular to a kind of inspection of heavy metal lead ion
Test agent box and its application, the side of detection kit and detection heavy metal lead ion more particularly, to heavy metal lead ion
Method
Background technology
With economic development and process of industrialization, heavy metal is widely used in Modern Industry Products, industrial waste
The discharge of object leads to the seriously polluted of heavy metal in soil and water resource.The heavy metal that the whole world is discharged into environment every year reaches tens thousand of
Ton, wherein most enters soil and water source, to pollute agricultural product and drinking water.
Heavy metal lead ion in environment can not degrade, and accumulate year by year, then enter human body by food chain, seriously
Human health is threatened, heavy metal lead is the very strong metallic element of cumulative effect, is easily stored in water source, water plant, aquatic products
Product.Therefore, the detection drinking water of rapid sensitive and the heavy metal lead ion residues in food are food securities and safeguard that the mankind are strong
The guarantee of health.
The remaining main method of traditional detection heavy metal ion has atomic absorption spectrography (AAS), gas chromatography, colorimetric
Method, inductively coupled plasma spectrometry method and its combination method etc..Expensive equipment needed for conventional method, it is complicated for operation, need professional people
Member.And with the development of immunoassay technology, the immunoassay such as ELISA, electricity of heavy metal ion are identified based on specific antibody
Chemiluminescence detection, immunochromatography detection etc. can be used for the detection of heavy-metal residual.It is existing currently used for heavy metal ion residual
Screening method, cannot meet the requirements very well because its sensitivity is low, it is therefore desirable to which exploitation can at the scene more accurately and quickly
Detection method.
Invention content
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention
It is to propose that a kind of detection kit of detection heavy metal lead ion, detection sensitivity height, high specificity, the energy of the kit are fast
Speed easily detects heavy metal lead ion.
In a first aspect, a kind of detection kit of claimed heavy metal lead ion.
The detection kit of heavy metal lead ion provided by the present invention, it is compound containing test strips and through superparamagnetism fluorescence
The first antibody of particle label；The first antibody being capable of specific bond heavy metal lead ion.
Wherein, the test strips by be sequentially connected and be fixed on basal layer sample pad, be equipped with detection line and nature controlling line
Coated film and water absorption pad composition.The detection line and the nature controlling line are separated from each other.It is coated with carrier at the detection line
Albumen coupling heavy metal lead ion antigen.Secondary antibody is coated at the nature controlling line, the secondary antibody is to resist resisting for the first antibody
Body.The detection line is located at the coated film close to one end of the sample pad.It is close that the nature controlling line is located at the coated film
One end of the water absorption pad.
Further, the first antibody through superparamagnetism fluorescence compound particle label is by the first antibody and institute
It states superparamagnetism fluorescence compound particle and combines the condensate formed by covalent peptide bonds.The first antibody can be specifically to tie
Close the polyclonal antibody or monoclonal antibody of heavy metal lead ion.The carrier protein couplet heavy metal lead ion antigen is to pass through
By the carrier protein couplet amino functional EDTA and chelate what heavy metal lead ion obtained.The sample pad can be glass fibers
The plain film of dimension, the coated film can be nitrocellulose filter.
Wherein, the carrier protein can be BSA, OVA, KLH etc..In specific embodiments of the present invention mode, the load
Body protein is specially bovine serum albumin(BSA) (BSA).
Further, the superparamagnetism fluorescence compound particle is superparamagnetism fluorescence complex microsphere.The superparamagnetic
Property fluorescence complex microsphere be Fe3O4Magnetic nano-particle and the coated SiO of CdSe/ZnS fluorescent materials2And surface modification carboxyl work(
The complex microsphere of group can be changed.The diameter of the superparamagnetism fluorescence complex microsphere can be 10-500nm, it is preferable that be 50-
400nm, it is highly preferred that being 100-300nm.The magnetic saturation intensity of the superparamagnetism fluorescence complex microsphere is 40~120emu/
G, corresponding external magnetic field response speed are 30~120 seconds, the magnetic saturation intensity of the superparamagnetism compound particle is specially 40~
80emu/g, corresponding external magnetic field response speed are 30~60 seconds, the superparamagnetism fluorescence complex microsphere functionalization group carboxyl
Content be 40~400 μm of ol/g, the content of the carboxyl is specially 60~100 μm of ol/g.
In the method, the carrier protein couplet heavy metal lead ion antigen can be according to the method included the following steps
It prepares：
(a1) carrier protein is dissolved in a concentration of 0.05M, pH value in 9.6 borate buffer solution, to obtain carrier
Protein solution；
The proportioning for the borate buffer solution that the carrier protein and a concentration of 0.05M, pH value are 9.6 is 100mg：
10mL。
(a2) Aminobenzyl-EDTA is dissolved in 1M hydrochloric acid, then 0 DEG C (ice bath) stirring is lower is added dropwise a concentration of 0.2M
NaNO2Solution, reaction, obtains Aminobenzyl-EDTA solution；
The NaNO of the Aminobenzyl-EDTA, the 1M hydrochloric acid and a concentration of 0.2M2The proportioning of solution is
9mg：4mL：150μL；
(a3) by the Aminobenzyl-EDTA solution that step (a2) obtains be added drop-wise to step (a1) obtain it is described
In carrier protein solution, 1M NaOH is used in combination to adjust pH to 8.5, then reaction uses 1M HCL to adjust pH to 7.5, be added a concentration of
The heavy metal lead solion of 0.5M, then react, obtain reaction solution；
The Aminobenzyl-EDTA solution, the carrier protein solution, a concentration of 0.5M heavy metal lead from
The proportioning of sub- solution is 4.15mL：10mL：90μL.
