CN106990237B - A kind of kanamycins quick detection test paper and its preparation method and application based on aptamer - Google Patents
A kind of kanamycins quick detection test paper and its preparation method and application based on aptamer Download PDFInfo
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Abstract
A kind of kanamycins quick detection test paper and its preparation method and application based on aptamer, belongs to analytical chemistry and medicine, environment and field of detection of food safety.The Test paper includes sample pad, gold-labelled pad, nitrocellulose filter, water absorption pad, PVC bottom plates, detection line and nature controlling line；This method is using the easily prepared modification of gold, silver nano-particle and the conformation change of signal dual amplification and aptamers being realized to, the strong and weak of optical signalling of detection line changes, and then realizes the detection to kanamycins；Test paper method detects kanamycins simplicity quickly, can detect whenever and wherever possible, and result visualization, detection time is short, test strips sample pad one end need to be only inserted into detection liquid, experimental result is can be obtained in 10min, detection efficiency can be greatly improved；Quantitative analysis can be carried out by colloidal gold quantitative instrument reading, is of great significance to yapamicin relict detection in animal derived food.
Description
Technical field
The kanamycins quick detection test paper and its preparation method and application based on aptamer that the present invention relates to a kind of,
Belong to analytical chemistry and medicine, environment and field of detection of food safety.
Background technology
Kanamycins（kanamycin）It is a kind of broad spectrum type aminoglycoside antibiotics, molecular formula C18H36N4O11, point
Son amount is 582.58, can effectively be treated by the infection caused by Gram-positive or gramnegative bacterium.However, excessively making
Contain the animal derived food of kanamycins with kanamycins or overconsumption, can cause kanamycins by food chain in human body
Interior accumulation, and some quite serious side effects may be caused, cochlea nerve is such as influenced, ear toxication is induced, causes permanently
Hearing loss；Certain influence may also be generated to neuromuscular and hemopoietic system simultaneously；In addition, kanamycins can also cause
Renal failure occurs for the damage to kidney.Therefore, one kind should be established as early as possible to can be used in detecting yapamicin relict in food
Effective ways, further increase the safety of animal derived food.
A variety of methods for detecting yapamicin relict, such as enzyme linked immunosorbent assay (ELISA) are worked out now
（ELISA）, Capillary Electrophoresis（CZE）, high efficiency liquid chromatography（HPLC）, surface plasma body resonant vibration（SPR）And electrochemical method
Deng.However, the application of the above method is still partly limited, such as time-consuming, needs large-scale instrument and equipment and consumption relatively great amount of
Reagent, it is of high cost, be easily disturbed the influence of object, be more demanding to operating personnel, being not suitable for field application etc..Therefore, one is established
Kind kanamycins is simple, and quickly, the method for economy and super sensitivity detection is particularly significant, and the development of Test paper is quickly detection
Provide possibility.
Invention content
The purpose of the present invention is overcoming above-mentioned shortcoming, a kind of kanamycins based on aptamer is provided and is quickly examined
Test paper and its preparation method and application, by colloid gold label chromatograph test strip technology, nano meter biomaterial constructing technology and core
The advantage of many technologies such as sour aptamers is joined together, prepare it is a kind of for easy, quick, at low cost, high sensitivity card that
The remaining Test paper of mycin is simultaneously applied to sample detection.
Technical scheme of the present invention：A kind of kanamycins quick detection test paper based on aptamer, including sample pad,
Gold-labelled pad, nitrocellulose filter, water absorption pad, PVC bottom plates, detection line and nature controlling line；Sample is pasted successively on the PVC bottom plates
Pad, gold-labelled pad, nitrocellulose filter, water absorption pad, wherein sample pad overlap gold-labelled pad, and nitrocellulose filter is directly arranged at PVC
On bottom plate, nitrocellulose filter one end overlaps gold-labelled pad, and the nitrocellulose filter other end overlaps water absorption pad；The nitrocellulose
Detection line and nature controlling line are additionally provided on film successively.
The sample pad, gold-labelled pad, nitrocellulose filter, water absorption pad, each clinch overlay segment are 2mm.
