CN106731007A - A kind of bisphenol-A aptamers affinity column and its production and use - Google Patents
A kind of bisphenol-A aptamers affinity column and its production and use Download PDFInfo
- Publication number
- CN106731007A CN106731007A CN201710048805.7A CN201710048805A CN106731007A CN 106731007 A CN106731007 A CN 106731007A CN 201710048805 A CN201710048805 A CN 201710048805A CN 106731007 A CN106731007 A CN 106731007A
- Authority
- CN
- China
- Prior art keywords
- aptamers
- bisphenol
- carrier
- affinity column
- gel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The present invention relates to a kind of bisphenol-A aptamers affinity column and its production and use.Then the affinity purification post carries out covalent coupling using the solid phase carrier of chemical modification with the aptamer and carrier of bisphenol-A.Then affinity column is loaded.The aptamers affinity column is mainly useful for the purifying of the bisphenol-A in water, former milk, milk powder and other various samples, in order to the later stage to the high performance liquid chromatography of bisphenol-A in sample（HPLC）Detection and fluoroscopic examination.
Description
Technical field
Aptamers affinity column the present invention relates to a kind of bisphenol-A and its production and use.Category food safety detection neck
Domain.
Background technology
Bisphenol-A is 2,2- double（4- hydroxy phenyls）Propane is commonly called as, also known as diphenol propane, by 2 molecule phenol and 1
The aldehydes matter of molecule acetone catalyzing and condensing in acid medium.It is production makrolon（Polycarbonate, PC）
And epoxy resin（EpoxyResin, EP）Primary raw material, phenolic resin, plasticity polyester, antioxidant and polychlorostyrene are can be used as again
Ethene（PVC）Stabilizer etc..In terms of packaging for foodstuff, adding the plastics of BPA has water white transparency, durable, light and anti-
The characteristic such as fall, have important purposes at aspects such as packaging material for food, container inner wall coating, especially with baby bottles, metal can
The undercoating of container generally existing the most.
During bisphenol-A can move to food or beverage by packaging for foodstuff and plastic sheeting, it is absorbed by the body, and then by
Step is detrimental to health, and can make internal system, immune system, the nervous system appearance exception of the mankind and wild animal, can also
The reproduction heredity function of severe jamming human and animal.Research report, bisphenol-A has the characteristic of female hormone, to liver, kidney
Dirty, mammary gland has certain damage;Bisphenol-A is also relevant with obesity, diabetes and male infertility simultaneously, and research is confirmed, low
The bisphenol-A (10 of dosage -7Mol/L) sertoli cell and spermatoblast can be caused dead.The harm of bisphenol-A has caused all
Many researchers and the extensive concern of the World Health Organization, many countries have promulgated relevant law, forbid in packaging for foodstuff material
Material of the addition containing bisphenol-A, especially articles for babies in material.2014, EFSA was proposed current bisphenol-A
Daily maximum intake is reduced to 0.005 mg/(kgd) by 0.05 mg/(kgd).1994, China announced
Sanitary standard in contained bisphenol-A in every liter of distilled water be defined as to bisphenol-A consumption be less than 0.05 mg.Although at present
To the limitation of bisphenol-A constantly reducing, but due to managing inadequate specification, people's food often edible in daily life is normal
Often the material using bisphenol-A as additive is used to pack.There are some researches show in some food, dixie cup, blood of human body, Human Urine
Bisphenol-A is can detect that in the samples such as water, feeding bottle in liquid, human saliva, nature.Because bisphenol-A is very harmful, therefore
Set up detection limit it is low, accurately and rapidly bisphenol-A detection method it is particularly important.
The detection method of bisphenol-A include fluorescence method, enzyme linked immunosorbent assay, ultraviolet spectrophotometry, gas-chromatography hair and
High performance liquid chromatography etc..
Ultraviolet spectrophotometry in 278nm wavelength is detected with absworption peak using bisphenol-A, and its advantage is simple to operate
It is convenient.But the method is very big by ambient influnence and solvent effect, and the degree of accuracy is not high, and it is larger to make a variation.