In the method, the first antibody through superparamagnetism fluorescence compound particle label can be according to including walking as follows
It is prepared by rapid method：
It (b1) will every 5mg superparamagnetism fluorescence compound particle, two Asia of 0.96mg1- ethyls-(3- dimethylaminopropyls) carbon
Amine (EDC), 1.15mg N- hydroxysuccinimides (NHS) and a concentration of 0.1M of 1mL, 2- (N- morpholines) second that pH value is 4.7
Sulfonic acid (MES) buffer solution mixing, reaction, magnetic particle after being activated；
(b2) magnetic particle after being activated per 5mg steps (b1), first antibody and 0.8mL are dense described in 0.1-0.2mg
Degree be 50mM pH be 8.5 borate buffer solution mixing, reaction, obtain containing be coupled after magnetic particle reaction solution；
(b3) BSA is added in the reaction solution obtained to step (b2) and is uniformly mixed so as to obtain mixed liquor, reaction, obtains containing after closing
The reaction solution of magnetic particle；Mass percentages of the BSA in the mixed liquor is 1%.
Further, in the preparation method of the carrier protein couplet heavy metal lead ion antigen：
In step (a2), the temperature of the reaction can be 0 DEG C, the time can be 15min；
In step (a3), the temperature of the reaction can be 4 DEG C, time 4h；The temperature reacted again can be 4 DEG C, when
Between be 12h；
Further, in the preparation method of the first antibody through superparamagnetism fluorescence compound particle label：
In step (b1), the temperature of the reaction can be 37 DEG C, time 0.5h；
In step (b2), the temperature of the reaction (being protected from light) can be 37 DEG C, and the time can be 2h；
In step (b3), the temperature of the reaction (being protected from light) can be 37 DEG C, and the time can be 0.5h.
Further, in the preparation method of the carrier protein couplet heavy metal lead ion antigen, in step (a3)
It may also include the steps of later (a4)：
(a4) reaction solution obtained by step (a3) is obtained into the carrier protein couplet heavy metal lead ion antigen through dialysis.
Wherein, it can be 0.02M PBS buffer solution that the dialysis, which is the dialyzate used, and dialysis time can be 48h.
Further, in the preparation method of the first antibody through superparamagnetism fluorescence compound particle label,
It may also include the steps of (b4) after step (b3)：
(b4) magnetic particle after the closing in the reaction solution containing magnetic particle after closing washed, suspended,
Obtain the first antibody marked through superparamagnetism fluorescence compound particle.
Wherein, the cleaning solution used when carrying out the washing and the suspension used when the suspension can be concentration
The PBS buffer solution for being 7.4 for 0.02M pH value.
In the specific embodiment party examination of the present invention, the first antibody is the mouse for capableing of specific bond heavy metal lead ion
The polyclonal antibody in source；Correspondingly, the secondary antibody can be the antibody (such as sheep anti-mouse igg) of anti-mouse IgG.
Further, the polyclonal antibody in the mouse source for capableing of specific bond heavy metal lead ion can according to including
The method of following steps is prepared：Mouse will be immunized after immunogen solution (a concentration of 5 μ g/mL) and adjuvant in equal volume mixing；
The immunogene is the conjugate of heavy metal lead ion haptens (lead (II) ion) and carrier protein (according to the above-mentioned " load
Body protein is coupled the preparation method of heavy metal lead ion antigen " be prepared)；The adjuvant is Quick Antibody-Mouse
3W adjuvants.Wherein, the carrier protein can be BSA, OVA, KLH etc..In specific embodiments of the present invention mode, the carrier
Albumen is specially ovalbumin (ovalbumin, OVA).
Further, it is described it is immune can be immunized twice, including just exempt from 2 weeks after the booster immunization that carries out；Each
Immunizing dose is 250ng, with the gauge of the heavy metal lead ion haptens and the conjugate of carrier protein.
More specifically, in the preparation of the polyclonal antibody in the mouse source for capableing of specific bond heavy metal lead ion
In method, it may also include the steps of：Ascites is prepared within the 10th day (1 week after booster immunization after carrying out booster immunization to mouse
Blood sampling detects antibody titers from serum and the specificity to heavy metal lead ion), ascites is collected, can specifically be tied described in acquisition
Close the polyclonal antibody in the mouse source of heavy metal lead ion.
Second aspect, the preparation method of claimed kit described previously.
The preparation method of kit provided by the present invention may include following steps：It prepares respectively in kit described previously
The test strips and it is described through superparamagnetism fluorescence compound particle mark first antibody.
Wherein, the method for preparing the test strips includes：
I, the sample pad and the coated film equipped with detection line and nature controlling line are prepared respectively；
II, by the sample pad that step I is obtained, step I obtain described in be equipped with the coated film of detection line and nature controlling line
It is pasted successively on the basal layer with the water absorption pad, obtains the test strips；
The coated film equipped with detection line and nature controlling line is prepared as follows：By previously described carrier protein idol
Connection heavy metal lead ion antigen and the secondary antibody are sprayed on the both ends different zones of the coated film respectively, formed the detection line and
The nature controlling line obtains the coated film for being equipped with detection line and nature controlling line；
The sample pad is prepared as follows：Glass fibre membrane is immersed in film process buffer solution, is taken after 0.5h
Go out and dries to get the sample pad.The film process buffer solution is the 0.02M PBS for the Tween20 for being 0.2% containing volume fraction
Solution (pH7.4).
The method for preparing the first antibody marked through superparamagnetism fluorescence compound particle sees above associated description.
The third aspect, application of the claimed kit described previously in detecting heavy metal lead ion.Wherein,
It is described to be detected as quantitative detection or qualitative detection.
