CN103130856A - Method for separating beta-estradiol and oestrone based on nanometer channel modified by aptamer - Google Patents
Method for separating beta-estradiol and oestrone based on nanometer channel modified by aptamer Download PDFInfo
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- CN103130856A CN103130856A CN2013100417918A CN201310041791A CN103130856A CN 103130856 A CN103130856 A CN 103130856A CN 2013100417918 A CN2013100417918 A CN 2013100417918A CN 201310041791 A CN201310041791 A CN 201310041791A CN 103130856 A CN103130856 A CN 103130856A
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- aptamer
- nanochannel
- film
- oestrone
- estradiol
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Abstract
The invention discloses a method for separating beta-estradiol and oestrone method based on a nanometer channel modified by an aptamer. A prepared array gold nanoparticle channel is used as a carrier and aptamer modifies beta-estradiol inside the gold nanoparticle channel so as to obtain a gold nanoparticle channel membrane which has special selectivity to the beta-estradiol. A difference of the beta-estradiol and the oestrone is utilized, transfer speed of the beta-estradiol and the oestrone in the nanometer channel which modifies the aptamer, the aptamer has the selectivity to the beta-estradiol, and comparatively passes through the modified nanometer channel, but the oestrone is not easy to pass through, and the separation of the beta-estradiol and the oestrone is achieved. The method can provide a novel idea for conducting compartment analysis to estrogens with different affinity of other aptamers.
Description
Technical field
The present invention relates to a kind of based on the molecular level membrane separation technique on the nanochannel mould material, specifically a kind of based on aptamer modified nanochannel separation beta estradiol and the method for oestrone; Material Field and separation technology field.
Background technology
There is the phenomenon of hormone abuse in edible animal raising and Cosmetics Production, causes microbiotic and hormone residues pollution problem in meat, eggs, Dairy products and makeup.Lot of documents shows, these hormonal substances particularly estrogenic chemicals are taken in human body and may be caused the tumour relevant to internal secretion and fertility defective, damage liver and kidney, also can to the baby and teen-age grow to cause have a strong impact on; In addition, hormone has special beauty functions and is often illegally added in all kinds of makeup by some cosmetics production business, health to consumers in general produces potential threat, so the problem of the oestrogenic hormon serious threat mankind's health day by day becomes the focus that people pay close attention to.
In order to contain the pollution of hormone in foods and cosmetics, strictly control quality product, guarantee to use safety, ensure that the people are physically and mentally healthy, in the urgent need to setting up estrogenic chemicals compartment analysis novel method.The method that often relates to for hormonal substance at present has radioimmunology, euzymelinked immunosorbent assay (ELISA), vapor-phase chromatography and high performance liquid chromatography, utilize these methods that the multiple estrogenic method for separating and analyzing such as trihydroxy-oestrin, estradiol, Norethisterone, oestrone, stilboestrol, Dienestrol and hexestrol in foods and cosmetics are studied, obtained effect preferably.But these methods are complicated, time-consuming, instrument carries inconvenience, and some also needs the sample derivatization treatment, and the cycle is longer.Beta estradiol (β-estradiol, female steroid-1,3,5(10)-triolefin-3,17-isoallopregnane-3β, C particularly
18H
24O
2) and oestrone (estrone, 3-hydroxyl female steroid-1,3,5(10)-triolefin-17-ketone, C
18H
22O
2) these two kinds of natural estrogens, structure is closely similar, and very difficult realization both separates, and therefore, development is accurate, quick, method sensitive and this pollutant of separation detection efficiently is significant to national health and Economic development.
Summary of the invention
The object of the present invention is to provide a kind of method of separating beta estradiol and oestrone based on aptamer modified nanochannel.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of separating beta estradiol and oestrone based on aptamer modified nanochannel, its step is as follows:
1) preparation of gold nano channel membrane:
Adopt chemical deposition deposited gold on porous polycarbonate (PC) film.The PC film is immersed in anhydrous methanol, and ultrasonic concussion 5min is to wash away the impurity that adsorbs on basement membrane and film is activated; Then the PC film after cleaning immerses 0.025mol/L SnCl
2Solution 45min, constantly slight agitation of solutions makes Sn
2+Be adsorbed on equably basement membrane and fenestra surperficial, take out and use water rinse 2 times; At N
2Under the gas protection, film is immersed 0.029M Ag (NH
3)
2 +15min in solution, taking-up washes with water 4 times, soaks 5～10min in methyl alcohol, and immersing concentration is in 5～10mmol/L gold sodium sulfide deposit solution (pH=10); Chemical gilding at 1～4 ℃ of temperature takes out after 5～6 hours and washes 4 times with water, then uses 25%HNO
3Soak 12h and remove unreacted Ag, namely obtain the Au nanochannel arrays.