The characteristics of Enzyme-linked Immunosorbent Assay technology law is using antigen and antibody specific combination is detected, with specific high, spirit
Quick property is strong, and the low advantage of testing cost.BPA is a kind of small molecule, it is impossible to be directly used in immune animal, but can be with antibody
Generation specific binding reaction.Competitive type enzyme linked immunosorbent assay analysis method（ELISA）It is exactly using this characteristic.Shi Chunhong [foods
Product industrial technology, 2011（01）, 290-292] etc. establish indirect competitive ELISA detect bisphenol-A method, detection be limited to 1
Ng/mL, the bottled water to PC materials does recovery test, and the rate of recovery is 92.5%~107.7%.But because bisphenol-A is small point
Son, immunogenicity is weak, it is difficult to obtain the antibody of high-affinity.So as to limit the exploitation of the method.
Atomic fluorescence spectrometry is to excite the fluorescence intensity of lower transmitting to carry out the transmitting of quantitative analysis in radiation energy with atom
[Treatment of Industrial Water, 2006,26 such as spectra methods Tang Shuya（3）：74-76 ］ utilize in the acid medium of pH=1, β-
The characteristics of cyclodextrin has humidification to BPA fluorescence intensities, the range of linearity for establishing fluorimetry the method is
0.4-300ug/L, relative standard deviation is 1.3%, and detection is limited to 0.02ug/L.
High performance liquid chromatography (HPLC) has the advantages that the degree of accuracy is high, sensitivity strong, can microdetermination, be at present often
For the method for food small molecular detection.Especially HPLC-MS technology is extensive in the detection of bisphenol-A
Using.But the requirement because of its method to sample small molecular purity is higher, cause that testing cost is high, the cycle is long, cannot meet
Batch samples are quickly the need for screening, so using being restricted.Solid-phase extraction column-the efficient liquid phase for growing up this year
Chromatogram（SPE-HPLC)）Solid phase extraction techniques are combined with high performance liquid chromatography.Make the use of HPLC more extensive.Solid phase extracts
It is a kind of new purification techniques to be taken as, using more and more extensive in Food Safety Analysis detection.It is to carry hydrophobic solid phase
Packed column pipe is prepared from body.When sample extracting solution passes through pillar, separated due to the difference of the polarity of material in sample
Come.Stepwise elution can just obtain purity purpose thing higher.
But due to complicated component in sample, many materials all have similar polarity, cause what SPE obtained to wash
Being temporarily released from one's regular work, usually component is more for thing.The bisphenol-A impurity for causing SPE to obtain is more, influences subsequent HPLC detections.Thus, need
Want a species specific bisphenol-A purification process.
Immune affinity column is to substitute the conventional purification means of solid-phase extraction column, using the affinity interaction of antigen and antibody specific
To separate object.It is the ideal chose for substituting solid-phase extraction column.Immune affinity column-high performance liquid chromatography combination（IAC-HPLC）
It is also the conventional detection technique of current food safety detection.Immune affinity column has simple to operate, specificity advantage high.But
Because antibody obtains difficult, immune affinity column is typically relatively expensive.And it is in itself a kind of protein due to antibody, its activity is received
To the influence of surrounding environment.Preserve improper or misoperation and easily cause antibody inactivation so as to influence the effect of immune affinity column
Rate.Because bisphenol-A is small-molecule substance, immunogenicity is poor, very big for preparing antibody difficulty.Lead to not obtain effective double
Phenol A antibody is used to prepare immune affinity column.
Aptamers are one section of nucleotide sequences, usually DNA or RNA sequence.Single-stranded nucleotide sequence can form two grades
Structure, so as to be specifically bound with target spot.By multiple enrichment and screening, can screen has high-affinity with target spot
Specific aptamers.Herman etc. describes the binding specificity of aptamer, [Science 287,820-825] nucleic acid
Aptamers are more stable for antibody, are not easily susceptible to the influence of environment, and aptamer can be with chemical synthesis, it is ensured that
The accuracy of sequence.[Song. Trends Analy. Chem 2008.27(2)].