Fourth aspect, the method for claimed detection heavy metal lead ion, specially following (A) or (B)：
(A) it is a kind of detection or auxiliary detection sample to be tested in whether the method containing metal lead ion, including：
A1) 1mL samples to be tested and 20 μ L are contained described through the compound grain of superparamagnetism fluorescence in kit described previously
The solution of the first antibody of son label mixes 2 minutes, after Magneto separate with 50 μ L containing volume fraction for 0.2% Tween20
0.02M PBS solutions (pH7.4) are resuspended, and are then added to the sample pad of the test strips, react 10min, then carry out
Fluoroscopic examination；
A2) according to the reaction result of the test strips, according to determining in the sample to be tested whether contain metallic lead as follows
Ion：If the detection line shows fluorescence with the nature controlling line, and the fluorescence intensity of the detection line is not less than the Quality Control
The fluorescence intensity of line does not then contain metal lead ion in the sample to be tested without containing metal lead ion or candidate；If the inspection
Survey line does not show that fluorescence and the nature controlling line show that fluorescence or the detection line show fluorescence but the inspection with the nature controlling line
The fluorescence intensity of survey line is less than the fluorescence intensity of the nature controlling line, then contains containing metal lead ion or candidate in the sample to be tested
There is metal lead ion.
(B) a kind of method detecting metal lead ion content in sample to be tested, including：
B1 standard curve) is drawn：The standard solution for preparing the metal lead ion of series concentration, the several pieces of gained are contained
There is the standard solution of the metal lead ion of various concentration that 1mL and 20 μ L is taken to contain the warp in kit described previously respectively
The solution of the first antibody of superparamagnetism fluorescence compound particle label mixes 2 minutes, is containing volume fraction with 50 μ L after Magneto separate
The 0.02M PBS solutions (pH7.4) of 0.2% Tween20 are resuspended, and obtain several pieces re-suspension liquid；Take several kits
In the ELISA test strip described in several pieces re-suspension liquid, a described re-suspension liquid of each described ELISA test strip, the inspection
Survey is：The re-suspension liquid is added to the sample pad of the test strips, reacts 10min；Described in being read with Fluorescent reader
The fluorescence intensity of the nature controlling line and the detection line of test strips；With the concentration of the standard items of the metal lead ion or its is right
Number is abscissa, using the ratio or its logarithm of the detection line and the fluorescence intensity of the nature controlling line as ordinate, draws standard
Curve graph obtains calibration curve equation；
B2) sample to be tested detects：The 1mL samples to be tested and 20 μ L are contained described through superparamagnetism fluorescence compound particle
The solution of the first antibody of label mixes 2 minutes, with the 0.02M of the 50 μ L Tween20 for being 0.2% containing volume fraction after Magneto separate
PBS solution (pH7.4) is resuspended, and is then added to the sample pad of the original test strips, reacts 10min；With glimmering
Photoreading instrument reads the fluorescence intensity of the nature controlling line and the detection line of the test strips, by the detection line and the matter
The ratio or its logarithm for controlling the fluorescence intensity of line substitute into step b1) gained calibration curve equation, the sample to be tested is calculated
In metal lead ion concentration value.
Detection kit and detection method provided by the present invention detect heavy metal lead ion, the high sensitivity of detection, spy
Anisotropic strong, detection time is short, and testing cost is low, easily operated and popularization.
Description of the drawings
Fig. 1 is the side structure schematic view according to the detection kit of the heavy metal lead ion of one embodiment of the invention.
100 indicate basal layer；200 indicate sample pad；310 indicate detection line (T)；320 indicate nature controlling line (C).
Fig. 2 is the main structure diagram according to the detection kit of the heavy metal lead ion of one embodiment of the invention.
200 indicate sample pad；310 indicate detection line (T)；320 indicate nature controlling line (C)；400 indicate water absorption pad.
Fig. 3 is the song according to the detected value and concentration of the detection kit of the heavy metal lead ion of one embodiment of the invention
Line schematic diagram.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment 1, carrier protein couplet heavy metal lead ion antigen
The specific steps are：
1) it weighs 100mg albumen (BSA, OVA or KLH) and is dissolved in the borate buffer solution of 10mL 0.05M (pH9.6), obtain
To protein solution.
2) 9mg Aminobenzyl-EDTA (eastern Renhua subject skill (Shanghai) Co., Ltd., M029) are weighed and are dissolved in 4mL1M
Hydrochloric acid, the lower NaNO that 150 a concentration of 0.2M of μ L are added dropwise of 0 DEG C (ice bath) stirring2Solution, and reacted 15 minutes in 0 DEG C.
3) Aminobenzyl-EDTA solution is slowly added dropwise in 10 minutes into protein solution, 1M concentration NaOH tune is used in combination
PH to 8.5,4 DEG C are reacted 4 hours, and 1M HCL tune pH to 7.5 are then used.
4) a concentration of 0.5M heavy metal ion solution (lead ion) of 90 μ L are added, 4 DEG C are reacted 12 hours.
5) reaction solution of step 4) is dialysed 48 hours through 0.02M PBS buffer solutions and obtains the weight of carrier protein couplet
Metal lead ion antigen.Gained sample is lyophilized with freeze dryer, in -20 DEG C of preservations.
The preparation method of " carrier protein couplet heavy metal lead ion antigen " provided in the embodiment be by parameter,
Result after condition, process optimization.
