CN108008128A - Aptamer identifies and the kanamycins quick detection test paper of functionalized magnetic microsphere separation and culture and its preparation and application - Google Patents
Aptamer identifies and the kanamycins quick detection test paper of functionalized magnetic microsphere separation and culture and its preparation and application Download PDFInfo
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- CN108008128A CN108008128A CN201711391701.2A CN201711391701A CN108008128A CN 108008128 A CN108008128 A CN 108008128A CN 201711391701 A CN201711391701 A CN 201711391701A CN 108008128 A CN108008128 A CN 108008128A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
Aptamer identifies and the kanamycins quick detection test paper of functionalized magnetic microsphere separation and culture and its preparation and application, belongs to environment, food and medicine analysis field.The present invention utilizes the good separating effect of magnetic microsphere, aptamer（Apt）High-affinity and gold nanoparticle between target（AuNPs）Cue mark characteristic, qualitative analysis to kanamycins is realized by the colour developing depth for observing detection line, while quantitative analysis can be carried out using colloidal gold quantitative instrument.CDNA can be mediated to form sandwich structure and realize in detection line and AuNPs is captured, and it is to cDNA and then linear related with the concentration of kanamycins to capture the quantity of AuNPs, so as to can differentiate kanamycins content according to the detection line colour developing depth.Test paper method detection kanamycins of the present invention is easy to be quick, detects the limitation from environmental condition, result visualization, detection process only needs 20 min, easy to expand and apply.
Description
Technical field
The present invention relates to a kind of kanamycins based on aptamer identification and functionalized magnetic microsphere separation and culture
Quick detection test paper and its preparation and application, belong to environment, food and medicine analysis field.
Background technology
Antibiotic（Antibiotics）It is the secondary metabolite being metabolized out by bacterium, fungi or animals and plants, has disease-resistant
The property of substance.Since aminoglycoside antibiotics contrasts advantage of lower cost, water-soluble effect for the antibiotic of other classifications
Preferably, has a broad antifungal spectrum, while there is good therapeutic effect, therefore extensive use to common bacteriosis.Kanamycins
（kanamycin）It is one of most frequently used aminoglycoside antibiotics, kanamycins is to pass through streptomycete
（Streptomyces kanamyceticus）What fermentation produced, it is widely used in bacterial treatment of infection.Kanamycins
Mechanism of action be to upset the conjunction of bacterioprotein by misreading genetic code and combining ribosomal 30S subunits and suppress translation
Into, and then the growth and breeding inhibited bacteria.Kanamycins is applied to animal doctor as two wires antibiotic in the form of injecting with capsule
Learn, effectively suppress the growth of Gram-negative bacteria and gram-positive bacteria.As kanamycins more and more frequently makes with excessive
With, cause it progressively to be accumulated in the food of animal origin, the health to us is a kind of harm, may trigger ear toxication, urine
Toxication and neural toxication etc..Therefore the problem of increasingly increasing on yapamicin relict in the world, becomes serious public
Health threatens.
Since the residual of kanamycins is there are many hazards, to improve food quality and safety, develop practical simple fast
The detection method of speed is very necessary.Now it has been reported that a variety of detection methods for antibiotic residue, such as micro- life
Analyte detection method, instrumental method, biochemical immunity detection method and electrochemical assay etc..There are one for the application of these detection methods
A little shortcomings, and be not suitable for Site Detection and amplification production, therefore one kind should be established as early as possible and can be used in detection food blocking that
Mycin is remaining easily and fast, economy, accuracy are good, high sensitivity detection method, can effectively improve animal derived food
Security.
The content of the invention
The purpose of the present invention is overcome above-mentioned deficiency, there is provided one kind is based on aptamer identification and functionalized magnetic microsphere
The kanamycins quick detection test paper of separation and culture and its preparation and application, by colloid gold label chromatograph test strip technology, magnetic
Property the quick separating of microballoon and the advantage of many technologies such as specificity of aptamer join together, prepare and a kind of be used for letter
Just, quickly, the Test paper of low, high sensitivity the yapamicin relict of cost and be applied to sample detection.
Technical scheme, a kind of card based on aptamer identification and functionalized magnetic microsphere separation and culture
That mycin quick detection test paper, its composition include sample pad, gold-labelled pad, nitrocellulose filter, water absorption pad, PVC bottom plates, detection line
And nature controlling line；Sample pad, gold-labelled pad, nitrocellulose filter and water absorption pad are pasted successively on the PVC bottom plates, and wherein sample pad is taken
Gold-labelled pad is connect, nitrocellulose filter is directly arranged on PVC bottom plates, nitrocellulose filter one end overlap joint gold-labelled pad, cellulose nitrate
The plain film other end overlaps water absorption pad；Detection line and nature controlling line are equipped with the nitrocellulose filter successively.
Each clinch overlay segment of the sample pad, gold-labelled pad, nitrocellulose filter, water absorption pad is 2mm.
