CN104248863A - Preparation method of aptamer affinity organic-silica hybrid capillary monolithic column - Google Patents
Preparation method of aptamer affinity organic-silica hybrid capillary monolithic column Download PDFInfo
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- CN104248863A CN104248863A CN201410305228.1A CN201410305228A CN104248863A CN 104248863 A CN104248863 A CN 104248863A CN 201410305228 A CN201410305228 A CN 201410305228A CN 104248863 A CN104248863 A CN 104248863A
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Abstract
A preparation method of an aptamer affinity organic-silica hybrid capillary monolithic column. The invention comprises the steps of: first tetramethoxysilane and 3-(methacryloyloxy) propyl trimethoxy silane is used as a precursor, then a thiol modified nucleic acid aptamer and 3-mercapto-1-propane sulfonate is injected into the pretreated capillary, and click chemical reaction of thiol-ene is carried out under mild conditions to prepare the affinity capillary monolithic column. compared with a traditional no-covalent bond binding affinity ligand method, the method provided by the invention has the advantages of low cost, simple preparation and high immobilization stability of affinity ligand. The nucleic acid aptamer affinity organic-silica hybrid monolithic capillary column provided by invention as a chromatography stationary phase can realize the separation, enrichment and purification of thrombin.
Description
[technical field]: the invention belongs to technical field of bioengineering, relate to a kind of affine organic-preparation of silica gel hybridization capillary tube monolithic column, specifically, relate to a kind of preparation fibrin ferment to the affinity capillary integral post of specific recognition.
[background technology]: integral post (monolithic column) has unique structure and excellent characteristic, makes it receive increasing concern as a kind of novel chromatographic stationary phases.Integral post can be divided into Organic Polymer Monolithic Columns, monolithic silica column and organic-silica gel hybridization integral post.The above two also also exist inevitable shortcoming while having series of advantages, and the advantage that organic-silica gel hybridization integral post (Organic-silicaHybridMonolithic Column) combines both as prepare simple, be easy to that modification, mechanical strength are high, the pH wide ranges that is suitable for and solvent resistance good, obtained and applied more and more widely.
Affinity chromatography utilizes to be modified at part chromatographic stationary phases with specific recognition function, there is efficient and single-minded interaction, thus realize the process of sample separation with analyte.It has comparatively high selectivity, has been widely used in now in the separation and concentration of complex sample and quantitative analysis.In sample, the retention time of analysans is determined by the affinity size between analysans and affinity ligand.When affinity between them is more weak, mobile phase can not be changed and just analysans can be eluted; And affinity between them very strong time, then need the composition changing mobile phase to be just able to the analysans wash-out of selective absorption.Aptamer affinity chromatography (Aptamer Affinity Chromatography) has obtained the concern of researcher as a kind of novel chromatographic isolation pattern, be successfully applied to and be separated in micromolecular compound, protein and cell.Aptamer based on cromoci has been combined in by non-covalent bond and Organic Polymer Monolithic Columns matrix has achieved being separated and detection (Zhao Q cromoci and fibrin ferment by the people such as Zhao, Li X F, Le X C.Aptamer Modified Monolithic Capillary Chromatography for Protein Separation and DetectionAnal.Chem.2008,80:3915-3920).
Click chemistry is the reaction that a class has high selectivity, and it has the advantage of some uniquenesses, for example (,) initial reactant is easy to get, productive rate is high, reaction condition is gentle, to oxygen and water insensitive and single product can be obtained.The click chemistry reaction had been found that is if the reaction of the nitrine of monovalence copper catalysis and alkynes, D-A cycloaddition reaction and a series of click-reaction based on mercaptan are as the reaction of mercaptan-alkene, mercaptan-alkynes, mercaptan-isocyanates, mercaptan-bromine.In these reactions, the nitrine of monovalence copper catalysis and being most widely used of alkyne reaction, but the introducing because of its heavy metal catalyst can be polluted response matrix and be reduced biocompatibility, so mercaptan-alkene addition reaction (Thiol-ene Chemistry) alternatively receives the favor of more and more researcher.As the people such as Wang have prepared C by the addition reaction of mercaptan-alkene
18modify organic-silica gel hybridization integral post, benzene and aromatic (Wang K Y are successfully separated, Chen Y Z, Yang H H, et al.Modification of VTMS hybrid monolith via thiol-ene click chemistry for capillary electrochromatography.Talanta, 2012,91:52-59).The people such as Chen use the click-reaction of mercaptan-alkene to prepare enzyme reactor (Chen Y Z based on insulin, Wu M H, Wang K Y, et al.Vmyl functionalized silica hybrid monolith-based trypsin microreactor on line digestion and separationviathiol-ene " click " strategy.JChromatogr A, 2011,1218:7982-7988).The new method of this Fast back-projection algorithm compound, has been successfully applied in the synthesis of new material and the rear modification of material.At present, the relevant report of integral post is not also prepared by the efficient fixed nucleic acid aptamers of the mode of click chemistry.