The preparation process of the gold-labelled pad is：Gold, silver nano-particle is prepared respectively and respectively by Au-S keys and Ag-S keys
Covalent effect Apt, DNA1 and gold, silver nano particle are coupled to the nano-particle to form functionalization；
Wherein Apt and DNA1 sequences are as follows：
Aptamers Apt：5'-HS-(CH2)6-TGGGGGTTGA GGCTAAGCCG A-Biotin-3'；
DNA1：5'-HS-(CH2)6-TCAGTCGGCT TAGCCGTCCA ACGTCAGATC C-3’。
One end Streptavidin and biotin being incubated in advance of the gold-labelled pad close on nitrocellulose filter
Modifying DNA 2 draws detection line, and a nature controlling line is being drawn with Streptavidin close to water absorption pad one end；
Wherein DNA2：5'-biotin-CCGATGGATC TGACGT-3' .
The preparation method of the kanamycins quick detection test paper based on aptamer, steps are as follows：Jenner's grain of rice
The preparation and functionalization of son, the preparation of Nano silver grain and functionalization, the pretreatment of gold-labelled pad and sample pad, nitrocellulose filter
Pretreatment, the assemblings of test strips, test strip develop the color situation；The specific steps are：
（1）The preparation of gold nanoparticle and functionalization：
All glass apparatus impregnate 30min with chloroazotic acid before preparing gold nanoparticle, are then cleaned with distilled water, surpass
Pure water impregnates 12h, is dried for standby；The gold chloride of 100mL mass concentrations 0.01% is added in 250mL round-bottomed flasks, stirring is simultaneously
It is heated to boiling, rapidly joins the trisodium citrate of 3.5mL mass concentrations 1% with vigorous stirring, continue heating stirring 15min
Solution becomes claret afterwards, stops heating, continues to stand after stirring 30min, room temperature natural cooling obtains the Jenner of grain size 13nm
Rice corpuscles solution, 4 DEG C save backup；
Above-mentioned solution of gold nanoparticles is continued to heat and is stirred continuously under the conditions of boiling water bath, makes moisture evaporation, until
Solution in flask stops boiling water bath after being 20mL, and room temperature natural cooling, the gold nanoparticle concentrated, 4 DEG C save backup；
The aptamers Apt of 100 μm of ol/L of three (2- carboxyethyls) phosphine TCEP and 30 μ L of 15 μ L 1mmol/L is mixed,
Mercaptolation is carried out, is then added in the gold nanoparticle of 1mL concentrations, stirs 1h at room temperature；Then by 30 μ L 15
The deoxyadenosine triphosphate dATP of mmol/L is added in above-mentioned solution, and 35min is stirred at room temperature；20 μ L are added after the completion of concussion
The NaCl solution of 1mol/L is placed at room temperature for 30min, and 4 DEG C preserve 6h to increase the stability of combination；4 DEG C, 8000r/min centrifugations
10min removes extra reagent；1mL is resuspended in containing 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100
20 mmol/L Na3PO4In buffer solution, 4 DEG C be kept in dark place it is spare；
（2）The preparation of Nano silver grain and its functionalization：
The silver nitrate of 0.2mmol/L, the NaTDC of 2.0mmol/L and the sodium borohydride of 20mmol/L are prepared respectively,
Silver nitrate and sodium deoxycholate solution are aged 24 h with the molar ratio of 1 ︰ 2 under conditions of pH7；Solution after ageing is added
Into round-bottomed flask, the sodium borohydride that Fresh is added dropwise dropwise in the case where condition of ice bath is vigorously stirred effect reacts 10 min, instead
Should after obtain yellow colloidal silver solution, 4 DEG C be kept in dark place it is spare；
Take 1mL colloidal silver solutions in the centrifuge tube of 1.5 mL, centrifuging 10 min under the conditions of 4 DEG C, 12000 r/min goes
Except supernatant, with 20 mmol/Ls of the 200 μ L containing 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100
Na3PO4Buffer solution can be obtained the colloidal silver solution of 5 times of concentrations after being resuspended.