2) modification of gold nano passage:
Adopt method deposited gold 3h on 50nm porous polycarbonate film of electroless plating, obtain functionalization gold nano channel array; The pH=7.4, the 0.5mmol/L Tris-HCl that the functionalization gold nano channel membrane for preparing are immersed in 1～3 μ mol/L aptamer are hatched 24h in buffered soln, make nanochannel modify upper aptamer; Then use with the buffered soln cleaning modified membrane of concentration and remove the not aptamer molecule of bonding, can obtain the nanochannel film of aptamer functionalization;
3) separated migrates of nanochannel film:
The nanochannel film of aptamer functionalization of preparation is placed in the middle of the U-shaped flow-through cell, the U-shaped flow-through cell is divided into sample inlet pool and diffusion cell, Chi Zhongjun has magnetic stirring apparatus to stir with identical speed, and effective transmission area of film is 1.96cm
2, sample inlet pool and diffusion cell volume are 5mL; Solution to be separated is placed in sample inlet pool, and diffusion cell is placed the blank solution (the Tris-HCl buffered soln that does not contain the same concentration of material to be analyzed) that does not contain material to be separated.
Step 2) in, the aperture of described functionalization gold nano channel array is about 20nm.
Step 2) in, described aptamer be by 25～80 based compositions can with oligonucleotide DNA or the RNA chain of the specific bindings such as protein, polypeptide, metal ion and small molecules.
In step 3), the nanochannel of aptamer functionalization seals with " zero " RunddichtringO.
The present invention carries out the self-assembly of covalent linkage individual layer by golden sulfide linkage.
The present invention be the array gold nano passage for preparing be carrier, modify the aptamer of beta estradiol in the gold nano passage, obtain that beta estradiol is had specific optionally gold nano channel membrane.Utilize the structural difference of beta estradiol and oestrone, both the rate of migration in the nanochannel of having modified aptamer is different, and aptamers has selectivity to beta estradiol, is easier to by the nanochannel after modifying, and oestrone be not easy by, realized separation both.The present invention can be the different oestrogenic hormon of other aptamer affinities of compartment analysis a new thinking is provided.
The aptamer Au nanochannel film of the present invention's preparation has specific selectivity to beta estradiol.Under same experimental conditions, beta estradiol and other compound such as genistein with hydroxyl, Daidezin and dihydroxyphenyl propane are relatively, beta estradiol is maximum by the amount of aptamer modified nanochannel, show that aptamer is treated the selective separation coefficient of separate substance high, sepn process is simple, has application prospect preferably.
The present invention is based on aptamer modified gold nano channel membrane technology, separate beta estradiol and oestrone, development is accurate, quick, method sensitive and this pollutant of separation detection efficiently is significant to national health and Economic development.
Description of drawings
Fig. 1 nanochannel membrane sepn moving apparatus structural representation;
In figure: 1, sample inlet pool 2, diffusion cell 3, the nanochannel film 4 of aptamer functionalization, magnetic stirring apparatus.
Fig. 2 is the FESEM figure of the former film of 50nm PC (Fig. 2 (a)) and the nanochannel (Fig. 2 (b)) that obtains through the 3h depositing time; This film CH
2Cl
2The gold nanotubes TEM(Fig. 2 (c) that obtains after dissolving, Fig. 2 (d)).
Fig. 3 is the migration amount temporal evolution figure in oestrone (E1), the nanochannel of beta estradiol (E2) before modification;
Fig. 4 E1, E2 blending ingredients be migration amount temporal evolution figure in the nanochannel of having modified aptamer (aperture is 20nm approximately);
Five kinds of estrogenic chemicalses of Fig. 5 compare in the infiltration capacity of the nanochannel of having modified aptamer.
Embodiment
Below in conjunction with specific examples, further set forth the present invention.Should be noted that, these examples only are used for explanation the present invention and do not limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's description, these equivalent form of values fall within the application's appended claims limited range equally.
The experimental technique of unreceipted actual conditions in following example, usually according to normal condition, as operational manual, or the condition of advising according to manufacturer.
The mixture of beta estradiol (E2) with oestrone (E1) separated
Compound concentration is 1.7647 * 10
-5The mixing solutions of the beta estradiol of mol/L (E2) and oestrone (E1) (in pH=7.4, Tris-HCl buffered soln), the beta estradiol (E2) and oestrone (E1) mixing solutions that add respectively 8mL to prepare in sample inlet pool add the Tris-HCl buffered soln of the same concentration that does not contain material to be analyzed in seepage basin.Under the effect of stirring, utilize the about nanochannel film in 20nm aperture to carry out both separation.Detect the content of seepage basin material with high performance liquid chromatograph at interval of 2h, test sample number of times totally 5 times, transition time is 12h altogether.
By Fig. 2 (c) and Fig. 2 (d) as seen, adopt the inventive method can get the good gold nanotubes of type shape.
As seen from Figure 3, both several indifferences of travelling speed in the gold nano passage of unmodified.
Fig. 4 and Fig. 3 relatively, the nanochannel of having modified aptamer obviously demonstrates the selectivity to beta estradiol (E2).
As seen from Figure 5, under these estrogenic chemicalses of genistein, Daidezin, dihydroxyphenyl propane and oestrone existed, the nanochannel of having modified aptamer was best to the selectivity of beta estradiol (E2).