Aptamer is applied to the detection of small molecule, in patent CN105505940A, describes a kind of aspergillus flavus
The aptamers DNA sensor of toxin B1.Prepare DNA and pass by the aptamers of AFB1, and a plurality of complementary probe sequences
Sensor, is reacted by zymolyte, realizes the detection of AFB1.In patent CN104399283, describe a kind of yellow bent
The aptamers affinity column of mould toxin B1.The agarose microbeads of the patent utilization epoxy-activated, by the adaptation of AFB1
Body is coupled, and is prepared into affinity column.Realize the separation of AFB1.
The content of the invention
It is an object of the invention to provide a kind of bisphenol-A aptamers affinity column and its production and use
In the present invention, we utilize SELEX systems, and one group of high specific, high-affinity are screened from aptamers library
New bisphenol-A single stranded DNA aptamers.The carrier activated with N-hydroxy-succinamide after aptamers modification is by covalent
Key is coupled.The idiosyncratic carrier of bisphenol-A is obtained by washing and closing.The double of high-affinity are formed after carrier dress post
Phenol A aptamers affinity columns
The affinity column is easy to operate, purifies bisphenol-A efficiency high.Organic solvent is resistant to, and can be reused.Sample passes through
Purifying is can be carried out after simple treatment, purity bisphenol-A very high is obtained.Examined for high performance liquid chromatography detection and mass spectrum
Survey.
Concrete operations are as follows：
Maximum advantage of the invention is exactly the characteristic that make use of aptamer, by steps such as enrichment, washing, the amplifications taken turns more
Suddenly, the specific aptamers for bisphenol-A are filtered out.The sequence of aptamers is obtained by sequencing
Aptamer for antibody, with adapt to environment extensively, be resistant to high temperature, be resistant to organic solvent and
Easy to operate the advantages of.Topmost, aptamers need not move through the process in animal body in preparation process, but by changing
Synthesis is learned to obtain.Therefore, it is with short production cycle, and the accuracy of sequence can be ensured by synthesis condition
The affinity column prepared based on aptamer has specificity good, and bisphenol-A binding capacity is big, the characteristics of purification efficiency is high
Bisphenol-A aptamers affinity column and preparation method thereof is described as follows
1. N-hydroxy-succinamide is selected（NHS）The agarose carrier sepharose4B of modification, is activated
The agarose of 1gN- HOSu NHSs modification is taken, the HCl with 50ml 1mM is added, after swelling 30min, gel is used
100ml 1mM HCl are washed 6 times, then use 100ml milli-Q waters
2. the Ago-Gel sepharose4B coupling buffers that will have been activated（The NaHCO of 0.1M3, 0.8M NaCl,
PH8.2）Washing 3 times.The amido modified bisphenol-A aptamers of 20mol/L, room temperature are added to be coupled 8 hours
3. the phosphate buffer PBS of the bisphenol-A aptamers that will be coupled-agarose carrier 20mM, PH7.4 is washed 3 times
4. close
The Tris.Cl PH8.0 of 100mmol/L, room temperature reaction 2 are added in the bisphenol-A aptamers-agarose carrier that will be coupled
Hour
5. the phosphate buffer PBS of the bisphenol-A aptamers that will have been closed-agarose carrier 20mM, PH7.4 is washed 3 times
6. post is filled
Bisphenol-A-agarose carrier is fitted into chromatographic column as needed, the bisphenol-A that can as needed prepare different capabilities is affine
Post.The structure of chromatographic column is as shown in Figure 1：
Compared with prior art, the invention has the advantages that：
1. the present invention sufficiently make use of the advantage of aptamer, by the screening and enrichment of many wheels.High specific is obtained
The bisphenol-A aptamer of high-affinity.The aptamers can be in specific combination sample bisphenol-A, effectively eliminate solid
The common impurity of phase extraction column is more, the shortcoming of complicated components.Reduce the cross reactivity that antibody affinity column is frequently encountered.Parent
Increased substantially with column efficiency
Aptamer is not influenceed by operating environment and organic solvent, be especially suitable for Aflatrem liposoluble substance it is pure