The TCEP of 15 μ L 1mmol/L is mixed with the DNA1 of 20 100 μm of ol/L of μ L, carries out mercaptolation, then
It is added to the colloidal silver solution of 5 times of concentrations of 1mL, stirs 1h at room temperature；Then the dATP of 30 μ L 15mmol/L is added to
It states in solution, 35min is stirred at room temperature；The NaCl solution that 20 μ L 1mol/L are added dropwise after the completion of concussion dropwise is placed at room temperature for 30min, and 4
DEG C preserving 6h increases the stability of combination；4 DEG C, 8000 r/min centrifuge 10 min remove extra reagent, be resuspended in 1mL and contain
0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100 20 mmol/L Na3PO4Buffer solution, 4 DEG C are protected from light
It saves backup；
（3）Sample pad, gold-labelled pad pretreatment：
Gold-labelled pad and sample pad are cut into after suitable size containing 1% NaCl, 0.5% Tween-20,1% BSA, 2%
Sucrose is completely soaked 30min in 0.1 mol/L Tris-HCl (pH 8.2) buffer solution of 1% TritonX-100, then puts
To after being dried in 37 DEG C of thermostatic drying chambers, drying condition preserves, is spare；
Functionalization gold, silver nano-particle, i.e. AuNPs-Apt and AgNPs-DNA1 are added dropwise in the gold-labelled pad handled well,
Middle DNA1 and Apt partial complementarities are dried in 37 DEG C of thermostatic drying chambers, and drying condition preserves, spare；
（4）Nitrocellulose filter pre-processes：
By the DNA2 of Streptavidin and biotin modification with the molar ratio of 1 ︰ 4 after mixing under conditions of 4 DEG C
2h is placed, the biotin modified on Streptavidin and DNA2 is made fully to combine；Take the 1 μ L of SA-biotin-DNA2 being combined
On nitrocellulose filter detection line T lines are drawn close to gold-labelled pad one end；1 μ L Streptavidins SA is taken to be leaned on nitrocellulose filter
Nature controlling line C line is drawn in nearly water absorption pad one end, the nitrocellulose filter for pulling T, C line is dried 0.5h at 37 DEG C, under drying condition
It saves backup；
（5）The assembling of test strips：The composition material of colloid gold chromatographic test paper strip includes mainly PVC bottom plates, sample pad, Jin Biao
Pad, nitrocellulose filter, five part of water absorption pad, it is light to press on the corresponding position that nitrocellulose filter is first pasted to PVC bottom plates,
Both make to combine closely, processed sample pad, gold-labelled pad be respectively adhered on the corresponding position of PVC bottom plates, sample pad with
Gold-labelled pad, each be overlapped 2mm of gold-labelled pad and nitrocellulose filter, finally pastes water absorption pad 2mm Chong Die with nitrocellulose filter
On PVC bottom plates, redundance is dismissed；Then each section firm pasting is cut into the test strips that specification is the mm of 65mm × 4, dried strip
Part preserves, spare.
The detection method of the kanamycins quick detection test paper based on aptamer, the test paper that will be prepared first
Item is placed in test strips cartridge, and solution to be detected is added dropwise after in sample pad in well, and liquid is with capillarity to suction
Water cushion moves, and liquid is by the detection line and nature controlling line on nitrocellulose filter；After test strips colour developing completely, T is visually observed
The line color depth can qualitative or semi-quantitative analysis, or test strips are placed in colloidal gold quantitative instrument and are scanned, obtain detection line
With the peak area value of nature controlling line, the kanamycins concentration of detection liquid is quantitative determined out according to standard curve.
It is as follows：Detection liquid is added dropwise in sample pad one end in the test strips prepared, due to the siphon of capillary
Effect detection liquid constantly can move through nitrocellulose filter from sample pad to water absorption pad, assemble Jenner in the position of T lines and C lines
Rice corpuscles on nitrocellulose filter so that form two red stripes；
When detect object kanamycins is not present in liquid when, the DNA2 on T lines can hybridize with DNA1 and DNA1 and the portions Apt
Point complementation, therefore the gold nanoparticle of functionalization can be captured on T lines and developed the color, the Streptavidin on C lines can basis
Streptomysin-biotin effect makes gold nanoparticle assemble on C lines and develop the color, at this point, T, C line develop the color；
When detecting there are when kanamycins in liquid, Apt can change its space conformation and specifically be combined with kanamycins, and
It is no longer combined with DNA1, AuNPs-Apt can be set to change, and so that the colour developing of T lines is shoaled or is not developed the color, Apt is combined with kanamycins
Its 3 ' end biotin is still exposed to outer afterwards, therefore can be captured by the Streptavidin on C lines to develop the color, at this point, T lines are aobvious
Color it is shallower or it is complete fade, the colour developing of C lines it is normal；
Test strips are finally placed in colloidal gold quantitative instrument to the colored intensity for detecting its T, C line, it can be according to the colour developing of T lines
Situation judges the concentration of detected kanamycins.