Functionalization gold nanoparticle AuNPs-DNA1 is added dropwise in the gold-labelled pad, capture probe 1 is modified in detection line
CapDNA1, modifies capture probe 2 capDNA2 on nature controlling line, and cDNA, which moves to mediate after detection line, forms complementary strand Sanming City
Structures capture AuNPs is controlled, unnecessary AuNPs is then captured in nature controlling line；Kanamycins concentration and the AuNPs captured in detection line
Correlation is presented in quantity and the colour developing depth.
The preparation process of the gold-labelled pad is：Prepare gold nanoparticle and respectively by the covalent effects of Au-S keys by DNA1
The gold nanoparticle AuNPs-DNA1 to form functionalization is coupled with gold nano grain；
Wherein DNA1 sequences are：5’-ATAGCACAAC CGTC-(CH2)6-HS-3’
It is described on nitrocellulose filter close to one end of gold-labelled pad with the Streptavidin and biotin modification being incubated in advance
CapDNA1 prepares detection line, and with Streptavidin and biotin modification capDNA2 Quality Control is being prepared close to water absorption pad one end
Line；
The sequence of wherein capDNA1 and capDNA2 is respectively：
capDNA1：5’-Biotin-TGACTGTAAG CCG-3’；
capDNA2：5’-GGTTGTGCTA TTATGA-Biotin-3’.
The kanamycins based on aptamer identification and functionalized magnetic microsphere separation and culture quickly detects examination
The preparation method of paper, step are as follows：
（1）The preparation of functionalized magnetic microsphere：One section of cDNA with aptamers Apt partial complementarities is designed first, by cDNA and Apt
Respectively in 95 DEG C of metal bath 3min, then cDNA and Apt are sufficiently mixed, 1h is incubated at 37 DEG C, makes Apt-cDNA and Apt abundant
Hybridization；Mixed after magnetic microsphere MBs is cleaned 5 times with the PBS buffer of 10mmol/L, pH 7.4 with above-mentioned hybrid dna, 37 DEG C
1h is shaken, forms functionalized magnetic microsphere MBs-Apt-cDNA, which cleans 3 removings with cleaning solution and do not tie
The Apt-cDNA on magnetic microsphere surface is closed, the functionalized magnetic microsphere MBs-Apt-cDNA that will have been pre-processed, 4 DEG C of preservations are standby
With；
Wherein, the sequence of Apt and cDNA is respectively：
Apt：5 '-Biotin-TGGGGGTTGA GGCTAAGCCG A-3 ',
cDNA：5’- TTGTGCTATC GGCTTAC-3’；
（2）The preparation of functionalization gold nanoparticle：All glass apparatus are soaked with chloroazotic acid before preparing gold nanoparticle
30min, is then cleaned with distilled water, and ultra-pure water immersion 12h, is dried for standby；The gold chloride of 100mL mass concentrations 0.01% is added
Enter in 250mL round-bottomed flasks, be stirred and heated to boiling, rapidly join the lemon of 3.5mL mass concentrations 1% with vigorous stirring
Sour trisodium, solution becomes claret after continuing heating stirring 15min, stops heating, continues to stand after stirring 30min, and room temperature is certainly
So cooling, obtains the solution of gold nanoparticles of particle diameter 13nm, 4 DEG C save backup；
Above-mentioned solution of gold nanoparticles is continued to heat and be stirred continuously under the conditions of boiling water bath, makes moisture evaporation, until flask
Interior liquor capacity is to boiling water bath is stopped after 20mL, and room temperature natural cooling, the gold nanoparticle concentrated, 4 DEG C save backup；
By the three of 15 μ L 1mmol/L（2- carboxyethyls）Phosphine TCEP is mixed with the DNA1 of 30 100 μm of ol/L of μ L, and it is anti-to carry out sulfhydrylation
Should, it is then added in the gold nanoparticle of 1mL concentrations, stirs 1h at room temperature；Then the triphosphoric acid of 30 μ L 15mmol/L is taken off
Oxygen adenosine dATP is added in above-mentioned solution, and 35min is stirred at room temperature；The NaCl solution of 20 μ L 1mol/L is added after the completion of concussion
Room temperature places 30min, and 4 DEG C preserve 6h to increase the stability of combination；4 DEG C, 8000r/min centrifugations 10min remove unnecessary examination
Agent；Be resuspended in 1mL containing 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100 20mmol/L Na3PO4
Obtain functionalization gold nanoparticle AuNPs-DNA1 in buffer solution, 4 DEG C be kept in dark place it is spare；
Wherein, the sequence of DNA1 is：5’-ATAGCACAACCGTC-(CH2)6- SH-3 ', with cDNA and capDNA2 parts mutually
Mend；
（3）Sample pad, gold-labelled pad pretreatment：Gold-labelled pad and sample pad are cut into after suitable size and contain 1% in pH8.2
NaCl, 0.5% Tween-20,1% BSA, 2% sucrose, 1% TritonX-100 0.1 mol/L Tris-HCl buffer solutions in it is complete
Complete immersion 30min, is then put into after being dried in 37 DEG C of thermostatic drying chambers, and drying condition preserves, is spare；
Functionalization gold nanoparticle AuNPs-DNA1 is added dropwise in the gold-labelled pad handled well, is dried in 37 DEG C of thermostatic drying chambers, does