The mostly anti-preparation of embodiment 2, preventing from heavy metal lead ion mouse
Balb/c mouse are immunized using tachyphylaxis：OVA-Pb antigens (are prepared using the method for embodiment 1
The conjugate of heavy metal lead ion and carrier protein OVA) be configured to 5 μ g/mL of concentration with normal saline dilution after with Quick
Antibody-Mouse 3W adjuvants 1:1 (each 50 μ L) is mixed, and Balb/c mouse are immunized in gaskin intramuscular injection, and the 14th day by same
Sample loading mode booster immunization mouse, each immunizing dose is 250ng, with the conjugate of heavy metal lead ion and carrier protein OVA
Gauge.It took a blood sample from mouse tail in the 21st day, antibody titers from serum and counterweight is detected with indirect elisa method after separation serum
The specificity of metal lead ion.Reach 1 in mice serum potency：104The 10th day after final immunization afterwards, using inducing in vivo
Method prepares ascites, in the intraperitoneal injection 0.5mL paraffin oils of immune mouse, when preparing ascites antibody, is injected in immune mouse peritoneal
Inoculation about 106A well-grown NS1 myeloma cell.Start to collect ascites after 7 days until dead mouse, ascites is through albumin A
The Mouse Polyclonal Antibody that affinitive layer purification is purified.It is frozen in -20 DEG C after antibody packing.
The preparation of embodiment 3, superparamagnetism fluorescence complex microsphere
1) the superparamagnetism Fe of 30 nanometers of 50mg grain sizes is taken3O4Nano particle, with the HCl processing of 0.1M, washing.Then will
It is scattered in the mixed liquor (volume ratio 10 of 55ml ethyl alcohol and water:1) in.It is (a concentration of that 0.5mL concentrated ammonia liquors are added under ultrasound condition
28%).0.03mL tetraethyl orthosilicates are added afterwards, 25 DEG C are reacted 24 hours.It is scattered in after washing in 100ml ethyl alcohol, 0.5mL is added
(0.3mL OTMS are then added in a concentration of 28%) to concentrated ammonia liquor, and 25 DEG C are reacted 24 hours, are scattered in chloroform after washing, then
1g PMA-ODE (octadecylene polyacrylic acid formicester) are added, ammonia solvent is added after dry, obtains surface carboxyl functionalized and wraps
Cover SiO2Superparamagnetism Fe3O4Particle.
2) by coated Si O2Superparamagnetism Fe3O4Particle is dissolved in 1% PDDA solution (Sigma-Aldrich, 522376)
In, ultrasound 10 minutes, and purification is washed with deionized.10 μM of water-soluble CdSes/ZnS quantum are then added into the solution
Point, ultrasound 10 minutes can be obtained superparamagnetism Fe after washing purification3O4Compound CdSe/ZnS fluorescent particles.
3) by superparamagnetism Fe3O4Compound CdSe/ZnS fluorescent particles are scattered in the mixed liquor (volume ratio of 55ml ethyl alcohol and water
10:1) in.0.5mL concentrated ammonia liquor (a concentration of 28%) is added under ultrasound condition.0.03mL tetraethyl orthosilicates, 25 DEG C of reactions are added afterwards
24 hours.It is scattered in after washing in 100ml ethyl alcohol, 0.5mL concentrated ammonia liquors are added, and (a concentration of 28%), is then added 0.3mL
OTMS, 25 DEG C are reacted 24 hours, are scattered in chloroform after washing, and 1g PMA-ODE are then added, ammonia solvent is added after dry,
Obtain surface carboxyl functionalized and coated Si O2 compound CdSe/ZnS fluorescent microspheres of Superparamagnetic Fe_3O_4, prepared superparamagnetic
Property fluorescence complex microsphere grain size be 100-300nm, magnetic saturation intensity is 40~80emu/g, and corresponding external magnetic field response speed is
30~60 seconds, the content of carboxyl was 60~100 μm of ol/g.
The preparation of embodiment 4, superparamagnetism fluorescence complex microsphere label heavy metal lead ion antibody
It is 100-300nm with average diameter, the superparamagnetism fluorescence complex microsphere of carboxyl modified (preparation of embodiment 3), resists
Heavy metal lead ion mouse is mostly anti-(preparation of embodiment 2), prepares superparamagnetism fluorescence complex microsphere label weight by the following method
Metal lead ion marks first antibody：
(1) the superparamagnetism fluorescence complex microsphere of the above-mentioned carboxyl modified of 5mg is taken to be washed with MES buffer solutions (0.1M, pH4.7)
It washs and after Magneto separate, is resuspended with 1ml MES buffer solutions (0.1M, pH4.7), 1- ethyls-(3- dimethylaminopropyls) carbon is added
Diimine (EDC) is extremely final concentration of to final concentration of 5mM (addition 0.96mg), addition NHS (N- hydroxysuccinimides)
10mM (addition 1.15mg), 37 DEG C are protected from light, and the superparamagnetism fluorescence of carboxyl modified is compound micro- after reaction half an hour is activated
Ball.
(2) the superparamagnetism fluorescence of carboxyl modified is compound micro- after washing the activation with the borate buffer solution of 50mM pH8.5
Ball takes the superparamagnetic of carboxyl modified after the above-mentioned preventing from heavy metal lead ion first antibodies to be marked of 0.1-0.2mg and the above-mentioned activation of 5mg
Property fluorescence complex microsphere, which is mixed into the borate buffer solution of 50mM pH8.5, to be mixed well.37 DEG C are protected from light lower reaction 2 hours, allow this
Antibody and superparamagnetism fluorescence complex microsphere form stable covalent peptide bonds and combine, obtain superparamagnetism fluorescence complex microsphere with again
The conjugate of metal lead ion antibody.After reaction, the BSA solution of final concentration of 1% (mass percentage) is added to super
Paramagnetism fluorescence complex microsphere is closed with residual activity carboxyl site on the conjugate of heavy metal lead ion antibody, and 37 DEG C are kept away
Light reaction 0.5 hour.After the completion, it washed with the 0.02M PBS buffer solution of pH7.4, be resuspended and obtain 5mg/ml superparamagnetism fluorescence
Complex microsphere marks heavy metal lead ion antibody liquid, and 4 DEG C preserve for use.
