CN104147927B - Preparation method of protein-adsorption-resistant covalently-bonded capillary electrophoresis coating coated column - Google Patents
Preparation method of protein-adsorption-resistant covalently-bonded capillary electrophoresis coating coated column Download PDFInfo
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- CN104147927B CN104147927B CN201310608172.2A CN201310608172A CN104147927B CN 104147927 B CN104147927 B CN 104147927B CN 201310608172 A CN201310608172 A CN 201310608172A CN 104147927 B CN104147927 B CN 104147927B
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Abstract
The invention provides a preparation method of a protein-adsorption-resistant covalently-bonded capillary electrophoresis coating coated column. By virtue of electrostatic self-assembly and photo-curing crosslinking reaction of poly(sodium-p-styrenesulfonate) and photopolymer diazoresin, a covalently-bonded protein-adsorption-resistant coating is built on the inner wall of a quartz capillary column. The capillary electrophoresis coating coated column has good protein-adsorption-resistant property, and compared with an uncoated quartz capillary column, the coated column has the advantage that the protein separating property is enhanced by over one time. According to the preparation method, the technological process is simple, the production efficiency is high, the production cost is low, the repeatability is good, nontoxic and environmental-friendly effects are achieved, and large-scale production is facilitated; the covalently-bonded capillary electrophoresis coating coated column has good chemical stability and protein-adsorption-resistant property and is applicable to electrophoretic separation detection and purification of biological samples of protein, glycoprotein, peptide chain, hormone, enzyme and the like.
Description
Technical field
The present invention relates to the preparation method of anti-protein adsorption capillary electrophoresis covalent bonding coating column, is specifically a kind of
Using the electrostatic self-assembled and photocured cross-linked reaction of Sodium Polystyrene Sulfonate and photopolymer diazo resin in quartzy capillary
The method that the anti-protein adsorption coating of covalent bonding is constructed on the inwall of tubing string.
Background technology
As capillary electrophoresis technique is in the development in each field of life science, antagonism protein adsorption capillary electrophoresis are applied
The coating performance of layer post requires also more and more higher, and preferable coating should both maintain the modification to capillary wall, suppression
Protein processed will not fall off and polluted to combination equipment again in the absorption of tube wall.With adsorbed in capillary with physics or ionic forces
Non-covalent bonding coatings on inside pipe wall are compared, and are by the permanent coating of covalent bonding advantage in this respect：Covalent bond
The coating life length of conjunction, good stability, separation efficiency be high, difficult for drop-off causes unnecessary dirt to the combination equipment such as mass spectrograph
Dye.
In recent years, by the use of the anti-protein adsorption covalent bonding coating column prepared by silylating reagent as coupling agent in capillary
It is widely used in electrophoresis tube.But its shortcoming is：Preparation process is complicated, is related to capillary tube silanization and trigger monomer
The multistep reactions such as polymerization；The toxicity of silylating reagent used is very big in preparation, very sensitive to the moisture content in solvent, and water easily occurs
Solution generates silicon dioxide granule and causes various coating quality problems；Polyreaction in capillary tube also is difficult to control, easily makes
Inhomogeneity, irregularity into coating, even block capillary tube.Such as：(1) Sun etc. exists《Biochemical
Engineering Journal》Magazine 2010,53,137-142 is reported using polyvinyl alcohol (PVA) and silylating reagent system
For the capillary electrophoresis covalent bonding coating column of anti-protein adsorption.Manufacturing process is divided into pretreatment, silylating reagent
Multiple steps such as coating, introducing functional molecular containing aldehyde radical, polymer coating acetalation；(2) Timperman etc. exists
《Analytical Chemistry》Magazine 2006,78,4326-4333 is reported using the silanization containing Polyethylene Glycol (PEG)
Reagent is prepared for the capillary core chip electrophoresis covalent bonding coating column of anti-protein adsorption.Preparation process is loaded down with trivial details, time-consuming longer, used
Silylating reagent there is toxicity, facile hydrolysiss, cost intensive.Sodium Polystyrene Sulfonate (PSS) is applied as a kind of non-covalent bonding
Layer material has also obtained certain application in capillary electrophoresis, such as：(3) Schlenoff etc. exists《Analytical
Chemistry》Magazine 1999,71,4007-4013 reports logical quiet using PSS and methyl diallyl ammonium chloride (PDADMAC)
The method of electric self assembly is prepared for the anti-protein adsorption coating of non-covalent bonding on capillary tube inner wall, but its shortcoming is coating
Cannot be by covalent bonding on capillary tube inner wall, short life, stability are poor.