Dry condition preserves, spare；
（4）Nitrocellulose filter pre-processes：First, by biotinylated capDNA1 and capDNA2 respectively with Streptavidin
Streptavidin is combined；By 10 μ L 1mg/mL Streptavidins respectively with 20 μ L, 10 μm of biotinylated capDNA1 of ol/L
And capDNA2 combines 2h under conditions of 4 DEG C after mixing, make Streptavidin and biotinylated capDNA1 and
CapDNA2 is fully coupled；
The Streptavidin-biotin-capDNA1 that 1 μ L are combined is taken to draw detection line T lines at the 6mm of gold-labelled pad one end；
1 μ L Streptavidin-biotin-capDNA2 are taken to draw nature controlling line C line at the 6mm of water absorption pad one end；T, C line will be pulled
Nitrocellulose filter dried at 37 DEG C, 4 DEG C save backup；
（5）The assembling of test strips：The composition material of test strips mainly includes PVC bottom plates, sample pad, gold-labelled pad, nitrocellulose
Film, five part of water absorption pad, on the correspondence position that nitrocellulose filter is first pasted to PVC bottom plates, light pressure, makes the two closely tie
Close, processed sample pad, gold-labelled pad are respectively adhered on the correspondence position of PVC bottom plates, sample pad and gold-labelled pad, Jin Biao
Pad and each overlapping 2 mm of nitrocellulose filter, finally paste water absorption pad 2mm overlapping with nitrocellulose filter on PVC bottom plates,
Redundance is dismissed；Each several part firm pasting is simultaneously cut into test strips of the specification for the mm of 60 mm × 4, and drying condition preserves, spare.
The kanamycins quick detection test paper based on aptamer and functionalized magnetic microsphere separation and culture
Using：After functionalized magnetic microsphere MBs-Apt-cDNA is cleaned 3 times with the PBS buffer of 10mmol/L, pH7.4, it is resuspended in
Combination buffer, adds sample to be tested thereto, 10 min of concussion make card contained in the Apt and sample on magnetic microsphere surface that
Mycin fully combines, by the cDNA being complementary to displacements into solution；
The combination buffer is specially EDTA containing 1mmol/L, 50mmol/L NaCl, the 50mmol/ of 0.05% Tween-20
L, the Tris-HCl buffer solutions of pH7.5；
Magnetic microsphere is adsorbed under magneticaction, takes the supernatant containing cDNA as ELISA test strip liquid；The test paper that will be prepared
Bar is placed in test strips cartridge, and solution to be detected is added dropwise in well；
When detect kanamycins is not present in liquid when, T lines do not develop the color, C lines colour developing；
When detecting there are during kanamycins in liquid, T, C line develop the color；10 min are stood, after test strips colour developing completely, pass through meat
Eye observation detection line shade can qualitative or semi-quantitative analysis, or test strips are placed in colloidal gold quantitative instrument and are scanned inspection
Survey, obtain the peak area value of detection line and nature controlling line, kanamycins concentration in detection liquid is quantitative determined out according to standard curve.
Beneficial effects of the present invention：1. this method is become using the conformation of the good separating effect of magnetic microsphere and aptamers
Change the change for realizing detection line optical strength, and then realize the detection to kanamycins；2. test paper method detection kanamycins is easy
Quickly, the limitation from environmental condition, result visualization, easy to expand and apply are detected.Detection process only needs 20 min
Experimental result is obtained, detection efficiency greatly improves, practical application effect substantially increases compared with the method for common detection kanamycins
By force；3. the detection method can also pass through colloidal gold by observing qualitative analysis of the color intensity realization to kanamycins
Quantitative instrument carries out quantitative detection, is of great significance to yapamicin relict detection in animal derived food.
Brief description of the drawings
Fig. 1 is based on aptamers and the separated kanamycins Test paper schematic diagram of functionalized magnetic microsphere.A, magnetic microsphere
Preprocessing process figure；B, test strips composition figure；C, test paper detection negative findings figure；D, test paper detection positive findings figure；1st, sample
Pad；2nd, gold-labelled pad；3rd, nitrocellulose filter；4th, water absorption pad；5th, PVC bottom plates；6th, detection line；7th, nature controlling line.
The detection figure of Fig. 2 ELISA test strip various concentrations kanamycins standard solution.
Fig. 3 T lines peak area and canonical plotting of kanamycins concentration in standard detection liquid.
The specificity verification figure of Fig. 4 ELISA test strip methods.