Change.Compare, with also in conjunction with specific immune affinity column, because antibody therein not organic solvent-resistant, organic solvent
The inactivation of antibody can usually be caused and make affine column efficiency reduction
Further, since organic solvent causes antibody to inactivate, therefore immune affinity column is usually single use.And aptamer
Affinity column can tolerate organic solvent, can be reused many times, and considerably reduce use cost
2. the aptamer that the present invention is used can be obtained by chemical synthesis, it is ensured that the correctness of sequence.Significantly
Degree reduces the variation between different batches.And comparatively, the antibody of different batches comes from different mouse or rabbit, lead
Qualitative variability is larger between causing antibody, affinity column quality is there is difference
3. easy to operate using present invention purifying bisphenol-A, several steps can be obtained by purity bisphenol-A higher.More convenient operator
Use
4. the bisphenol-A purity for being obtained using product of the invention is very high, subsequently just can be direct without doing other purification process again
For high performance liquid chromatography detection or fluoroscopic examination.Save time and the expense of operator.
Brief description of the drawings
Fig. 1：Bisphenol-A aptamers affinity column structural component schematic diagram
A：Injection port plug；B：Piston adaptor；C：Stable ferrule；D：Upper sieve plate；E：Cylinder；F：Carrier filler；G：Lower sieve plate；H：Go out
Sample mouthful plug
Fig. 2：Bisphenol-A aptamers affinity column appearance assumption diagram
A：Injection port plug；B：Piston adaptor；C：Stable ferrule；D：Upper sieve plate；E：Cylinder；F：Carrier filler； G：Lower sieve plate；H：
Outlet plug
Fig. 3：The Mass Spectrometer Method figure of bisphenol-A detection in water
Fig. 4：The Mass Spectrometer Method figure of bisphenol-A detection in milk powder sample.
Specific embodiment
Embodiment 1：Affinity column is prepared using amido modified bisphenol-A aptamers
The preferred embodiment that the present invention prepares bisphenol-A aptamers affinity column is as follows：
1. support-activated
Selection N-hydroxy-succinamide（NHS）The agarose carrier sepharose4B of modification, is activated
The agarose of 1gN- HOSu NHSs modification is taken, the HCl with 50ml 1mM is added, after swelling 30min, gel is used
100ml 1mM HCl are washed 6 times, then use 100ml milli-Q waters
2. the Ago-Gel sepharose4B coupling buffers that will have been activated（The NaHCO of 0.1M3, 0.8M NaCl,
PH8.2）Washing 3 times.The amido modified bisphenol-A aptamers of 20mol/L, room temperature are added to be coupled 8 hours
3. the phosphate buffer PBS of the bisphenol-A aptamers that will be coupled-agarose carrier 20mM, PH7.4 is washed 3 times
4. close
The Tris.Cl PH8.0 of 100mmol/L, room temperature reaction 2 are added in the bisphenol-A aptamers-agarose carrier that will be coupled
Hour
5. the phosphate buffer PBS of the bisphenol-A aptamers that will have been closed-agarose carrier 20mM, PH7.4 is washed 3 times
6. post is filled
Bisphenol-A-Ago-Gel after crosslinking is resuspended with 10 milliliters of 20mM PBS PH7.4, it is then charged into the affinity purification of sky
In post cylinder
1) cylinder of sky is taken, lower screening deck is added, 1mlPBS is added, it is drained off naturally
2) lower section outlet plug is added, the bisphenol-A aptamers-Ago-Gel carrier after adding the above-mentioned treatment of 1ml in cylinder,
Static 5 minutes, make carrier natural subsidence
3) upper screening deck is added, sieve plate is pressed, it is reached carrier top
4) piston adaptor is added, adapter has a pressuring action, upper screening deck is close to carrier
5) stable ferrule is enclosed within cylinder from below, it is close to injection port
6) outlet plug is pulled out, 5mlPBS is taken with syringe, syringe is connected on injection port, PBS is slowly injected into parent
In post, use liquid speed degree and be maintained at 1-2 drops/sec.Until whole liquid injection affinity columns
7) outlet plug is put, injection port plug is put, the bisphenol-A aptamers affinity column that will be prepared is put into 4 DEG C of preservations.