Embodiment 5, the preparation for detecting heavy metal lead Ion reagent box
The detection kit of heavy metal lead ion provided by the present invention, it is compound containing test strips and through superparamagnetism fluorescence
The first antibody (preparation of embodiment 4) of particle label.
Sample pad of the test strips by being sequentially connected and being fixed on basal layer, the coating equipped with detection line and nature controlling line
Film and water absorption pad composition.The detection line and the nature controlling line are separated from each other；Carrier protein idol is coated at the detection line
Join heavy metal lead ion antigen；It is coated with sheep anti-mouse igg antibody at the nature controlling line.The detection line is located at the coated film and leans on
One end of the nearly sample pad；The nature controlling line is located at the coated film close to one end of the water absorption pad.
Wherein, the preparation method of the test strips is specific as follows：
1, heavy metal lead ion antigen (the heavy metal lead ion being prepared using the method for embodiment 1 is coupled with BSA
With the conjugate of carrier protein BSA) it is used as envelope antigen, coated film is prepared with sheep anti-mouse igg antibody, the specific method is as follows：
(a) the 0.02M PBS buffer solution for using pH7.4, (the limited public affairs of excellent biotechnology are won in Changsha by sheep anti-mouse igg antibody
Department, ABGAM-0500) it is formulated as concentration 0.5mg/ml solution, it is 0.5- by BSA coupling heavy metal lead ion antigen compound concentrations
The solution of 1mg/ml.
(b) it selects the XYZ3050 spray membranous systems of BioDot that step (a) is obtained sheep anti-mouse igg antibody solution and is sprayed onto coating
BSA coupling heavy metal lead ion antigen coat buffer solutions are sprayed onto detection by nature controlling line (C lines) position of film (nitrocellulose filter)
Line (T lines) position is that 20% drying plant below carries out dried for standby after dehumidifier 4 hours in relative humidity, obtains having inspection
The coated film of survey line and nature controlling line.
2, it is impregnated with film process buffer solution (the 0.02M PBS solutions containing the Tween20 that volume fraction is 0.2%, pH7.4)
The temperature of sample pad all-glass paper half an hour, immersion are 37 DEG C, after same dehumidifier condition carries out dehumidifier 4 hours.10
Above-mentioned dried coated film, sample pad, the water absorption pad with detection line and nature controlling line in ten thousand grades of cleanings and dry workshop
(sample pad, coated film and water absorption pad are pasted into basal layer successively by collocation assembling is carried out shown in Fig. 1 and Fig. 2 with basal layer
On) after, it uses the CM4000 cutting systems of BioDot by the Paperboard cutting posted for the width of 3.5mm/ items, is packed into detection and presss from both sides
Piece is for use.
The application method of kit prepared by embodiment 6, embodiment 5
One, qualitative detection
The salpeter solution that 5mL concentration 1% (% indicates mass fraction) is added into sample to be tested extracts 5-20 minutes, mistake
Filter, filtrate take the 0.02M PBS that 1mL is 7.4 with pH of 10 times of volumes containing complexing agent (Aminobenzyl-EDTA, 2 μM of content)
Buffer solution dilutes, and takes containing through superparamagnetism fluorescence in the detection kit of heavy metal lead ion described in 1mL solution and 20 μ L
The solution of the first antibody of compound particle label mixes 2 minutes, and through Magneto separate, containing volume fraction is 0.2% with 50 μ L
The 0.02M PBS solutions (pH7.4) of Tween20 are resuspended, and are then added drop-wise to the sample-adding end of detection heavy metal lead Ion reagent box (i.e.
At sample pad) in, room temperature reaction after ten minutes, carries out fluoroscopic examination.
According to the reaction result of the test strips, according to determine as follows in the sample to be tested whether containing heavy metal lead from
Son：Test strips after reaction are placed under ultraviolet lamp and are irradiated, if the detection line is displayed in red fluorescence (institute with the nature controlling line
The fluorescence intensity for stating detection line is not significantly lower than the fluorescence intensity of the nature controlling line), then testing result is feminine gender, that is, thinks described
Heavy metal lead ion is not contained in sample to be tested；If the detection line is not displayed in red fluorescence or the faint (detection line of colour developing
Fluorescence intensity be less than the fluorescence intensity of the nature controlling line), and the nature controlling line is displayed in red fluorescence, then testing result is sun
Property, that is, think to contain heavy metal lead ion in the sample to be tested.
Two, quantitative detection
1, standard curve is drawn
By heavy metal lead ion standard items (Sigma-Aldrich, 51777) with containing complexing agent (Aminobenzyl-EDTA,
2 μM of content) pH be 7.4 0.02M PBS buffer solution be configured to series concentration (0,0.01,0.05,0.1,0.5,1,5,10 μ
G/L), for the heavy metal lead ion standard solution of each dilution, heavy metal described in 1mL standard solutions and 20 μ L is taken
The solution containing the first antibody marked through superparamagnetism fluorescence compound particle in the detection kit of lead ion mixes 2 points
Clock, and through Magneto separate, the 0.02M PBS solutions (pH7.4) with 50 μ L containing volume fraction for 0.2% Tween20 are resuspended, then
It is added drop-wise in the sample-adding end (i.e. at sample pad) of detection heavy metal lead Ion reagent box, reacts at room temperature after ten minutes, using fluorescence
Detector detects.The excitation and launch wavelength used when detection is respectively 365nm and 615nm, reads the described of the test strips
The fluorescence intensity of nature controlling line and the detection line；Using the logarithm of the heavy metal lead ion standard concentration as abscissa (X), with
The fluorescence intensity ratio logarithm of the detection line and the nature controlling line is ordinate (Y), draws canonical plotting, obtains standard song
Line equation.The fluorescence intensity level of nature controlling line is used for judging whether test strips are effective.