Beneficial effects of the present invention：1. this method utilizes the easily prepared modification of gold, silver nano-particle and can be by signal
The conformation change of dual amplification and aptamers realizes the strong and weak variation of the optical signalling of detection line, and then realizes to kanamycins
Detection；2. test paper method detects, kanamycins is easy can quickly to be detected whenever and wherever possible, and result visualization, detection time is short, uses
This method need to only be inserted into test strips sample pad one end in detection liquid, experimental result is can be obtained in 10min, can be substantially
The raising detection efficiency of degree；3. quantitative analysis can be carried out by colloidal gold quantitative instrument reading, it is mould to blocking that in animal derived food
Plain residue detection is of great significance.
Description of the drawings
Fig. 1 test strips assembling figures of the present invention.1, sample pad；2, gold-labelled pad；3, nitrocellulose filter；4, water absorption pad；5,
PVC bottom plates；6, detection line；7, nature controlling line.
Kanamycins colloidal gold fast detecting test paper detection negative schematic diagrams of the Fig. 2 based on aptamer.
Kanamycins colloidal gold fast detecting test paper detection positive schematic diagrams of the Fig. 3 based on aptamer.
Detection figure of Fig. 4 test strips to various concentration kanamycins standard solution.
Fig. 5 is in standard detection liquid, the canonical plotting of T lines peak area and kanamycins concentration.
Detection figure of Fig. 6 test strips to the milk sample of the kanamycins containing various concentration.
Specific implementation mode
A kind of kanamycins quick detection test paper based on aptamer of embodiment 1
The preparation of gold nanoparticle and functionalization, the preparation of Nano silver grain and functionalization, gold-labelled pad and sample pad it is pre-
Processing, the pretreatment of nitrocellulose filter, the assembling of test strips, test strip colour developing situation.The specific steps are：
（1）The preparation of gold nanoparticle and functionalization：
All glass apparatus impregnate 30min with chloroazotic acid before preparing gold nanoparticle, are then cleaned with distilled water, surpass
Pure water impregnates 12h, is dried for standby；The gold chloride of 100mL 0.01% is added in 250mL round-bottomed flasks, boiling is stirred and heated to
It rises, rapidly joins the trisodium citrate of 3.5mL 1% with vigorous stirring, continuing solution after heating stirring 15min, to become wine red
Color stops heating, continues to stand after stirring 30min, room temperature natural cooling obtains the solution of gold nanoparticles of grain size 13nm, 4 DEG C
It saves backup；
Above-mentioned solution of gold nanoparticles is continued to heat and is stirred continuously under the conditions of boiling water bath, makes moisture evaporation, until
Solution in flask stops boiling water bath after being 20mL, and room temperature natural cooling, the gold nanoparticle concentrated, 4 DEG C save backup；
The aptamers Apt of 100 μm of ol/L of three (2- carboxyethyls) phosphine TCEP and 30 μ L of 15 μ L 1mmol/L is mixed,
Mercaptolation is carried out, is then added in the gold nanoparticle of 1mL concentrations, stirs 1h at room temperature；Then by 30 μ L 15
The deoxyadenosine triphosphate dATP of mmol/L is added in above-mentioned solution, and 35min is stirred at room temperature；20 μ L are added after the completion of concussion
The NaCl solution of 1mol/L is placed at room temperature for 30min, and 4 DEG C preserve 6h to increase the stability of combination；4 DEG C, 8000r/min centrifugations
10min removes extra reagent；It is resuspended in 1mL and contains 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100
20 mmol/L Na3PO4In buffer solution, 4 DEG C be kept in dark place it is spare；
（2）The preparation of Nano silver grain and its functionalization：
The silver nitrate of 0.2mmol/L, the NaTDC of 2.0mmol/L and the sodium borohydride of 20mmol/L are prepared respectively,
Silver nitrate and sodium deoxycholate solution are aged 24 h with the molar ratio of 1 ︰ 2 under conditions of pH7；Solution after ageing is added
Into round-bottomed flask, the sodium borohydride that Fresh is added dropwise dropwise in the case where condition of ice bath is vigorously stirred effect reacts 10 min, instead
Should after obtain yellow colloidal silver solution, 4 DEG C be kept in dark place it is spare；
Take 1mL colloidal silver solutions in the centrifuge tube of 1.5 mL, centrifuging 10 min under the conditions of 4 DEG C, 12000 r/min goes
Except supernatant, contain 0.5% Tween-20 with 200 μ L, 5% BSA, 2% sucrose, 20 mmol/L's of 0.5% TritonX-100
Na3PO4Buffer solution can be obtained the colloidal silver solution of 5 times of concentrations after being resuspended.