Claims (3)
1. method of separating beta estradiol and oestrone based on aptamer modified nanochannel, it is characterized in that: its step is as follows:
1) preparation of gold nano channel membrane:
The PC film is immersed in anhydrous methanol, and ultrasonic concussion 5min is to wash away the impurity that adsorbs on basement membrane and film is activated; Then the PC film after cleaning immerses 0.025mol/L SnCl
2Solution 45min, constantly slight agitation of solutions makes Sn
2+Be adsorbed on equably basement membrane and fenestra surperficial, take out and use water rinse 2 times; At N
2Under the gas protection, film is immersed 0.029M Ag (NH
3)
2 +15min in solution, taking-up washes with water 4 times, soaks 5～10min in methyl alcohol, and immersion concentration is in the gold sodium sulfide deposit solution of 5～10mmol/L, pH=10; Chemical gilding at 1～4 ℃ of temperature takes out after 5～6 hours and washes 4 times with water, then uses 25%HNO
3Soak 12h and remove unreacted Ag, namely obtain the Au nanochannel arrays;
2) modification of gold nano passage:
Adopt method deposited gold 3h on 50nm porous polycarbonate film of electroless plating, obtain functionalization gold nano channel array; The pH=7.4, the 0.5mmol/L Tris-HCl that the functionalization gold nano channel membrane for preparing are immersed in 1～3 μ mol/L aptamer are hatched 24h in buffered soln, make nanochannel modify upper aptamer; Then use with the buffered soln cleaning modified membrane of concentration and remove the not aptamer molecule of bonding, can obtain the nanochannel film of aptamer functionalization;
3) separated migrates of nanochannel film:
The nanochannel film of aptamer functionalization of preparation is placed in the middle of the U-shaped flow-through cell, the U-shaped flow-through cell is divided into sample inlet pool and diffusion cell, Chi Zhongjun has magnetic stirring apparatus to stir with identical speed, and effective transmission area of film is 1.96cm
2, sample inlet pool and diffusion cell volume are 5mL; Solution to be separated is placed in sample inlet pool, and diffusion cell is placed the blank solution that does not contain material to be separated.
2. a kind of method of separating beta estradiol and oestrone based on aptamer modified nanochannel according to claim 1, is characterized in that: step 2) in, the aperture of described functionalization gold nano channel array is about 20nm.
3. according to claim 1 a kind of based on aptamer modified nanochannel separation beta estradiol and the method for oestrone, it is characterized in that: in step 3), the nanochannel of aptamer functionalization seals with " zero " RunddichtringO.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940867A (en) * | 2014-04-11 | 2014-07-23 | 同济大学 | Method for preparing photoelectric adapter sensor for detecting 17beta-estradiol |
| CN104119244A (en) * | 2014-06-27 | 2014-10-29 | 上海师范大学 | DL tyrosine chiral resolution and on-line detection method based on functional nano channel arrays |
| CN110879243A (en) * | 2019-12-04 | 2020-03-13 | 江南大学 | Preparation method and application of functional biological inorganic nano composite membrane |
| CN111705062A (en) * | 2020-07-07 | 2020-09-25 | 华侨大学 | Stilbene estrogen aptamer and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090081427A1 (en) * | 2007-09-21 | 2009-03-26 | Fujifilm Corporation | Method for producing an immobilization substrate and immobilization substrate produced by the method |
| CN101788463A (en) * | 2009-12-30 | 2010-07-28 | 上海师范大学 | Method for separating and detecting atrazine and paraquat |
-
2013
- 2013-02-01 CN CN201310041791.8A patent/CN103130856B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090081427A1 (en) * | 2007-09-21 | 2009-03-26 | Fujifilm Corporation | Method for producing an immobilization substrate and immobilization substrate produced by the method |
| CN101788463A (en) * | 2009-12-30 | 2010-07-28 | 上海师范大学 | Method for separating and detecting atrazine and paraquat |
Non-Patent Citations (1)
| Title |
|---|
| 沈健等: "基于Au纳米通道膜分离测定芦丁", 《化学学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940867A (en) * | 2014-04-11 | 2014-07-23 | 同济大学 | Method for preparing photoelectric adapter sensor for detecting 17beta-estradiol |
| CN103940867B (en) * | 2014-04-11 | 2016-02-17 | 同济大学 | A kind of preparation method of the photoelectricity aptamer sensor for detecting 17 beta estradiols |
| CN104119244A (en) * | 2014-06-27 | 2014-10-29 | 上海师范大学 | DL tyrosine chiral resolution and on-line detection method based on functional nano channel arrays |
| CN104119244B (en) * | 2014-06-27 | 2016-09-07 | 上海师范大学 | Chiral resolution and the method for on-line checking of DL tyrosine is realized based on functional nano channel array |
| CN110879243A (en) * | 2019-12-04 | 2020-03-13 | 江南大学 | Preparation method and application of functional biological inorganic nano composite membrane |
| CN111705062A (en) * | 2020-07-07 | 2020-09-25 | 华侨大学 | Stilbene estrogen aptamer and application thereof |
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| CN103130856B (en) | 2015-07-01 |
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