[summary of the invention]: the object of the invention is the fixing means expanding current aptamer.Traditional fixing means is interacted aptamer to be fixed in integral post matrix by the form of non-covalent bond, and aptamer is combined by covalent bond by this method application click chemical reaction, achieve the fixing means of low cost, high efficiency, high stability.Organic-silica gel hybridization capillary tube monolithic column that the aptamer obtained is affine can realize separation to fibrin ferment, enrichment and purifying.
The preparation method of organic-silica gel hybridization capillary tube monolithic column that aptamer provided by the invention is affine, comprises the following steps:
The first, by tetramethoxy-silicane and 3-(methacryloxypropyl) propyl trimethoxy silicane urea, polyethylene glycol and acetic acid mixing, stir and form homogeneous pre-polymerization liquid in 4 hours under 0 DEG C of ice bath;
The second, the pre-polymerization liquid ultrasonic degas upper step formed, logical nitrogen deoxygenation, injects pretreated capillary, puts into 45 DEG C of baking oven thermal-initiated polymerization reactions, rinse removing pore-foaming agent and unreacted reactant after the sealing of post two ends Rubber end;
Three, the aptamer (30 μMs of sulfydryl modification is got, 50 μ tL), 90 DEG C are heated 3 minutes with again moulding, 3-sulfydryl-1-propane sulfonate and 2 are added after being cooled to room temperature, 2 '-azo diisobutyl amidine dihydrochloride also ultrasonicly to mix, inject the capillary monolithic column obtained, keep 25 DEG C, 5 hours;
Four, rinse the aptamer and the 3-sulfydryl-1-propane sulfonate that remove non-bonding, obtain capillary monolithic column;
Wherein,
Silylating reagent described in the first step is tetramethoxy-silicane and 3-(methacryloxypropyl) propyl trimethoxy silicane; Catalyst is acetic acid; Pore-foaming agent is urea and polyethylene glycol; Action time is 4 hours;
The ultrasonic degas time described in second step is 10min; The letting nitrogen in and deoxidizing time is 10min; Action time is 12 hours;
Function monomer described in 3rd step is aptamer and the 3-sulfydryl-1-propane sulfonate of sulfydryl modification; Initator is 2,2 '-azo diisobutyl amidine dihydrochloride; Described hot initiation conditions is 25 DEG C of room temperature reactions 5 hours.
Volume ratio between described silylating reagent is 2: 1,3: 1,4: 1 or 5: 1; Temperature is 43 DEG C, 45 DEG C or 47 DEG C; The mass ratio of polyethylene glycol and urea is respectively 5: 6,5: 7,1: 1 or 6: 7.
The total consumption of pore-foaming agent is 0.3g, and initiator amount is 0.05mg.
Quartz capillary column described in second step, overall length is 35cm, and detection window is 8.5cm to the length of integral post end, and effective length is 27cm.
Cleaning fluid described in second step is respectively water and methyl alcohol; Cleaning fluid described in 4th step is Tris-HCl buffer solution (50mM, pH7.4).
Advantage of the present invention and good effect:
The affine integral post of covalently immobolization aptamer prepared by the present invention, compared with the affine integral post of traditional non-covalent bond fixed nucleic acid aptamers, has following advantage:
1, preparation process is simple to operation, and cost is low;
2, reaction yield is high, mild condition, no coupling product;
3, affinity ligand immobilization stability is high.
Consider from using value, the affinity capillary integral post of aptamer provided by the present invention, as a kind of chromatographic stationary phases, can realize the separation to fibrin ferment high selectivity, enrichment and purifying.And by changing different aptamers, the range of application of affine integral post can be expanded, realize the compartment analysis to particular organisms protein.
[accompanying drawing explanation]:
Fig. 1-2 is the light microscope figure of the affine hybrid integral post under the ratio of Different Silicon Alkylators, different temperatures, different pore-foaming agent ratio.Fig. 3 is the infrared spectrogram of the affine integral post of aptamer and double bond matrix integral post.
[detailed description of the invention]:
Example 1.