The TCEP of 15 μ L 1mmol/L is mixed with the DNA1 of 20 100 μm of ol/L of μ L, carries out mercaptolation, then
It is added to the colloidal silver solution of 5 times of concentrations of 1mL, stirs 1h at room temperature；Then the dATP of 30 μ L 15mmol/L is added to
It states in solution, 35min is stirred at room temperature；The NaCl solution that 20 μ L 1mol/L are added dropwise after the completion of concussion dropwise is placed at room temperature for 30min, and 4
DEG C preserving 6h increases the stability of combination；4 DEG C, 8000 r/min centrifuge 10 min remove extra reagent, be resuspended in 1mL and contain
0.5% Tween-20,5% BSA, 2% sucrose, the Na of 20 mmol/L of 0.5% TritonX-1003PO4Buffer solution, 4 DEG C are protected from light
It saves backup；
（3）Sample pad, gold-labelled pad pretreatment：
Gold-labelled pad and sample pad are cut into after suitable size containing 1% NaCl, 0.5% Tween-20,1% BSA, 2%
Sucrose is completely soaked 30min in 0.1 mol/L Tris-HCl (pH 8.2) buffer solution of 1% TritonX-100, then puts
To after being dried in 37 DEG C of thermostatic drying chambers, drying condition preserves, is spare；
Functionalization gold, silver nano-particle, i.e. AuNPs-Apt and AgNPs-DNA1 are added dropwise in the gold-labelled pad handled well,
Middle DNA1 and Apt partial complementarities are dried in 37 DEG C of thermostatic drying chambers, and drying condition preserves, spare；
（4）Nitrocellulose filter pre-processes：
By the DNA2 of Streptavidin and biotin modification with the molar ratio of 1 ︰ 4 after mixing under conditions of 4 DEG C
2h is placed, the biotin modified on Streptavidin and DNA2 is made fully to combine；Take the 1 μ L of SA-biotin-DNA2 being combined
On nitrocellulose filter detection line T lines are drawn close to gold-labelled pad one end；1 μ L Streptavidins SA is taken to be leaned on nitrocellulose filter
Nature controlling line C line is drawn in nearly water absorption pad one end, the nitrocellulose filter for pulling T, C line is dried 0.5h at 37 DEG C, under drying condition
It saves backup；
（5）The assembling of test strips：The composition material of colloid gold chromatographic test paper strip includes mainly PVC bottom plates, sample pad, Jin Biao
Pad, nitrocellulose filter, five part of water absorption pad, it is light to press on the corresponding position that nitrocellulose filter is first pasted to PVC bottom plates,
Both make to combine closely, processed sample pad, gold-labelled pad be respectively adhered on the corresponding position of PVC bottom plates, sample pad with
Gold-labelled pad, each be overlapped 2mm of gold-labelled pad and nitrocellulose filter, finally pastes water absorption pad 2mm Chong Die with nitrocellulose filter
On PVC bottom plates, redundance is dismissed；Then each section firm pasting is cut into the test strips that specification is the mm of 65mm × 4, dried strip
Part preserves, spare.
Embodiment 2 is with test strips to the measurement of kanamycins standard solution
Kanamycins is diluted to different concentration respectively with 2mL centrifuge tubes, often 100 μ L of pipe.It will prepare as stated above
Good test strips sample pad one end is inserted respectively into various concentration kanamycins standard solution, reacts 10 min at room temperature.From
As can be seen that T line colors do not become substantially between kanamycins concentration 0~1 nmol/L and 35~400 nmol/L in Fig. 4
Change, and T line colors shoal with the increase of kanamycins concentration between 1~35 nmol/L, and examined after 35 nmol/L
Survey line is substantially without color.Thus the test paper is limited to 35 nmol/L to the naked eyes detection of kanamycins.By completely reacted test strips
It is put into the colour developing situation for detecting T, C line in plastic clip with colloidal gold quantitative instrument, obtain detection line peak area as shown in Figure 5 and blocks that
The variation relation curve of mycin concentration.Wherein in 1~30 nmol/L concentration ranges, T lines colored intensity exists linear with concentration
Relationship, equation of linear regression are y=- 44.5115x+2295.2893, R2=0.9830, y is detection line peak area in formula, and x is card
The concentration of that mycin（nmol/L）.