The canonical plotting of gained is as shown in figure 3, calibration curve equation is：
Y=-0.1178X2- 0.5576X-0.7493, R2=0.9981.
2, sample to be tested detects
The salpeter solution that 5mL concentration 1% (% indicates mass fraction) is added into sample to be tested extracts 5-20 minutes, mistake
Filter, filtrate take the 0.02M PBS that 1mL is 7.4 with pH of 10 times of volumes containing complexing agent (Aminobenzyl-EDTA, 2 μM of content)
Buffer solution dilutes, and takes containing through superparamagnetism fluorescence in the detection kit of heavy metal lead ion described in 1mL solution and 20 μ L
The solution of the first antibody of compound particle label mixes 2 minutes, and through Magneto separate, containing volume fraction is 0.2% with 50 μ L
The 0.02M PBS solutions (pH7.4) of Tween20 are resuspended, and are then added drop-wise to the sample-adding end of detection heavy metal lead Ion reagent box (i.e.
At sample pad) in, room temperature reaction after ten minutes, is detected using fluorescence detector.The excitation used when detection and launch wavelength point
Not Wei 365nm and 615nm, read the fluorescence intensity of the nature controlling line and the detection line of the test strips；By the detection
The logarithm of the fluorescence intensity ratio of line and the nature controlling line substitutes into the calibration curve equation of step 1, is calculated described to be measured
The concentration value of heavy metal lead ion in sample.
Embodiment 7, the Performance Evaluation for detecting heavy metal lead Ion reagent box
The detection heavy metal lead Ion reagent box that embodiment 5 obtains is assessed, the specific method is as follows：
1, detection sensitivity
With heavy metal lead ion standard items (Sigma-Aldrich, 51777) embodiment 5 is measured as sample to be tested
Detect the sensitivity of heavy metal lead Ion reagent box.
Standard curve is drawn according to the method in 6 step 2 of embodiment, canonical plotting is as shown in Figure 3.It will detect sensitive
Degree is set to heavy metal lead ion concentration when 50% inhibiting rate, and heavy metal lead ion when detection to be limited to 90% inhibiting rate is dense
Degree, the results showed that, the sensitivity that heavy metal lead ion fluorescence Test paper detects heavy metal lead ion is 0.4 μ g/L, detection limit
For 0.02 μ g/L.
2, accuracy and accuracy detection
Rice sample and pure water sample are detected, adds the heavy metal lead ion of various concentration respectively.Weigh sample to be tested
1g, solid sample need to grind broken, and the salpeter solution that fluid sample can be directly added into 5mL concentration 1% (% indicate mass fraction) carries
It takes 5-20 minutes, filters, filtrate takes 1mL dilute for 7.4 0.02M PBS buffer solution with pH of 10 times of volumes containing complexing agent (being same as above)
It releases, takes containing through superparamagnetism fluorescence compound particle in the detection kit of heavy metal lead ion described in 1mL solution and 20 μ L
The solution of the first antibody of label mixes 2 minutes, and through Magneto separate, the Tween20 for being 0.2% containing volume fraction with 50 μ L
0.02M PBS solutions (pH7.4) are resuspended, and are then added drop-wise to the sample-adding end of detection heavy metal lead Ion reagent box (i.e. at sample pad)
In, reaction after ten minutes, is detected using fluorescence detector.Each addition sample test 5 times, and calculate the rate of recovery.Measurement result
It is shown in Table 1, heavy metal lead ion TIANZHU XINGNAO Capsul is 87.3%~102.7%, and accuracy is preferable；The coefficient of variation 6.9%~
12.9%, average coefficient of variation is less than 15%.
1 accuracy of table and accuracy detection
3, cross reaction detects
5 obtained heavy metal lead Ion reagent box of embodiment is subjected to cross reaction survey to different metal ions respectively
It is fixed, and calculate cross reacting rate (cross reacting rate (%)=[IC50 (Pb)/IC50 (waiting for measured ion)] × 100).As a result it shows
(table 2) low with the cross reacting rate of remaining metal ion.Heavy metal lead Ion reagent box has specificity to heavy metal lead ion.
2 cross reaction of table detects
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of being detached from the principle of the present invention and objective a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The range of invention is limited by claim and its equivalent.
Claims (10)
1. a kind of detection kit of heavy metal lead ion, the containing test strips and through superparamagnetism fluorescence compound particle label
One antibody；The first antibody being capable of specific bond heavy metal lead ion；
Sample pad of the test strips by being sequentially connected and being fixed on basal layer, the coated film equipped with detection line and nature controlling line,
And water absorption pad composition；
The detection line and the nature controlling line are separated from each other；
Carrier protein couplet heavy metal lead ion antigen is coated at the detection line；
Secondary antibody is coated at the nature controlling line, the secondary antibody is the antibody for resisting the first antibody；
The detection line is located at the coated film close to one end of the sample pad；
The nature controlling line is located at the coated film close to one end of the water absorption pad.