First by silylating reagent: tetramethoxy-silicane 0.45mL and 3-(methacryloxypropyl) propyl trimethoxy silicane 0.15mL (volume ratio 3: 1), pore-foaming agent: polyethylene glycol (0.125g) and urea (0.175g), acetic acid mixes, stir under 0 DEG C of ice bath and form homogeneous pre-polymerization liquid in 4 hours, be injected in pretreated capillary after ultrasonic degas 10min and logical nitrogen deoxygenation 10min, after the sealing of post two ends Rubber end, put into 45 DEG C of baking oven reactions 12 hours.Then pore-foaming agent and unreacted reactant is rinsed out.Get the aptamer (30 μMs of sulfydryl modification, 50 μ L), 90 DEG C are heated 3 minutes with again moulding, 3-sulfydryl-1-propane sulfonate (50mM is added after being cooled to room temperature, 50 μ L) and initator 2,2 '-azo diisobutyl amidine dihydrochloride also ultrasonicly to mix, and injects the capillary monolithic column obtained with syringe pump with the speed of 20 μ L/h, keep 25 DEG C, 5 hours.Finally rinse out aptamer and the 3-sulfydryl-1-propane sulfonate of non-bonding, obtain affinity capillary integral post 1.
Example 2.
Tetramethoxy-silicane 0.4mL and 3-(methacryloxypropyl) propyl trimethoxy silicane 0.2mL (volume ratio 2: 1), other can obtain affinity capillary integral post 2 with example 1.
Example 3.
Tetramethoxy-silicane 0.48mL and 3-(methacryloxypropyl) propyl trimethoxy silicane 0.12mL (volume ratio 4: 1), other can obtain affinity capillary integral post 3 with example 1.
Example 4.
Tetramethoxy-silicane 0.5mL and 3-(methacryloxypropyl) propyl trimethoxy silicane 0.1mL (volume ratio 5: 1), other can obtain affinity capillary integral post 4 with example 1.
Example 5.
Polymeric reaction temperature is 43 DEG C, and other can obtain affinity capillary integral post 5 with example 1.
Example 6.
Polymeric reaction temperature is 47 DEG C, and other can obtain affinity capillary integral post 6 with example 1.
Example 7.
Add pore-foaming agent: polyethylene glycol (0.125g), urea (0.15g), other can obtain affinity capillary integral post 7 with example 1.
Example 8.
Add pore-foaming agent: polyethylene glycol (0.15g), urea (0.15g), other can obtain affinity capillary integral post 8 with example 1.
Example 9.
Add pore-foaming agent: polyethylene glycol (0.15g), urea (0.175g), other can obtain affinity capillary integral post 9 with example 1.
As seen in Figure 1, at 45 DEG C the ratio of silylating reagent be 3: 1 and 4: 1 ratio can obtain full and homogeneous skeleton, and as seen in Figure 2, comparatively 4: 1 more can obtain homogeneous full skeleton at 3: 1, and the integral post shape characteristic obtained when PEG consumption be 0.125g, Urea consumption is 0.175g and permeability best.As seen in Figure 3, the stretching vibration peak of the C=C of the integral post of modification of nucleic acids aptamers obviously reduces, and illustrates that the carbon-carbon double bond overwhelming majority in skeleton all take part in click chemistry reaction.689.92cm
-1and 652.21cm
-1for the vibration peak of C-S key, the aptamer that these peaks all demonstrate sulfydryl modification is reacted by click chemistry in the skeleton being successfully grafted to hybrid integral post.