The detection of yapamicin relict in 3 milk sample of embodiment
It is obtained respectively by the way of adding normal concentration kanamycins into milk and preparing artificial contamination's milk herein and contains 5
Nmol/L, 10 nmol/L, 20 nmol/L, 30 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L kanamycins
Milk sample.20% trichloroacetic acid is added dropwise into milk sample, adjusts 4.6,45 DEG C of 10 min of water-bath of pH and precipitates junket egg
In vain, the protein and fat that 25 min remove condensation are centrifuged with the rotating speed of 10000 r/min, with 0.22 μm of membrane filtration, most
PH is adjusted again afterwards to neutrality, obtains milk sample after pretreatment.The card containing various concentration is detected by aforesaid operations step
The milk sample of that mycin.Obtain the ELISA test strip figure of the milk sample of the kanamycins containing various concentration shown in Fig. 6.In Fig. 6
In it can be seen that with kanamycins concentration increase, the lighter of T lines, T lines after kanamycins reaches 100 nmol/L
Substantially without color, therefore we set 100 nmol/L and are examined as test strips to the visualization of kanamycins in milk sample
Survey lower limit.Experiments have shown that the detection method can detect the kanamycins by pretreated milk mesostroma.
Claims (3)
1. a kind of preparation method of the kanamycins quick detection test paper based on aptamer, it is characterized in that steps are as follows：Gold
The preparation of nano-particle and functionalization, the preparation of Nano silver grain and functionalization, the pretreatment of gold-labelled pad and sample pad, nitric acid are fine
Tie up the pretreatment of film, the assembling of test strips, test strip colour developing situation；The specific steps are：
（1）The preparation of gold nanoparticle and functionalization：
All glass apparatus impregnate 30min with chloroazotic acid before preparing gold nanoparticle, are then cleaned with distilled water, ultra-pure water
12h is impregnated, is dried for standby；The gold chloride of 100mL mass concentrations 0.01% is added in 250mL round-bottomed flasks, stirs and heats
To boiling, the trisodium citrate of 3.5mL mass concentrations 1% is rapidly joined with vigorous stirring, continue molten after heating stirring 15min
Liquid becomes claret, stops heating, continues to stand after stirring 30min, room temperature natural cooling obtains Jenner's grain of rice of grain size 13nm
Sub- solution, 4 DEG C save backup；
Above-mentioned solution of gold nanoparticles is continued to heat and is stirred continuously under the conditions of boiling water bath, makes moisture evaporation, until flask
Interior solution stops boiling water bath after being 20mL, and room temperature natural cooling, the gold nanoparticle concentrated, 4 DEG C save backup；
The aptamers Apt of 100 μm of ol/L of three (2- carboxyethyls) phosphine TCEP and 30 μ L of 15 μ L 1mmol/L is mixed, is carried out
Mercaptolation is then added in the gold nanoparticle of 1mL concentrations, stirs 1h at room temperature；Then by 30 μ L, 15 mmol/L
Deoxyadenosine triphosphate dATP be added in above-mentioned solution, 35min is stirred at room temperature；20 μ L 1mol/L are added after the completion of concussion
NaCl solution be placed at room temperature for 30min, 4 DEG C preserve 6h to increase the stability of combination；4 DEG C, 8000r/min centrifugation 10min go
Except extra reagent；Be resuspended in 1mL containing 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100 20
The Na of mmol/L3PO4In buffer solution, 4 DEG C be kept in dark place it is spare；
（2）The preparation of Nano silver grain and its functionalization：
The silver nitrate of 0.2mmol/L, the NaTDC of 2.0mmol/L and the sodium borohydride of 20mmol/L are prepared respectively, by nitre
Sour silver and sodium deoxycholate solution are aged 24 h with the molar ratio of 1 ︰ 2 under conditions of pH7；Solution after ageing is added to circle
In the flask of bottom, the sodium borohydride that Fresh is added dropwise dropwise in the case where condition of ice bath is vigorously stirred effect reacts 10 min, after reaction
Obtain yellow colloidal silver solution, 4 DEG C be kept in dark place it is spare；