The stability proof diagram of Fig. 5 ELISA test strip methods.
The actual sample detection figure of Fig. 6 test strips.A, milk；B, honey；C, milk powder.
Embodiment
Kanamycins quick detection test paper of the embodiment 1 based on aptamer and magnetic microsphere separation and culture
The preparation and functionalization of pretreatment, gold nanoparticle including magnetic microsphere, the pretreatment of gold-labelled pad and sample pad, nitric acid
The pretreatment of cellulose membrane, the assembling of test strips, test strip colour developing situation；Concretely comprise the following steps：
（1）The preparation of functionalized magnetic microsphere
One section and the cDNA of aptamers Apt partial complementarities are designed first, by cDNA and Apt respectively in 95 DEG C of 3 min of metal bath,
Open the complementary portion of cDNA, formation one is single-stranded, easy to the Complementary hybridization of next step cDNA and Apt.CDNA and Apt are filled
Divide mixing, 1 h is incubated in 37 DEG C of constant incubators, cDNA and Apt is fully combined.Since magnetic microsphere surface modification has chain
Mould Avidin, Apt 5 ' it is terminal modified have a biotin, therefore Apt can be coupled to by Streptavidin-biotin effect it is magnetic micro-
Ball surface.After magnetic microsphere is cleaned 5 times with the PBS buffer of 10 mmol/L, pH 7.4 with above-mentioned Apt-cDNA mixed liquors knot
Close, 1 h is shaken at 37 DEG C, avoids magnetic microsphere from settling, form MBs-Apt-cDNA.The magnetic microsphere cleaning solution being combined
3 removings of cleaning are not bonded to the Apt-cDNA on magnetic microsphere surface.The functionalized magnetic microsphere MBs-Apt- that will have been pre-processed
CDNA, 4 DEG C save backup.
（2）The preparation of gold nanoparticle and functionalization
All glass apparatus soak 30 min with chloroazotic acid before preparing gold nanoparticle, are then cleaned with distilled water, ultrapure
Water soaks 12 h, is dried for standby；The gold chloride of 100 mL mass concentrations 0.01% is added in 250 mL round-bottomed flasks, stirring is simultaneously
It is heated to seething with excitement, rapidly joins the trisodium citrate of 3.5 mL mass concentrations 1% with vigorous stirring, continues heating stirring 15
Solution becomes claret after min, stops heating, continues to stand after stirring 30 min, room temperature natural cooling, obtains 13 nm of particle diameter
Solution of gold nanoparticles, 4 DEG C save backup；
Above-mentioned solution of gold nanoparticles is continued to heat and be stirred continuously under the conditions of boiling water bath, makes moisture evaporation, until flask
Interior solution stops boiling water bath after being 20 mL, and room temperature natural cooling, the gold nanoparticle concentrated, 4 DEG C save backup；
By the three of 15 μ L, 1 mmol/L（2- carboxyethyls）Phosphine TCEP is mixed with the DNA1 of 30 100 μm of ol/L of μ L, carries out sulfydryl
Change reaction, be then added in the gold nanoparticle of 1 mL concentrations, stir 1 h at room temperature；Then by 30 μ L, 15 mmol/L's
Deoxyadenosine triphosphate dATP is added in above-mentioned solution, and 35 min are stirred at room temperature；20 μ L, 1 mol/L are added after the completion of concussion
NaCl solution room temperature place 30 min, 4 DEG C preserve 6 h and increase the stability of combination；4 DEG C, 8000 r/min centrifugations 10
Min removes unnecessary reagent；1 mL is resuspended in containing 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100
The Na of 20 mmol/L3PO4In buffer solution, 4 DEG C be kept in dark place it is spare；
（3）Sample pad, gold-labelled pad pretreatment
By gold-labelled pad and sample pad be cut into after suitable size pH 8.2 containing 1% NaCl, 0.5% Tween-20,1% BSA,
2% sucrose, 1% TritonX-100 0.1 mol/L Tris-HCl buffer solutions in be completely soaked 30 min, be then put into 37 DEG C
After being dried in thermostatic drying chamber, drying condition preserves, is spare；
Be added dropwise functionalization gold nanoparticle in the gold-labelled pad handled well, i.e. AuNPs-DNA1, wherein DNA1 and cDNA and
The equal partial complementarities of capDNA2, are dried in 37 DEG C of thermostatic drying chambers, and drying condition preserves, spare；
（4）Nitrocellulose filter pre-processes
First, biotinylated capDNA1 and capDNA2 are combined with Streptavidin.10 μ L, 1 mg/mL strepto-s is affine
Element combines 2 under conditions of 4 DEG C after mixing with the biotinylated capDNA1 and capDNA2 of 20 μ L, 10 μm of ol/L respectively
H, can make Streptavidin and biotinylated capDNA1 and capDNA2 with reference to power by force between Streptavidin and biotin
Fully coupling.The Streptavidin-biotin-capDNA1 that 1 μ L are combined is taken to draw inspection at 6 mm of gold-labelled pad one end
Survey line（T lines）.1 μ L Streptavidin-biotin-capDNA2 are taken to draw nature controlling line at 6 mm of water absorption pad one end（C
Line）.The NC films for pulling T, C line are dried at 37 DEG C, lower 4 DEG C of drying condition saves backup.