2. product according to claim 1, it is characterised in that：First marked through superparamagnetism fluorescence compound particle
Antibody is that the first antibody is combined the condensate to be formed by covalent peptide bonds with the superparamagnetism fluorescence compound particle；
And/or
The first antibody is the polyclonal antibody or monoclonal antibody for capableing of specific bond heavy metal lead ion；
And/or
The carrier protein couplet heavy metal lead ion antigen be by by the carrier protein couplet amino functional EDTA simultaneously
Chelating heavy metal lead ion obtains；
And/or
The sample pad is glass fibre element film, and the coated film is nitrocellulose filter.
3. product according to claim 1 or 2, it is characterised in that：The superparamagnetism fluorescence compound particle is superparamagnetic
Property fluorescence complex microsphere；
Further, the superparamagnetism fluorescence complex microsphere is Fe3O4Magnetic nano-particle is with CdSe/ZnS fluorescent materials through packet
Cover SiO2And the complex microsphere of surface modification carboxyl-functional group；And/or
A diameter of 10-500nm of the superparamagnetism fluorescence complex microsphere, it is preferable that be 50-400nm, it is highly preferred that being
100-300nm；
The magnetic saturation intensity of the superparamagnetism fluorescence complex microsphere is 40~120emu/g, and corresponding external magnetic field response speed is
30~120 seconds；Further, the magnetic saturation intensity of the superparamagnetism compound particle is 40~80emu/g, corresponding external magnetic field
Response speed is 30~60 seconds；
The content of the superparamagnetism fluorescence complex microsphere functionalization group carboxyl is 40~400 μm of ol/g；Further, described
The content of carboxyl is 60~100 μm of ol/g.
4. according to any product in claim 1-3, it is characterised in that：
The carrier protein couplet heavy metal lead ion antigen is prepared according to the method included the following steps：
(a1) carrier protein is dissolved in a concentration of 0.05M, pH value in 9.6 borate buffer solution, to obtain carrier protein
Solution；
The proportioning for the borate buffer solution that the carrier protein and a concentration of 0.05M, pH value are 9.6 is 100mg：10mL；
(a2) Aminobenzyl-EDTA is dissolved in 1M hydrochloric acid, then the lower NaNO that a concentration of 0.2M is added dropwise of 0 DEG C of stirring2Solution,
Reaction, obtains Aminobenzyl-EDTA solution；
The NaNO of the Aminobenzyl-EDTA, the 1M hydrochloric acid and a concentration of 0.2M2The proportioning of solution is 9mg：4mL：
150μL；
(a3) the Aminobenzyl-EDTA solution that step (a2) obtains is added drop-wise to the carrier that step (a1) obtains
It in protein solution, is used in combination and adjusts pH to 8.5, then reaction adjusts pH to 7.5, the heavy metal lead ion of a concentration of 0.5M is added
Solution, then react, obtain reaction solution；
The Aminobenzyl-EDTA solution, the carrier protein solution, the heavy metal lead ion of a concentration of 0.5M are molten
The proportioning of liquid is 4.15mL：10mL：90μL；
And/or
The first antibody through superparamagnetism fluorescence compound particle label is prepared according to the method included the following steps：
(b1) will per 5mg superparamagnetism fluorescence compound particle, 0.96mg1- ethyls-(3- dimethylaminopropyls) carbodiimide,
1.15mg N- hydroxysuccinimides and a concentration of 0.1M of 1mL, 2- (N- morpholines) ethanesulfonic acid buffer that pH value is 4.7 mix
It is even, reaction, magnetic particle after being activated；
(b2) magnetic particle after being activated per 5mg steps (b1), first antibody and 0.8mL are a concentration of described in 0.1-0.2mg
50mM pH be 8.5 borate buffer solution mixing, reaction, obtain containing be coupled after magnetic particle reaction solution；
(b3) BSA is added in the reaction solution obtained to step (b2) and is uniformly mixed so as to obtain mixed liquor, reaction, obtains containing magnetic after closing
The reaction solution of particle；Mass percentages of the BSA in the mixed liquor is 1%.
5. kit according to claim 4, it is characterised in that：
In the preparation method of the carrier protein couplet heavy metal lead ion antigen：
In step (a2), the temperature of the reaction is 0 DEG C, time 15min；
In step (a3), the temperature of the reaction is 4 DEG C, time 4h；The temperature reacted again is 4 DEG C, time 12h；
In the preparation method of the first antibody through superparamagnetism fluorescence compound particle label：
In step (b1), the temperature of the reaction is 37 DEG C, time 0.5h；
In step (b2), the temperature of the reaction is 37 DEG C, time 2h；
In step (b3), the temperature of the reaction is 37 DEG C, time 0.5h.
6. kit according to claim 4 or 5, it is characterised in that：
Further include walking as follows after step (a3) in the preparation method of the carrier protein couplet heavy metal lead ion antigen
Suddenly (a4)：
(a4) reaction solution obtained by step (a3) is obtained into the carrier protein couplet heavy metal lead ion antigen through dialysis；
Further, the dialysis is the dialyzate that uses for 0.02M PBS buffer solution, dialysis time 48h；
In the preparation method of the first antibody through superparamagnetism fluorescence compound particle label, also wrapped after step (b3)
Include following steps (b4)：
(b4) magnetic particle after the closing in the reaction solution containing magnetic particle after closing washed, suspended, obtained
The first antibody marked through superparamagnetism fluorescence compound particle；
Further, the cleaning solution used when carrying out the washing and the suspension used when the suspension are a concentration of
The PBS buffer solution that 0.02M pH value is 7.4.