It takes 1mL colloidal silver solutions in the centrifuge tube of 1.5 mL, is centrifuged under the conditions of 4 DEG C, 12000 r/min in 10 min removals
Clearly, with 200 μ L containing 0.5% Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100 20 mmol/L Na3PO4
Buffer solution can be obtained the colloidal silver solution of 5 times of concentrations after being resuspended；
The TCEP of 15 μ L 1mmol/L is mixed with the DNA1 of 20 100 μm of ol/L of μ L, mercaptolation is carried out, is then added
To the colloidal silver solution of 5 times of concentrations of 1mL, 1h is stirred at room temperature；Then the dATP of 30 μ L 15mmol/L is added to above-mentioned molten
In liquid, 35min is stirred at room temperature；The NaCl solution that 20 μ L 1mol/L are added dropwise after the completion of concussion dropwise is placed at room temperature for 30min, 4 DEG C of guarantors
6h is deposited to increase the stability of combination；4 DEG C, 8000 r/min centrifuge 10 min remove extra reagent, be resuspended in 1mL contain 0.5%
Tween-20,5% BSA, 2% sucrose, 0.5% TritonX-100 20 mmol/L Na3PO4Buffer solution, 4 DEG C are 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 30min, be then put into 37 DEG C of perseverances
After being dried in warm drying box, drying condition preserves, is spare；
Functionalization gold, silver nano-particle, i.e. AuNPs-Apt and AgNPs-DNA1 are added dropwise in the gold-labelled pad handled well, wherein
DNA1 and Apt partial complementarities are dried in 37 DEG C of thermostatic drying chambers, and drying condition preserves, spare；
Wherein Apt and DNA1 sequences are as follows：
Aptamers Apt：5'-HS-(CH2)6-TGGGGGTTGA GGCTAAGCCG A-Biotin-3'；
DNA1：5'-HS-(CH2)6-TCAGTCGGCT TAGCCGTCCA ACGTCAGATC C-3'；
（4）Nitrocellulose filter pre-processes：
The DNA2 of Streptavidin and biotin modification is placed under conditions of 4 DEG C after mixing with the molar ratio of 1 ︰ 4
2h makes the biotin modified on Streptavidin and DNA2 fully combine；Take the 1 μ L of SA-biotin-DNA2 being combined in nitre
On acid cellulose film detection line T lines are drawn close to gold-labelled pad one end；Take 1 μ L Streptavidins SA on nitrocellulose filter close to suction
Nature controlling line C line is drawn in water cushion one end, and the nitrocellulose filter for pulling T, C line is dried 0.5h at 37 DEG C, is preserved under drying condition
It is spare；
（5）The assembling of test strips：The composition material of colloid gold chromatographic test paper strip mainly include PVC bottom plates, sample pad, gold-labelled pad,
Nitrocellulose filter, five part of water absorption pad, it is light to press on the corresponding position that nitrocellulose filter is first pasted to PVC bottom plates, make two
Person combines closely, and processed sample pad, gold-labelled pad are respectively adhered on the corresponding position of PVC bottom plates, and sample pad is marked with gold
Pad, each be overlapped 2mm of gold-labelled pad and nitrocellulose filter, finally pastes the bottoms PVC by water absorption pad 2mm Chong Die with nitrocellulose filter
On plate, redundance is dismissed；Then each section firm pasting, is cut into the test strips that specification is the mm of 65mm × 4, and drying condition is protected
It deposits, it is spare；
Test strips after assembling include sample pad（1）, gold-labelled pad（2）, nitrocellulose filter（3）, water absorption pad（4）, PVC bottom plates
（5）, detection line（6）And nature controlling line（7）；The PVC bottom plates（5）On paste sample pad successively（1）, gold-labelled pad（2）, cellulose nitrate
Plain film（3）, 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, nitrocellulose filter（3）One end overlaps gold-labelled pad（2）, nitrocellulose filter（3）The other end overlaps water absorption pad（4）；Institute
State nitrocellulose filter（3）On be additionally provided with detection line successively（6）And nature controlling line（7）；
The sample pad（1）, gold-labelled pad（2）, nitrocellulose filter（3）, water absorption pad（4）, each clinch overlay segment be 2mm；
It is described in nitrocellulose filter（3）Upper close gold-labelled pad（2）One end Streptavidin and biology being incubated in advance
Plain modifying DNA 2 draws detection line（6）, close to water absorption pad（4）Nature controlling line is drawn with Streptavidin in one end（7）；
Wherein DNA2：5'-biotin-CCGATGGATC TGACGT-3' .