Embodiment 2：Affinity column is prepared using the bisphenol-A aptamers of biotin modification
The preferred embodiment that the present invention prepares bisphenol-A aptamers affinity column is as follows：
1. 1ml Streptavidins are taken（SA）The Ago-Gel sepharose4B of modification, with 10ml milli-Q waters 3 times
2. will（SA）The Ago-Gel sepharose4B coupling buffers of modification（The PBS of 0.02M, 0.8M NaCl,
PH7.4）Washing 3 times.The bisphenol-A aptamers of the biotin modification of 20mol/L, room temperature are added to be coupled 4 hours
3. the phosphate buffer PBS of the bisphenol-A aptamers that will be coupled-agarose carrier 20mM, PH7.4 is washed 3 times
4. post is filled
Bisphenol-A-agarose carrier is fitted into chromatographic column as needed, the bisphenol-A that can as needed prepare different capabilities is affine
Purification column
1) cylinder of sky is taken, lower screening deck is added, 1mlPBS is added, it is drained off naturally
2) lower section outlet plug is added, the bisphenol-A aptamers-Ago-Gel carrier after adding the above-mentioned treatment of 1ml in cylinder,
Static 5 minutes, make carrier natural subsidence
3) upper screening deck is added, sieve plate is pressed, it is reached carrier top
4) piston adaptor is added, one pressuring action of adapter makes upper screening deck be close to carrier
5) stable ferrule is enclosed within cylinder from below, it is close to injection port
6) outlet plug is pulled out, 5mlPBS is taken with syringe, syringe is connected on injection port, PBS is slowly injected into parent
In post, use liquid speed degree and be maintained at 1-2 drops/sec.Until whole liquid injection affinity columns
7) outlet plug is put, injection port plug is put, the bisphenol-A aptamers affinity column that will be prepared is put into 4 DEG C of preservations.
Embodiment 3：Using the bisphenol-A in the purifying of bisphenol-A aptamers affinity column and water
Then the present embodiment is entered using the standard items that bisphenol-A is added in normal purifying water sample with bisphenol-A aptamers affinity column
Row purifying, uses high performance liquid chromatography detection after purification.Determine the rate of recovery
1. sample treatment
According to every milliliter of standard of water sample 10ng, bisphenol-A standard items are added in water；
Be connected to immune affinity column under 10.0 ml disposable syringes by w
W accurately pipettes the water sample after 10ml adds bisphenol-A standard items, in injection disposable syringe, by air pressure pump and glass
Glass syringe connection regulation switch, makes liquid be flowed out with 1~2 drop/sec of speed
W washs 10mL, then washed with distilled water or deionized water with the 0.1%Tween-20 aqueous solution first after after drain
10mL, 2~3 drops/sec of flow velocity
W connects eluent, 1 drop/sec of flow velocity after after drain, more renewing syringe, loading 2ml acetonitriles with scale test tube；
Liquid nitrogen is blown near dry under the conditions of 30 DEG C after w wash-outs
10% acetonitrile solutions of w dissolve and constant volume is to 1ml
W eluents are transferred to sample bottle and are analyzed for HPLC after being filtered with 0.22 μm of millipore filter
2. eluted product is detected with high-efficient liquid phase chromatogram HPLC
High performance liquid chromatography detection result shows, from testing result as can be seen that adding the bis-phenol of 100ng in 10 milliliters of water
A, can be returned by 92.4ng with aptamers affinity column of the invention, and the rate of recovery is 92.4%
Mass spectrogram is shown in accompanying drawing 3.