The preparation method of above listed capillary electrophoresis covalent bonding coating column, universal technological process is complicated, operates bar
Part is harsh, and production process toxicity is larger, and production efficiency is low, and production cost is higher, prepared anti-protein adsorption capillary electrophoresis
The quality of covalent bonding coating column is unstable, limits their applications in Separation of Proteins analysis.
The content of the invention
It is an object of the invention to provide a kind of preparation for simplifying anti-protein adsorption capillary electrophoresis covalent bonding coating column
Method, solution preparation technology flow process is complicated, operating condition is harsh, production process toxicity is big, low production efficiency, production cost are high,
The not good problem of prepared anti-protein adsorption capillary electrophoresis covalent bonding coating column quality.
The technical scheme is that：A kind of preparation method of anti-protein adsorption capillary electrophoresis covalent bonding coating column,
It is characterized in that by the use of water miscible photographic diazoresin as a kind of coupling agent of positively charged by electronegative polystyrene sulphur
By electrostatic interaction LBL self-assembly on the inwall of quartz capillary column, diazo resin is same after ultraviolet lighting for sour sodium
When with quartz capillary inwall on silicone hydroxyl and the sulfonic group of Sodium Polystyrene Sulfonate there is photocured cross-linked reaction, so as to
Stable diazo resin-Sodium Polystyrene Sulfonate composite construction covalent bonding coating is formed on quartz capillary inwall.
The molecular weight of the diazo resin used by the present invention is 400 to 100000, and molecular weight is too low, then self assembly paintability
Not good, the too high then water solublity of molecular weight is bad, and preferred molecular weight is 750 to 5000.
The molecular weight of the Sodium Polystyrene Sulfonate used by the present invention is 5000 to 600000, and molecular weight is too low, then self assembly
Paintability is not good, and the too high then water solublity of molecular weight is bad, and preferred molecular weight is 10000 to 100000.
The concentration of diazo resin and Sodium Polystyrene Sulfonate aqueous solution used by the present invention is 0.01mg/ml extremely
1000mg/ml, concentration is too low, then self assembly paintability is not good, and the too high then mobility of concentration is deteriorated, and preferred concentration is
0.1mg/ml to 100mg/ml.
The temperature of the diazo resin used by the present invention-Sodium Polystyrene Sulfonate LBL self-assembly at 1 to 50 DEG C, self assembly temperature
Spend low, then aqueous solution easily freezes, and self assembly temperature is too high, and diazo resin is easily pyrolyzed, and preferred self assembly temperature is 1
To 35 DEG C.
The number of plies of the diazo resin used by the present invention-Sodium Polystyrene Sulfonate LBL self-assembly composite structure coating 2 to
12 layers, very little, then anti-protein adsorption performance is not good for the number of plies, and too much then production efficiency is reduced the number of plies, and preferred LBL self-assembly is answered
The number of plies for closing structure coating is 2 to 8 layers.
In 193nm to 400nm, exposure dose is 5mJ/cm to uv-exposure optical source wavelength used by the present invention2Extremely
20000mJ/cm2.Exposure light source wavelength is too short or oversize, and exposure dose is too high or too low, can all cause coating photocured cross-linked
Quality Down, in 248nm to 365nm, exposure dose is 30mJ/cm to preferred uv-exposure optical source wavelength2To 6000mJ/
cm2。
Compared with prior art, it is an advantage of the current invention that：
(1) adopt the method for the present invention, without using high toxicity, expensive silylating reagent as coupling agent, letter
The preparation flow of capillary electrophoresis covalent bonding coating column is changed, process equipment is simple, reproducible, raw materials used to be easy to get, raw
Produce low cost, production process environmental protection；
(2) adopting the self assembly coating procedure of the method for the present invention, coating can be carried out in water phase, be overcome due to silane
The coating quality problem changed reagent itself hydrolysis and cause, the covalent bonding of coating can utilize the light of photopolymer diazo resin
Curing cross-linking reaction easily and reliably realizes, is less prone to the quality problems such as coating shedding and capillary blockage；
(3) the inventive method prepare anti-protein adsorption capillary electrophoresis covalent bonding coating column, can be used for protein,
The occasion such as the electrophoretic separation detection of the biological sample such as glycoprotein, peptide chain, hormone, enzyme and purification.
Description of the drawings
Accompanying drawing is typical anti-protein adsorption covalent bonding coating column and the naked post of quartz capillary to four kinds of common proteins
The comparison of electrophoretic separation performance.