（5）The assembling of test strips
The composition material of colloid gold chromatographic test paper strip mainly includes PVC bottom plates, sample pad, gold-labelled pad, nitrocellulose filter, water suction
Five part of pad, on the correspondence position that nitrocellulose filter is first pasted to PVC bottom plates, light pressure, makes the two combine closely, will handle
Sample pad, the gold-labelled pad crossed, are respectively adhered on the correspondence position of PVC bottom plates, sample pad and gold-labelled pad, and gold-labelled pad is fine with nitric acid
Dimension each overlapping 2 mm of plain film, finally pastes water absorption pad 2 mms overlapping with nitrocellulose filter on PVC bottom plates, redundance is cut out
Fall；Then each several part firm pasting, is cut into test strips of the specification for the mm of 60 mm × 4, drying condition preserves, spare.
Measure of the embodiment 2 with test strips to kanamycins standard solution
（1）The pretreatment of magnetic microsphere
One section and the cDNA of aptamers Apt partial complementarities are designed first, by cDNA and Apt respectively in 95 DEG C of metal bath 3min, are made
The complementary portion of cDNA is opened, and formation one is single-stranded, easy to the Complementary hybridization of next step cDNA and Apt.CDNA and Apt is abundant
Mixing, is incubated 1h in 37 DEG C of constant incubators, cDNA and Apt is fully combined.Since magnetic microsphere surface modification has strepto-
Avidin, Apt 5 ' it is terminal modified have a biotin, therefore Apt can be coupled to magnetic microsphere by Streptavidin-biotin effect
Surface.Combined after magnetic microsphere is cleaned 5 times with the PBS buffer of 10mmol/L, pH 7.4 with above-mentioned Apt-cDNA mixed liquors,
To shake 1 h at 37 DEG C, avoid magnetic microsphere from settling, form MBs-Apt-cDNA.Magnetic microsphere cleaning after being combined
Liquid cleans 3 times and removes the Apt-cDNA for being not bonded to magnetic microsphere surface.The functionalized magnetic microsphere MBs- that will have been pre-processed
4 DEG C of Apt-cDNA is saved backup.
（2）The test strips that will be prepared as stated above, for qualitative or sxemiquantitative analysis and with colloidal gold quantitative instrument
Quantitative detection kanamycins.First handling the card of the mmol/L containing 0-5 respectively by the pre-treatment step of above-mentioned magnetic microsphere, that is mould
Element, the test strips then prepared with the same terms detect the cDNA replaced, since amount and the kanamycins of cDNA are dense
Spend it is directly proportional, it is thus determined that the concentration of cDNA is assured that the concentration of kanamycins.As shown in Fig. 2, do not deposited when in detection liquid
In kanamycins, since cDNA is not set to change, the capDNA1 in detection line cannot capture Jenner in no cDNA
Rice corpuscles, the capDNA2 on nature controlling line can capture gold nanoparticle, therefore there was only nature controlling line colour developing, detection line in test strips
Do not develop the color.When the concentration of kanamycins gradually increases, the color of C lines does not change substantially, and the color of T lines gradually strengthens, when
When the concentration of kanamycins reaches 50 nmol/L, naked eyes are it is observed that detection line has slight colour developing, therefore sets 50
Nmol/L is that the Visual retrieval of the detection method limits.With the peak area of colloidal gold quantitative instrument test strip detection line, determine
The kanamycins of various concentrations and the relation of detection line peak area, as shown in figure 3, with the increase of kanamycins concentration, detection
The peak area of line gradually increases, when the concentration of kanamycins is between 5-500 nmol/L, the concentration of kanamycins and detection
There are linear relationship, equation of linear regression between the peak area of line to be：Y=678.2684x+263.9928, R2=0.9917, x generation
The concentration of table kanamycins, y represent the peak area of detection line.The theoretical detection calculated according to linear relationship is limited to 4.958
nmol/L (S/N=3).This represents that the ELISA test strip has higher sensitivity.
The specificity verification of 3 ELISA test strip method of embodiment
The test strips that the same terms are prepared come the streptomysin (STR) that detectable concentration is 5 mmol/L, ammonia benzyl mould respectively
Plain (AMP), neomycin (NEO), gentamicin (GEN), kanamycins (KAN) and water, the colour developing feelings of contrasting detection line after detection
Condition and peak area size verify the specificity of the ELISA test strip, and variety classes antibiotic is used for the detection line peak face after detecting
Product figure, as shown in Figure 4.In Fig. 4 it can be seen that the peak area of detection line is several apparently higher than other when detecting kanamycins
Antibiotic, hence it is demonstrated that detection of the detection method to kanamycins has higher specificity.