Embodiment 4：The bisphenol-A in powdered milk sample is purified and detected using bisphenol-A aptamers affinity column
1. pre-treatment
According to every gram of standard of powdered milk sample 100ng, bisphenol-A standard items are added in powdered milk sample
W takes 1g milk powder samples, adds 100ng bisphenol-A standard items, adds 10ml 10mmolPBSPH7.4 buffer solutions, fully shaking
Mix
W is filtered with fast qualitative filter paper, collects filtrate
W whole filtrates are used as the excessively affine column purification of sample solution
2. purify
Be connected to affinity column under 10.0 ml disposable syringes by w.According to pre-treatment requirement, the sample of respective volume is accurately pipetted
In product extract solution injection disposable syringe, air pressure pump is connected regulation switch with glass syringe, makes liquid with 1~2
Drop/sec speed outflow
W is washed 2 times, each 10mL after after drain with distilled water or deionized water
W after after drain, collect eluent and be settled to 1mL by loading 1mL methyl alcohol, 1 drop/sec of flow velocity
W eluents are transferred to sample bottle and are analyzed for HPLC after being filtered with 0.22 μm of millipore filter
Testing result shows that the addition 100ng bisphenol-A standard items in 1g milk powder reclaim 89.8ng, the rate of recovery 89.8%
Mass spectrogram is shown in accompanying drawing 4.
Embodiment 5：Bisphenol-A aptamers affinity column reuses the change of column capacity
Then the present embodiment is purified using the standard items of quantitative bisphenol-A with bisphenol-A aptamers affinity column.On affinity column
Bisphenol-A with organic solvent elute after, with its concentration of high performance liquid chromatography detection.Affinity column repeats loading and washes after rebalancing
It is de-, determine the bisphenol A concentration of wash-out.Main purpose is to test the reusability of bisphenol-A aptamers affinity column
1. the bisphenol-A standard items of 1ug/ml are prepared
2. bisphenol-A aptamers affinity column is taken out, injection port plug is opened, injection port is connected with injector syringe, and syringe is linked into
On gas control crosshead
3. outlet plug is opened, affinity column is washed 3 times with 10mmol/L PBS PH7.4,10 milliliters every time, regulation stomata operation
Frame air pump pressure, makes liquid be flowed out with 3 drops/sec of flow velocity
4. the bisphenol-A standard items of the above-mentioned preparations of 100ul are taken, total amount 100ng, in adding affinity column post, regulation flow to 1-2 drops/
Second.Until sample all flows out affinity column
5. distillation water washing purification column 3 times, every time 5 milliliters is used
6. 1ml methyl alcohol is added, eluted product is collected
7. eluted product is detected with high-efficient liquid phase chromatogram HPLC
8. affinity column milli-Q water 3 times, then each 10ml washs affinity column 3 times, often with 10mmol/L PBS PH7.4
Secondary 10 milliliters
9. loading 100ul bisphenol-As standard items, total amount 100ng again
10. washed according to aforesaid operations and eluted, eluted product its concentration of high performance liquid chromatography detection
11. repeat step 8.9.10 tri- times.Detect the concentration of eluted product
The rate of recovery of the 12. bisphenol-A aptamers affinity column purifying for calculating 5 times altogether
High performance liquid chromatography detection the results are shown in Table 4：
It can be seen from the results above that after bisphenol-A aptamers affinity column reuses 5 times, its binding ability does not drop significantly
It is low.
Bisphenol-A aptamers sequence 1：
5'-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAGCGCATCACGGGTTCGCACCA-3'
Bisphenol-A aptamers sequence 2：
5'-CCGCCGTTGGTGTGGTGGGCCTAGGGCCGGCGGCGCACAGCTGTTATAGACGTCTCCAGC-3'
Bisphenol-A aptamers sequence 3：
5'-CCGCCGTTGGTGTGGTGGGCCTAGGGCCGGCGGCGCACAGCTGTTATAGACGCCTCCAGC-3'
Claims (12)
1. a kind of affinity column of bisphenol-A, it is characterised in that include carrier and bisphenol-A aptamers.