Specific embodiment
The present invention is described in further detail with reference to embodiments.
Separation of Proteins performance is measured by CL-1020 type capillary electrophoresis apparatus, four kinds of protein (Ox blood serums in analyte
Albumen, lysozyme, cytochrome C, ribonucleotidase A) concentration be 0.5mg/ml, ultraviolet detection wavelength is 214nm,
Covalent bonding coating column is 41cm, internal diameter and is 75 μm with the effective length of the naked post of quartz capillary, and column temperature is 25 DEG C, point
Ionization voltage is+15kV, and separating medium is the phosphate buffered solution (pH=3.0) of 40mM concentration.
Embodiment 1
At 1 DEG C, by the new naked post of quartz capillary first with the diazo resin (molecular weight 400) that concentration is 0.01mg/ml
Aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again.Then with the Sodium Polystyrene Sulfonate that concentration is 0.01mg/ml
(molecular weight 5000) aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again, this completes 1 electrostatic self-assembled
Circulation.Capillary tube is dried up with compressed air after 1 circulation of assembling, is placed under the uviol lamp that wavelength is 193nm and is exposed, expose agent
Measure as 5mJ/cm2, you can form the anti-protein adsorption covalent bond with 2 layers of composite construction of diazo resin-Sodium Polystyrene Sulfonate
Close coating column.Because the silicone hydroxyl on quartz capillary inwall is shielded with interlayer generation covalent cross-linking reaction is applied, therefore should
Covalent bonding coating column has good anti-protein adsorption performance.
Embodiment 2
At 50 DEG C, by the new naked post of quartz capillary first with the diazo resin (molecular weight that concentration is 1000mg/ml
100000) aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again.Then with the polyphenyl second that concentration is 1000mg/ml
Alkene sodium sulfonate (molecular weight 600000) aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again, this completes 1
Electrostatic self-assembled is circulated.Repeat to dry up capillary tube with compressed air after 6 circulations of assembling, be placed in wavelength for the ultraviolet of 400nm
Expose under lamp, exposure dose is 20000mJ/cm2, you can formed and there are 12 layers of composite junction of diazo resin-Sodium Polystyrene Sulfonate
The anti-protein adsorption covalent bonding coating column of structure.Because there is covalent cross-linking with interlayer is applied in the silicone hydroxyl on quartz capillary inwall
React and shielded, therefore the covalent bonding coating column has good anti-protein adsorption performance.
Embodiment 3
At 20 DEG C, by the new naked post of quartz capillary first with the diazo resin (molecular weight 750) that concentration is 0.1mg/ml
Aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again.Then with the Sodium Polystyrene Sulfonate that concentration is 0.1mg/ml
(molecular weight 10000) aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again, this completes 1 electrostatic from group
Dress circulation.Repeat to dry up capillary tube with compressed air after 5 circulations of assembling, be placed under the uviol lamp that wavelength is 248nm and expose,
Exposure dose is 30mJ/cm2, you can form the anti-albumen with 10 layers of composite construction of diazo resin-Sodium Polystyrene Sulfonate and inhale
Attached covalent bonding coating column.Because the silicone hydroxyl on quartz capillary inwall is shielded with interlayer generation covalent cross-linking reaction is applied
Cover, therefore the covalent bonding coating column has good anti-protein adsorption performance.
Embodiment 4
At 35 DEG C, by the new naked post of quartz capillary first with the diazo resin (molecular weight 5000) that concentration is 100mg/ml
Aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again.Then with the Sodium Polystyrene Sulfonate that concentration is 100mg/ml
(molecular weight 100000) aqueous solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again, this completes 1 electrostatic from group
Dress circulation.Repeat to dry up capillary tube with compressed air after 4 circulations of assembling, be placed under the uviol lamp that wavelength is 365nm and expose,
Exposure dose is 6000mJ/cm2, you can form the anti-albumen with 8 layers of composite construction of diazo resin-Sodium Polystyrene Sulfonate and inhale
Attached covalent bonding coating column.Because the silicone hydroxyl on quartz capillary inwall is shielded with interlayer generation covalent cross-linking reaction is applied
Cover, therefore the covalent bonding coating column has good anti-protein adsorption performance.