The stability verification of 4 ELISA test strip method of embodiment
For determine the test strips stability, test strips are completed be placed on 4 DEG C preserve different number of days respectively after be used for
Detection.This experiment detects the kanamycins of various concentrations respectively after test strips are stored respectively 0,3,7,15,30,60 and 90 day
(0,30, μm ol/L of 100nmol/L, 10,100,500), the optical strength of the detection line of different holding time test strips determines this
The stability result of detection method is shown as shown in Figure 5.In Figure 5 it can be seen that holding time of the detection method at 3 months
The peak area change of interior detection same concentrations kanamycins is small, and the Weaken degree of the peak area of detection line is in 3.60%-9.84%
Between.Show that the test strips stability is preferable.
The detection of yapamicin relict in 5 actual sample of embodiment
In order to verify the practicality of the detection method, the kanamycins in milk, honey and milk powder have detected respectively.First to ox
In milk, honey and powdered milk sample add kanamycins to concentration be respectively 0,30 nmol/L, 50 nmol/L, 100 nmol/L,
200 nmol/L, 500 nmol/L, 1 μm of ol/L, 10 μm of ol/L and 100 μm of ol/L, then pre-process them, to
20% solution of trichloroacetic acid is added dropwise in milk dropwise, the pH of milk is adjusted to 4.6, then 45 DEG C of 10 min of water-bath are to precipitate
Albumen, 10000 r/min centrifuge 25 min and remove the protein and fat condensed, obtain milk sample after pretreatment.Bee
Honey dilutes 10 times with magnetic microsphere combination liquid.Take 2 g milk powder to add 5 mL of magnetic microsphere combination liquid to mix.10000 r/
Min centrifuges 20 min removing fat deposits and takes supernatant, and supernatant then is carried out ten times of dilutions with magnetic microsphere combination liquid, is obtained
Powdered milk sample after pretreatment.Pretreated milk, honey and powdered milk sample and MBs-Apt-cDNA are sufficiently mixed, instead
Should after with test strips detect the cDNA for being set to change in solution.The appearance feelings of detection line are checked with the detection of colloidal gold quantitative instrument
Condition.In figure 6 it can be seen that being respectively 50 with the test limit of the kanamycins in ELISA test strip milk, honey, milk powder
Nmol/L, 50 nmol/L and 100 nmol/L.The test limit of different actual samples is different, detects milk and honey knot
Fruit is substantially consistent with standard items result, and when detecting milk powder, test limit is also within the acceptable range.Therefore, this test paper
Bar detection has good application prospect in actual sample detection.
The comparison of 6 ELISA test strip method of embodiment and HPLC detection methods
High performance liquid chromatography is a kind of common method of antibiotic detection, is compared herein using HPLC and ELISA test strip technology
The accuracy of test strips method is verified more afterwards.The kanamycins standard items of various concentrations are detected with test strips and HPLC respectively, this
The detector of HPLC used is the evaporation photodetector of Shimadzu in invention, and mobile phase is 0.2% trifluoroacetic acid-methanol（95:5）
Solution, 1.0 mL/min of flow velocity, 30 DEG C of column temperature, 20 μ L of sample size, 80 DEG C of drift tube temperature, 10 min of detection time.Contrast is flat
Equal concentration, RSD values determine the accuracy of the detection method.As shown in table 1, from the angle analysis of mean concentration, the examination of foundation
Paper slip detection technique and HPLC and the concentration comparable of the kanamycins standard items added during detection, show that both detection methods have
There is good accuracy；From the angle analysis of RSD values, the RSD values of ELISA test strip are between 2.15%-4.96%, the RSD of HPLC
In 0.25%-0.59%, it was demonstrated that though the accuracy of ELISA test strip technology is slightly inferior to HPLC, performance is still good, while test paper
Bar detection method is significantly better than HPLC detection methods in terms of sensitivity and test limit.