2. the affinity column according to claim 1, it is characterised in that aptamers are coupled on carrier by chemical bond.
3. the affinity column according to claim 1, it is characterised in that described aptamers are aptamers, more specifically
It is oligodeoxynucleotide aptamers.
4. the aptamer according to claim 3, it is characterised in that described aptamers sequence is the institute of sequence 1,2,3
Any one in the nucleotide sequence of description.
5. aptamers according to described in claim 3, it is characterised in that aptamers are by the aptamers of chemical modification.
6. aptamers according to described in claim 5, it is characterised in that modification mode is including but not limited to amido modified, carboxylic
Base modification, sulfydryl modification and biotin modification.
7. the aptamers affinity column according to claim 1, it is characterised in that described carrier be Ago-Gel or
The solid phase carrier of chemical synthesis.
8. the affinity column according to claim 5, it is characterised in that described Ago-Gel is including but not limited to N- hydroxyls
The succinimide activated Ago-Gel of base, the Ago-Gel of cyanogen bromide-activated, Ago-Gel, the polyacrylic acid of crosslinking
Ago-Gel.
9. the solid phase carrier carrier of the chemical synthesis according to described in claim 5, including but not limited to polystyrene, porous
Polystyrene and cross-linked porous polystyrene filler.
It is 10. a kind of to purify the affine column preparation method of bisphenol-A, it is characterised in that
Carrier after selection N-hydroxy-succinamide activation, carrier dry powder is lived with the hydrochloric acid soaked overnight of 1mmol/L
Change
Every gram of carrier of activation adds the aptamers of 30-200nmol, in 0.1M NaHCO3, the buffer solution of 0.5M NaCl PH8.3
In, room temperature is coupled 2 hours；Or 4 DEG C of couplings are overnight
Coupled product 8.0 room temperature reactions of 1M Tris.HCl PH 2 hours
Carrier-the aptamers being coupled, are washed with the PBS of 20mM PH7.4
Bisphenol-A aptamers-carrier conjugation product is washed with the 10mM PBS PH7.4 containing 0.01% thimerosal, post is filled, 4 are put in
DEG C preserve.
A kind of 11. affinity columns of bisphenol-A, its purposes is the enrichment and purifying to the bisphenol-A in thing to be checked.
12. things to be checked according to claim 11, it is characterised in that thing to be checked is including but not limited to water, former milk and milk powder etc.
Food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710048805.7A CN106731007A (en) | 2017-01-23 | 2017-01-23 | A kind of bisphenol-A aptamers affinity column and its production and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710048805.7A CN106731007A (en) | 2017-01-23 | 2017-01-23 | A kind of bisphenol-A aptamers affinity column and its production and use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106731007A true CN106731007A (en) | 2017-05-31 |
Family
ID=58942222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710048805.7A Pending CN106731007A (en) | 2017-01-23 | 2017-01-23 | A kind of bisphenol-A aptamers affinity column and its production and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106731007A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030064530A1 (en) * | 2001-07-04 | 2003-04-03 | Nitto Denko Corporation | Aptamer capable of specifically adsorbing to bisphenol A and method for obtaining the aptamer |
| CN101031344A (en) * | 2004-07-19 | 2007-09-05 | 格雷斯股份有限两合公司 | Apparatus components and methods of using apparatus components to detect the presence of an analyte |
| US20120015354A1 (en) * | 2009-02-16 | 2012-01-19 | So Youn Kim | Nucleic acid aptamer binder specifically to bisphenol a |
| CN103983774A (en) * | 2014-06-03 | 2014-08-13 | 合肥工业大学 | Probe capable of specifically identifying bisphenol A nucleic acid aptamer and test strip detection application of probe |
| CN104606919A (en) * | 2014-12-31 | 2015-05-13 | 华南师范大学 | Preparation method of novel nucleic acid aptamer/nano-gold modified solid phase microextraction quartz capillary tube |