7. according to any kit in claim 1-6, it is characterised in that：The first antibody is being capable of specific bond
The polyclonal antibody in the mouse source of heavy metal lead ion；
Further, the polyclonal antibody in the mouse source for capableing of specific bond heavy metal lead ion according to including walking as follows
Rapid method is prepared：Mouse will be immunized after immunogen solution and adjuvant in equal volume mixing；The immunogene is heavy metal lead
The conjugate of ion haptens and carrier protein；The adjuvant is Quick Antibody-Mouse 3W adjuvants；
Further, described to be immunized to be immunized twice, including just exempt from 2 weeks after the booster immunization that carries out；Each immunizing agent
Amount is 250ng, with the gauge of the heavy metal lead ion haptens and the conjugate of carrier protein.
8. kit according to claim 7, it is characterised in that：The mouse for capableing of specific bond heavy metal lead ion
Further include following steps in the preparation method of the polyclonal antibody in source：Making for the 10th day after carrying out booster immunization to mouse
Standby ascites collects ascites, the polyclonal antibody in the mouse source of specific bond heavy metal lead ion is capable of described in acquisition.
9. the preparation method of any kit, includes the following steps in claim 1-8：Claim 1-8 is prepared respectively
In test strips in any kit and the first antibody marked through superparamagnetism fluorescence compound particle；
The method for preparing the test strips includes：
I, the sample pad and the coated film equipped with detection line and nature controlling line are prepared respectively；
II, by the sample pad that step I is obtained, step I obtain described in be equipped with coated film and the institute of detection line and nature controlling line
It states water absorption pad to paste successively on the basal layer, obtains the test strips；
The coated film equipped with detection line and nature controlling line is prepared as follows：By claim 1-8 it is any described in
Carrier protein couplet heavy metal lead ion antigen and the secondary antibody are sprayed on the both ends different zones of the coated film respectively, form institute
Detection line and the nature controlling line are stated, the coated film for being equipped with detection line and nature controlling line is obtained；
The method for preparing the first antibody marked through superparamagnetism fluorescence compound particle is described in claim 4-8 is any
The preparation method of first antibody through superparamagnetism fluorescence compound particle label.
10. application or method：
The application is application of any kit in detecting heavy metal lead ion in claim 1-8；
The method is following (A) or (B)：
(A) it is a kind of detection or auxiliary detection sample to be tested in whether the method containing metal lead ion, including：
It a1) will be described through superparamagnetism fluorescence compound particle mark in sample to be tested and any kits of claim 1-8
After the first antibody mixing of note, Magneto separate, then the 0.02M pH7.4PBS with the Tween20 for being 0.2% containing volume fraction are molten
Liquid is resuspended, and is then added to the sample pad of the test strips, reacts 10min, then carries out fluoroscopic examination；
A2) according to the reaction result of the test strips, according to determining in the sample to be tested whether contain metal lead ion as follows：
If the detection line shows fluorescence with the nature controlling line, and the fluorescence intensity of the detection line is glimmering not less than the nature controlling line
Luminous intensity does not then contain metal lead ion in the sample to be tested without containing metal lead ion or candidate；If the detection line is not
Display fluorescence and the nature controlling line show that fluorescence or the detection line and the nature controlling line show fluorescence but the detection line
Fluorescence intensity is less than the fluorescence intensity of the nature controlling line, then contains metal containing metal lead ion or candidate in the sample to be tested
Lead ion；
(B) a kind of method detecting metal lead ion content in sample to be tested, including：
B1 standard curve) is drawn：The standard solution for preparing the metal lead ion of series concentration, the several pieces of gained are contained not
With concentration metal lead ion standard solution respectively with it is described through superparamagnetic in any kits of claim 1-8
Property fluorescence compound particle label first antibody mixing, Magneto separate, then with the Tween20 for being 0.2% containing volume fraction
0.02M pH7.4 PBS solutions are resuspended, and obtain several pieces re-suspension liquid；Take the ELISA test strip in several described kits
The several pieces re-suspension liquid, a re-suspension liquid of each described ELISA test strip are described to be detected as：The re-suspension liquid is added
Enter the sample pad to the test strips, reacts 10min；With Fluorescent reader read the test strips the nature controlling line and
The fluorescence intensity of the detection line；Using the concentration of the standard items of the metal lead ion or its logarithm as abscissa, with the inspection
The ratio of the fluorescence intensity of survey line and the nature controlling line or its logarithm are ordinate, draw canonical plotting, obtain standard curve
Equation；
B2) sample to be tested detects：By the sample to be tested and the first antibody marked through superparamagnetism fluorescence compound particle
After mixing, then Magneto separate is resuspended, then with the 0.02M pH7.4 PBS solutions for the Tween20 for being 0.2% containing volume fraction
It is added to the sample pad of the original test strips, reacts 10min；The test strips are read with Fluorescent reader
The fluorescence intensity of the nature controlling line and the detection line, by the ratio of the detection line and the fluorescence intensity of the nature controlling line or its
Logarithm substitutes into step b1) gained calibration curve equation, the concentration value of the metal lead ion in the sample to be tested is calculated.
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| CN110286107A (en) * | 2019-06-26 | 2019-09-27 | 湖北工业大学 | The detection method of heavy metal lead ion |
| CN110672834A (en) * | 2019-09-26 | 2020-01-10 | 北京丹大生物技术有限公司 | Kit for rapidly detecting lead content in sample |
| CN112526122A (en) * | 2020-11-13 | 2021-03-19 | 山东美正生物科技有限公司 | Fluorescent quantitative test strip for rapidly detecting heavy metal lead |
| CN115950877A (en) * | 2022-10-20 | 2023-04-11 | 国家粮食和物资储备局科学研究院 | Magnetic particle chemiluminescence kit for detecting heavy metals |
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