1 ELISA test strip method of table and HPLC methods detection kanamycins results contrast
。
Sequence table
<110>Southern Yangtze University
<120>Aptamer identify and functionalized magnetic microsphere separation and culture kanamycins quick detection test paper and its
Prepare and apply
<130> 201712031103
<141> 2017-12-21
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> DNA
<213>DNA1 sequences (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
atagcacaac cgtc 14
<210> 2
<211> 13
<212> DNA
<213> capDNA1(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
tgactgtaag ccg 13
<210> 3
<211> 16
<212> DNA
<213> capDNA2(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
ggttgtgcta ttatga 16
<210> 4
<211> 21
<212> DNA
<213> Apt(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
tgggggttga ggctaagccg a 21
<210> 5
<211> 17
<212> DNA
<213> cDNA(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
ttgtgctatc ggcttac 17
Claims (5)
1. a kind of kanamycins quick detection test paper based on aptamer identification and functionalized magnetic microsphere separation and culture,
It is characterized in that：The composition of test paper includes sample pad（1）, gold-labelled pad（2）, nitrocellulose filter（3）, water absorption pad（4）, PVC bottoms
Plate（5）, detection line（6）And nature controlling line（7）；
The PVC bottom plates of the test paper（5）On paste sample pad successively（1）, gold-labelled pad（2）, nitrocellulose filter（3）And water absorption pad
（4）, wherein sample pad（1）Overlap gold-labelled pad（2）, nitrocellulose filter（3）It is directly arranged at PVC bottom plates（5）On, cellulose nitrate
Plain film（3）One end overlaps gold-labelled pad（2）, other end overlap joint water absorption pad（4）；The nitrocellulose filter（3）On successively be equipped with inspection
Survey line（6）And nature controlling line（7）；Sample pad（1）, gold-labelled pad（2）, nitrocellulose filter（3）And water absorption pad（4）Each overlap weight
Folded section is 2mm；
The gold-labelled pad（2）Upper dropwise addition functionalization gold nanoparticle AuNPs-DNA1, detection line（6）Upper modification capture probe 1
CapDNA1, nature controlling line（7）Upper modification capture probe 2 capDNA2, cDNA, which move to mediate after detection line, forms complementary strand three
Mingzhi's structures capture AuNPs, unnecessary AuNPs is then in nature controlling line（7）It is captured；Kanamycins concentration and detection line（6）On catch
Correlation is presented in the AuNPs quantity and the colour developing depth obtained.
2. the kanamycins based on aptamer identification and functionalized magnetic microsphere separation and culture according to claim 1
Quick detection test paper, it is characterised in that the gold-labelled pad（2）Preparation process is：Prepare gold nanoparticle and being total to by Au-S keys
DNA1 and gold nano grain are coupled to form functionalization gold nanoparticle AuNPs- DNA1 by valency effect；
Wherein DNA1 sequences are：5’-ATAGCACAAC CGTC-(CH2)6-HS-3’。
3. the kanamycins based on aptamer identification and functionalized magnetic microsphere separation and culture according to claim 1
Quick detection test paper, it is characterised in that：It is described in nitrocellulose filter（3）Upper close gold-labelled pad（2）One end with advance be incubated
The Streptavidin and biotin modification capDNA1 crossed prepares detection line（6）, close to water absorption pad（4）One end is close with strepto-
Nature controlling line is prepared with element and biotin modification capDNA2（7）；
The sequence of wherein capDNA1 and capDNA2 is respectively：
capDNA1：5’-Biotin-TGACTGTAAG CCG-3’；
capDNA2：5’-GGTTGTGCTA TTATGA-Biotin-3’.
4. the kanamycins based on aptamer identification and functionalized magnetic microsphere separation and culture described in claim 1 is quick
The preparation method of Test paper, it is characterised in that step is as follows：
（1）The preparation of functionalized magnetic microsphere：One section of cDNA with aptamers Apt partial complementarities is designed first, by cDNA and Apt
Respectively in 95 DEG C of metal bath 3min, then cDNA and Apt are sufficiently mixed, 1h is incubated at 37 DEG C, makes cDNA and Apt fully miscellaneous
Hand over；Mixed after magnetic microsphere MBs is cleaned 5 times with the PBS buffer of 10mmol/L, pH 7.4 with above-mentioned hybridization Apt-cDNA,
37 DEG C of concussion 1h, form functionalized magnetic microsphere MBs-Apt-cDNA, which cleans 3 removings with cleaning solution
The Apt-cDNA on magnetic microsphere surface is not bonded to, the functionalized magnetic microsphere MBs-Apt-cDNA that will have been pre-processed, 4 DEG C of preservations
It is spare；
Wherein, the sequence of Apt and cDNA is respectively：
Apt：5 '-Biotin-TGGGGGTTGA GGCTAAGCCG A-3 ',
cDNA：5’- TTGTGCTATC GGCTTAC-3’；
（2）The preparation of functionalization gold nanoparticle：All glass apparatus are soaked with chloroazotic acid before preparing gold nanoparticle
30min, is then cleaned with distilled water, and ultra-pure water immersion 12h, is dried for standby；The gold chloride of 100mL mass concentrations 0.01% is added
Enter in 250mL round-bottomed flasks, be stirred and heated to boiling, rapidly join the lemon of 3.5mL mass concentrations 1% with vigorous stirring
Sour trisodium, solution becomes claret after continuing heating stirring 15min, stops heating, continues to stand after stirring 30min, and room temperature is certainly