| CN104730172A (en) * | 2013-12-20 | 2015-06-24 | 中国医学科学院药用植物研究所 | New purification method and testing technology of ochratoxin A in traditional Chinese medicines |
-
2017
- 2017-01-23 CN CN201710048805.7A patent/CN106731007A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030064530A1 (en) * | 2001-07-04 | 2003-04-03 | Nitto Denko Corporation | Aptamer capable of specifically adsorbing to bisphenol A and method for obtaining the aptamer |
| CN101031344A (en) * | 2004-07-19 | 2007-09-05 | 格雷斯股份有限两合公司 | Apparatus components and methods of using apparatus components to detect the presence of an analyte |
| US20120015354A1 (en) * | 2009-02-16 | 2012-01-19 | So Youn Kim | Nucleic acid aptamer binder specifically to bisphenol a |
| CN104730172A (en) * | 2013-12-20 | 2015-06-24 | 中国医学科学院药用植物研究所 | New purification method and testing technology of ochratoxin A in traditional Chinese medicines |
| CN103983774A (en) * | 2014-06-03 | 2014-08-13 | 合肥工业大学 | Probe capable of specifically identifying bisphenol A nucleic acid aptamer and test strip detection application of probe |
| CN104606919A (en) * | 2014-12-31 | 2015-05-13 | 华南师范大学 | Preparation method of novel nucleic acid aptamer/nano-gold modified solid phase microextraction quartz capillary tube |
Non-Patent Citations (1)
| Title |
|---|
| 丁金凤等: "《基因分析和生物芯片技术》", 31 January 2004, 湖北科学技术出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xu et al. | Separation and screening of compounds of biological origin using molecularly imprinted polymers | |
| US8021889B2 (en) | Chromatographic material for the absorption of proteins at physiological ionic strength | |
| CN101865924B (en) | Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay | |
| CN104655759B (en) | A kind of method of aptamers functional magnetic nano material Magneto separate bisphenol-A | |
| US20210178290A1 (en) | Systems and method for detection of analytes in high volumetric flow applications | |
| CN106970172A (en) | A kind of aflatoxin aptamers affinity column and its production and use | |
| CN103160471B (en) | Ochratoxin A hybridoma cell strain, antibody, compound immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit | |
| CN103038631A (en) | Method for recognition and quantification of multiple analytes in a single analysis | |
| WO2007024825A2 (en) | Immunoaffinity separation and analysis compositions and methods | |
| CN107063820A (en) | A kind of T2 toxin aptamers affinity column and its production and use | |
| CN103575826A (en) | Efficient liquid-phase chromatographic detection method for content of polyamines in goose tissue as well as application thereof | |
| CN103949228B (en) | A kind of preparation method of molecular engram magnetic silica gel microball of surface and hydrophilic outer | |
| CN107262074A (en) | A kind of deoxynivalenol aptamers affinity column and its production and use | |
| CN102279232A (en) | Method for efficiently separating and purifying multiresidue sulfonamide antibiotics in biological sample | |
| CN104117227B (en) | A kind of deoxynivalenol immune affinity column and its production and use | |
| EP2522996B1 (en) | Method for analysing aflatoxins in milk and milk by-products | |
| CN106693443A (en) | Vitamin B12 aptamer affinity column, and preparation method and application thereof | |
| CN104174185A (en) | Malachite green immunoaffinity column, and making method and use thereof | |
| CN104784972B (en) | A kind of preparation and its application of aurantiamarin para-immunity affinity column | |
| CN103160472B (en) | Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit | |
| CN105891387B (en) | A kind of enrichment method for being used to detect hydrophilic amino acid | |
| CN106731007A (en) | A kind of bisphenol-A aptamers affinity column and its production and use | |
| CN106984067A (en) | A kind of ochratoxin aptamers affinity column and its production and use | |
| CN112114074B (en) | Lactoferrin aptamer affinity column and preparation method and application thereof | |
| CN106940347A (en) | The method that simultaneous quantitative detects sea-tangle bioconversion broth small molecular blood pressure lowering peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |
|
| RJ01 | Rejection of invention patent application after publication |