Claims (5)
1. aptamer affine organic-preparation method of silica gel hybridization capillary tube monolithic column, it is characterized in that, the method comprises the following steps:
The first, by tetramethoxy-silicane and 3-(methacryloxypropyl) propyl trimethoxy silicane, urea, polyethylene glycol and acetic acid mixing, stir and form homogeneous pre-polymerization liquid in 4 hours under 0 DEG C of ice bath;
The second, the pre-polymerization liquid ultrasonic degas upper step formed, logical nitrogen deoxygenation, injects pretreated capillary, puts into 45 DEG C of baking oven thermal-initiated polymerization reactions, rinse removing pore-foaming agent and unreacted reactant after the sealing of post two ends Rubber end;
Three, the aptamer (30 μMs of sulfydryl modification is got, 50 μ L), 90 DEG C are heated 3 minutes with again moulding, 3-sulfydryl 1-propane sulfonate (50mM is added after being cooled to room temperature, 50 μ L) and 2,2 '-azo diisobutyl amidine dihydrochloride also ultrasonicly to mix, and injects the capillary monolithic column obtained, keep 25 DEG C, 5 hours;
Four, rinse the aptamer and the 3-sulfydryl-1-propane sulfonate that remove non-bonding, obtain capillary monolithic column;
Wherein,
Silylating reagent described in the first step is tetramethoxy-silicane and 3-(methacryloxypropyl) propyl trimethoxy silicane; Catalyst is acetic acid; Pore-foaming agent is urea and polyethylene glycol; Action time is 4 hours;
The ultrasonic degas time described in second step is 10min; The letting nitrogen in and deoxidizing time is 10min; Action time is 12 hours;
Function monomer described in 3rd step is aptamer and the 3-sulfydryl-1-propane sulfonate of sulfydryl modification; Initator is 2,2 '-azo diisobutyl amidine dihydrochloride; Described hot initiation conditions is 25 DEG C of room temperature reactions 5 hours.
2. method according to claim 1, is characterized in that the volume ratio between silylating reagent is 2: 1,3: 1,4: 1 or 5: 1; Temperature is 43 DEG C, 45 DEG C or 47 DEG C; The mass ratio of polyethylene glycol and urea is respectively 5: 6,5: 7,1: 1 or 6: 7.
3. method according to claim 1 and 2, it is characterized in that the total consumption of pore-foaming agent is 0.3g, initiator amount is 0.05mg.
4. method according to claim 1 and 2, is characterized in that the cleaning fluid described in second step is respectively water and methyl alcohol; Cleaning fluid described in 4th step is Tris-HCl buffer solution (50mM, pH7.4).
5. method according to claim 1 and 2, is characterized in that the quartz capillary column described in second step, and overall length is 35cm, and detection window is 8.5cm to the length of integral post end, and effective length is 27cm.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107308923A (en) * | 2017-08-03 | 2017-11-03 | 西南大学 | A kind of Stationary Phase of HPLC preparation method and chromatographic column |
| CN107349636A (en) * | 2017-07-12 | 2017-11-17 | 北京大学 | Capillary and its preparation and application of the biomaterial as interaction phase |
| CN108107144A (en) * | 2017-12-29 | 2018-06-01 | 福州大学 | A kind of POSS of aptamer functionalization is crosslinked organic-silica gel hybridization integral post and preparation method thereof |
| CN109337811A (en) * | 2018-10-15 | 2019-02-15 | 天津医科大学 | Eutectic solvent integral post enzyme reactor and preparation method thereof |
| CN110102270A (en) * | 2019-06-03 | 2019-08-09 | 福州大学 | A kind of affine integral post of aptamer of specific recognition F2 toxin and preparation method thereof |
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| CN101306263A (en) * | 2008-01-22 | 2008-11-19 | 广西师范大学 | Alkylamine silica gel capillary monolithic column and preparation method and use thereof |
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| CN101306263A (en) * | 2008-01-22 | 2008-11-19 | 广西师范大学 | Alkylamine silica gel capillary monolithic column and preparation method and use thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107349636A (en) * | 2017-07-12 | 2017-11-17 | 北京大学 | Capillary and its preparation and application of the biomaterial as interaction phase |
| CN107349636B (en) * | 2017-07-12 | 2020-01-10 | 北京大学 | Capillary tube with biological material as interaction phase and preparation and application thereof |
| CN107308923A (en) * | 2017-08-03 | 2017-11-03 | 西南大学 | A kind of Stationary Phase of HPLC preparation method and chromatographic column |
| CN108107144A (en) * | 2017-12-29 | 2018-06-01 | 福州大学 | A kind of POSS of aptamer functionalization is crosslinked organic-silica gel hybridization integral post and preparation method thereof |
| CN109337811A (en) * | 2018-10-15 | 2019-02-15 | 天津医科大学 | Eutectic solvent integral post enzyme reactor and preparation method thereof |
| CN109337811B (en) * | 2018-10-15 | 2021-10-12 | 天津医科大学 | Eutectic solvent monolithic column enzyme reactor and preparation method thereof |
| CN110102270A (en) * | 2019-06-03 | 2019-08-09 | 福州大学 | A kind of affine integral post of aptamer of specific recognition F2 toxin and preparation method thereof |
| CN110102270B (en) * | 2019-06-03 | 2021-06-01 | 福州大学 | Aptamer affinity monolithic column for specifically recognizing F2 toxin and preparation method thereof |
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Application publication date: 20141231 |