2. with the detection side of the kanamycins quick detection test paper based on aptamer prepared by claim 1 the method
Method, it is characterised in that：The test strips prepared are placed in test strips cartridge first, well be added dropwise solution to be detected in
After in sample pad, liquid is moved with capillarity to water absorption pad, and liquid passes through detection line and Quality Control on nitrocellulose filter
Line；After test strips colour developing completely, visually observe the T line colors depth can qualitative or semi-quantitative analysis, or test strips are placed in glue
It is scanned in body gold quantitative instrument, obtains the peak area value of detection line and nature controlling line, detection is quantitative determined out according to standard curve
The kanamycins concentration of liquid.
3. the detection method of the kanamycins quick detection test paper based on aptamer, feature exist as claimed in claim 2
In being as follows：Detection liquid is added dropwise in sample pad one end in test strips in cartridge, since the siphonage of capillary is examined
Survey liquid constantly can move through nitrocellulose filter from sample pad to water absorption pad；
When detecting there is no when object kanamycins in liquid, the DNA2 on T lines can hybridize with DNA1 and the parts DNA1 and Apt are mutual
It mends, therefore the gold nanoparticle of functionalization can be captured on T lines and developed the color, the Streptavidin on C lines is according to streptomysin-life
The effect of object element makes gold nanoparticle assemble on C lines and develop the color, at this point, T, C line develop the color；
When detecting there are when kanamycins in liquid, Apt can change its space conformation and specifically be combined with kanamycins, and no longer
Combined with DNA1, AuNPs-Apt can be set to change, make T lines colour developing shoal or do not develop the color, after Apt is combined with kanamycins its
3 ' end biotins are still exposed to outer, therefore can be captured by the Streptavidin on C lines to develop the color, at this point, the colour developing of T lines compared with
Shallow or complete colour fading, the colour developing of C lines are normal；
Test strips are finally placed in colloidal gold quantitative instrument to the colored intensity for detecting its T, C line, are sentenced according to the colour developing situation of T lines
The concentration of disconnected detected kanamycins.
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| CN108196048A (en) * | 2017-09-28 | 2018-06-22 | 浙江省产品质量安全检测研究院 | A kind of bisphenol-A fluorescence detection test strip, preparation method and application |
| CN108008128B (en) * | 2017-12-21 | 2020-03-31 | 江南大学 | Kanamycin rapid detection test paper for nucleic acid aptamer recognition and functional magnetic microsphere separation pretreatment, and preparation and application thereof |
| CN108303415B (en) * | 2018-01-18 | 2020-10-27 | 北京勤邦生物技术有限公司 | Aptamer test strip for detecting aflatoxin B1, and preparation method and application thereof |
| CN110320356B (en) * | 2018-03-29 | 2021-10-12 | 南京农业大学 | Colorimetric detection method for multiple antibiotic residues in milk |
| CN108802014B (en) * | 2018-06-08 | 2019-07-12 | 西安交通大学 | Effluent test paper detection device and preparation method thereof |
| CN108956743B (en) * | 2018-07-24 | 2021-01-08 | 哈尔滨工程大学 | Detection method of field effect transistor biosensor enhanced by AuNPs |
| CN112180083A (en) * | 2020-09-30 | 2021-01-05 | 厦门稀土材料研究所 | IgM antibody detection kit, detection card thereof and preparation method of detection card |
| CN113341151B (en) * | 2021-03-19 | 2023-07-14 | 合肥工业大学 | Detection card for crustacean allergen and preparation method and application thereof |
| CN113406329B (en) * | 2021-06-15 | 2024-05-28 | 江南大学 | Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances |
| CN113820482A (en) * | 2021-09-03 | 2021-12-21 | 大连理工大学 | Paper-based device for colorimetric detection of kanamycin through protease controlled release and preparation method and application thereof |
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