So cooling, obtains the solution of gold nanoparticles of particle diameter 13nm, 4 DEG C save backup；
Above-mentioned solution of gold nanoparticles is continued to heat and be stirred continuously under the conditions of boiling water bath, makes moisture evaporation, until flask
Interior liquor capacity is to boiling water bath is stopped after 20mL, and room temperature natural cooling, the gold nanoparticle concentrated, 4 DEG C save backup；
By the three of 15 μ L 1mmol/L（2- carboxyethyls）Phosphine TCEP is mixed with the DNA1 of 30 100 μm of ol/L of μ L, and it is anti-to carry out sulfhydrylation
Should, it is then added in the gold nanoparticle of 1mL concentrations, stirs 1h at room temperature；Then the triphosphoric acid of 30 μ L 15mmol/L is taken off
Oxygen adenosine dATP is added in above-mentioned solution, and 35min is stirred at room temperature；The NaCl solution of 20 μ L 1mol/L is added after the completion of concussion
Room temperature places 30min, and 4 DEG C preserve 6h to increase the stability of combination；4 DEG C, 8000r/min centrifugations 10min remove unnecessary examination
Agent；Be resuspended in 1mL containing 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100 20mmol/L Na3PO4
Obtain functionalization gold nanoparticle AuNPs-DNA1 in buffer solution, 4 DEG C be kept in dark place it is spare；
Wherein, the sequence of DNA1 is：5’-ATAGCACAAC CGTC-(CH2)6- SH-3 ', with cDNA and capDNA2 parts mutually
Mend；
（3）Sample pad, gold-labelled pad pretreatment：Gold-labelled pad and sample pad are cut into after suitable size and contain 1% in pH8.2
NaCl, 0.5% Tween-20,1% BSA, 2% sucrose, 1% TritonX-100 0.1 mol/L Tris-HCl buffer solutions in it is complete
Complete immersion 30min, is then put into after being dried in 37 DEG C of thermostatic drying chambers, and drying condition preserves, is spare；
Functionalization gold nanoparticle AuNPs-DNA1 is added dropwise in the gold-labelled pad handled well, is dried in 37 DEG C of thermostatic drying chambers, does
Dry condition preserves, spare；
（4）Nitrocellulose filter pre-processes：First, by biotinylated capDNA1 and capDNA2 respectively with Streptavidin
Streptavidin is combined；By 10 μ L 1mg/mL Streptavidins respectively with 20 μ L, 10 μm of biotinylated capDNA1 of ol/L
And capDNA2 combines 2h under conditions of 4 DEG C after mixing, make Streptavidin and biotinylated capDNA1 and
CapDNA2 is fully coupled；
The Streptavidin-biotin-capDNA1 that 1 μ L are combined is taken to draw detection line T lines at the 6mm of gold-labelled pad one end；
1 μ L Streptavidin-biotin-capDNA2 are taken to draw nature controlling line C line at the 6mm of water absorption pad one end；T, C line will be pulled
Nitrocellulose filter dried at 37 DEG C, 4 DEG C save backup；
（5）The assembling of test strips：The composition material of test strips mainly includes PVC bottom plates（5）, sample pad（1）, gold-labelled pad（2）, nitre
Acid cellulose film（3）, water absorption pad（4）Five parts, on the correspondence position that nitrocellulose filter is first pasted to PVC bottom plates, light pressure,
The two is combined closely, processed sample pad, gold-labelled pad be respectively adhered on the correspondence position of PVC bottom plates, sample pad with
Gold-labelled pad, gold-labelled pad and each overlapping 2 mm of nitrocellulose filter, finally paste water absorption pad 2mm overlapping with nitrocellulose filter
On PVC bottom plates, redundance is dismissed；Each several part firm pasting is simultaneously cut into test strips of the specification for the mm of 60 mm × 4, drying condition
Preserve, it is spare.
5. the kanamycins based on aptamer and functionalized magnetic microsphere separation and culture described in claim 1 quickly detects
The application of test paper, it is characterised in that：By the PBS buffer of functionalized magnetic microsphere MBs-Apt-cDNA 10mmol/L, pH7.4
After cleaning 3 times, combination buffer is resuspended in, adds sample to be tested thereto, 10 min of concussion make functionalized magnetic microsphere surface
Apt fully combined with kanamycins contained in sample, by the cDNA being complementary to displacements into solution；
The combination buffer is specially EDTA containing 1mmol/L, 50mmol/L NaCl, the 50mmol/ of 0.05% Tween-20
L, the Tris-HCl buffer solutions of pH7.5；
Magnetic microsphere is adsorbed under magneticaction, takes the supernatant containing cDNA as ELISA test strip liquid；The test paper that will be prepared
Bar is placed in test strips cartridge, and solution to be detected is added dropwise in well；
When detect kanamycins is not present in liquid when, T lines do not develop the color, C lines colour developing；
When detecting there are during kanamycins in liquid, T, C line develop the color；10 min are stood, after test strips colour developing completely, pass through meat
Eye observation detection line shade can qualitative or semi-quantitative analysis, or test strips are placed in colloidal gold quantitative instrument and are scanned inspection
Survey, obtain the peak area value of detection line and nature controlling line, kanamycins concentration in detection liquid is quantitative determined out according to standard curve.
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