Embodiment 5
At 25 DEG C, by the new naked post of quartz capillary first with diazo resin (molecular weight 2000) water that concentration is 1mg/ml
Solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again.Then with the Sodium Polystyrene Sulfonate (molecule that concentration is 1mg/ml
50000) aqueous solution is rinsed 5 minutes amount, then uses distilled water flushing 1 minute again, be this completes 1 electrostatic self-assembled and is followed
Ring.Repeat to dry up capillary tube with compressed air after 2 circulations of assembling, be placed under the uviol lamp that wavelength is 300nm and expose, exposure
Dosage is 2000mJ/cm2, you can form the anti-protein adsorption with 4 layers of composite construction of diazo resin-Sodium Polystyrene Sulfonate and be total to
Valence link closes coating column.Because the silicone hydroxyl on quartz capillary inwall is shielded with interlayer generation covalent cross-linking reaction is applied, because
This covalent bonding coating column has good anti-protein adsorption performance.
Embodiment 6
At 30 DEG C, by the new naked post of quartz capillary first with diazo resin (molecular weight 5000) water that concentration is 5mg/ml
Solution is rinsed 5 minutes, then uses distilled water flushing 1 minute again.Then with the Sodium Polystyrene Sulfonate (molecule that concentration is 2mg/ml
20000) aqueous solution is rinsed 5 minutes amount, then uses distilled water flushing 1 minute again, be this completes 1 electrostatic self-assembled and is followed
Ring.Repeat to dry up capillary tube with compressed air after 3 circulations of assembling, be placed under the uviol lamp that wavelength is 365nm and expose, exposure
Dosage is 4000mJ/cm2, you can form the anti-protein adsorption with 6 layers of composite construction of diazo resin-Sodium Polystyrene Sulfonate and be total to
Valence link closes coating column.Because the silicone hydroxyl on quartz capillary inwall is shielded with interlayer generation covalent cross-linking reaction is applied, because
This covalent bonding coating column has good anti-protein adsorption performance.
Claims (5)
1. a kind of preparation method of anti-protein adsorption capillary electrophoresis covalent bonding coating column, it is characterised in that using water miscible
Electronegative Sodium Polystyrene Sulfonate is passed through electrostatic interaction layer by photographic diazoresin as a kind of coupling agent of positively charged
Layer self assembly on the inwall of quartz capillary column, after ultraviolet lighting diazo resin simultaneously with quartz capillary inwall on
There is photocured cross-linked reaction in the sulfonic group of silicone hydroxyl and Sodium Polystyrene Sulfonate, so as to form steady on quartz capillary inwall
Fixed diazo resin-Sodium Polystyrene Sulfonate composite construction covalent bonding coating, described diazo resin-Sodium Polystyrene Sulfonate
The temperature of LBL self-assembly is at 1 to 50 DEG C；In 193nm to 400nm, exposure dose is 5mJ/ to described uv-exposure optical source wavelength
cm2To 20000mJ/cm2。
2. preparation method according to claim 1, it is characterised in that the molecular weight of described diazo resin 400 to
100000, the concentration of aqueous solution is 0.01mg/ml to 1000mg/ml.
3. preparation method according to claim 1, it is characterised in that the molecular weight of described Sodium Polystyrene Sulfonate exists
5000 to 600000, the concentration of aqueous solution is 0.01mg/ml to 1000mg/ml.
4. preparation method according to claim 1, it is characterised in that described diazo resin-Sodium Polystyrene Sulfonate is layer by layer
The number of plies of self assembly composite structure coating is at 2 to 12 layers.
5. the preparation method according to Claims 2 or 3 or 4, it is characterised in that Optimal technique process is：Diazo resin point
Son amount is 750 to 5000, and the molecular weight of Sodium Polystyrene Sulfonate is 10000 to 100000, diazo resin and polystyrolsulfon acid
The concentration of sodium water solution is 0.1mg/ml to 100mg/ml, the temperature of electrostatic self-assembled at 1 to 35 DEG C, LBL self-assembly coating
The number of plies be 2 to 8 layers, in 248nm to 365nm, exposure dose is 30mJ/cm to uv-exposure optical source wavelength2To 6000mJ/cm2。
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| CN107402248B (en) * | 2016-05-18 | 2019-06-25 | 青岛大学 | A kind of renewable capillary of Thermo-sensitive, preparation method and application |
| CN107398094A (en) * | 2016-05-19 | 2017-11-28 | 青岛大学 | A kind of capillary column of anti-protein adsorption and preparation method thereof |
| CN107449819B (en) * | 2016-06-06 | 2020-12-22 | 青岛大学 | Anti-protein adsorption capillary column and preparation method thereof |
| CN107469407B (en) * | 2016-06-14 | 2019-11-08 | 青岛大学 | The capillary column and preparation method thereof of anti-protein adsorption |
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